Keywords: Plasmodium falciparum, cerebral malaria, uncomplicated malaria, var genes, domain cassette, transcript abundance, cytoadherence, CD36, ICAM-1, CSPG

Aims of the study 42

Chapter (2)

Manuscript (1) Gene transcriptomic pattern of Plasmodium falciparum in children with cerebral malaria and asymptomatic carriers 44

Chapter (3)

Acknowledgment

First of all and from the depth of my heart I should thank those, who were always beside me despite the huge distance between us. Without your spiritual support and encouragement I would never ever attain this goal. Thank you my lovely family.

I would like to thank my advisor, Dr. Rachida Tahar who provided me with necessary knowledge that I needed for my work.

I wish to express my sincere thanks to my thesis committee: Dr. George Snounou, Dr. Frédéric Ariey, Dr. Marie-José Butel and Dr. Benoît Gamain, who have accepted my invitation to be members of my thesis jury.

Thanks to Dr. Nicaise Ndam Tuikue, who helped me a lot during my missions in Benin and with whom the discussion was always interesting.

I am grateful to Dr. Philippe Deloron, who welcomed me to his laboratory and without his advices I could not be able to accomplish the statistical analysis in the second part of my thesis.

I place on record my gratitude to all the unit members in France and Benin for the nice moments we spent together especially during "Pot de Noël": Célia, Alexandra, Justin, Samad, Sèm, Firmine, Kossiwa, Azzizzath, Charles, Simon, Florentin, Pierre, Gwladys, Violetta, Géraud, Julie, Laure, Kadiatou, Emmanuelle, Aicha, Adrian, Sayeh, Pascale, Murielle, Jean-Gerard, Anaïs, Jérôme, Audrey, Nadine, Florence, Roman, Valérie, Michel, Isabelle, Jean Yves, Achille, André, Gilles, Magalie, Brigitte, Carmen, Ibrahim, Komi, Odilon, Aurax, Rodolphe, Atikath, Adicath, Pépin, Rafiou, Carine, Nadia, Bienvenu, Darius, Bienvenu, Manfred, Aziz, Honoré, Silvestre, Patrick, Erasme, Silvain, Paulin, Sévérin, Joseph, Jacques, Pépin, Ginette and Sophie.

Immense thanks to all the patients and their parents who made this thesis possible.

Summary

Plasmodium falciparum is the most deadly species, causing broad spectrum of disease manifestations ranging from asymptomatic infection to symptomatic outcomes, including uncomplicated and severe malaria. This latter comprises various clinical forms, notably cerebral malaria. The biological processes involved in the heterogeneity of these clinical outcomes are not well understood. We hypothesized that differential gene expressions contribute to phenotypic variation of parasites leading to specific interaction with the host which subsequently induces several clinical categories of malaria. In order to verify this hypothesis, we investigated the transcriptomes of parasites isolated from Cameroonian children with asymptomatic (AM), uncomplicated (UM), and cerebral malaria (CM). We also analyzed the transcriptomes of the 3D7 line and the selected 3D7-Lib line. The latter has been selected by several rounds of incubation with anti-IgG of Liberian hyperimmune plasma. It has the ability to express group A var and neighboring rif genes, which have been described in several studies to be associated with severe disease and cerebral malaria.

Our results of transcriptomic study revealed the up-regulation of several genes in CMassociated isolates and the 3D7-Lib line compared to AM-associated isolates, and the 3D7 line. Gene Ontology term analysis shows that a majority of differentially expressed genes are mainly implicated in pathogenesis, cytoadherence to host endothelial tissues, and erythrocyte aggregation. Among up-regulated genes in CM-associated isolates compared to AM-associated isolates, there were genes encoding for surface antigens, Maurer's cleft proteins, transcriptional factors, and antigens involved in protein transport to the host cell surface. In addition, an important variation in the expression of variant surface polymorphic families like var, rif and phist genes has been observed. The most remarkable outcomes were the up-regulation of UPS A and UPS B var genes containing architectural Domains Cassettes DC4, DC5, DC13, and DC8 and their neighboring rif genes in isolates from CM and in the 3D7-Lib line compared with isolates from AM and the unselected 3D7 line, respectively.

Several studies have shown that group A and B var genes containing DC13 and DC8 are implicated in severe malaria, including cerebral malaria. However, the transcriptome profiles were similar in CM and UM isolates, and between AM and UM parasites. The lack of differences in genes expression between CM and UM parasites may be due to similar transcription patterns between isolates of these two clinical groups, or to the inability of microarrays to detect trivial variations. Therefore, in order to confirm the specific transcription profiles of these genes in CM isolates, we examined by RT-qPCR the expression abundance of UPS A1, A3, and B1 var groups found to be highly up-regulated in the CM group, using new samples collected from Beninese children with CM or UM. These genes were significantly highly transcribed in CM compared to UM group. The involvement of these genes in the parasite virulence rises from the ability of their encoded proteins to mediate the cytoadherence of infected erythrocytes to post capillary endothelial cells. Cytoadherence is essential for parasite life to evade spleen-dependent clearance. However, the clinical consequences of this cytoadherence differ among infected individuals. Several endothelial receptors that are involved in the cytoadherence of IEs to venular endothelia have been identified, and few studies attempted to correlate a particular binding phenotype of parasites with a given clinical presentation of malaria. Of these receptors, CD36 and Inter Cellular Adhesion Molecule-1 (ICAM-1) were found as the most commonly used by patient parasites. The implication of these two receptors carried out along with PfEMP-1 ligands in the pathogenesis of cerebral malaria needs to be more elucidated. We examined the adhesive phenotype and the transcription patterns of Pfemp-1 variants of fresh isolates from Beninese children with cerebral malaria and uncomplicated malaria by static binding assay and RT-qPCR respectively, to identify a relationship between the Pfemp-1 variants expressed by the parasites and their cytoadherence phenotype to a specific receptor. The binding levels of IEs were investigated to CD36, ICAM-1 and CSPG (CSA) and the transcription level was assessed for groups A, B, var2, var3, DC8, and DC13, up-regulated in CM isolates in our transcriptomic study.

Our findings reported for the first time that isolates from CM patients have a significant preference for CD36-binding compared with those from UM cases. No differences were observed in binding levels to ICAM-1 or CSPG between these two groups. Furthermore, cerebral malaria isolates transcribed groups A, B, var2, var3, DC8 and DC13 of var genes to higher levels than UM isolates. Interestingly, the high transcription levels of group B in CM parasites correlated with their raised level of binding to CD36, supporting the implication of this receptor along with group B in cerebral malaria pathogenesis. In contrary, the expression profiles of a specific var group and the binding phenotype of isolates to ICAM-1 and to CSPG were not correlated.

The molecular basis of interaction between CD36 and PfEMP-1 variants of group B need to be further investigated for better understanding of cerebral malaria pathogenesis. This interaction could be targeted by specific antibodies or suitable agonist molecules to prevent P. falciparum severe complicated malaria as cerebral malaria.

In addition to PfEMP1 group A1, A3 and B1 subset, other genes encoding exported proteins as well as non-exported proteins, were up-regulated in CM-associated parasites. The potential role of these genes in CM pathogenesis deserves to be investigated and may allow the identification of new targets for malaria control. Hippocrates was one of the first to notice and describe malaria symptoms such as poor health, fever and enlarged spleens in the 5 th century B.C. The first documented treatment against malaria was brought from Peru in the 1600s A.C by the Jesuits. The natives at that time were using the Cinchona barks as a traditional treatment which then had been transported to Europe under the name of "Jesuit's powder" to treat patients experiencing fever "agues" (Fig. 1). In 1820, Pelletier and Caventou, French pharmacists, were able to successfully isolate the quinine and the quinidine from Cinchona barks that were used later by French workers in Senegal and in the United States of America.

The discovery of malaria parasite was made in 1880 by Alphonse Laveran, a French surgeon in the French army posted in Constantine city, Algeria. He observed parasites in blood samples of patients with fever that were absent in healthy individual blood. He also noticed the disappearance of parasites after administration of quinine. These observations encouraged Laveran to identify and describe the causative agent of malaria, "the parasite". For this precious finding, Laveran has been attributed Nobel prize of medicine in 1907.

In 1885, Marchiafava, an Italian physician, named the genus of malaria parasites "Plasmodium", and this was followed by the identification of P. vivax and P. malariae by Camillo Golgi and P. falciparum by Marchiafava. The exflagellation property of male gametocytes and the ability to enter inside female gametocyte of P. falciparum parasites has been recognized by MacCallum in 1897. The fourth species of human malaria parasites P. ovale had been identified in 1922 by Stephens.

The incrimination of mosquito in malaria transmission was made in 1897, when Ronald Ross in India observed different stages of development such as oocytes in the stomach wall as well as sporozoites in the salivary glands inside mosquitoes that had fed on infected birds, and independently by Grassi in Italy who incriminated Anopheles for the transmission of P. falciparum. Ross was was awarded the Nobel Prize of medicine in 1902.

The sporogonic cycle of Plasmodium parasites inside the female mosquito was observed in 1899 by Grassi, Bastianelli and Bignami. Using blood samples from naïve volunteers infected with mosquito fed on malarial patients, they were able to describe the life cycle inside the anopheles and the biological cycle of P. falciparum, P. vivax and P. malariae. In 1948, Shortt and Garnham discovered the pre-erythrocytic stage of Plasmodium parasites, which takes place inside the human host. Finally, in 1982, Krotoski was able to characterize the hypnozoite or the latent phase of P. cynomolgi and then of P. vivax [START_REF] Krotoski | Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection[END_REF].

The discovery of natural infections by a 5 th species, Plasmodium knowlesi, came late in 2007 after cross infection from Macaca monkey to human being. During the past years, hundreds of cases had been diagnosed with malaria caused by P. knowlesi infection. This latter morphologically resembles to P. malariae, However, P. knowlesi infections, unlike those of P. malariae, can be fatal to humans [START_REF] Cox | History of the discovery of the malaria parasites and their vectors[END_REF].

Malaria burden worldwide

Malaria, the implacable enemy, is one of the most serious health problems in the world (Fig. 2). In 2010, the World health organization estimated 216 million acute cases of malaria in 106 endemic countries and up to 655,000 deaths annually (WHO 2011). The highest rate of malaria cases (81%) and deaths (91%) took place in sub-Saharan Africa, though other continents including Latin America, the Middle East and Asia are at risk as well [START_REF] Murray | Global malaria mortality between 1980 and 2010: a systematic analysis[END_REF]. Children and pregnant women in endemic regions bear the brunt. While the majority of deaths occur among children under five years of age, most surviving patients suffer from grave complications, mainly severe anemia. Pregnancy associated malaria (PAM) has undesirable birth consequences such as premature delivery, spontaneous abortion, still birth and low birth weight [START_REF] Steketee | The burden of malaria in pregnancy in malaria-endemic areas[END_REF]. This disease affects negatively on the economic growth rate of endemic areas, where it is estimated to decrease by 1.3% annually compared to that in malaria-free countries [START_REF] Sachs | The economic and social burden of malaria[END_REF]. Thus, malaria has social and economic impact on needy people and indigent populations who are not able to afford treatment costs or have restrained access to public health care [START_REF] Orem | Impact of malaria morbidity on gross domestic product in Uganda[END_REF]. Insecticidal bed nets and indoor residual spraying (pyrethroids-vector control) and anti malarial drugs against parasites (notably artemisinins) have significantly favored the reduction of malaria cases and death rate (WHO, 2011). However, these strategies have been thwarted by the emergence of resistance in both mosquitoes and parasites, and by human migration and the recent financial crisis [START_REF] Leach-Kemon | The global financial crisis has led to a slowdown in growth of funding to improve health in many developing countries[END_REF]. All these causes are imminent threats to individuals living in endemic regions (WHO 2010). Another strategy to fight against malaria is a vaccine, which, if available, could decrease the malaria incidence by protecting the population at high risks (children and women) from the serious complications of disease; thereby it would reduce the medical expenses and support the reconstructive and developmental projects in the affected countries. However, all the vaccine candidates tested during the last decade were not sufficiently efficient [START_REF] Snounou | The vaccine is dead-long live the vaccine[END_REF].

Malaria burden in Benin

The republic of Benin is localized in West Africa, bordered by Nigeria on the East, Togo on the West, Burkina Faso and Niger on the north and the Atlantic Ocean on the south (Fig. 3). The surface area of Benin is 112,600 km² with a population density estimated to 10, 100,000 habitants (WHO, 2013). The climate is tropical in the south and soudanian in the north with two distinct dry and rainy seasons. The rainfall is not stable; it varies between regions and over the years with a range between 1400 mm in the south and around 800 mm in the north (Fink et al, (9) MPETUS Atlas du Bénin; http://www.impetus.uni-koeln.de/fr/impetusatlas/impetus-atlas-benin). In Benin, 100% of people are at risk of malaria (WHO, 2013). In 2006, the estimated death rate in infants under 5 years was 125 deaths/1000 live births (Demographic and Health Survey, 2006; http://dhsprogram.com/pubs/pdf/FR197/FR197.pdf). Plasmodium falciparum is the principal cause of malaria, transmitted by three major mosquito vectors: An. gambiae and An. melas and An. funestus [START_REF] Akogbeto | Infectivity of Anopheles melas vis-a-vis Plasmodium falciparum in the coastal lagoon area of Benin][END_REF][START_REF] Moiroux | Changes in Anopheles funestus biting behavior following universal coverage of long-lasting insecticidal nets in Benin[END_REF]. The epidemiological studies have been concentrated in the coastal areas south of Benin, where the economic and the administrative capitals are situated. Cotonou is the economic capital, and it is characterized by two rainy seasons from April to July and from September to November during which malaria infection is exclusively caused by P. falciparum parasites with approximately 33 infected bites per individual annually [START_REF] Akogbeto | Entomological study on the malaria transmission in coastal and lagoon areas: the case of a village built on a brackish lake][END_REF] (Fig. 3). In this city, the average parasite density in children is from 3000 to 6000 parasites/mm 3 and less than 1000 parasites/mm 3 in adults (Chippaux et al. 1991). A study conducted at CNHU hospital in Cotonou, showed that 20% of medical visits was due to malaria and 44% of cases were children between 6-23 months [START_REF] Boulard | A study of malaria morbidity in a pediatric hospital service in Benin (West Africa) in 1988 and 1989[END_REF]). Another study reported that the mean number of fever episodes occurred every year in Beninese children under 3 years was 2.4 per children and 33% of those cases were infected with P. falciparum with an estimated mortality rate of 8/1000 cases yearly [START_REF] Velema | Malaria morbidity and mortality in children under three years of age on the coast of Benin, West Africa[END_REF].

Part of my thesis work was carried out in Cotonou where field isolates had been collected from three hospitals located in this city, as it is described in the Materials and Methods in the second manuscript. (http://www.pmi.gov/countries/profiles/benin.html)

Human Plasmodium species

The etiological agent of malaria is a eukaryotic protozoan parasite of the genus Plasmodium belonging to the apicomplexan family. Parasites are transmitted to human beings by Anopheles mosquitos' bites. To date, more than 400 species of anopheline mosquitoes have been distinguished, but only 40 are recognized as malaria vectors [START_REF] Service | Community participation in vector-borne disease control[END_REF]. Some of these mosquitoes e.g. Anopheles gambiae, prefer human being to animals so-called "anthropophilic", making them highly active malaria transmitters to human hosts. There are five common species of Plasmpdium that could infect humans: P. falciparum (Welch 1887) (Schaudin 1802), P. vivax (Grassi and Feletti 1890), P. malariae (Grassi and Feletti 1892), P. ovale (Stephens 1922) and P. knowlesi which has recently been identified in Malaysia as the 5 th human infecting species after cross infection from Macaca monkey [START_REF] Sabbatani | The emerging of the fifth malaria parasite (Plasmodium knowlesi): a public health concern?[END_REF]). Among these species, P. falciparum is the most virulent species since it is associated with severe malaria and high mortality in sub-Saharan Africa.

Plasmpdium falciparum life cycle

The Plasmodium falciparum life cycle is complex, and the parasite needs to develop in two different hosts to complete its cycle. The infection is initiated when infected female mosquito bites an individual and inoculates sporozoites with the saliva into the skin. Within one hour the parasites move to the liver and invade hepatocytes. The sporozoites spend 6-10 days inside hepatocytes during which they differentiate and undergo asexual replication yielding upto ~40,000 merozoites that then burst from the infected liver cell into the blood stream. The merozoites invade erythrocytes and go through several morphological stages, beginning with the ring and followed with the trophozoite stages, where the parasites ingest part of erythrocyte cytoplasm and degrade its hemoglobin into amino acids to be used for protein synthesis. Schizonts are produced after multiple round of nuclear multiplication resulting in 4-32 daughter merozoites that are released into the circulation after the infected erythrocyte (IE) bursts. Each merozoite is able to infect a new erythrocyte within 30 seconds and reinitiates the cycle. The duration of the intra-erythrocytic stage of P. falciparum is 48 hours, and clinical symptoms of malaria, fever and chills correlate with IEs bursting. After 10-11 days, another form of parasites can be found in the peripheral blood, namely gametocytes. In fact, some of erythrocytic parasites do not develop into schizonts and instead they differentiate into sexual forms, male and female gametocytes, which are not harmful to human host, but are infectious to mosquitoes [START_REF] Mouchet | Biodiversité du paludisme dans le monde[END_REF][START_REF] Tilley | The Plasmodium falciparum-infected red blood cell[END_REF]. Once in the mosquito midgut, following a blood meal, the parasites encounter environmental change, low temperature, modified pH and blood digestive factors such as chymotrypsins and serine proteases. After 30 minutes, the gametocytes mature, where the males transform into eight-motile microgametes and the females into round shaped macrogametes that then emerge from IEs and fertilize to produce a zygote. At this stage the genome of the parasite is diploid and recombination events can lead to an extensive genetic diversity. The sexual life cycle continues inside the mosquito and the zygote pursues its development, as it becomes motile ookinete that enters the stomach wall and resides between basal lamina and epithelial cells. This is followed by the differentiation into an oocyte containing thousands of parasites harboring haploid genomes. The mature oocyte bursts and releases thousand of sporozoites that then migrate to the mosquito salivary glands where they are ready to be injected into a human host [START_REF] Cox | History of the discovery of the malaria parasites and their vectors[END_REF][START_REF] Vizioli | Blood digestion in the malaria mosquito Anopheles gambiae: molecular cloning and biochemical characterization of two inducible chymotrypsins[END_REF]. (1) Sporozoites injected by mosquito, migrate through the circulatory system to the liver, where they invade hepatocytes, multiply then released to blood stream as merozoites which are able to invade erythrocytes. Parasites enter asexual intraerythrocytic multiplications (2), which take 48 hours for P. falciparum. Each schizont gives birth to 30 daughter merozoites. Once erythrocyte ruptures, these merozoites infect other erythrocytes. Some merozoites differentiate to male and female gametocytes, which are taken by mosquito with blood meal, to be fertilized, differentiate and replicate resulting in sporozoites stages ready to infect human host. Source: Modified from [START_REF] Bousema | Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination[END_REF].

Selected clinical outcomes of P. falciparum malaria infection

The clinical manifestations of P. falciparum malaria vary from asymptomatic infection to uncomplicated malaria and severe malaria including cerebral malaria. The disease symptoms range from fever to severe complications.

Mild form of malaria is mainly manifested as fever and sometimes is accompanied with gastro-intestinal troubles, while severe or complicated malaria consists of life threatening disease. The most vulnerable populations are individuals with non-developed immunity such as children and travelers in endemic areas, or people with reduced acquired immunity like primigravidae women. In malaria endemic regions, asymptomatic infections with low parasite densities are prevalent.

The clinical consequences of malaria take place during the asexual blood cycle of parasite life cycle. The clinical manifestations of malaria are classified as uncomplicated malaria (UM); severe malaria (SM) including severe malaria anemia (SMA) and cerebral malaria (CM). Malaria infection in pregnant women is considered as complicated malaria and known as pregnancy associated malaria (PAM).

During my PhD thesis i have been interested in P. falciparum genetic factors implicated in the pathogenesis of falciparum malaria. For that, i used isolates from Cameroun and Benin collected by our team from children less than six years of age. These children presented distinct clinical manifestation such as CM and UM. A group of children with asymptomatic infections had been collected and used as well.

Uncomplicated malaria (UM)

The most frequent features of uncomplicated malaria infection are chills, fever, sweat, headache, asthenia, weariness, myalgia, nausea and vomiting due to the liberation of hemozoin and the toxic bilirubin followed IE rupture. In case of delayed or subcurative treatment, the high temperature and the high parasite density would worsen the clinical case.

Treatment of uncomplicated malaria aims to eliminate the parasites from the body as rapidly as possible in order to prevent the progression of disease to severe form. An adequate treatment of the infection can attenuate its transmission to others and reduce the emergence of resistance to antimalarial agents.

The first line treatment for infants and young children with UM is artemisinin-based combination therapy (ACTs). A combination of two or more antimalarial medicines with different mechanisms of action and contrasting pharmacokinetics such as artemether / lumefantrine, artesunate / amodiaquine, artesunate / mefloquine, artesunate / sulfadoxinepyrimethamine, and dihydrortemisinin / piperaquine is the treatment of choice for uncomplicated falciparum malaria. Alternative therapy such as artesunate / tetracycline or doxycycline or clindamycin, quinine plus tetracycline or doxycycline or clindamycin should be administered for seven days (WHO, 2010).

Severe malaria (SM)

In tropical endemic areas, severe malaria occurs primarily in children under 5 years of age whereas in industrialized countries, non-immune adults returning back from endemic zones are susceptible to develop severe malaria [START_REF] Hatz | Malaria--chemoprophylaxis 2001[END_REF][START_REF] Leder | Malaria in travelers: a review of the GeoSentinel surveillance network[END_REF]. Several predisposing factors participate in this type of malaria and may lead to death, involving age more than 65 years, naïve non immune cases, female sex (especially pregnant), lack of prophylaxis, delay in medical treatment, degree of disease severity at admission and coexisting other health problems [START_REF] Bruneel | The clinical spectrum of severe imported falciparum malaria in the intensive care unit: report of 188 cases in adults[END_REF][START_REF] Schwartz | Age as a risk factor for severe Plasmodium falciparum malaria in nonimmune patients[END_REF]). Severe P. falciparum infections cover a broad spectrum of complications, defined by the WHO as coma; generalized convulsions; cardio-vascular collapse; hemorrhagic syndrome; severe anemia (Hb<6 g/dl); pulmonary edema; hypoglycemia (<2.2 mmol/l); hypouresis (<400 ml/day); hemoglobinuria; and acidosis (PH <7.25). Of these, severe anemia, respiratory distress and cerebral malaria are the major grave outcomes of severe malaria (WHO, 2000 S1). In the absence of appropriate treatment, any of these complications could evolve rapidly and become fatal within hours or days.

Severe malaria anemia (SMA) It is the most common clinical complication of severe P. falciparum malaria and the main cause of hospitalization of children in endemic areas. Severe anemia is characterized by arthralgia, paleness and low hemoglobin level <5 g/dl with parasite density > 10.000 p/µl (WHO, 2000 S1). This is probably a consequence of multiple causes such as erythrocyte hemolysis, suppressed synthesis of erythrocytes [START_REF] Phillips | The importance of anaemia in cerebral and uncomplicated falciparum malaria: role of complications, dyserythropoiesis and iron sequestration[END_REF]) and increased spleen clearance for both uninfected and infected erythrocytes [START_REF] Dondorp | Red blood cell deformability as a predictor of anemia in severe falciparum malaria[END_REF].

Respiratory distress (RD)

This outcome is possibly due to fluid retention and it is described by unusual or frequent deep breathing as a result of metabolic acidosis. This latter is enhanced by the low level of oxygen as a consequence of anemia and decreased microvascular blood flow arising from parasite sequestration and rosetting (Kaul et al.1991;[START_REF] Marsh | The pathogenesis of severe malaria in African children[END_REF].

Cerebral malaria (CM)

Patient with this form is characterized by last long unrousable coma accompanied with multiple seizures; as a result of blood brain barrier deterioration [START_REF] Adams | Breaking down the blood-brain barrier: signaling a path to cerebral malaria?[END_REF] and fever that may surpass 41°C. In the case of cerebral malaria, careshould be taken to exclude other causes of coma, notably meningitis. This form is responsible for 15-20% of infant mortality and is considered as the principle cause of long-term neurocognitive impairments in African children [START_REF] Idro | Cerebral malaria: mechanisms of brain injury and strategies for improved neurocognitive outcome[END_REF]. In order to evaluate the state of consciousness of infected patient, the World Health Organization has adopted Glasgw coma scale (score between 6-7) for adult infected patients and Blantyre coma scale for children< 5 years of age. Blantyre score ranges from 0-5 and children with score ≤ 2 are diagnosed as cerebral malaria cases (Table 1, WHO 2000). The main goal of the treatment of severe malaria is to prevent death. In cerebral malaria cases, it aims to prevent neurological deficit.

In the case of cerebral malaria, parenteral full doses of any effective antimalarial drugs should be given as rapidly as possible. For adults and children artesunate IV or IM is indicated for a minimum of 24 hours. If it is not available, artemether or quinine is acceptable. After that, treatment should be completed with one of the following agents: ACT; artesunate plus clindamycin or doxycycline; or quinine plus clindamycin or doxycycline.

A pre-referral treatment such as rectal artesunate, quinine IM or artesunate IM could be given, and then patient should be referred immediately to an appropriate health care for further treatment (WHO, 2010) Pregnancy associated malaria (PAM)

In spite of the acquired immunity before pregnancy; pregnant women in malaria endemic zones are highly susceptible to malaria infections particularly during the first pregnancy where immunity decreases temporarily. During this period the placenta of pregnant woman offers a convenient environment for parasites with high affinity to placental receptors. P. falciparum parasites via VAR2CSA, the predominant parasite surface-expressed PfEMP1 protein in PAM, have an elevated capacity to adhere to chondroitin sulfate A (CSA) in the syncytiotrophoblasts of the placenta (Achur et al. 2008), provoking blood flow obstruction and disturbing the key source of nutrition for the fetus [START_REF] Dorman | Impaired uteroplacental blood flow in pregnancies complicated by falciparum malaria[END_REF]. This phenomenon results in maternal anemia which itself has undesirable consequences as maternal death, premature delivery, spontaneous abortion, still birth and low birth weight (<2.500g) [START_REF] Steketee | The burden of malaria in pregnancy in malaria-endemic areas[END_REF].

The principal objective of treatment in pregnancy associated malaria case, is to save the life of the mother. During the first trimester, administration of quinine plus clindamycin for seven days has been indicated by the World Health Organization. In case of treatment failure, a combination of artesunate plus clindamycin for 7 days is indicated. During the second and third trimesters, sulfadoxine/pyrimethamine combination is recommended as a profylaxis. However, a treatment artemesinin combination therapy (ACT) is indicated.

The virulence of P. falciparum is partially due the capacity of IEs to cytoadher to endothelial cells of microvasculature of deep organs leading to obstraction of these vessels and organs disfunctions.

Cytoadherence property of P. falciparum

In order to pursue its erythrocyte life cycle, P. falciparum parasite shows an amazing ability to trick the host immune system and evade spleen-dependent clearance. One such phenomenon is called "cytoadherence" [START_REF] Udeinya | Falciparum malaria-infected erythrocytes specifically bind to cultured human endothelial cells[END_REF]. It occurs once the erythrocytic parasites reach tthe mature stages (i.e. late-trphozoite and schizont), where they disappear from the peripheral blood and adhere to different venular endothelial cells of various organs including the brain [START_REF] Macpherson | Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration[END_REF][START_REF] Pongponratn | Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study[END_REF].

This sequestration of parasitized erythrocytes has sequels. It enhances the release of soluble mediators and activates the caspase pathways which in turn lead to vascular, neuronal and tissue apoptosis and blood brain barrier damage (BBB) in some cases of malaria [START_REF] Pino | Plasmodium falciparum-infected erythrocyte adhesion induces caspase activation and apoptosis in human endothelial cells[END_REF][START_REF] Wilson | Soluble factors from Plasmodium falciparum-infected erythrocytes induce apoptosis in human brain vascular endothelial and neuroglia cells[END_REF]). In addition, the accumulation of infected erythrocytes results in microvascular clogging, ischemia, hypoxia, lactic acidosis and activation of inflammatory cytokines which increase endothelial cell adherence molecules (eCAM) and accelerate the adherence of additional populations of infected erythrocytes (van der [START_REF] Van Der Heyde | A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction[END_REF]).

However, the impact of sequestration on the host depends on host immunity, the affected tissues and parasite density.

During pregnancy-associated malaria the target of parasite sequestration is the placenta. Histological analysis of parasitized placenta showed inflammatory lesions with fibrinoid deposition leading to chronic intervillositis. This correlates with negative consequences such as spontaneous abortion, decreased intrauterine growth and increased fetal death [START_REF] Rota | Perinatal prognosis of pregnancies complicated by placental chronic intervillitis[END_REF].

In cerebral malaria the preferred niche of IEs is the brain microvasculature. There is mounting evidence that cytoadherence of IEs to brain microvascular endothelia is linked to cerebral malaria, which was confirmed by postmortem studies [START_REF] Molyneux | Cerebral malaria in children: clinical implications of cytoadherence[END_REF]). Analysis of brain tissues from 24 Thai patients who died of malaria revealed that the post capillary venules of the brain were packed with IEs compared to the other parts of the body including the lungs and the heart [START_REF] Pongponratn | Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study[END_REF]. Similarly, another post mortem study of 21 Vietnamese individuals reported that accumulation of IEs within the microvascular endothelium of the brain was correlated with microvascular congestion and that was associated with premortem coma and shorter time to death [START_REF] Ponsford | Sequestration and microvascular congestion are associated with coma in human cerebral malaria[END_REF].

The most important mediators of cytoadherence are variant surface antigens (VSA), mainly PfEMP-1 members of VAR family. These proteins are exposed at the surface of IEs allowing them to cling to various post capillary endothelial cells [START_REF] Baruch | Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes[END_REF].

The P. falciparum parasite has different ways to cytoadhere within the host. For that new terms have been coined to describe these mechanisms. The first one is known as "Rosetting", where the infected erythrocytes bind spontaneously to uninfected erythrocytes and form rosettes resembling aggregates [START_REF] Udomsangpetch | Plasmodium falciparuminfected erythrocytes form spontaneous erythrocyte rosettes[END_REF]. Rosettes in blood group A or B infected blood are larger and stronger that those in group O blood [START_REF] Rowe | Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting[END_REF][START_REF] Udomsangpetch | The effects of hemoglobin genotype and ABO blood group on the formation of rosettes by Plasmodium falciparum-infected red blood cells[END_REF]. Several lines of evidence have indicated that rosetting phenomenon is associated with severe malaria in sub-saharan Africa [START_REF] Carlson | Human cerebral malaria: association with erythrocyte rosetting and lack of anti-rosetting antibodies[END_REF][START_REF] Treutiger | Rosette formation in Plasmodium falciparum isolates and anti-rosette activity of sera from Gambians with cerebral or uncomplicated malaria[END_REF], but not in South East Asia [START_REF] Ho | Rosette formation of Plasmodium falciparum-infected erythrocytes from patients with acute malaria[END_REF].

A comparative study of 79 Malian isolates from children with different clinical complications of severe malaria, has reported a contribution of rosetting in severe malaria pathogenesis compared to 91 patients with uncomplicated malaria [START_REF] Doumbo | High levels of Plasmodium falciparum rosetting in all clinical forms of severe malaria in African children[END_REF]. Similarly, another study of 111 isolates of Kenyan children with severe malaria and mild malaria found an increased capacity to form rosettes and giant rosettes among isolates from children with severe malaria compared to mild cases [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF]). Horata et al identified a semiconserved feature represented by a position of limited variability named (cyc/PolV) that is located in DBLα of PfEMP-1 and associated with high rate of rosettes formation in both severe malaria and mild malaria groups [START_REF] Horata | Sequence variation of PfEMP1-DBLalpha in association with rosette formation in Plasmodium falciparum isolates causing severe and uncomplicated malaria[END_REF]).

The second term is "Clumping" where infected erythrocytes bind to platelets and form clumps [START_REF] Pain | Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria[END_REF]. In vivo, this phenomenon could promote blood vessels occlusion and it is found to be associated with severe malaria [START_REF] Mayor | Association of severe malaria outcomes with platelet-mediated clumping and adhesion to a novel host receptor[END_REF], particularly cerebral malaria since platelets express CD36 at their surfaces and induce the parasites binding even in absence of venular endothelial CD36 in the brain, thus they act as a bridge between brain endothelium and IEs. This mechanism of sequestration probably reorients the cytoadherence property of parasites and takes part in cerebral malaria pathogenesis [START_REF] Wassmer | Platelets reorient Plasmodium falciparum-infected erythrocyte cytoadhesion to activated endothelial cells[END_REF]). It has been suggested that thrombocytopenia could confer a protective mechanism for the host during cerebral malaria by decreasing platelet-mediated clumping of IEs [START_REF] Wassmer | Platelet-induced clumping of Plasmodium falciparum-infected erythrocytes from Malawian patients with cerebral malariapossible modulation in vivo by thrombocytopenia[END_REF].

Additional terms to express the distinct mechanisms of cytoadherence ability of infected erythrocyte comprise "binding to cells of immune system" such as plasma B cells, IgM, lymphocytes, monocytes, and dendritic cells [START_REF] Donati | Identification of a polyclonal B-cell activator in Plasmodium falciparum[END_REF][START_REF] Ockenhouse | Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor[END_REF][START_REF] Urban | Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells[END_REF])

Receptors contribute to cytoadherence of infected erythrocyte

A diverse array of endothelial receptors that are likely to participate in the adherence of IE to microvasculature endothelia has been identified. Several investigations attempted to describe a specific binding phenotype of isolates from severe malaria patients that is less frequent in uncomplicated malaria parasites. So far, no definitive candidate has been identified apart from chondroitin sulfate proteoglycan (CSPG) also known as CSA. It is believed that CSA is the main receptor of the VAR2CSA protein that is expressed at the surface of IE and can adhere to the syncytiotrophoblasts leading to placental sequestration of IEs during malaria in pregnant women [START_REF] Fried | Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta[END_REF][START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF].

Other potential receptors are: Thrombospondin [START_REF] Roberts | Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence[END_REF]; Cluster Differentiation 36 (CD36) [START_REF] Barnwell | A human 88-kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes[END_REF][START_REF] Oquendo | CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes[END_REF]; Inter Cellular Adhesion Molecule-1 (ICAM-1) [START_REF] Berendt | Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum[END_REF] ; Vascular Cell Adhesion Molecule-1 (VCAM-1) [START_REF] Ockenhouse | Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus[END_REF]); Complement Receptor-1 (CR-1) [START_REF] Rowe | P. falciparum rosetting mediated by a parasitevariant erythrocyte membrane protein and complement-receptor 1[END_REF]; Platelet endothelial Cell Adhesion Molecule-1 (PECAM-1) [START_REF] Treutiger | PECAM-1/CD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes[END_REF]); Integrin αvβ3 [START_REF] Siano | Short report: Plasmodium falciparum: cytoadherence to alpha(v)beta3 on human microvascular endothelial cells[END_REF]); Pselectin [START_REF] Ho | Characterization of Plasmodium falciparum-infected erythrocyte and P-selectin interaction under flow conditions[END_REF]; Heparan sulfate [START_REF] Vogt | Heparan sulfate on endothelial cells mediates the binding of Plasmodium falciparum-infected erythrocytes via the DBL1alpha domain of PfEMP1[END_REF]; Fractalkine [START_REF] Hatabu | Binding of Plasmodium falciparum-infected erythrocytes to the membrane-bound form of Fractalkine/CX3CL1[END_REF]; Fibronectin [START_REF] Eda | Plasmodium falciparum-infected erythrocytes bind to the RGD motif of fibronectin via the band 3-related adhesin[END_REF]); Neural Cell Adhesion Molecule (NCAM) [START_REF] Pouvelle | Neural cell adhesion molecule, a new cytoadhesion receptor for Plasmodium falciparum-infected erythrocytes capable of aggregation[END_REF]); gC1qR-HABP1-P32 [START_REF] Biswas | Plasmodium falciparum uses gC1qR/HABP1/p32 as a receptor to bind to vascular endothelium and for platelet-mediated clumping[END_REF]) and Endothelial Protein C Receptor (EPCR) [START_REF] Turner | Severe malaria is associated with parasite binding to endothelial protein C receptor[END_REF]). Most of these receptors have not been intensively explored and the role of P. falciparum ligands with each receptor in severe malaria is not well established.

Among these candidates, CD36 and ICAM-1 have been extensively investigated and were shown to be the most commonly used by parasite isolates [START_REF] Udomsangpetch | Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1[END_REF]. However, our knowledge about the implication of these receptors in cytoadherence of IE comes from the human genetic polymorphisms associated with malaria severity as well as from cytoadherence assays under static or flow conditions by employing recombinant proteins coated on plastic dishes, cell line, receptor-bound beads or fluorescently labeled proteins.

Cluster Differentiation 36 (CD36)

This transmembrane glycoprotein (88-KD) is encoded by cd36 gene on chromosome 7 and expressed on various cell types including phagocytes, microvascular endothelia; platelets; hepatocytes; retinal pigment epithelium; adipocytes and specialized epithelia of breast, kidney and gut. Thus, a broad spectrum of biological processes would be expected. It acts as anti-angiogenic factor; cellular receptor for thrombospondin, collagen, modified low-density lipoproteins and long-chain fatty. It is also involved in sensory perception [START_REF] Silverstein | CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior[END_REF]. The binding site in CD36 for P. falciparum ligands is located within 97-110 and 139-184 amino acids regions, as suggested by monoclonal antibodies and peptides against this protein [START_REF] Baruch | CD36 peptides that block cytoadherence define the CD36 binding region for Plasmodium falciparum-infected erythrocytes[END_REF][START_REF] Daviet | Characterization of two vaccinia CD36 recombinant-virus-generated monoclonal antibodies (10/5, 13/10): effects on malarial cytoadherence and platelet functions[END_REF]. The parasite ligands for this receptor are PfEMP-1 variants [START_REF] Baruch | Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1[END_REF] encoded by group B and C of var genes through their N-terminal CIDR1α domain [START_REF] Miller | Definition of the minimal domain of CIDR1alpha of Plasmodium falciparum PfEMP1 for binding CD36[END_REF][START_REF] Robinson | Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome[END_REF].

CD36 is considered as the major host receptor for P. falciparum field isolates [START_REF] Chaiyaroj | Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor[END_REF][START_REF] Newbold | Receptor-specific adhesion and clinical disease in Plasmodium falciparum[END_REF], though its pivotal role in malaria pathogenesis is still unclear. Whereas several laboratory assays did not note differences in CD36-binding patterns between field isolates from severe and mild malaria cases living in Africa [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF][START_REF] Mayor | Association of severe malaria outcomes with platelet-mediated clumping and adhesion to a novel host receptor[END_REF][START_REF] Rogerson | Cytoadherence characteristics of Plasmodium falciparum-infected erythrocytes from Malawian children with severe and uncomplicated malaria[END_REF], others have supported a correlation between uncomplicated malaria and binding to CD36 [START_REF] Newbold | Receptor-specific adhesion and clinical disease in Plasmodium falciparum[END_REF][START_REF] Ochola | Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome[END_REF]. Conversely, researchers in Asian endemic regions have shown that CD36 may play a part in malaria severity [START_REF] Ho | Rosette formation of Plasmodium falciparum-infected erythrocytes from patients with acute malaria[END_REF][START_REF] Udomsangpetch | Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1[END_REF] as it facilitates the binding of high proportion of parasites throughout the body, promoting systemic infection that contributes to the severity of the disease. The role of CD36 in the interaction between IEs and host immune system is elusive. While, adherence of IEs to macrophages via CD36 induces phagocytosis and enhances IEs clearance, [START_REF] Mcgilvray | Nonopsonic monocyte/macrophage phagocytosis of Plasmodium falciparum-parasitized erythrocytes: a role for CD36 in malarial clearance[END_REF], binding to dentritic cell (DC) via CD36 yields to DC dysfunction decreasing the adaptive immune response, thus exacerbating the infection [START_REF] Urban | A role for CD36 in the regulation of dendritic cell function[END_REF]. However, other finding suggests that IEs-binding to CD36 is not necessary to inhibit DC maturation; instead high parasite density can induce immunosuppression [START_REF] Elliott | Inhibition of dendritic cell maturation by malaria is dose dependent and does not require Plasmodium falciparum erythrocyte membrane protein 1[END_REF].

It has been shown that CD36-binding is not involved in the rosetting process [START_REF] Rowe | Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates[END_REF]; however, it is essential but not sufficient for the clumping phenomenon [START_REF] Arman | A method for positive and negative selection of Plasmodium falciparum platelet-mediated clumping parasites and investigation of the role of CD36[END_REF].

1.5.2.1. Role of cd36 gene polymorphism in malaria Several studies suggested an important role of human gene polymorphism in the malaria severity but these investigations have reported contradictory conclusions.

In Thailand, a correlation between cd36 polymorphisms and protection against severe malaria has been detected. The frequencies of 14T to C allele in the upstream promoter region and 53G to T in the downstream promoter region of exon 8, were significantly higher in mild malaria than those of cerebral malaria cases, suggesting that such cd36 polymorphism may protect against cerebral malaria. Similarly, the frequency of repeat 12TG in intron 3 [in3 (TG) 12], which is linked with CD36 deficiency, was higher in uncomplicated malaria than in cerebral malaria cases. This polymorphism could alter the binding affinity of CD36 isoform to IEs, leading to decrease the risk of cerebral malaria [START_REF] Omi | CD36 polymorphism is associated with protection from cerebral malaria[END_REF].

In Africa however, researches led to confounding outcomes. Non-sense substitution mutation of T to G in cd36 gene at nucleic acid 188 of exon 10 leading to CD36 deficiency was found to bestow protection against severe malarial anemia (SMA), probably through decreased binding of IEs to microvascular endothelia [START_REF] Chilongola | CD36 deficiency protects against malarial anaemia in children by reducing Plasmodium falciparum-infected red blood cell adherence to vascular endothelium[END_REF][START_REF] Pain | Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria[END_REF]). However, this finding contradicted with another study of African and Middle Eastern populations in which no correlation between CD36 deficiency and protection from severe malaria was observed [START_REF] Fry | Positive selection of a CD36 nonsense variant in sub-Saharan Africa, but no association with severe malaria phenotypes[END_REF]). These results concluded that the existence of distinct cd36 mutations in Africans and Asians populations might be related to alternative or additional selection pressures on cd36 and coevolution between human host and parasite.

Inter Cellular Adhesion Molecule-1 (ICAM-1 CD54)

It is a transmembrane glycoprotein (90-115-KD) and a member of the immunoglobulin like superfamily. It is constituted of five immunoglobulin (Ig)-like domains. This protein is found as a dimer at the surface of cells and its N-terminal Ig-like domain displays binding sites for fibrinogen, the Lymphocyte Function-associated Antigen-1 (LFA-1), cell-entrance site of human rhinouvirous and P. falciparum PfEMP-1. This latter is situated on the BED side in the core of ICAM-1 dimer (Fig. 6) [START_REF] Jun | Dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion[END_REF][START_REF] Ockenhouse | Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus[END_REF]). Thus, ICAM-1 takes part in recognition and adherence between cells.

The parasite ligands for this receptor are PfEMP-1s via DBLβ-C2 of group B, C and DBLβ3 of group A [START_REF] Bengtsson | A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies[END_REF][START_REF] Howell | Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors[END_REF][START_REF] Oleinikov | High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies[END_REF]. ICAM-1 is expressed at a restricted level on several types of tissues such as leucocytes, vascular endothelial cells, mucous epithelia of the tonsils, hepatic sinusoids lining cells, spleen, thymus epitheliums, and some fibroblasts [START_REF] Dustin | Induction by IL 1 and interferongamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1)[END_REF]. Upon pro-inflammatory cytokine stimulation e.g. TNF-α, IL-6 and INF-y, ICAM-1 is up-regulated in various cell lineage including vascular endothelial cells, macrophages and leucocytes. The activated leucocytes bind to ICAM-1/LFA-1 and transmigrate in to the sites of inflammation [START_REF] Caldenhoven | Stimulation of the human intercellular adhesion molecule-1 promoter by interleukin-6 and interferon-gamma involves binding of distinct factors to a palindromic response element[END_REF].

Postmortem brain histopathology examination has reported high levels of ICAM-1 and colocalaization with IEs in cerebral blood vessels [START_REF] Turner | An immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration[END_REF]. Furthermore, during malaria infection, ICAM-1 was found to be up-regulated on endothelial cells [START_REF] Turner | Systemic endothelial activation occurs in both mild and severe malaria. Correlating dermal microvascular endothelial cell phenotype and soluble cell adhesion molecules with disease severity[END_REF]. However, in vitro cytoadherence assays led to contradictory outcomes about the implication of this receptor in malaria pathology. While several outcomes did not note any difference in binding pattern of ICAM-1 between parasites form severe and mild cases [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF][START_REF] Rogerson | Cytoadherence characteristics of Plasmodium falciparum-infected erythrocytes from Malawian children with severe and uncomplicated malaria[END_REF], others pointed to an association between disease severity and binding to ICAM-1 [START_REF] Newbold | Receptor-specific adhesion and clinical disease in Plasmodium falciparum[END_REF][START_REF] Ochola | Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome[END_REF]).

However, it has been shown that clinical malaria isolates including from cerebral malaria, bound to CD36 10-fold higher than to ICAM-1; and 80% of ICAM-1-selected IEs bind to CD36 suggesting a crucial role of CD36 in IEs sequestration [START_REF] Udomsangpetch | Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1[END_REF]. There is rising evidence that CD36 and ICAM-1 act in synergy to strengthen IEs sequestration in post capillaries endothelial cells [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF][START_REF] Mccormick | Intercellular adhesion molecule-1 and CD36 synergize to mediate adherence of Plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells[END_REF][START_REF] Yipp | Synergism of multiple adhesion molecules in mediating cytoadherence of Plasmodium falciparum-infected erythrocytes to microvascular endothelial cells under flow[END_REF]. ICAM-1 promotes rolling and static binding of infected erythrocytes whereas CD36 provides stronger stationary adherence, mentioning to possible complementary role between these receptors [START_REF] Cooke | Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin[END_REF].

Role of icam-1 gene polymorphism in malaria

Polymorphism analyses of icam-1 gene, found on chromosome 19, and its relation with malaria are inconclusive and contradictory. Fernandez-Reyes et al have identified a mutation in the N-terminal domain of ICAM-1 (known as ICAM-1 Kilifi ) with a gene frequency >30% associated with an increased risk of developing cerebral malaria in Kenya [START_REF] Fernandez-Reyes | A high frequency African coding polymorphism in the N-terminal domain of ICAM-1 predisposing to cerebral malaria in Kenya[END_REF]. This polymorphism involves A to T substitution at position 179 resulting in a lysine to methionine replacement at position 29 in the BC-loop that interacts with rhinoviruses, lymphocytes, and infected erythrocytes. Similarly, Amodu et al found that the presence of the G allele at ICAM-1 exon 6 increase the risk of severe malaria in Nigerian children by 3.6 fold [START_REF] Amodu | Plasmodium falciparum malaria in south-west Nigerian children: is the polymorphism of ICAM-1 and E-selectin genes contributing to the clinical severity of malaria?[END_REF]. Conversely, three studies did not agree with those findings since no correlation between ICAM-1 Kilifi polymorphism frequency and severe malaria has been detected [START_REF] Bellamy | Absence of an association between intercellular adhesion molecule 1, complement receptor 1 and interleukin 1 receptor antagonist gene polymorphisms and severe malaria in a West African population[END_REF][START_REF] Fry | Variation in the ICAM1 gene is not associated with severe malaria phenotypes[END_REF][START_REF] Ohashi | Absence of association between the allele coding methionine at position 29 in the N-terminal domain of ICAM-1 (ICAM-1(Kilifi)) and severe malaria in the northwest of Thailand[END_REF]. Surprisingly, a study in Gabon has shown that 55% of children with mild malaria were carriers to ICAM-1 Kilifi whereas only 39% of those with severe malaria were carriers to this mutation and 52% of healthy children were ICAM-1 Kilifi carriers, suggesting a protective role for this polymorphism [START_REF] Kun | Association of the ICAM-1Kilifi mutation with protection against severe malaria in Lambarene, Gabon[END_REF].

Finally, mutagenesis analysis of ICAM-1 binding site for P. falciparum targeting alanine residues in the binding region resulted in 25 mutant proteins, e.g ICAM-1 S22/A where alanine residue was replaced by serine at position 22 [START_REF] Tse | Divergent binding sites on intercellular adhesion molecule-1 (ICAM-1) for variant Plasmodium falciparum isolates[END_REF]. In this study three different parasite lines (ItG, JDP8, A4) with different binding ability to wild type ICAM-1, (ICAM-1 ref )

were used to evaluate their adherence-capacities to a panel of mutant ICAM-1 proteins under flow and static conditions. The outcomes revealed that these parasite lines shared a similar binding region BED of ICAM-1, but they used different residues of ICAM-1 and showed variable binding phenotypes. This might be due to the variant PfEMP-1 expressed on the surface of each parasite line. The strands of the β-barrel are named from A to G. Some residues have been numbered for wild type ICAM-1. Source: [START_REF] Tse | Divergent binding sites on intercellular adhesion molecule-1 (ICAM-1) for variant Plasmodium falciparum isolates[END_REF]).

Platelet endothelial Cell Adhesion Molecule-1 (PECAM-1 CD31)

Human PECAM-1 gene maps to chromosome 17, and the 130-kD protein is extensively expressed on monocytes, platelets and endothelial cells where it participates in endothelial cell-cell junction stabilization and mediates the transmigration of leucocytes into sites of inflammation [START_REF] Gumina | The human PECAM1 gene maps to 17q23[END_REF]. It has been demonstrated that 54% of Kenyan isolates of cerebral malaria patients bound to PECAM-1 [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF]), but in another study no association of this binding with severe malaria had been found [START_REF] Newbold | Receptor-specific adhesion and clinical disease in Plasmodium falciparum[END_REF]. The parasite ligand is PfEMP-1 through its CIDRα and DBL2δ domains [START_REF] Chen | The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors[END_REF], which bind to the N-terminal segment of Ig-like domains (the first four domains) [START_REF] Treutiger | PECAM-1/CD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes[END_REF]).

Among the four existing polymorphisms at loci 125 and 563 [125 valine (V)-563 asparagine (N); 125V-563 serine (S); 125 leucine (L)-563N; and 125L-563S], The frequency Valine to asparagine (125 V/V-563 N/N) in PECAM-1 was found to raise the risk of cerebral malaria in Thailand [START_REF] Kikuchi | Association of adhesion molecule PECAM-1/CD31 polymorphism with susceptibility to cerebral malaria in Thais[END_REF], and Leucine to Valine polymorphism at codon 125 was not found to confer protection against severe malaria in Kenya or Papua New Guinea [START_REF] Casals-Pascual | Short report: codon 125 polymorphism of CD31 and susceptibility to malaria[END_REF].

Complement Receptor-1 (CR1)

Complement regulatory protein-1 (160-250kD) is expressed on infected erythrocytes, leucocytes and dentritc cells [START_REF] Khera | Complement Receptor 1: disease associations and therapeutic implications[END_REF]. In vivo, CR1 binds to complement components such as C3b and C4b resulting in several essential functions such as phagocytosis and elimination of immune complexes from the blood circulation [START_REF] Ahearn | Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21)[END_REF]. CR1 has been defined as rosetting-mediating receptor because in the absence of CR1, Infected erythrocytes were unable to form rosettes [START_REF] Rowe | P. falciparum rosetting mediated by a parasitevariant erythrocyte membrane protein and complement-receptor 1[END_REF]. Moreover, monoclonal antibodies against this receptor inhibited rosettes formation [START_REF] Rowe | Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates[END_REF].

The parasite ligand is PfEMP-1 through its DBLα and the binding site is C3b of CR1 [START_REF] Rowe | Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates[END_REF]. CR1 gene is harbored on chromosome 1 and it has been shown that Thai individuals with T-T genotype in promoter region are protected from cerebral malaria by enhancing the expression of erythrocyte-CR1 which in turn increases the clearance of immune complexes [START_REF] Teeranaipong | A functional singlenucleotide polymorphism in the CR1 promoter region contributes to protection against cerebral malaria[END_REF]. By contrast, CR1 deficiency of human erythrocyte decreased the rosetting in Papua New Guinea and protected against severe malaria [START_REF] Cockburn | A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria[END_REF]).

1.5.6. Endothelial Protein C Receptor (EPCR)

EPCR has recently been reported as a new mediator of infected erythrocytes adherence to microvascular endothelial cells via CDRα1 of domain cassette 8 and DC13 of PfEMP-1. It is encoded by proc gene on chromosome 20 and expressed primarily on arteries and veins endothelia and at low level on brain capillaries endothelia [START_REF] Simmonds | Structural and functional implications of the intron/exon organization of the human endothelial cell protein C/activated protein C receptor (EPCR) gene: comparison with the structure of CD1/major histocompatibility complex alpha1 and alpha2 domains[END_REF]. This receptor (46-kD) provides anti-inflammatory and cytoprotective effects to endothelial cells by the assistance of activated protein C (APC)-EPCR signaling pathway. Further, antithrombotic activity and conservation of vascular integrity are also promoted by the same pathway supported by this protein [START_REF] Mosnier | The cytoprotective protein C pathway[END_REF]). However, disruption of EPCR expression in the brain has been observed in cerebral malaria cases which gave rise to localized microvascular thrombosis and inflammation at site of sequestration [START_REF] Moxon | Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children[END_REF]). Interestingly, it has been shown that PfEMP1 binds to EPCR near or at the same region of activated protein C leading to abortion of cytoprotective effects mediated by APC-EPCR on endothelial cells (Turner L. 2013). These findings are shedding the light on a novel therapeutic strategy.

1.6. Plasmodium falciparum genome 1.6.1. Composition and organization of genome

The nuclear genome of P. falciparum is haploid with 23 mega bases (Mb) dispersed throughout 14 chromosomes that vary in size from 0.643-3.29 Mb. Up to now, 5542 proteincoding genes have been identified. The average length of intonless genes is 2.3 Kb, with introns of an average size of 950 bp found in 54% of the genes [START_REF] Gardner | Genome sequence of the human malaria parasite Plasmodium falciparum[END_REF]. This genome is distinct from the other eukaryotes as it rich in adenine (A) and thymine (T) bases that represent 80.6% of parasite genome. This confers instability to the genome and makes DNA arduous to handle. The genome holds 43 transfer RNAs (tRNA) and a few genes of ribosomal RNA (rRNA) that are classified according to the expression stage of parasite life cycle. Type A rRNAs are mainly expressed in human host whereas type S rRNAs are preferentially expressed in the Anopheles host [START_REF] Waters | The ribosomal RNA genes of Plasmodium[END_REF].

Apart from its nuclear genome, P. falciparum parasite has a small mitochondrial (6-kb) and an apicoplast (35-kb) genome. The latter is indispensable for parasite existence, since this organelle contributes to fatty acids, isoprenoids and haeme metabolism. In addition, about ten percent of proteins encoded by nuclear genes are targeted to the apicoplast [START_REF] Gardner | Genome sequence of the human malaria parasite Plasmodium falciparum[END_REF].

Chromosome structure

One of the properties of P. falciparum genome is the complexity of sub-telomeric regions that are composed of 5 blocks (SB 1-5), the localization and the composition of these blocks promotes mitotic recombination between telomers [START_REF] Gardner | Genome sequence of the human malaria parasite Plasmodium falciparum[END_REF]. The parasite chromosomes carry conserved and polymorphic domains. The conserved domains are located within the centromeric regions where the house-keeping genes are dominant. The polymorphic domains are situated in subtelomeric blocks, rich in adenine and thymine and so likely to play a role in recombination, gene conversion between telomere proximal genes and chromosome pairing [START_REF] De Bruin | The polymorphic subtelomeric regions of Plasmodium falciparum chromosomes contain arrays of repetitive sequence elements[END_REF].

At least three variant gene families (rif, stevor, var) are implicated in the antigenic variation phenomenon. They are preferentially found in the sub-telomeric regions and separated from the telomers by many conserved tandem repeats from 1-6 namely telomere-associated repetitive elements (TARE) each with 21 base pair (Repititives-20) [START_REF] Oquendo | Characterisation of a repetitive DNA sequence from the malaria parasite, Plasmodium falciparum[END_REF]) (Fig. 9).

Sixty percent of P. falciparum genes code for hypothetical proteins with unknown function.

A considerable proportion of products with specific function are proteins dedicated for antigenic variation followed by restricted repertoire of membrane transporters and metabolic enzymes [START_REF] Gardner | Genome sequence of the human malaria parasite Plasmodium falciparum[END_REF].

Variant surface antigens (VSA)

The spectacular ability of P. falciparum parasite to fool the host immune system, thus to ensure a chronic infection is associated with a set of variant surface antigens. VSA is a term used to describe variable surface antigens that are encoded by multigenic families and expressed at the surface of infected erythrocyte. These VSA are implicated in antigenic variation as well as in cytoadherence of IEs to host cells that lead to severe malaria under certain circumstances.

However, in malaria endemic regions, immunity to non-cerebral severe malaria is acquired after few malarial episodes [START_REF] Gupta | Immunity to non-cerebral severe malaria is acquired after one or two infections[END_REF]) and it appears to be directed against P. falciparum VSA. Indeed, parasites associated with severe malaria in non-immune young children, expressed limited and conserved subset of VSA which are recognized by IgG from adults more than the VSA expressed by parasites causing mild or asymptomatic infections [START_REF] Nielsen | Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity[END_REF].

So far, five families of VSA have been identified var (~60), rif (~150), stevor (~30), Pfmc2TM (~11) and surfin (~10) [START_REF] Baruch | Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes[END_REF][START_REF] Blythe | Characterization of the repertoire diversity of the Plasmodium falciparum stevor multigene family in laboratory and field isolates[END_REF][START_REF] Kyes | Rifins: a second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum[END_REF][START_REF] Lavazec | Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum[END_REF][START_REF] Winter | SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes[END_REF]) each with variable members of genes, predominantly localized in subtelomeric sites, the chromosomal regions subjected to gene recombination [START_REF] Hernandez-Rivas | Compartmentalization of genes coding for immunodominant antigens to fragile chromosome ends leads to dispersed subtelomeric gene families and rapid gene evolution in Plasmodium falciparum[END_REF][START_REF] Freitas-Junior | Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum[END_REF]. Furthermore, var genes family is the bestcharacterized VSA member and is found to play a crucial role in malaria pathogenesis.

STEVOR and RIFIN

Although these two hyper variable families share sequence homology, stevor genes seem to be more conserved than rifin ones [START_REF] Gardner | The malaria genome sequencing project[END_REF]. The 3D7 genome holds approximately 150 rif genes; most of which are organized head to head with var genes, and about 30 stevor genes. The majority of stevor genes are localized at the chromosome ends; contrary to var and rif which can be found in the center of parasite chromosomes [START_REF] Gardner | Genome sequence of the human malaria parasite Plasmodium falciparum[END_REF]. The proteins encoded by these genes, RIFIN and STEVOR, are small polypeptides ranging from 25 to 45 kD and characterized by putative signal sequence, followed by a semi-conserved domain, an hyper variable region (HVR) and a conserved C-terminal domain [START_REF] Cheng | stevor and rif are Plasmodium falciparum multicopy gene families which potentially encode variant antigens[END_REF]. These proteins are exported to the surface of infected erythrocytes [START_REF] Blythe | Plasmodium falciparum STEVOR proteins are highly expressed in patient isolates and located in the surface membranes of infected red blood cells and the apical tips of merozoites[END_REF][START_REF] Kyes | Antigenic variation at the infected red cell surface in malaria[END_REF]) and display highly polymorphic amino acid sequences in P. falciparum isolates [START_REF] Albrecht | Extense variant gene family repertoire overlap in Western Amazon Plasmodium falciparum isolates[END_REF][START_REF] Blythe | Plasmodium falciparum STEVOR proteins are highly expressed in patient isolates and located in the surface membranes of infected red blood cells and the apical tips of merozoites[END_REF], suggesting a potential implication in antigenic variation and hence in evading the defense mechanism [START_REF] Fernandez | Small, clonally variant antigens expressed on the surface of the Plasmodium falciparum-infected erythrocyte are encoded by the rif gene family and are the target of human immune responses[END_REF][START_REF] Niang | The Plasmodium falciparum STEVOR multigene family mediates antigenic variation of the infected erythrocyte[END_REF]).

The RIFIN family members have been classified into two sub-groups, type-A and type-B which further could be divided into B1, B2, B3 clusters and can be transcribed simultaneously with type-A [START_REF] Joannin | Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family[END_REF][START_REF] Petter | Variant proteins of the Plasmodium falciparum RIFIN family show distinct subcellular localization and developmental expression patterns[END_REF]). Type-A RIFINs are larger than those of type-B due to the presence of 25 amino acids motif within the semi-conserved region (C1) of type-A. Indeed, this motif is situated at approximately 66-amino acids downstream of Plasmodium export element motif (PEXEL), but it is not found in B-type RIFINs (Fig. 7). Moreover, while type-A group contains 10 conserved cysteine residues, only 6 of them are located in type-B RIFINs [START_REF] Joannin | Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family[END_REF]. Furthermore, solely type-A RIFINs are trafficked to the surface of parasitized erythrocytes via Maurer's clefts whereas type-B RIFINs are retained within the parasites [START_REF] Petter | Variant proteins of the Plasmodium falciparum RIFIN family show distinct subcellular localization and developmental expression patterns[END_REF].

A comparison analysis of transcript abundance of rif and var using P. falciparum 3D7 and NF54 clones showed a distinct rif transcript pattern during gametocytes and sporozoites stages comparing to rif and var expression profiles during the asexual stages. In addition, a single variant of B1-type RIFIN dominated the sexual stages of P. falciparum 3D7 and NF54 suggesting an important role of this gene in the adherence during this stage and shedding the light on this protein as a potential transmission blocking vaccine candidate [START_REF] Wang | Identification of a major rif transcript common to gametocytes and sporozoites of Plasmodium falciparum[END_REF].

As RIFIN proteins, STEVOR antigens display two transmembrane domains; one of which is hypothetical (Fig. 7). Antibodies directed against STEVORs found that these proteins are expressed at the surface of the IE at schizogony, resulting in antigenic variability during this stage [START_REF] Niang | The Plasmodium falciparum STEVOR multigene family mediates antigenic variation of the infected erythrocyte[END_REF]). These antigens are found in the rhoptries and at the surface of merozoites as well as among released component in the invading merozoites, suggesting a potential role during invasion and probably in immune evasion in the erythrocyte invasion process [START_REF] Khattab | Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion[END_REF].

Transcription profiles of rif-A, rif-B and stevor at different stages of parasite life cycle in the clinical isolates cultivated in vitro compared to 3D7 clone had been investigated by real time PCR [START_REF] Bachmann | Temporal expression and localization patterns of variant surface antigens in clinical Plasmodium falciparum isolates during erythrocyte schizogony[END_REF]. The results revealed two peaks of transcription of rif-A and stevor during the ring and trophozoite stages. In addition, clinical isolates showed tremendous transcript abundance of these variant surface antigens comparing with 3D7. Immunoflouresence analysis of these isolates detected the localization of RIFIN and STEVOR at young trophozoites, shiozonts and merozoites. The findings led to propose a complex model of the expression of these polymorphic families throughout the asexual life cycle of parasite which might be in relation with clinical outcomes of infection [START_REF] Bachmann | Temporal expression and localization patterns of variant surface antigens in clinical Plasmodium falciparum isolates during erythrocyte schizogony[END_REF].

Var genes and PfEMP-1

The proteins encoded by the hyper variable var genes family are Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP-1) that range in size from 200-400 kDa [START_REF] Baruch | Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes[END_REF][START_REF] Smith | Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes[END_REF][START_REF] Su | The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes[END_REF]. These antigens are trafficked to the surface of infected erythrocyte where they are anchored into electron dense protrusions, namely knobs [START_REF] Igarashi | Knob antigen deposition in cerebral malaria[END_REF] with the assistance of other proteins, mainly Knob Associated Histidine Rich Proteins (KAHRP) [START_REF] Pologe | Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum[END_REF]. These antigens play a role of mediator between IEs and endothelial receptors, leading to sequestration of IEs throughout the body organs and bringing about severe complications. PfEMP-1 proteins are major targets of host defense. Pre-exposure to anti PfEMP-1 antibodies promotes selection and expression of specific antigens during a novel infection [START_REF] Bull | Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria[END_REF]. Each individual parasite harbors ~ 60 copies of var genes dispersed throughout 14 chromosomes with a majority localized in the subtelomeric regions and the rest in the center of chromosomes. All these genes consist of two exons separated by a conserved intron. The larger one, exon I (3.5-9 kb) encodes the polymorphic extracellular region implicated in cytoadherence phenomenon which includes N-terminal segment (NTS); multiple domains of duffy binding like domain (DBL), one or two cysteine rich inter domain regions (CIDR) and a transmembrane domain (TM), while exon II (1-1.3 kb) codes for a semi-conserved cytoplasmic tail with an acidic terminal segment (ATS) (Fig. 8) [START_REF] Gardner | Genome sequence of the human malaria parasite Plasmodium falciparum[END_REF][START_REF] Su | The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes[END_REF]. Based on sequence homology of coding and non coding regions, chromosomal location, direction of transcription and domain structure of encoded proteins, var genes have been categorized into three major groups A, B and C, three conserved groups var1, var2 and var3, and intermediate groups B/A & B/C [START_REF] Kraemer | Evidence for the importance of genetic structuring to the structural and functional specialization of the Plasmodium falciparum var gene family[END_REF][START_REF] Lavstsen | Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions[END_REF]. All group A var genes (10 genes) possess ups A flanking sequence, situated in the sub telomeric sites and transcribed toward the telomere in the opposite direction of group B var genes (22 genes) which have a ups B sequence and are found near the telomere and their transcription is headed to the centromere. Group C var genes (13 genes) are flanked by ups C, located near the centromere and oriented toward the telomere. The intermediate groups B/A (4 genes) and B/C (10 genes) have upstream B sequence followed by the coding regions of group A or C respectively. Var1 ups D and var2csa ups E are highly conserved groups each with a unique sub telomeric gene; var3 is a conserved subfamily of group A with three small genes (Fig. 9) [START_REF] Lavstsen | Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions[END_REF]. Global organization of subtelomeric regions of the 14 linear chromosomes shows that they all share non coding repeats of various sizes called TARE 1-6 (telomere associated repeat elements) situated beside telomere repeats. Subtelomeric site is followed by a member of the group B var genes, usually accompanied by another group A var genes transcribed in the opposite direction. Group C var genes are clustering in the chromosome center and rif genes are often interspersed with var genes. Source: Modified from [START_REF] Scherf | Antigenic variation in Plasmodium falciparum[END_REF].

The extra cellular parts of PfEMP-1 proteins are composed of multiple Duffy binding-like domains (DBL) and one or two cysteine rich inter domain regions (CIDR), each of 300-650 amino acids. Each copy of var gene has its own length according to the number and types of domains including in its sequence (3.9-13 kb). However, four proteins, VAR2CSA and VAR3 PfEMP-1 members, do not belong CIDR domains [START_REF] Rask | Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer[END_REF][START_REF] Smith | Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family[END_REF] (Fig. 10).

Based on sequence similarity, DBL domains are categorized into DBL α, β, ϒ, ϵ, δ ,ζ and five smaller distinct classes (four DBL domains of VAR2CSA and a DBL α/ ζ of VAR3 ) [START_REF] Rask | Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer[END_REF][START_REF] Smith | Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family[END_REF]. CIDR domains are classified into five distinct domains α, β, ϒ, δ, and pam [START_REF] Rask | Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer[END_REF][START_REF] Smith | Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family[END_REF]. These subclasses can be further sub categorized into 147 subtypes (e.g. DBLα 1.1). In general, the subtypes of these domains are located in the N-terminal part of PfEMP-1 (e.g. DBLαCIDRαDBLβ/DBLϒ) and they are associated with ups of var groups, which is not the case of domains associated with C-terminal part (e.g. DBLδCIDRβ/ϒDBLϵDBLζ) [START_REF] Rask | Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer[END_REF]. It has previously been shown that group A and B of var genes are differentially transcribed in severe malaria isolates, including cerebral malaria, as compared to parasites from uncomplicated malaria (UM) or asymptomatic infections [START_REF] Jensen | Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes[END_REF][START_REF] Kyriacou | Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia[END_REF][START_REF] Rottmann | Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children[END_REF]). Moreover, group A var genes code for large and complex multi domains PfEMP-1, in agreement with studies that found that parasite isolated from patients with cerebral malaria express high molecular weight PfEMP-1 as compared to those from non severe malaria isolates [START_REF] Bian | Expression of Plasmodium falciparum-infected erythrocyte membrane protein from cerebral malaria patients[END_REF]. Recently, It has been shown that the high level of group A var genes is correlated with severe malaria and rosetting phenotype in African children [START_REF] Warimwe | Prognostic indicators of lifethreatening malaria are associated with distinct parasite variant antigen profiles[END_REF]. This latter was found to be associated with respiratory distress while non-rosetting group A PfEMP-1 isolates was associated with impaired consciousness, thus the clinical outcomes of malaria infection are affected by specific group A var gene expressed by infecting parasites.

A remarkable up-regulation of var2csa by P. falciparum parasite isolates selected in vitro for adherence to CSA receptor was shown using real time PCR [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF]. This unique gene codes for VAR2CSA protein, expressed at the surface of parasitized erythrocyte infected placenta [START_REF] Magistrado | VAR2CSA expression on the surface of placenta-derived Plasmodium falciparum-infected erythrocytes[END_REF]. Currently, VAR2CSA is considered as the major ligand for parasite adherence to placenta intervillous space of pregnant women, and high plasma level of antibodies directed against VAR2CSA in pregnant women found to decrease the risk of low birth weight at delivery [START_REF] Salanti | Evidence for the involvement of VAR2CSA in pregnancy-associated malaria[END_REF].

A recent study employed alignment and distance tree analysis of 399 known domains of PfEMP-1 from seven genomes has identified 628 conserved minimal homology blocks describing most of PfEMP-1 sequences. In addition, 23 conserved domains, namely domain cassettes have been categorized [START_REF] Rask | Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer[END_REF]). Some of these domains have been described previously such as DC1 for var1 [START_REF] Salanti | A sub-family of common and highly conserved Plasmodium falciparum var genes[END_REF][START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF], DC2 for var2csa [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF], and DC3 for var3 [START_REF] Trimnell | Global genetic diversity and evolution of var genes associated with placental and severe childhood malaria[END_REF]. A domain cassette is identified as a tandem of 2 to 4 domains of DBL and CIDR semi-conserved involved in many var genes and are located exclusively in N or C-terminal parts of PfEMP-1 [START_REF] Rask | Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer[END_REF].

Var genes encoding domain cassette 8 (DC8) and domain cassette 13 (DC13) are of clinical significance. DC8 involves genes with upstream B followed by four domains NTSβDBLα2CIDRα1.1DBLβ12DBLγ4/6, whereas DC13 found in group A var genes and it is characterized by the tandem domains of DBLα1.7CIDRα1.4 (Fig. 11).

The transcript abundance of these domain cassettes found to be higher for all severe malaria forms including cerebral malaria than mild malaria [START_REF] Bertin | Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin[END_REF][START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]. Moreover, Tanzanian children revealed serological recognition for recombinant PfEMP-1 domains belonging to DC8 similar to group A domains and higher than group B and C domains [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]. It has been demonstrated that P. falciparum parasites selected for binding to human brain microvascular endothelial cells (HBMEC) showed significantexpression levels of PfEMP-1 DC8 genes [START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF]). In addition, plasma from convalescent Kenyan children exhibited higher antibody recognition profiles to HBMEC-selected parasites than those of unselected ones [START_REF] Avril | A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells[END_REF], suggesting the importance of these PfEMP-1 motifs in building protecting immunity to malaria.

PfEMP-1 proteins are seen as the major determinants in cytoaherence affinity of IEs to different receptors and that the binding sites of some receptors have been mapped to specific DBL and CIDR domains of PfEMP-1 (Fig. 12) [START_REF] Baruch | Identification of a region of PfEMP1 that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: conserved function with variant sequence[END_REF][START_REF] Chen | The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors[END_REF][START_REF] Joergensen | Surface co-expression of two different PfEMP1 antigens on single plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1[END_REF].

The head structure of CIDR1α domain of group B and C PfEMP-1 demonstrates a great ability to bind multiple receptors including CD36; PECAM-1 (along with DBL2δ) [START_REF] Chen | The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors[END_REF][START_REF] Janes | Investigating the host binding signature on the Plasmodium falciparum PfEMP1 protein family[END_REF][START_REF] Robinson | Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome[END_REF].

Likewise, DBL domains show wide spectrum of binding capacity to different receptors such as heparin sulfate (HS) and blood group A antigen. They also mediate the adherence of infected erythrocytes to CR1 of uninfected erythrocytes through NTS-DBLα-CIDRϒ of varO (group A PfEMP-1) resulting in rosettes formation [START_REF] Rowe | P. falciparum rosetting mediated by a parasitevariant erythrocyte membrane protein and complement-receptor 1[END_REF][START_REF] Vigan-Womas | Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting[END_REF].

The infected erythrocytes bind to ICAM-1 through DBLβ-C2 domains of group B and C PfEMP-1 and the DBLβ3 of group A PfEMP-1 [START_REF] Bengtsson | A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies[END_REF][START_REF] Howell | Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors[END_REF][START_REF] Oleinikov | High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies[END_REF]).

The tight regulation of PfEMP-1s expression endows the parasite a high degree of fitness. PfEMP-1s are employed in several biological processes allowing the parasite to modulate the host immune response and to set a long lasting infection to ensure its transmission and survival. For instance, PfEMP-1 binds to dendritic cells, and inhibits their maturation resulting in decreased ability to induce T cell response, thus, decreasing the immune defense [START_REF] Urban | A role for CD36 in the regulation of dendritic cell function[END_REF].

PfEMP-1 via its CIDR1α domain can also bind and induce B-lymphocytes of naïve donors. This interaction elicits the multiplication, augments the size of B lymphocytes and stimulates the secretion of non-immune IgM and cytokines (TNFa & IL-6). In addition, CIDR1α binds to Fab and FC fragments of human Ig [START_REF] Donati | Identification of a polyclonal B-cell activator in Plasmodium falciparum[END_REF].

Recently, a new mechanism used by parasites from pregnant women to by-pass the recognition of host's antibodies has been described. The parasites employ non-functional epitopes of VAR2CSA to bind non-immune IgM which would mask PfEMP-1-specific IgG epitopes and confer a protection to IE from phagocytosis without affecting the binding of VAR2CSA to CSA in placenta [START_REF] Barfod | Evasion of immunity to Plasmodium falciparum malaria by IgM masking of protective IgG epitopes in infected erythrocyte surface-exposed PfEMP1[END_REF]. It has previously been shown that of the hyper variable repertoires of var genes, trophozoite parasite chooses single PfEMP-1 to be transcribed and expressed on the surface of infected erythrocytes at each life cycle [START_REF] Dzikowski | Mutually exclusive expression of virulence genes by malaria parasites is regulated independently of antigen production[END_REF][START_REF] Voss | A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria[END_REF]). However, these observations have been contradicted with recent results which reported an ability of P. falciparum parasite to co-express two different PfEMP-1 antigens at the surface of IE [START_REF] Joergensen | Surface co-expression of two different PfEMP1 antigens on single plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1[END_REF].

Mutually exclusive expression and switching properties

The mutually exclusive transcription of P. falciparum var genes and the ability to switch between members are essential properties for the parasite life in order to avoid the host immune defense and to ensure chronic infection. The underlying strategies contributing to these mechanisms have not been clearly illustrated. However, several studies aimed to test the implication of epigenetic factors or specific genetic elements that may control monoallelic expression.

Mutually exclusive expression property

Mutually exclusive expression of single var gene and keeping the rest of family members in a transcriptionally silent state is thought to be epigenetically controlled and it is linked to histone modification [START_REF] Chookajorn | Epigenetic memory at malaria virulence genes[END_REF][START_REF] Lopez-Rubio | 5' flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites[END_REF]. During the ring stage of intraerythrocyte life cycle, the 5' upstream of the actively transcribed var gene is extensively supplemented with di and trimethylation histone 3 lysine4 (H3K4me2 and H3K4me3) [START_REF] Lopez-Rubio | 5' flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites[END_REF]) and with acetylated trimethylation histone3 lysine 9 (H3K9me3ac) [START_REF] Chookajorn | Epigenetic memory at malaria virulence genes[END_REF]. At the late stage of parasite life this gene loses its richness in H3K4me3 and H3K9me3ac and becomes temporarily silent, however it keeps H3K4me2 probably to transmit memory of the active gene to the following asexual life cycles. Conversely, the 5' flanking region of stably silent var genes are enriching with trimethylation histone3 lysin 9 (H3K9me3), which is highly associated with gene silencing by heterochromatin in the other eukaryotes. As yet, the shift of a silenced var gene from heterochromatin to a euchromatin nuclear region has not been clarified. However, these results suggest a competition between the two processes methylation and acetylation of H3K9 at the 5' upstream which affects the transcription activity and shed some light on the important role of 5' flanking region [START_REF] Chookajorn | Epigenetic memory at malaria virulence genes[END_REF][START_REF] Lopez-Rubio | 5' flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites[END_REF]. Another gene activation marker of the euchromatin is the alternative histone PfH2A.E, which is dynamically enriched in active var genes promoters during the ring stage of asexual life cycle, but it is not found in silent var genes promoters referring to its implication in the transcriptional activity of var genes [START_REF] Petter | Expression of P. falciparum var genes involves exchange of the histone variant H2A.Z at the promoter[END_REF].

Sterile transcript mediated by the conserved var gene intron between exon1 and exon 2 is presumably involved in the regulatory mechanism of transcription. It has been shown that var gene silencing requires an interaction between var intron, which has promoter activity and another promoter within the upstream of coding region [START_REF] Calderwood | Plasmodium falciparum var genes are regulated by two regions with separate promoters, one upstream of the coding region and a second within the intron[END_REF]. Furthermore, each intron is able to repress only a single promoter, thus only one var gene is silenced without influencing the transcriptional mechanism of adjacent var promoters. This finding suggests that one-to-one pairing is indispensable for silencing and that mechanism of var gene silencing is controlled for each individual gene instead of whole silent chromatin over a chromosomal region [START_REF] Frank | Strict pairing of var promoters and introns is required for var gene silencing in the malaria parasite Plasmodium falciparum[END_REF]. It has been reported that during the late stage of intraerythrocyte life cycle, the non coding RNA (ncRNA) transcripts which initiated from a bi-directional promoter inside the conserved intron of var gene are physically associated with chromatin at var loci and they may act as transcription silencer [START_REF] Epp | Chromatin associated sense and antisense noncoding RNAs are transcribed from the var gene family of virulence genes of the malaria parasite Plasmodium falciparum[END_REF].

Switching property of Plasmodium falciparum

The dynamic of antigenic variation known as switching rate, is one of unanswered questions about P. falciparum life and properties. Whereas in vitro investigation in the lack of immune pressure found that the parasites have the capacity to switch the expression of var genes immediately at a rate of 2% [START_REF] Roberts | Rapid switching to multiple antigenic and adhesive phenotypes in malaria[END_REF], switching in human malaria parasites from infected volunteers showed that only one var gene type transcribed predominantly in different isolates with high switching rate estimated around 16% per generation [START_REF] Peters | High diversity and rapid changeover of expressed var genes during the acute phase of Plasmodium falciparum infections in human volunteers[END_REF]. The data suggested that var gene switching property is due to a "reset" pattern i.e the same var gene is re-expressed as merozoites left the liver. In addition, switching from the first var gene to another might take place quickly after leaving hepatocytes, but the subsequent change-overs are carried out at slower rate.

Another switching model based on in vitro cultures where clones of parasites with selected specific cytoadherence pattern were used to measure "on rate" (switching to active state) and "off rate" (switching to inactive state). Results indicated that each var gene has its own on and off switching rate "ordered switch model" contrarily to "random switch model" which supposes that all var genes have the same switch rate. The outcomes showed that switching is not different between subtelomeric and central var genes [START_REF] Horrocks | Variable var transition rates underlie antigenic variation in malaria[END_REF]).

1.9. Potential vaccine targets P. falciparum undergoes a fascinating and complicated life cycle. The parasites spend most of their life inside human host cells and they are extracellular solely for a short period of time as sporozoites, merozoites. For each stage of development, only a small proportion of parasites are required to persist and to complete the parasite life cycle. In addition, at every step of life, P. falciparum is able to express a set of antigens that are principle contributors to host-immune escape. Up to date, a P. falciparum vaccine remains elusive. However, through the last decade; several trials have been attempted to interrupt the life cycle of the parasite by targeting multiple antigens.

The ultimate aim of our study is to find out P. falciparum genetic factors implicated in the pathogenesis of severe malaria, especially cerebral malaria. The proteins encoded by these genes could be interesting vaccine candidates. A suitable vaccine should be cost-effective and reduce the risk of antimalarial drug resistance developing. This vaccine candidate should confer protection or at least prevent the progression towards the severe form of malaria; consequently the rate of mortality among African children due to severe malaria would be reduced.

The known vaccine candidates could be divided into three groups.

1) Pre erythrocytic candidates -CircumSporozoite protein (CS): is considered as the principal antigen expressed at the sporozoite stage and it is necessary for parasite development. Region II plus in this protein plays a key role in sporozoite motility and hepatocyte invasion where it binds to heparin sulfate proteoglycan [START_REF] Agnandji | First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children[END_REF][START_REF] Aly | Malaria parasite development in the mosquito and infection of the mammalian host[END_REF][START_REF] Cerami | Binding of malarial circumsporozoite protein to sulfatides [Gal(3-SO4)beta 1-Cer] and cholesterol-3-sulfate and its dependence on disulfide bond formation between cysteines in region II[END_REF]. Recently, this protein has been targeted by the RTS,S/AS01 vaccine. Preliminary outcomes of phase III trial in African children found that the vaccinees showed approximately 50% less clinical malaria episodes for the first year. Conclusive results are expected later in 2014 [START_REF] Agnandji | First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children[END_REF]).

-Thrombospondin Related Anonymous Protein (TRAP) of P. falciparum is located within the micronemes and on the surface of sporozoites. It holds an amino acid sequence that provides sporozoites with the ability to recognize and invade host hepatocytes [START_REF] Muller | Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum binds specifically to sulfated glycoconjugates and to HepG2 hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes[END_REF]).

-Liver Stage Antigen1 (LSA1) is a P. falciparum protein specifically expressed at liver stage. It is indispensable for exoerythrocytic schizogony. Indeed, without this antigen, schizontes loss their ability to differentiate into merozoites and to express the apical membrane antigen1 (AMA1) which is a crucial protein involved in erythrocyte invasion [START_REF] Mikolajczak | Disruption of the Plasmodium falciparum liver-stage antigen-1 locus causes a differentiation defect in late liver-stage parasites[END_REF]). The first Phase I/II trial for LSA-NRC recombinant protein with aluminum hydroxide adjuvant, reported good safety profile, robust antibody-antigen specificity and CD4+ T-cell responses. However, it could not confer protective immunity against experimental malaria challenge in the tested naïve volunteers [START_REF] Cummings | Recombinant Liver Stage Antigen-1 (LSA-1) formulated with AS01 or AS02 is safe, elicits high titer antibody and induces IFN-gamma/IL-2 CD4+ T cells but does not protect against experimental Plasmodium falciparum infection[END_REF].

2) Blood stage targets -The Glutamate-Rich Protein (GLURP) of P. falciparum is expressed in both liver and erythrocytic stages. Naïve participants have been inoculated with long synthetic peptide LSP-GLURP 85-213 formulated with aluminum hydroxide or montandine ISA720 for Phase I vaccine trial. The peptide was able to induce a specific antibody response and cytophilic IgG1 antibodies against GLURP 85-213 which could inhibit parasite growth in vitro [START_REF] Hermsen | Glutamate-rich protein (GLURP) induces antibodies that inhibit in vitro growth of Plasmodium falciparum in a phase 1 malaria vaccine trial[END_REF]).

-The Merozoite Surface Protein1 (MSP1) is one of blood stage parasite proteins that are required for erythrocyte invasion. A vaccine based on the 42kDa C-terminal fragment of MSP1 (known as falciparum malaria protein1, FMP1/AS02) was administered to healthy Kenyan children for Phase IIb clinical trial. Although it was well-tolerated and showed a favorable safety profile in those infants, FMP1 was not an efficient vaccine because it did not provide a protection against first falciparum malaria episode and was not able to decrease the parasitemia [START_REF] Ogutu | Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya[END_REF]).

-Merozoite Surface Protein2 (MSP2): A combination B vaccine containing part of the 3D7 MSP1, MSP2 and the ring infected erythrocyte surface antigen (RESA) combined with montandine ISA720, has been evaluated for Phase I-IIb clinical trial. The product decreased the parasitemia by 62% in children living in Papua New Guinea, but it was not efficient to reduce the frequency of malaria episodes [START_REF] Genton | A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea[END_REF].

-Merozoite Surface Protein3 (MSP3): A long synthetic peptide containing the conserved region of MSP3 (MSP3-LSP) adjuvanted with montandine ISA720 or aluminum hydroxide, had been produced to immunize healthy volunteers for Phase I trial. MSP3-LSP was well tolerated and without severe side effects. It was also capable to trigger a strong and a specific immune response including T-cells, INF-y and cytophilic IgG1 [START_REF] Audran | Phase I malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein 3 antigen[END_REF][START_REF] Druilhe | A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum[END_REF].

-GMZ: is a fusion protein of MSP3-GLURP with aluminum hydroxide was produced for Phase Ia clinical trial. It demonstrated a capacity to elicit a specific antibody-antigen reaction and to induce memory B cells which were recognized during the 12 months follow-up of the study [START_REF] Esen | Safety and immunogenicity of GMZ2 -a MSP3-GLURP fusion protein malaria vaccine candidate[END_REF]).

-The Apical membrane protein1 (AMA1) is located at P. falciparum surface and expressed during the asexual blood stage of parasite life. Phase II study was conducted in healthy Malian children to investigate the immunogenicity and the safety of AMA1-Combination 1 (C1) comprising two recombinant allelic proteins (AMA-1FVO and AMA-13D7) with Alhydrogel ® adjuvant. Results showed that in spite of the desirable safety profile of this combination, it was moderately immunogenic and had no influence on the level of parasitemia [START_REF] Sagara | A randomized and controlled Phase 1 study of the safety and immunogenicity of the AMA1-C1/Alhydrogel + CPG 7909 vaccine for Plasmodium falciparum malaria in semi-immune Malian adults[END_REF]).

-The Erythrocyte binding antigen 175 (EBA175) is a P. falciparum conserved protein that mediates the parasites binding to the host's erythrocytes. A recombinant EBA175 vaccine combined with aluminum phosphate was inoculated into malaria naïve adults from the United States. This vaccine was safe and well tolerated. It was capable to elicit antibody response and to inhibit parasite growth in vitro (El [START_REF] Sahly | Safety and immunogenicity of a recombinant nonglycosylated erythrocyte binding antigen 175 Region II malaria vaccine in healthy adults living in an area where malaria is not endemic[END_REF]).

-The Reticulocyte binding protein homologue 5 (Rh5) is a special member of the reticulocyte binding homologue family (PfRh) which is involved in erythrocyte invasion and adherence to infected erythrocyte surface. This antigen is distinct from the other members as it is smaller and has no C-terminal transmembrane domain. It is located in the rhoptries of merozoites and associated to the tight junction of the parasite, suggesting a crucial role in the penetration of merozoites into the infected erythrocyte [START_REF] Baum | Reticulocyte-binding protein homologue 5 -an essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum[END_REF].

P. falciparum merozoite has multiple alternative pathways to invade the infected erythrocyte, which hampers the researches to define and develop an adequate invasionblocking vaccine targets (Baum et al. 2005).

-Serine repeat antigen 5 (SERA5) is an asexual intra erythrocytic stage protein expressed in the mature parasites i.e. late trophozoites and schizonts and released into the lumen of parasitophorous vacule. Phase Ia clinical trial evaluated the capacity of SE36/AHG, which is a recombinant molecule of SERA5 with aluminum hydroxide gel, in healthy adults. It showed a favorable safety profile and highly sero conversion against SE36 with increasing age, but it did not induce an antibody response during the primary malaria infections [START_REF] Horii | Evidences of protection against blood-stage infection of Plasmodium falciparum by the novel protein vaccine SE36[END_REF].

-Other set of asexual intra erythrocytic stage targets: Members of variant surface antigen (VSA) families which are highly polymorphic and include: VAR (PfEMP-1), STEVOR, RIFIN, SURFIN and PFMC2TM. Of these protein families VAR, RIFIN and STEVOR families have been investigated. The majority of these proteins are expressed and transported to the surface of infected erythrocytes. Members of VSA were found to mediate the cytoadherence of IEs to endothelial host receptors throughout the body. Therefore these antigens are potential vaccine candidates against malaria pathogenesis.

The most well studied member of the PfEMP-1 as a vaccine candidate is VAR2CSA. This protein is considered as a major antigen in parasites infecting pregnant women, conferring the parasites a high tropism towards syncytiotrophoblasts of the placenta. The sequestration of IEs in the placenta is at the origin of severe clinical effects on mothers and children [START_REF] Salanti | Evidence for the involvement of VAR2CSA in pregnancy-associated malaria[END_REF]). It has been shown that multigravidae women acquire adherenceblocking-antibodies against VAR2CSA which prevent from harmful consequences of Pregnancy Associated Malaria (PAM) supporting the idea of a vaccine targeting VAR2CSA protein [START_REF] Salanti | Evidence for the involvement of VAR2CSA in pregnancy-associated malaria[END_REF]). Several domains combinations of VAR2CSA with suitable adjuvants have been used to immunize animals and to test the binding inhibitory efficacy of elicited IgG. These domains include DBL4ϵ-ID4, NTS-DBL1X, NTS-DBL1X-Id1, Id1, Id1-DBL2X and DBL2X which were able to trigger high antibodies responses with similar efficacy as pooled plasma from immune multigravidae African women [START_REF] Bordbar | Identification of Id1-DBL2X of VAR2CSA as a key domain inducing highly inhibitory and cross-reactive antibodies[END_REF][START_REF] Magistrado | High efficacy of anti DBL4varepsilon-VAR2CSA antibodies in inhibition of CSA-binding Plasmodium falciparum-infected erythrocytes from pregnant women[END_REF][START_REF] Pinto | The effect of adjuvants on the immune response induced by a DBL4varepsilon-ID4 VAR2CSA based Plasmodium falciparum vaccine against placental malaria[END_REF]. These antigens will be tested for their safety and efficacy in vaccine trials against PAM in the next two years.

The vaccine that we seek should be able to induce a specific antibody response against specific PfEMP-1 types exported at the surface of IE that would prevent the cytoadherence of IEs to microvascular endothelial receptors in the deep organs such as the brain. As a consequence, the non-adhered IEs would circulate in the peripheral blood where they would be filtered out by the spleen. Thus, this vaccine could be very efficient to hinder the progression to complicated forms of malaria and reduce the risk of developing cerebral malaria.

3) Sexual stages targets or Transmission Blocking Vaccine (TBV) -Pfs25: is a 25 kDa protein located at the surface of zygotes and ookinetes. Because Pfs25 is expressed solely during the parasites life in the mosquito midgut, no immunity against this protein can be acquired naturally [START_REF] Kaslow | Minimal variation in a vaccine candidate from the sexual stage of Plasmodium falciparum[END_REF]). However, Pfs25 with montadin ISA51 (Pfs25/ISA51) was a vaccine candidate for Phase I clinical trial in American naïve volunteers. Although the results found an outstanding antibody response to this protein and that specific antibodies could inhibit the transmission of parasites to mosquitoes in ex vivo assays, this vaccine was astonishingly reactogenic and was not well-tolerated by participants [START_REF] Wu | Phase 1 trial of malaria transmission blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide ISA 51[END_REF]).

-Pfs230: is a large protein, expressed in early gametocytes for few hours after leaving the human infected erythrocyte to the mosquito midgut. Recombinant protein spanning part of this antigen was able to elicit antibodies responses that recognized the gametocytes surfaces and reduced the capacity of parasites to infect mosquito by 71.2-89.8% [START_REF] Williamson | Recombinant Pfs230, a Plasmodium falciparum gametocyte protein, induces antisera that reduce the infectivity of Plasmodium falciparum to mosquitoes[END_REF].

-Pfs48/45 is specifically expressed by gametocytes inside the human host and located at the surface of macrogamets P. falciparum parasites. Without this antigen, parasites fertilization could not take place [START_REF] Outchkourov | Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice[END_REF]).

Thus, the complexity of P. falciparum parasites hinders the efficacy of these vaccine candidates and incites investigators to explore the parasite proteins implicated in pathogenesis to identify candidates that could be efficient against clinical malaria.

Aims of study

Plasmodium falciparum malaria causes diverse clinical manifestations ranging from asymptomatic infection to uncomplicated and severe malaria including cerebral malaria. The heterogeneity of these clinical outcomes is due to both host and parasite factors. Though, the biological mechanisms implicated in the progression from mild malaria towards severe clinical state are poorly known, the variation in the genetic and epigenetic background of parasite populations may be involved the parasites virulence thus, leading to several clinical manifestations of malaria. Therefore, the relationships between parasite genetic characteristics and malaria-related pathology need to be further investigated. The cytoadherence of infected erythrocytes to microvascular endothelial cells in deep organs is believed to be an important virulence factor. This process results from the interaction between parasite proteins named variant surface antigens (VSA) expressed on the surface of infected erythrocytes (IEs) and human receptors expressed at sites of cytoadherence. Yet, the cytoadherence of IEs to a given receptor has not been attributed to a distinct clinical manifestation of malaria. Several endothelial receptors that may participate in the cytoadherence of IEs to microvascular endothelia have been identified, and few studies attempted to correlate a particular binding phenotype of IEs with a given clinical presentation of malaria. Of these receptors, CD36 and Inter Cellular Adhesion Molecule-1 (ICAM-1) were shown to be used by the majority of patient-derived parasites. We hypothesized that differential in gene expression could be at the origin of phenotypic variation of parasites resulting in specific interaction with the host which may lead to a particular manifestation of malaria. The overall aim of this PhD thesis was to gain increased knowledge about the parasite genetic factors implicated in cerebral malaria. The specific objectives of this work were 1) to analyze the transcriptomes of isolates obtained from asymptomatic carriers and patients with uncomplicated or cerebral malaria. We also investigated the transcriptomes of the 3D7 clone and the 3D7-Lib line that expresses severe malaria associated-variant surface antigen. 2) To examine the potential role of a set of up-regulated genes revealed by arrays analysis in IEs cytoadherence phenotype to a particular receptor. Firstly, we applied DNA microarrays hybridization analysis to compare gene expression profiles of isolates from Cameroonian children with cerebral malaria; uncomplicated malaria and asymptomatic carriers at ring and mature stages. We identified and confirmed by RT-qPCR deferentially transcribed genes in CM and 3D7-Lib line comparing to AM and 3D7 clone, (Manuscript 1) Secondly, among the genes in up-regulated CM-associated parasites as compared to AMassociated parasites, UPS A1, A3, B and domain cassettes DC8 and DC13 var genes groups were found to be highly induced. Hence, we aimed to elucidate their potential implication in the pathogenesis of cerebral malaria by promoting increased cytoadherence to specific receptors CD36, ICAM-1 and CSPG (CSA). Therefore, we studied the adhesive phenotype of clinical fresh isolates, collected during two field missions (June-September 2012 and May-July 2013), from Beninese children with cerebral malaria and uncomplicated malaria by static binding assay. We then performed RT-qPCR to assess the transcription level of var groups A, B, var2, var3, domain cassettes DC8 and DC13 of these isolates to identify a relationship between the cytoadherence phenotype to a specific receptor with the transcription pattern of particular Pfemp-1 variant, (Manuscript 2).

Abstract

The mechanisms underlying the heterogeneity of clinical malaria remain largely unknown. We hypothesized that differential gene expression contributes to phenotypic variation of parasites which results in a specific interaction with the host, leading to different clinical features of malaria. In this study, we analyzed the transcriptomes of isolates obtained from asymptomatic carriers, and from patients with uncomplicated or cerebral malaria. We also investigated the transcriptomes of the 3D7 clone and that of the 3D7-Lib line that expresses severe malaria associated-variant surface antigens. Our findings revealed a specific up-regulation of genes invloved in pathogenesis, adhesion to host cell and erythrocyte aggregation in parasites from patients with cerebral malaria and 3D7-Lib line. We did not find any significant difference between the transcriptome of parasites from cerebral and uncomplicated malaria. The difference between isolates from asymptomatic children and cerebral malaria concerned genes coding for surface antigens, Maurer's cleft proteins, transcriptional factor proteins, and proteins implicated in protein transport. We also found an important variation in the expression of variant surface multigenic families like var, rif and phist genes. The most outstanding difference was the up-regulation of UPS A1, A3 and UPS B1 of var genes containing architectural domains of DC4, DC5, DC8, DC13 and their neighboring rif genes in isolates from cerebral malaria and 3D7-lib. The predominance of type A var gene transcripts containing DC 4, DC5, DC8, and DC13 in parasites from children with cerebral malaria strengthens the association of these var genes with cerebral malaria pathogenesis. Therefore, deciphering the mechanisms by which proteins encoded by these genes are implicated in malaria pathology may provide new opportunities to control malaria disease. In addition, the concomitant regulation of these genes with other genes in their chromosomal neighborhood suggests a common mechanism of gene regulation in P. falciparum.

Keywords:

Plasmodium falciparum, transcriptome, cerebral malaria, asymptomatic parasitemia, microarrays-hybridization, variant surface antigens

Background

Of the five malaria species that infect humans, Plasmodium falciparum is the most common in Africa, and is the species that causes the most severe disease. According to the World Health Organization (WHO), the majority (86%) of the estimated 660 000 annual deaths related to malaria are due to P. falciparum infections that occur in children under the age of five years old living in Africa [START_REF] Greenwood | Why do some African children develop severe malaria?[END_REF][START_REF] Murray | Global malaria mortality between 1980 and 2010: a systematic analysis[END_REF][START_REF] Organization | Severe faciparum malaria. Communicable Diseases Cluster[END_REF][START_REF] Snow | The global distribution of clinical episodes of Plasmodium falciparum malaria[END_REF]. Despite an adequate antimalarial treatment, 10 to 30% of patients with severe malaria die, and 25% of survivors of cerebral malaria develop chronic neuro-cognitive impairment ( N e w t o n e t a l . 1 9 9 8 ) . Indeed, the dramatic loss of lives due to malaria and, the negative economic impact associated with the disease lead to the loss of approximately 412 billion US dollars in African countries yearly, further retarding their economic development [START_REF] Sachs | The economic and social burden of malaria[END_REF]. At present, the only means to effectively control malaria in human hosts remains the use of antimalarial drugs. However, malaria parasites are capable of developing resistance to all currently available drugs, and only a few promising novel drug candidates are under development. Furthermore, despite several candidates undergoing early phases of clinical development, a highly effective vaccine against malaria parasites is still unavailable. The identification of new therapeutic targets or vaccine candidates is hampered by the lack of knowledge on various molecular mechanisms used by malaria parasites to survive within the human host. P. falciparum infection causes a large spectrum of disease manifestations, ranging from asymptomatic infection (AM) to uncomplicated malaria (UM) and severe and complicated malaria. Only a small proportion of individuals (Organization 2011) develop severe disease, including cerebral malaria (CM), severe malaria-associated anemia (SMA), and other life-threatening clinical syndromes, such as acute renal failure, acute respiratory distress, pulmonary edema, hemoglobinuria, disseminated intravascular coagulation, and circulatory collapse [START_REF] Pongponratn | Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study[END_REF]. In areas of low transmission, severe malaria also occurs in older children (i.e. > 5 years old) and adults. After repeated malaria episodes, individuals develop natural protective immunity and considerably decrease their risk to develop severe and complicated malaria, suggesting acquired immunity and protection against severe disease [START_REF] Marsh | Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection[END_REF][START_REF] Bull | Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria[END_REF][START_REF] Dodoo | Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children[END_REF] . The heterogeneity of clinical manifestations is due to both human and parasite factors [START_REF] Mackintosh | Clinical features and pathogenesis of severe malaria[END_REF][START_REF] Ranjit | Distribution of Plasmodium falciparum genotypes in clinically mild and severe malaria cases in Orissa, India[END_REF][START_REF] Kwiatkowski | How malaria has affected the human genome and what human genetics can teach us about malaria[END_REF]. Although the biological processes involved in the progression from acute uncomplicated malaria towards severe clinical state are mostly unknown, we hypothesized that the variation of clinical manifestations depends on parasite gene expression changes.

The relationship between parasite genetic profiles and malaria-related pathologies needs to be established to further understand the pathogenesis of severe and complicated malaria. Genomic research on P. falciparum has advanced considerebly since the entire genome sequence of the P. falciparum reference clone 3D7 was determined. The P. falciparum 23-Mb genome is AT-rich and comprises 14 nuclear chromosomes, a 6-kb mitochondrial genome, and a 35-kb plastid-like apicoplast genome ( Gardne r e t al. 2 00 2) . The genome sequencing project revealed a large proportion of predicted genes which are likely to be involved in the parasite evasion mechanisms from the human immune system, and host-parasite interactions. The majority of these latter genes encode amplified gene families, such as the hypervariable variant surface antigen (VSA) genes (var, rif, stevor, and pfmc2TM) that mediate evasion from the host immune system, and genes involved in host cell invasion pathways. Gene transcription profiling of the intraerythrocytic developmental cell cycle, proteomic studies, as well as in silico sequence computational analysis have led to a substantial progress in defining gene functions and genetic regulatory networks (Le [START_REF] Roch | Discovery of gene function by expression profiling of the malaria parasite life cycle[END_REF][START_REF] Bozdech | The transcriptome of the Intra erythrocytic developmental cycle of Plasmodium falciparum[END_REF][START_REF] Kuss | Quantitative proteomics reveals new insights into erythrocyte invasion by Plasmodium falciparum[END_REF][START_REF] Patra | Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum[END_REF]). However, the absence of data from a large number of P. falciparum field isolates associated with different clinical manifestations, ranging from asymptomatic carriage to uncomplicated malaria and severe disease, tends to limit our knowledge on the biological processes involved in parasite virulence. It has been shown that parasites isolated from children with severe malaria (SM) express a limited and conserved set of VSA antigens (VSA-SM) that are more strongly and more commonly recognized by IgG from malaria-exposed individuals than VSA expressed by parasites infecting children with uncomplicated malaria (VSA-UM) [START_REF] Staalsoe | In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children[END_REF]. Therefore, the identification of VSA-SM encoding genes may reveal new therapeutic and vaccine targets for malaria control.

The aim of this study was to analyze and compare the transcriptomes of P. falciparum parasites isolates from asymptomatic carriers and patients with clinical malaria, including UM and CM, by DNA microarray hybridization, in order to investigate potential virulence factors associated with severe clinical manifestations. As controls for our experiments, we analyzed and compared the transcriptome of the 3D7 reference clone to that of the 3D7 Lib-line that expresses VSA-SM related to severe pathogenesis [START_REF] Staalsoe | In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children[END_REF].

Patients, materials and methods

Patients

After obtaining verbal informed consent from children parents or guardians, Cameroonian schoolchildren aged <12 years old were mass-screened in November 2009 to identify asymptomatic P. falciparum malaria parasite carriers (AM) in Ekoundouma, a village located about 5 km west of Yaoundé (Latitude:3.8644652°, Longitude:11.5138617°). Thick smears prepared from fingerpricked capillary blood samples were stained with 10% Giemsa, and the parasite density of P. falciparum was determined by microscopy. Children with positive thick blood smear who had not taken antimalarial treatment within the previous two weeks and who were afebrile at the time of mass screening and during the previous one week were enrolled after obtention of verbal informed consent from their parents or their caretakers. Children with gametocytemia, mixed infections with P. ovale and/or P. malariae, or fever (axillary temperature > 37.5°C) were excluded. Schoolchildren with >1,000 asexual parasites/µL of blood and symptoms associated with malaria were treated with artesunateamodiaquine combination and paracetamol, as recommended by the Cameroonian Ministry of Public Health.

Symptomatic patients with acute uncomplicated malaria (UM) were enrolled among patients consulting spontaneously at the Nlongkak Catholic missionary dispensary in Yaoundé from January to December 2009. The inclusion criteria were as follows: age < 12 years old, P. falciparum parasitemia ≥ 0.1% (or > 5,000 asexual parasites/µL of blood), absence of other Plasmodium species, fever (axillary temperature > 37.5°C) at the time of consultation or history of fever within 24 h preceding consultation, absence of signs and symptoms of severe and complicated malaria, as defined by the WHO, and denial of recent self-medication with an antimalarial drug. The enrolled patients were treated with artesunate-amodiaquine and paracetamol. Children presenting cerebral malaria (CM) were enrolled at Olembe hospital in Yaoundé, Cameroon, between April 2008 and December 2009, and at the Centre National Hospitalier Universitaire Hubert Koutoucou Mega (CNHU-HKM) in Cotonou, Benin, in 2012 and 2013 between June and August. CM was defined as a Blantyre coma score of ≤ 2 persisting for 30 min and/or at least two seizures within 24 h preceding consultation, and no other obvious cause of coma. The patients were given the appropriate treatment and necessary medical care at the hospital, according to the current guidelines for the treatment of severe and complicated malaria recommended by the Cameroonian and the Beninese Ministries of Health. Clinical history, physical and neurological examinations, as well as laboratory examinations, including P. falciparum parasitemia, creatinine, glycemia, and C-reactive protein were registered on an ad-hoc data form. After written informed consent was obtained from symptomatic children's parents or legal guardian, P. falciparum-infected blood was collected by venipuncture. Approximately 3-5 mL of blood was collected in ethylene diamine tetraacetic acid (EDTA)-coated tubes. The study protocol was reviewed and approved by the Cameroonian National Ethics Committee and the Cameroonian Ministry of Public (Health authorization N° 028/CNE/DNM/07) as well as by the ethics committee of the Research Institute of Applied Biomedical Sciences, Cotonou, Benin (N°006/CER/ISBA/12 and N°21/CER/ISBA/13).

We obtained verbal informed consent from all parents or gardiants of schoolchildren who participated in the current study, including those for which venous blood was not collected. Verbal informed consent was in agreement with the national ethic committee rules and recommendations at the time the study was performed. For all symptomatic children enrolled in the study, written informed consent was obtained from their parents or legal guardians. The consents were recorded on paper sheets. The written informed consent and the way of its recording were approved by the two ethic committees.

Cultivation of P. falciparum parasites

Two laboratory-adapted parasite lines were used. Cryopreserved stocks of 3D7 reference clone and 3D7-Lib line derived from 3D7 [START_REF] Wang | Preferential transcription of conserved rif genes in two phenotypically distinct Plasmodium falciparum parasite lines[END_REF] were cultivated for 10 days using the standard method developed by Trager and Jensen [START_REF] Trager | Human malaria parasites in continuous culture[END_REF]). Parasites were synchronized, and ring and late trophozoite stages were isolated using plasmagel [START_REF] Jensen | Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum[END_REF]. The 3D7-Lib line was obtained by several rounds of incubation with Liberian hyper-immune plasma using anti-IgG coupled with streptavidine-conjugated Dynabeads, as previously described [START_REF] Staalsoe | In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children[END_REF]. The 3D7-Lib line was shown to express group A var and neighboring rif genes, suggesting that it mainly expresses variable surface antigens (VSAs) associated with severe disease [START_REF] Kirchgatter | Association of severe noncerebral Plasmodium falciparum malaria in Brazil with expressed PfEMP1 DBL1 alpha sequences lacking cysteine residues[END_REF][START_REF] Bull | Plasmodium falciparum antigenic variation: relationships between in vivo selection, acquired antibody response, and disease severity[END_REF][START_REF] Kyriacou | Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia[END_REF].

Plasmodium falciparum isolates maturation

Venous samples containing late trophozoites were excluded, and only samples with 100% ring stage parasites were used in the present study. Samples were washed in cold phosphate-buffered saline (PBS) by centrifugation and divided into two fractions of 200 µL cell pellets. One fraction was immediately stored at -80°C in Trizol (Life Technologies) for RNA extraction. The second aliquot was cultivated for 24 to 38 hours to obtain late trophozoites. Isolates that developed into at least 85% of late trophozoite stages were harvested and stored at -80°C in Trizol.

Microarray design and hybridization

For each clinical group, three samples were constituted by pooling equivalent amounts of parasite RNA from six isolates to perform array hybridization. To ensure that the same quantity of RNA from each isolate was analyzed, we used isolates of approximately the same parasitemia. For the controls (3D7 clone and 3D7-Lib line), three samples (at either ring or late trophozoite stage) were obtained from three independent parasite cultures.

Microarrays were manufactured by Agilent Technologies using SurePrint in situ synthesis technology (Agilent Technologies France SAS, Les Ulis, France). Fifteen slides of eight arrays each containing 14,882 oligonucleotides covering 5,534 Open Reading Frame (ORF) sequences derived from the P. falciparum database were used. The oligonucleotides were designed using oligoarray2 software [START_REF] Rouillard | OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach[END_REF]) by setting the general parameters to 55-60 bases for oligonucleotides length with a melting temperature between 79 and 81°C, and with a 3' bias of 1,500 nucleotides since RNases preferentially act from the 5' end. The melting temperature range was relaxed for genes for which no oligonucleotide could be found with the default parameters. The array contained two sets of oligonucleotides. The first set was designed on the basis of the 3D7 sequence genome. These oligonucleotides were chosen within the coding sequences (CDS) of protein-coding genes in the nuclear genome. A minimum of three oligonucleotides per gene were designed when possible. To avoid cross-hybridization with human genes, cross-hybridization was computed using both 3D7 and human genome libraries. The second set of oligonucleotides was designed to hybridize to genes exhibiting large sequence variations, including var, rif, stevor and Pfmc-2TM multigenic families. For these genes, all sequences available in the GenBank and those kindly provided by our collaborators (T. Lavstsen, Centre for Medical Parasitology, University of Copenhagen, Denmark) were retrieved and added to the array. Sequence alignment using Clustal W and Clustal X version 2.0 [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF]) was performed for each family or sub-family, and specific oligonucleotides were designed. For the other genes with high sequence variations (i.e., genes with high numbers of single nucleotide polymorphisms [SNPs]), sequences were retrieved from PlasmoDB, and specific oligonucleotides were designed.

Total RNA from 200 µl of P. falciparum pellet and laboratory-adapted parasites (3D7 and 3D7-lib) was extracted using chloroform-isopropanol method as recommended by the manufacturer. The integrity of the total RNA from each sample was checked using Agilent 2100 Bioanalyser (Agilent Technologies, France). For reverse transcriptase (RT)-real time PCR (rtPCR), RNA was treated with DNAse at 37°C for 15 min. The absence of contaminating DNA was assessed by 40 cycles of rtPCR using primers targeting seryl tRNA transferase and fructose biphosphate aldolase genes, which are highly conserved P. falciparum housekeeping genes. Total RNA (6-10 µg) was transcribed using the SuperScript First-Strand Synthesis System® (Life Technologies) and random hexamer oligonucleotides mixture. The newly synthesized cDNA was labeled using SuperScript® indirect cDNA labeling system (Invitrogen, Carlsbad, CA) to generate green fluorescent Cyanin-3 labeled cDNA according to the manufacturer's instructions. The quantity of labeled cDNA was measured using Nanodrop® (Thermo Fisher Scientific, Villebon-sur-Yvette, France) to ensure that the same quantity of labeled cDNA was deposited for each array hybridization. After quantification of the fluorescent specific activity of Cy3-labeled cDNA, the quantities of targets were normalized at 100 pmol of Cy3, and hybridized for 24 h at 65°C. The slide was washed with water and dried at room temperature. Slides were scanned using GenePix 4000 A scanner (Axon), and the images were analysed using (GenePix5 software, Axon). Dusted and poorly fluorescent spots (below the background level) were excluded from analysis. After array gridding, data were exported as gpr files for statistical analysis.

Quantitative real-time rtPCR

Quantitative rtPCR was performed to confirm array results. For each target gene, a pair of primers was designed to obtain specific DNA sequence fragments that span less than 350 bp. Primer sequences are presented in Table 1 supplemented Data S1. Individual real-time rtPCR amplifications were carried out in a final volume of 20 µl in a Frame Star 384-well plate (4titude® France, Bagneux, France) containing 1× final concentration of SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma-Aldrich, Saint-Quentin Fallavier, France), 100 nM of gene-specific primers and 1 µl cDNA. rtPCR was performed on using 7900 HT (Applied Biosystems®, Saint Aubin, France). The thermal cycle program was as follows: 10 minutes of pre-incubation at 95°C followed by 95°C x 15s and one minute at 60°C x 1 min for 40 cycles. All samples were run in triplicate. Threshold cycles (Ct) and melting curves were analyzed using Applied Biosystems ABI SDS software 2.3. The level of amplification referred to the number of cycles at which the PCR product was detectable and confirmed by the melting curve for each gene. Normalized data were used to quantify the relative levels of a given mRNA between samples using the ΔΔCt analysis [START_REF] Hooper | Molecular analysis of commensal host-microbial relationships in the intestine[END_REF]. Before proceeding to the relative quantification using this method, the similarity in PCR amplification efficiency of the target gene and a house keeping gene (argenyl tRNA synthetase, PF3D7_1218600 / PFL0900c) used as internal controls was checked. To validate the array results by rt-qPCR, a set of 14 genes were chosen randomly from genes that were differentially expressed between the clinical groups and between 3D7 and 3D7-Lib laboratoryadapted parasites. These genes were grouped into three categories, depending on their fold change on the arrays: down-regulated genes defined as Fold change [FC] value < 2 units of expression), moderately up-regulated genes (FC value, 2-7 units of expression), and highly up-regulated genes (FC value > 7 units of expression). To further confirm our findings, another rt-qPCR was performed for a set of var gene family found to be up-regulated by arrays analysis using a new batch of parasite RNAs isolated from children with CM or UM. The relative expression between the two conditions was calculated by the 2 -ΔΔCt method {Hooper, 2001 #6151} and the transcript abundance of var genes was expressed as Transcript units (T u ), defined as T u = 2 (5-ΔCt) [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF].

Statistical analysis

Data generated from the arrays were analyzed using the R software 2.14.1 R Core Team (2012) http://www.R-project.org/ [START_REF] Gentleman | Bioconductor: open software development for computational biology and bioinformatics[END_REF]) and the Bioconductor limma package [START_REF] Smyth | Use of within-array replicate spots for assessing differential expression in microarray experiments[END_REF]. Since hybridizations included only one condition per array, the resulting one-color hybridization signal was first loaded in a limma-like RG structure with the red foreground signal set to 1. Background signals were set to 0 for both colors, and the RG data structure was transformed into MA one using the RG.MA function, with logarithmic ratios in log 2 (green signal/red signal). A quantile normalization between arrays was applied to the full data set [START_REF] Smyth | Normalization of cDNA microarray data[END_REF]. After exclusion of flagged (invalid) and control spots, differentially expressed genes were extracted using linear models and the empirical Bayes method implemented in limma [START_REF] Smyth | Linear models and empirical Bayes methods for assessing differential expression in microarray experiments[END_REF]), followed by a Benjamini and Yekutieli (BY) P-value adjustment with error value set to 0.05 [START_REF] Benjamini | Controlling the false discovery rate in behavior genetics research[END_REF].

Mean fold changes of differential expression between pairs of conditions were computed and a cut-off value of 2 was applied. Transcripts were considered as differentially expressed when all their probes had a fold change higher than 5 and a p-value lower than 0.05. The rank-based nonparametric Spearman's test (Rho) was used to correlate qPCR and array results, and the Kendall test was used to test the correlation between our results and the published arrays data. The means of continuous variables were compared using the one-way analysis of variance, followed by Bonferroni multiple comparison post-test. Statistical tests were performed using SigmaStat 3.5 (Systat Software, Inc. Point Richmond, CA). The significance level was set at P < 0.05.

Bioinformatics analysis

Gene Ontology term analysis of differentially genes set: Functional analysis of the differentially expressed genes was performed using the GOstats package in Bioconductor with default settings [START_REF] Falcon | Using GOstats to test gene lists for GO term association[END_REF]) and Sanger GeneDB P. falciparum gene annotations. The supplementary file lists the top eight most overrepresented GO terms in each set (table 2).

Clustering analysis: Differentially expressed genes were clustered according to their fold change using the neighbor joining algorithm with Lance-Williams dissimilarity updates (hclust function from R 2.15.1) starting from euclidian distance on fold changes.

Results

Characteristics of patients

A total of 54 patients were enrolled in the study: 18 children with cerebral malaria (CM), 18 children with acute uncomplicated malaria (UM), and 18 children with asymptomatic malaria (AM). The clinical and biological characteristics of these patients are summarized in Table 1. The mean age differences between patients of the three clinical groups were statistically significant (P < 0.05). At enrolment, mean body temperatures were comparable between CM and UM groups (P > 0.05). As expected, the asymptomatic carriers were all afebrile, and their mean body temperature was significantly lower (P < 0.05) than that of the other two clinical groups. The CM group had significantly lower hemoglobin values than the UM group (P < 0.05). The geometric mean parasite densities of CM and UM groups were not significantly different (P > 0.05). Asymptomatic carriers had a significantly lower parasite density compared with that of other patient groups (P < 0.05). These differences in parasite densities were corrected before performing the arrays. To avoid bias in the expression pattern of abundant transcripts due to high parasitemia, RNAs from highly parasitized CM and UM isolates were diluted to obtain similar concentrations of RNA as in AM isolates.

Gene expression pattern of field isolates

For each clinical group, three pools of six isolates were built and hybridized on arrays at both ring and late trophozoite stages. These isolates were collected from children with CM, UM, or AM. The transcriptome of isolates at the same asexual developmental stage was compared to that of the 3D7 reference clone. Furthermore, the transcriptome of clinical isolates from children with CM was also compared to that from children with AM. We also compared the transcriptome of the parent clone 3D7 to that of the selected 3D7-Lib. Figure 1 presents the MA plot of differentially expressed genes between these two conditions. Our transcriptomic data from 3D7 reference clone was also compared to those of the published database [START_REF] Bartfai | H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3[END_REF], which yielded a high correlation between the two transcriptomic data. The transcriptome of the rings was obtained by hybridization of cDNA from a mixture of 80% rings, 5% late trophozoites, and 15% schizonts for laboratory-adapted parasite lines. All field isolates consisted of 100% ring stages. In parallel, the transcriptome of late trophozoites of both laboratoryadapted parasites and clinical isolates was analyzed by cDNA hybridization using a mixture of 85% late trophozoites, 10% schizonts, and 5% rings.

Using one-color labeling method, the mean green fluorescence intensity after removing the background signal reflects the presence or absence of cDNA corresponding to each gene among the 5,534 analyzed ORFs. The analysis of transcriptomic data of 3D7 and 3D7-Lib ring stage parasites provided 168 transcripts with a mean fluorescence level below the background, which were therefore considered to be untranscribed. Likewise, in late trophozoite stages, 61 of these 168 transcripts were also not transcribed. However, the transcriptome data of field isolates at late trophozoites stage showed only 38 transcripts with fluorescence intensities below the background level. Transcriptomic data from 3D7 reference clone and field isolates at the same developmental stages were highly correlated (r = 0.79) in most (4,837 to 5,344) transcribed ORFs. These results suggest that we detected the majority of isolate transcripts at both ring and late trophozoite stages. The lists of non-transcribed genes in 3D7, 3D7-Lib, CM-associated isolates, and AM-associated isolates are provided in supplementary information Tables 2, 3, 4, SI, respectively.

Differentially expressed genes in the 3D7-Lib and 3D7

3D7-Lib expresses VSAs that are potentially implicated in severe and complicated malaria, including cerebral malaria. The comparison of this selected 3D7-Lib to the parent 3D7 reference clone showed some differences in the expression profile. A total of 395 transcripts were up-regulated in 3D7-Lib late trophozoites, as compared to the corresponding developmental stage of 3D7. Of these 395 genes, 52 displayed 3-to 9-fold higher expression in 3D7-Lib. Some of these genes encod ring exported proteins REX1, REX2, REX3, erythrocyte membrane protein PfEMP1 members, membraneassociated histidine-rich protein (MAHRP1), histidine-rich protein II (HRPII), Plasmodium exported proteins (PHISTa, PHISTb, PHISTc), DnaJ protein, putative serine/threonine protein kinase family (FIKK4.2) and hypothetical proteins. A total of 296 genes were slightly down-regulated, exhibiting 2to 3-fold change in the expression level. The comparison of ring-stage transcriptomes revealed 30 up-regulated genes, ranging from 4-to 30-fold increased expression, in 3D7-Lib, and five downregulated genes. The majority of the overexpressed genes encoded proteins belonging to PfEMP1 and RIFIN families, exported proteins, knob-associated histidine-rich protein (KAHRP), proteins with DNAJ domain, and some hypothetical proteins. The group A var genes [PF3D7_1100200 / PF11_0008, PF3D7_1150400 / PF11_0521), PF3D7_1300300 / PF13_0003), and PF3D7_0425800 /PFD1235w], and neighboring rif genes [PF3D7_1100300 / PF11_0009, PF3D7_1150300 / PF11_0520, PF3D7_1300400 / PF13_0004, and rif PF3D7_0425700 / PFD1230c] were up-regulated in 3D7-Lib, as compared to 3D7, with an expression level ranging from 2.5 to 26-fold (Table 5, SI). These genes were found to be up-regulated in 3D7-Lib in a previous study [START_REF] Wang | Preferential transcription of conserved rif genes in two phenotypically distinct Plasmodium falciparum parasite lines[END_REF]).

Identification of differentially expressed genes in CM compared with AM.

Only the comparison between late trophozoites from CM and AM patients showed statistically significant differentially expressed genes, leading to 99 up-regulated transcripts and 135 down-regulated transcripts (Table 5, SI). However, the comparison between parasites from CM and UM patients, or from AM and UM patients showed no significant differences in the transcriptomic pattern. Up-regulated genes in CM parasites compared to AM parasites exhibited 3 to 14 higher expression rates, while down-regulated genes showed 3-to 22-fold change. The 15 most upregulated genes encode Plasmodium exported proteins, glycophorin-binding proteins (GBP), hypothetical proteins, proteins encoding VSA, such as PfEMP1, RIFIN, other surface proteins like tryptophan-and threonine-rich antigen, and ring-infected erythrocyte surface antigen (RESA). Of the 99 up-regulated genes in CM isolates, 39% were also up-regulated in 3D7-Lib, and include members of rif A1 and var A group, some of them being topologically located in the neighborhood. In parallel, 135 transcripts were down-regulated in CM parasites, as compared to AM parasites. Among these 135 transcripts, 36 showed a 5-to 22-fold decrease in transcription and correspond to genes encoding rhoptry-associated proteins (RAP1, RAP2, and RAP3), merozoite surface proteins (MSP3, MSP7, and MSP9), Maurer's cleft 2 transmembrane domain protein, and hypothetical proteins. We then compared CM and AM-associated isolates transcriptomes to the laboratory-adapted 3D7 one. Significant differences in gene expression between field maturated trophozoites from individuals with CM and those with AM, compared to the 3D7 were observed. In CM-associated isolates, 182 transcripts were up-regulated and 85 were down-regulated, while in AM-associated parasites 40 were up-regulated and 41 were down-regulated. The top list of up-regulated transcripts in CM parasites presented 2 to 9-fold changes and the most down-regulated genes presented fold changes ranging from 2 to 16 for the majority of transcripts with only one transcript that was not transcribed. 5 0 to 67% of up-regulated genes in both CM-and AM-associated parasites encode exported proteins that are located on the RBC surface: PHIST, RIFIN, STEVOR, PfMc-2TM and PfEMP1 members. In addition to the up-regulation of VSA-encoding genes, like var and rif, we observed the up-regulation of genes encoding Skeleton Binding Protein1 (SBP1), HRPIII, glycophorin binding protein (GBPH2), as well as genes encoding Plasmodium conserved proteins for which no function has been assigned yet. Down-regulated genes in CM-associated parasite isolates include other members of Plasmodium exported proteins such as Pfmsp11 and genes encoding variant surface proteins belonging to RIFIN, PfMc-2TM, STEVOR, and PfEMP1 as well as other genes [PF11_0442, PF13_0090, PFC0185w, PF11_0381] encoding transcription factors with AP2 domain, ADP-ribosylation factor, membrane skeletal protein IMC1, respectively. PF10_0343 coding for S antigen was the most repressed transcript in CM-and AM-associated parasites, respectively. This gene might not be transcribed in these isolates. The non-significant differences in the transcription pattern between CM and UM parasites may result from a similar transcription pattern between these two parasite populations or may be due to the inability of arrays to detect slight variations. Therefore, in order to confirm the specific regulation pattern of these genes in CM -associated parasites, we examined the transcription pattern of UPS A1, A3, and B1 var groups found to be highly up-regulated in another set of RNAs collected from children presenting CM or UM. We found that these genes were significantly up-regulated in CM-associated parasites, as compared to UM-associated parasites (Fig 3) and that our results were in agreement with our arrays outcomes.

Confirmation of array results using quantitative real-time rt-PCR

The differential transcription level obtained by DNA hybridization arrays was further confirmed by quantitative PCR using cDNA from the samples used for array hybridization (Fig. 2) and cDNA from a second set of samples (Fig. 3). A highly significant correlation (rho = 0.775; P < 0.0011, Spearman's rank correlation) between array and qPCR results was found, indicating that both methodological approaches yielded similar results and identified the genes that were either down-or upregulated with accuracy (Fig. 2; S1).

Bioinformatics analysis of differentially expressed genes

Gene Ontology term analysis of up-regulated genes in CM-associated parasites compared to AM-associated parasites, as well as in 3D7-Lib at ring and late trophozoite stages compared to 3D7 revealed GO terms of pathogenesis as the most overrepresented term (Table 2). Other over represented GO terms included cytoadherence to microvasculature mediated by symbiont protein, antigenic variation, or interaction with host.

Clustering analysis performed for up-regulated genes according to their fold change exhibited a network of two clusters in up-regulated gene sets for the three compared conditions, CM versus AM, 3D7-Lib T versus 3D7 T, and 3D7-lib R versus 3D7 R. For CM up-regulated genes, the first cluster contained three genes with PF3D7_0201700 / PFD0085c; encoding acylcoA gene separated from PF3D7_1301200 / PF13_0010 gene encoding GBPH2 and PF3D7_0701900 / PF07_0004 encoding Plasmodium exported protein. Similarly, comparion of the first cluster of 3D7-Lib T versus 3D7 contained PF10_0038; gene encoding 40S ribosomal protein (S20e) separated from PF3D7_0935900 / PFI1735c REX1, PF3D7_1301700 / MAL13P1.61 Plasmodium exported protein, (GEXP07), PF3D7_1001500 / PF10_0019 early transcribed membrane protein 10.1 (ETRAMP10), as well. The first cluster of 3D7-Lib R up-regulated genes also contained four genes with KAHRP-encoding gene with the highest fold change followed by three PfEMP1 encoding genes; PF13_0003, PF11_0521 and PFD1235w.

In the three conditions, the second cluster contained the remaining up-regulated genes within two sub-networks in which some of the connected genes were grouped according to their chromosomal location or sequence and functional similarities, as PF3D7_0201600 / PFB0080c, encoding PHISTb, PF3D7_0201800/PFB0090c, encoding RESA-like protein with PHIST and DNAJ domains.

Discussion

Microarray-based transcriptomic studies are powerful approaches that have been successfully applied to identify many virulence genes in bacterial and cancer cell systems ( M a h a n e t a l . 1 9 9 3 ; A l i z a d e h e t a l . 2 0 0 0 ) . Due to the limited amount of RNA that can be obtained from clinical samples of P. falciparum-infected individuals, only a few studies have investigated whole-transcriptomes of clinical isolates [START_REF] Daily | In vivo transcriptional profiling of Plasmodium falciparum[END_REF][START_REF] Daily | In vivo transcriptome of Plasmodium falciparum reveals overexpression of transcripts that encode surface proteins[END_REF][START_REF] Daily | Distinct physiological states of Plasmodium falciparum in malaria-infected patients[END_REF][START_REF] Ndam | Plasmodium falciparum transcriptome analysis reveals pregnancy malaria associated gene expression[END_REF][START_REF] Vignali | NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children[END_REF]). These studies have demonstrated substantial differences in gene expression patterns between clinical isolates and laboratory-adapted P. falciparum strains. This difference in gene transcription between in vitro and in vivo conditions is probably related to the biological processes and adaptation that the parasites undergo to survive within the human host or in artificial in vitro environment. The host-parasite interactions that require gene activation include parasite sequestration and immune system evasion, sexual differentiation for parasite transmission, as well as various metabolic processes that are essential for in vivo parasite survival. The present study aimed to compare and analyze the whole transcriptome of P. falciparum parasites isolated from patients with distinct clinical features, including asymptomatic carriage, acute uncomplicated malaria (UM), and cerebral malaria (CM), in order to gain new insights into regulation of gene transcription underlying malaria pathology. Our results indicate noticeable variation in gene transcription pattern of parasite isolates compared to 3D7, as well as between the parent 3D7 and selected 3D7-Lib. Interestingly, we observed a consistent variation in the gene transcription pattern between parasites isolated from children with CM and parasites isolated from asymptomatic children. Although the array hybridization failed to detect differentially expressed genes between CM and UM, and between UM and AM parasites, our rt-PCR results indicate that the up-regulation of specific var gene groups in CM-associated parasites, which was not observed in AM-and UM-associated parasites, might be of biological significance. It is likely that genes differentially expressed in parasites associated with CM, compared to those from asymptomatic carriers, are implicated in the biological mechanisms involved in CM pathogenesis, such as cytoadherence of parasitized erythrocytes to brain endothelial cells, leading to an enhanced inflammation, both locally at the parasite-host cell binding site and elsewhere far from the binding site via the action of cytokines. These assumptions are reinforced by our test for enrichment on gene ontology categories of up-regulated genes in 3D7-Lib and CM-associated parasites, which yielded the overrepresentation of pathogenesis, regulation of cell adhesion, cytoadherence to microvasculature, erythrocytes aggregation, and antigenic variation GO terms.

We then focused our attention on genes induced both in isolates from children with CM and in isolates from children with AM. These genes were also up-regulated in 3D7-Lib, compared to 3D7. This finding allowed us to develop an in vitro model for further extensive studies on these gene candidates. We also compared our transcriptomic results to published data and found that the majority of transcripts that were not transcribed in our study in ring and trophozoite stages of 3D7 and 3D7-Lib were also absent in previous array data ( [START_REF] Roch | Discovery of gene function by expression profiling of the malaria parasite life cycle[END_REF][START_REF] Bozdech | The transcriptome of the Intra erythrocytic developmental cycle of Plasmodium falciparum[END_REF], suggesting untranscribed ORFs in in vitro cultivated parasites. However, many transcripts absent from the 3D7 transcriptome were present in our transcriptomic data of field isolates and in the transcriptome of other clinical isolates [START_REF] Vignali | NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children[END_REF], suggesting that these transcripts are not essential for asexual in vitro survival within the RBC. Among the undetected transcripts in ring and trophozoite stages in 3D7 clone, most were amplified gene families that encode (PfEMP1, RIFIN, STEVOR, and PFMC2TM) and unknown hypothetical proteins. The difference in the expression of amplified gene families may be due to an inherent variable expression between isolates or to the high number of distinct parasite populations in field isolates. In our previous study in the same study site, 60% of infections were multiclonal and harbored up to seven distinct parasite populations [START_REF] Basco | Molecular epidemiology of malaria in Cameroon. XVIII. Polymorphisms of the Plasmodium falciparum merozoite surface antigen-2 gene in isolates from symptomatic patients[END_REF]. It is also likely that these represent vatiants that doe not exist in 3D7

We also compared our transcriptomic data from ring stage and late trophozoite stage of 3D7, as well as from parasite isolates, to those of the corresponding stages of 3D7 obtained by RNA sequencing [START_REF] Bartfai | H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3[END_REF]) and found a highly significant correlation (p = 2.2 e-16 , Kendall test) between these data. These results demonstrate the robustness of the array hybridization method to provide consistent data that are comparable to RNA sequencing and reflect the conserved nature of basic biological processes, including general metabolism and cell cycle, in both in vivo and in vitro conditions. This high correlation is consistent with the predominance of ring stage parasites in the peripheral blood of infected individuals and the changes in the transcription pattern leading to the progression and development towards the late trophozoite stage.

Differentially transcribed genes in parasites from CM patients compared to those of AMassociated parasites are expected to provide insights into parasite-associated virulence factors. We investigated the function of 99 up-regulated genes in parasites from children with CM compared to the parasites from children with AM, and found that 28% of up-regulated genes encode exported proteins, with the majority (67%) of them being VSA-encoding genes, such as var, rif, phist, and surfin. These genes are implicated in parasite sequestration and immune evasion. Interestingly, var group A/Ups A1, A3, and/or UpsB1, [PF3D7_1150400 / PF11_0521, PF3D7_1300300 / PF13_0003, PF3D7_0937600 / PFI1820w, PF3D7_0632800 / PFF1595c, PF3D7_0425800 / PFD1235w] were upregulated in CM-associated isolates and in the 3D7-Lib line, while [PF3D7_0200300 / PFB0020c PF3D7_0400400 / PFD0020c] were up-regulated only in CM-associated parasites. 3D7 PF3D7_1150400 / PF11_0521 displays DBLα-1.7 and CIDR-1.4, while PF3D7_0400400 / PFD0020c displays CIDRα-1.1, DBL12, DBL6. These architectural domains characterize Domain Cassettes DC13 and DC8, respectively, which were shown to be transcribed at higher levels in parasites from children with severe malaria in Tanzania [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF] and Benin [START_REF] Bertin | Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin[END_REF], as compared to the parasites from children with uncomplicated malaria. Similarly, PF3D7_1300300 / PF13_0003, PF3D7_0425800 / PFD1235w contain DC5 which also seems to be associated with severe malaria [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]) and parasite binding to the human receptor PECAM1 [START_REF] Berger | Plasmodium falciparum expressing domain cassette 5 type PfEMP1 (DC5-PfEMP1) bind PECAM1[END_REF]. Our findings are consistent with these earlier results. Furthermore, the transcription of PF3D7_0400400 / PFD0020c and that of its orthologs IT4var19, HB3var3, and IT4var7 were also shown to be highly induced in parasite lines selected for binding to human brain endothelial cells (HBEC) ( C l a e s s e n s e t a l . 2 0 1 2 ; A v r i l e t a l . 2 0 1 2 . These genes also encode DC8 and DC13containing PfEMP1s and antibodies raised against the NTS-DBLα1 and the NTS-DBLα1-CIDRα1 domains of HB3var3 inhibit the binding of infected erythrocytes on HBEC, providing further support of their implication in cytoadherence (C l a e s s e n s e t a l . 2 0 1 2 ) . In addition, recently the endothelial protein C receptor (EPCR) was identified as receptor for DC8 and DC13-containing PfEMP1s, suggesting a crucial role of EPCR binding in the pathology of malaria (Turner L. 2013). Interestingly, anti-PfEMP1 IgG antibodies targeting the recombinant DBLβ9 of PF13_0003, DBLβ3, DBLγ13, CIDRα1.6 of PFD1235w, CIDRα1.4 of PF11_0521 were also found in children exposed to malaria [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF], suggesting a selective expression of these PfEMP1 variants by parasites infecting children that lead to an activation of the immune system and response by antibodies synthesis.

Similarly, 3D7 PF13_0003, which is characterized by DBL1α1 and CIDR1γ that are shared with other genes, such as PaloAlto89F5 VarO, IT4/I29, and IT4var60, is implicated in rosetting (Vigan -Womas et al. 2008;[START_REF] Albrecht | var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2[END_REF] . This gene was up-regulated in parasites from children with CM and was highly induced in 3D7-Lib. The N-terminal region of DBL1α1 plays a role in the formation of pseudo-rosettes on the surface of a fibroblast-like cell line COS and baculovirusinfected cells expressing individual PfEMP1 domains (Vigan-Womas et al. 2008;[START_REF] Rowe | P. falciparum rosetting mediated by a parasitevariant erythrocyte membrane protein and complement-receptor 1[END_REF]). In the present study, the up-regulation of exclusively UPS A1 and UPS A3 var groups in isolates from children with CM is in strong agreement with the results obtained in other studies [START_REF] Kirchgatter | Association of severe noncerebral Plasmodium falciparum malaria in Brazil with expressed PfEMP1 DBL1 alpha sequences lacking cysteine residues[END_REF][START_REF] Kyriacou | Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia[END_REF][START_REF] Jensen | Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes[END_REF][START_REF] Warimwe | Prognostic indicators of lifethreatening malaria are associated with distinct parasite variant antigen profiles[END_REF]) reinforcing the relationship between specific PfEMP1 protein expression and the development of CM. Var genes are known to be ubiquitously transcribed at the early ring stage with restriction to only one transcript during mature trophozoites stage [START_REF] Scherf | Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intraerythrocytic development in Plasmodium falciparum[END_REF][START_REF] Chen | Developmental selection of var gene expression in Plasmodium falciparum[END_REF]. Our finding on the differential up-regulation of var genes in late trophozoïtes may be inherent to the high proportion of ring stages in our mRNA preparation from cultivated isolates as well as in laboratory-adapted parasites. Rif genes PF3D7_0631800/ PFF1545w, PF3D7_1041000 / PF10_0403, PF3D7_1041000 / PFC1100w PF3D7_1150300 /PF11_00520, PF3D7_0425700 / PFD1230c, PF3D7_1100300 / PF11_0009, phist a, b, c PF3D7_1478000 / PF14_0752, PF3D7_1253300 / PFL2565w, PF3D7_0702100 / MAL7P1.7, PF3D7_0936900 / PFI1785w, PF3D7_0830600 / MAL8P1.4, PF3D7_0219700 / PFB0900c, and PF3D7_1300300 / PF13_0003 pfemp1 were also up-regulated in 3D7-Lib and/or in both 3D7-Lib and parasites from CM patients. The phist a PF3D7_1478000 / PF14_0752 was previously reported to be up-regulated in parasite lines selected for binding to HBEC [START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF]. The rif genes are composed of a large family of about 150 clonally variant genes grouped into two subfamilies, A-and B-type RIFINs, according to their subcellular localization and 25-amino acid sequence, which occurs only in A-type RIFINs ( Joinnin e t al. 2008) . The A-type RIFINs are thought to be trafficked to the surface of the infected erythrocyte [START_REF] Khattab | Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion[END_REF], while some B-type RIFINs have been associated with the extracellular stages, merozoites and gametocytes [START_REF] Mwakalinga | Expression of a type B RIFIN in Plasmodium falciparum merozoites and gametes[END_REF]. The majority of the gene family members are located near the var genes within the sub-telomeric regions of the chromosomes. These regions are subjected to recombination, increasing their diversity. In 3D7-Lib, the rif PF3D7_1150300 / PF11_00520, PF3D7_1100300 / PF11_0009, PF3D7_0425700 / PFD1230c, PF3D7_1300400 / PF13_0004, were concomitantly upregulated with their neighboring var genes PF3D7_1150400 / PF11_0521), PF3D7_1100200 / PF11_0008, PF3D7_0425800 / PFD1235w, PF3D7_1300300 / PF13_0003. These genes may be under the control of the same promoter region.

Other genes, such as PF3D7_1301200/PF13_0010, PF3D7_1401000 /PF14_0010, and PF3D7_0501300/PFE0065w, were also found to be highly up-regulated in parasites from children with CM. PF3D7_1401000/PF14_0010 and PF3D7_1301200/PF13_0010, encode GBPH and GBPH2, respectively. These proteins belong to Maurer's cleft proteins [START_REF] Lanzer | Maurer's clefts: a novel multifunctional organelle in the cytoplasm of Plasmodium falciparum-infected erythrocytes[END_REF], either transiently during their export to the infected erythrocyte membrane, like PfEMP1, RIFIN [START_REF] Petter | Variant proteins of the Plasmodium falciparum RIFIN family show distinct subcellular localization and developmental expression patterns[END_REF], SURFIN [START_REF] Winter | SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes[END_REF], STEVOR [START_REF] Kaviratne | Small variant STEVOR antigen is uniquely located within Maurer's clefts in Plasmodium falciparum-infected red blood cells[END_REF]), PfMC-2TM (Sam-Yellowe et al. 2004), or are constitutive proteins of the Maurer's cleft itself, as Plasmodium skeleton-binding protein 1 (PfSBP1) which is encoded by PF3D7_0501300/PFE0065w [START_REF] Sam-Yellowe | A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling[END_REF]). PfSBP1 was demonstrated to be implicated in PfEMP1 trafficking to the surface of erythrocytes [START_REF] Spycher | Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes[END_REF]. PF3D7_1478000 / Pf 14_0752 encodes MAHRP1 (Phist a), a Maurer's cleft resident protein also essential for PFEMP1 trafficking to the surface of infected red blood cells [START_REF] Spycher | Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes[END_REF]); [START_REF] Spycher | Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes[END_REF]). These two genes were transcribed at higher levels in field isolates, compared to 3D7. MAHRP1 was among the 15 most up-regulated genes in parasite lines selected to adhere on HBEC [START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF]). Among the regulated genes, some code for antigenic proteins, such as P. falciparum sporozoite surface threonine-and asparagine-rich protein (STARP) (PF3D7_07023005 / PF07_0006, P. falciparum ring-infected erythrocyte surface antigen (RESA) PF3D7_1149200 / PF11_0509. These genes were found to be up-regulated in clinical isolates, as compared to laboratory-adapted strains [START_REF] Mackinnon | Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates[END_REF]). The genes implicated in the transcription process, including PF3D7_0318200 / PFC0805w DNA-directed RNA polymerase II, PF3D7_1433500 / PF14_0316 putative DNA topoisomerase II, PF3D7_1220100 / PFL0970w PF3D7_1231600 / PFL1525c pre-mRNA splicing factors were transcribed at higher levels in CM-associated parasites, compared to AM-associated parasites and 3D7. PF3D7_1220100 / PFL0970w was highly expressed in parasites from children and pregnant women [START_REF] Vignali | NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children[END_REF]. However, the transcription factors with ApiAp2 domains PF3D7_1439500 / PF14_0374, PF3D7_1143100 / PF11_0442 were slightly down-regulated in CM-and AM-associated parasites, compared to 3D7 which is consistent with the findings of Vignali et al [START_REF] Vignali | NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children[END_REF].

Several differentially expressed genes in the present study were similarly regulated in other studies [START_REF] Mackinnon | Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates[END_REF], supporting the variable nature of expression pattern of these genes between field isolates and 3D7 clone and the robustness of array hybridization method to produce reproducible results despite technical differences between laboratories. For instance, (REX1) ringexported protein 1, PF3D7_0935900 / PFI1735c, (REX2) ring-exported protein 2, PF3D7_0936000 / PFI1740; (GIG) gametocytogenesis-implicated protein PF3D7_0935600 / PFI1720w, (STARP) sporozoite threonine-and asparagine-rich protein PF3D7_0702300 / PF07_0006; lysophospholipase, putative PF3D7_0702200 / PF07_0005, conserved Plasmodium protein with unknown function PF3D7_0702400 /PF07_0007, (Pfg27) gamete antigen 27/25 PF3D7_1302100 / PF13_0011 were all up-regulated in AM-and CM-associated isolates analyzed in our study and were also among the top 20 up-regulated genes in clinical isolates in a previous study [START_REF] Mackinnon | Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates[END_REF]). REX1, REX2 and GIG genes are located in close proximity in chromosome 9, while STARP and putative lysophospholipase as well as a conserved Plasmodium gene are located within a close distance in chromosome 7, suggesting coregulation of gene transcription due to their proximity. Similarly, when examining the topological location of all up-regulated genes in parasites from CM patients compared to those from AM patients, three to 12 genes were located on the same chromosome at a distance ranging from 5 to 100 kb, a distance at which the variation of expression can be controlled by a single promoter or the same epigenetic mechanism.

In conclusion, we identified a set of specifically up-regulated genes in p a r a s i t e s c o l l e c t e d f r o m CM patients, which are likely to be implicated in the pathogenesis of cerebral malaria. Furthermore, our findings on type A var genes containing DC5, DC8 and DC13 in isolates from children with CM and in the 3D7-Lib are in agreement with other published studies ( L a v s t s e n e t a l . 2 0 1 2 ; C l a e s s e n s e t a l . 2 0 1 2 ; A v r i l e t a l . 2 0 1 2 ; W a r i m w e e t a l . 2 0 1 2 ; N o r m a r k e t a l . 2 0 0 7 ) , supporting the potential implication of these genes in CM.

These results call for further investigations on PfEMP1 members related with severe malaria for a better understanding of malaria-associated pathologies. In addition to PfEMP1 group A1, A3 and B1 subset, other genes encoding exported proteins as well as non-exported proteins, were up-regulated in CM-associated parasites. The potential role of these genes in CM deserves to be further investigated and may allow the identification of new targets for malaria control.

Authors contribution

RT, PD, NTN, AA, CR, MAD, and JYC conceived the study design. TA carried out RNA extraction, and RT performed qPCR experiments and participated in the analysis of results. GN and MAD carried out statistical analysis. EB and NTN designed oligonucleotides used in the arrays. ME carried out gene ontology term analysis. CWW performed selection of parasite strains. GNA participated in patient recruitment. RT performed sample collection, in vitro cultivation of isolates, arrays hybridization, analysis of results. TA, RT, LKB, CWW, EB, and PD wrote the manuscript. All authors have read, corrected, and approved the final manuscript. (38,200-65,600; 12,800-109,000) 3,460 (2,400-5,000; 1,120-9,630) Mean Body Temperature (°C) 39.0 ± 1.1 38.9 ± 0.8 36.8 ± 0.4

Mean hematocrit ± SD(%)

24.6 ± 3.7 29.9 ± 5.7 ND 1 mean Blantyre score, 2/5; mean blood glucose level ± SD, 1.17 ± 0.29 g/L; mean creatinine ± SD, 12.7 ± 33.7 mg/dL. In two cases, symptoms of cerebral malaria occurred concomitantly With acute respiratory distress or hemoglobinuria. ND, not determined.

Table2 :Top eight significant GO terms for Gene Ontology term analysis of differentially expressed genes set in cerebral malaria isolates and 3D7-Lib compared to asymptomatic malaria isolates and 3D7 clone, respectively.

S1 appendix: List of up-regulated and down-regulated genes in 3D7 versus 3D7-Lib and in isolates from children with cerebral malaria versus isolates from asymptomatic children. Lists of untranscibed genes in 3D7, 3D7-Lib, CM ans AM. The increase or decrease (fold change) and the Bonferroni P-value correction are presented.

List of up-regulated genes in 3D7-LibR vs 3D7R: Genes untranscribed in 3D7T:

[Nesw Gene ID] [Previous ID] [Product Description] PF3D7_0114900 PFA0720w

Introduction

Cerebral malaria (CM) is the most serious manifestation of falciparum malaria. It is responsible for 15-20 % of infant mortality [START_REF] Idro | Cerebral malaria: mechanisms of brain injury and strategies for improved neurocognitive outcome[END_REF]. There is strong evidence that sequestration of IEs within the brain microvascular endothelia plays a key role in CM pathogenesis [START_REF] Macpherson | Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration[END_REF][START_REF] Pongponratn | Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study[END_REF]. Such phenomenon protects the mature parasites from spleen-dependent clearance (van der [START_REF] Van Der Heyde | A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction[END_REF].

The adhesive properties of IE are attributed to P. falciparum Erythrocyte Membrane Protein-1 (PfEMP-1) expressed at the surface of IE [START_REF] Su | The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes[END_REF]. Encoded by ~60 members of var genes family, Pfemp-1s are divided into three main groups (A, B, C) and three relatively conserved groups var1, var2, and var3 [START_REF] Baruch | Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes[END_REF][START_REF] Kraemer | A family affair: var genes, PfEMP1 binding, and malaria disease[END_REF][START_REF] Su | The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes[END_REF].

It has been shown that groups A and B var genes are differentially transcribed in severe malaria isolates compared with parasites from uncomplicated malaria (UM) or asymptomatic infections [START_REF] Jensen | Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes[END_REF][START_REF] Kyriacou | Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia[END_REF][START_REF] Rottmann | Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children[END_REF].

Recent studies have shown that PfEMP-1s are characterized by 23 conserved architectural motifs named domain cassettes (DC), and that DC8 and DC13 are of clinical significance [START_REF] Rask | Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer[END_REF]. The transcription levels of these DCs are higher in parasites from severe malaria, including CM, than in those from UM [START_REF] Bertin | Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin[END_REF][START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]. Moreover, P. falciparum parasites selected for binding to human brain microvascular endothelial cells (HBMEC) express high levels PfEMP-1 DC8 genes [START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF], and plasma from malaria convalescent children exhibited higher antibody recognition profiles to HBMECselected parasites than unselected ones, suggesting the importance of PfEMP-1 motifs in building protective immunity to clinical malaria [START_REF] Avril | A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells[END_REF][START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF].

In vitro experiments identified a few endothelial receptors that participate in the adherence of IEs to the microvasculature. CD36 and Inter Cellular Adhesion Molecule-1 (ICAM-1) are the most commonly used by patient parasites [START_REF] Chaiyaroj | Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor[END_REF]. As yet, no definitive receptor implicated in the pathogenesis of malaria has been named, apart from chondroitin sulphate proteoglycan (CSPG) in the case of parasites isolated from pregnant women [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF]. CD36 has been presented as a major receptor involved in the cytoadherence for P. falciparum isolates [START_REF] Ho | Rosette formation of Plasmodium falciparum-infected erythrocytes from patients with acute malaria[END_REF], Newbold et al. 1997). Few studies have found no difference in CD36-binding patterns between parasites from severe malaria and those from UM patients [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF][START_REF] Mayor | Association of severe malaria outcomes with platelet-mediated clumping and adhesion to a novel host receptor[END_REF], Rogerson et al. 1999), others have reported an association between UM and high binding level to CD36 [START_REF] Newbold | Receptor-specific adhesion and clinical disease in Plasmodium falciparum[END_REF][START_REF] Ochola | Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome[END_REF]. Postmortem brain examination has reported high levels of ICAM-1, which co-localized with IEs in cerebral blood vessels (Turner G. D. et al. 1994). However, other studies did not detect any difference in binding to ICAM-1 in parasite isolates from severe or mild cases [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF], Rogerson et al. 1999), others suggested an association between disease severity and ICAM-1-binding [START_REF] Newbold | Receptor-specific adhesion and clinical disease in Plasmodium falciparum[END_REF][START_REF] Ochola | Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome[END_REF].

The implication of CD36 and ICAM-1 in the pathogenesis of CM, and their roles in combination with PfEMP-1 variants expression needs to be clarified. We combined binding phenotype examination by static binding assay and transcript abundance analysis of parasites freshly collected from Beninese children with CM or UM to investigate the relationship between parasite PfEMP-1 variants expression and binding phenotypes to CD36, ICAM-1 and CSPG.

Materials and methods

Ethics statement

This study was approved by the ethical committee of the research Institute of applied biomedical sciences, Cotonou, Benin (N°006/CER/ISBA/12 and N°21/CER/ISBA/13).

Study design, malaria patients

This study was conducted in Cotonou in southern Benin, during the 2012 and 2013 malaria transmission periods (June-September and May-July, respectively). This area is characterized by two rainy seasons during which malaria infection is mainly caused by P. falciparum, with approximately 33 infected bites per individual annually [START_REF] Akogbeto | Entomological study on the malaria transmission in coastal and lagoon areas: the case of a village built on a brackish lake][END_REF].

Children under five years of age presenting to Hôpital Mère-enfant de la lagune (HOMEL), Centre National Hospitalier Universitaire Hubert Koutoucou Mega (CNHU-HKM), or to Hôpital Suru-Léré were screened on arrival by the malaria rapid diagnostic test (DiaQuick Malaria P. falciparum Cassette, Dialab®) and were recruited in the study if they presented CM or UM. CM was defined as a microscopically confirmed P. falciparum infection and a Blantyre coma score ≤ 2, with the exclusion of any other cause of coma. UM was defined as a P. falciparum parasitemia accompanied by fever, headache or myalgia without signs of severity and/or evidence of vital organ dysfunction, as defined by the WHO. After obtaining informed and written consent from parents or guardians, 2 to 4 ml of venous blood samples were collected into tubes containing Citrate Phosphate Dextrose Adenine (CPDA). P. falciparum infections were confirmed by Giemsa-stained thick blood smears and parasitemia was recorded as the number of parasites/µL of blood. After diagnosis, all participants were treated according to the national malaria program policy.

Sample preparation and parasite culture

Fresh parasitized blood samples were treated as follows: plasma and buffy coat were removed from the blood, and erythrocytes pellet was washed twice in RPMI-gentamicin. One hundred µl were conserved in TRIzol® reagent (Life Technologies), and stored at -80°C for RNA extraction. Another 100 µl were spotted on Whatman 3MM filter paper, and stored at room temperature for DNA extraction and msp genotyping. Five hundred µl were cultured in RPMI1640 medium (LONZA). Parasites were cultured for less than 48 hours until they reached mature stage, then they were purified using magnetic columns (MACS, Milteny Biotec) for binding assay.

Binding assay

Static binding assays were carried out for 27 UM and 38 CM fresh isolates at 20 % parasitemia in 100 x 15-mm Petri dishes as described [START_REF] Bigey | The NTS-DBL2X region of VAR2CSA induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A[END_REF]. Twenty µl of each recombinant protein diluted in PBS were spotted on Petri dish and incubated overnight at 4° C in a sealed humid container. The following recombinant proteins were used: CSPG at 5 µg/ml (Decorin, Sigma-Aldrich, France); CD36 and ICAM-I at 10 µg/ml (R&D, Lille, France). Spots were then blocked with 3 % bovine serum albumin (BSA) in PBS for 30 minutes at 37° C in a humid chamber. Purified parasites were washed with PBS, centrifuged for 10 min at 2500 rpm, resuspended in 3 % BSA-RPMI, and 20 µl of parasite suspension were added to each spot, and incubated for 12 minutes at room temperature. Unbound erythrocytes were washed off gently with PBS using an automated washing system. Bound cells were fixed with 1.5 % gluteraldehyde in PBS for 10 minutes, Giemsa-stained, and counted microscopically.

The number of IEs bound to each receptor was determined by counting 10 fields using a 40x objective, and expressed as number of IEs bound per mm². For each sample, the binding assay was done in duplicate to all proteins in the same plate. All results shown are averages of the binding level of duplicate spots per sample. As a negative control, 1 % BSA was used to assess nonspecific binding.

Parasite RNA extraction and cDNA synthesis cDNAs were synthesized from 36 UM and 51 CM thawed samples preserved in TRIzol® reagent, following the manufacturer's instructions. RNAs were then treated with DNAse I (BioLabs), and absence of genomic DNA was assessed by 40 cycles of RT-PCR with fructosebiphosphate aldolase primers [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF]. Reverse transcription of RNA was performed by Thermoscript® (Life Technologies) with random hexamers and oligo dt primers, following manufacturer's recommendations.

Transcript abundance of var genes

Individual RT-PCR reactions were carried out on a Rotorgene® thermal cycler (Corbett Research) using 1 X SYBR Green® (Bioline), and 1.25 µM of specific primer pairs for group A (A1, A2, A3), group B (B1), var2csa and var3 of var genes. A set of primers to target DC8 and DC13 was also used [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]. The endogenous controls used were fructosebisphosphate aldolase and seryl-tRNA synthetase [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF]. Cycling conditions were 95° C for 1 minute, followed by 40 cycles of 95° C for 30 seconds, 54° C for 40 seconds, and 68° C for 50 seconds. Data were analyzed by the Rotorgene software for which the threshold cycle was set at 0.025. The specificity of each PCR product was verified by a specific melting curve for each pair of primers. The transcript abundance was calculated by relative quantification using two housekeeping genes (∆Ct var_primer = Ct var_primer -Ct Genomic DNA extraction and Pfmsp1 & Pfmsp2 genotyping Genomic DNA was extracted from 36 UM and 51 CM filter papers using the Chelex 100 resin method, as described [START_REF] Plowe | Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa[END_REF]. Specific primers were used to amplify block 2 Pfmsp1 and block 3 Pfmsp2 by PCR [START_REF] Snounou | Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand[END_REF]. Multiplicity of infection (MOI) was determined as the highest Pfmsp1 or Pfmsp2 allele number found in each sample.

Statistical analysis

Binding data were analyzed qualitatively from the number of IEs bound to a given receptor minus the number of IEs bound to BSA. Then, for each receptor, isolates were divided into two groups of low binders and high binders, according to the median binding level of CM and UM samples together. The distribution of low and high binders to a given receptor in both CM and UM groups was compared by Chi-square test. The Mann-Whitney U test was used to compare the transcript abundance of var group genes and DC8 and DC13 between CM and UM groups. Correlation between binding profile and transcript level of dominant var gene which was transcribed in more than 50% of samples was achieved by the Spearman's rank test. Data were plotted and statistical tests were performed by GraphPad, V5 (Prism software). Differences of each comparison were statistically significant when P-value <0.05.

Results

A total of 88 children were enrolled, of those 52 children presented with CM and 36 with UM. Of 52 children with CM, 21 presented CM alone, while the others had overlapping clinical syndromes: 25 children also presented with severe anemia (SA), and 16 with hyperparasitemia (HP) (10 children with CM presented both SA and HP). The clinical and biological features of these patients are summarized in Table 1. There was no difference in sex ratio, age, and mean body temperature between the two groups (all P > 0.05). The hemoglobin level was lower (P < 0.0001) while parasite density was higher in the CM as compared to the UM children (P < 0.01). All children with UM survived, while 19 patients (36.5%) with CM died. Multiple infections were recorded in both groups with a mean range of multiplicity of infection of 3.4 clones in CM and 2.2 clones in UM infections (P < 0.0001).

Binding profiles

Due to the low parasitemia of UM isolates and probably to pre-collection anti malarial treatment for CM children, some parasites cultures failed to reach mature stages. Therefore, we could only investigate the binding phenotypes for 27 of the 36 UM and 38 of the 52 CM isolates. We found that all isolates, except two (63 of 65), bound to both CD36 and ICAM-1, and 48 isolates bound weakly to CSPG (Table 2).

The distribution of low and high binders isolates to a given receptor in both CM and UM groups shows that the prevalence of CM-high binders to CD36 was significantly higher than UM-high binders (P = 0.007), while no significant difference in the binding to ICAM-1 (P = 0.4) or CSPG (P = 0.4) was noted between the parasites from these two clinical groups (Figure 1).

Transcript levels of var genes groups and DC8 & DC13

Transcription level (Tu) of A, B, var2, var3, DC8 and DC13 var genes were analyzed by RT-qPCR in 36 UM and 51 CM isolates. Group A, B, var2, var3, DC8 and DC13 were significantly transcribed to a higher level in CM than in UM isolates (Table 3, Figures 2 and3). However, no significant difference in the transcription levels of the A2, DBLγ4/6 and DBLαCIDRα genes was observed between CM and UM isolates .

Correlation between binding phenotype and transcription level of var genes

We examined the relationship between binding profiles and transcription levels of var genes groups and DCs for each isolate considering only highly transcribed genes determined as "dominants transcripts".

Group B var genes were the predominant transcripts in 51 % of samples. Tu of group B var genes was positively correlated with the binding level to CD36 (r = 0.46, P = 0.005), but not to ICAM-1 (r = 0.05, P = 0.7), nor to CSPG (r = 0.1, P = 0.2). Group A var genes were the predominant transcripts in 49% of samples, and no significant correlation was detected between the Tu and the binding levels to CD36 (r = 0.2, P = 0.2), ICAM-1 (r = 0.1, P = 0.4) and CSPG (r = -0.02, P = 0.1).

Likewise, DC8 and DC13 genes were predominant in 78.5% and in 21.5% of samples respectively. Tu of DC8 and DC13 were not correlated with binding levels to CD36 (r = -0.005, P = 0.9, and r = 0.09, P = 0.7, respectively), ICAM-1 (r = 0.1, P = 0.4, and r = -0.06, P = 0.8, respectively) and CSPG (r = 0.05, P = 0.7, and r = -0.1, P = 0.5, respectively).

Discussion

Cytoadherence of IEs to cerebral microvasculature cell lining is considered to be a major factor in CM pathogenesis [START_REF] Macpherson | Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration[END_REF][START_REF] Raventos-Suarez | Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes[END_REF]. This mechanism is probably mediated by PfEMP-1 variants expressed at the surface of IEs [START_REF] Baruch | Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1[END_REF]. However, the ligand-receptor interactions leading to IE-binding to brain microvascular endothelium are not well understood.

In order to investigate the potential role of PfEMP-1 variants in cytoadherence to host receptor, we used clinical isolates from individuals with cerebral or uncomplicated malaria to examine the cytoadherence phenotypes to ICAM-1, CD36 and CSPG, and analyzed the transcription profiles of the var genes.

We show that CD36 is the main binding receptor used by clinical isolates irrespective of the degree of disease severity, in agreement with earlier findings considering a strong implication of CD36 in the cytoadherence phenomenon [START_REF] Chaiyaroj | Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor[END_REF][START_REF] Ho | Rosette formation of Plasmodium falciparum-infected erythrocytes from patients with acute malaria[END_REF], Newbold et al. 1997). By contrast to previous studies, CD36-binding level was higher in CM than in UM isolates, suggesting an important role of CD36 in the parasite sequestration during CM.

Conversely, in agreement with previous studies using static binding assay [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF], Rogerson et al. 1999), binding phenotypes to ICAM-1 or CSPG were similar in CM and UM isolates. A higher binding level to ICAM-1 in CM than in UM isolates was seen under flow conditions, as probably flow assay better simulates physiological wall shear stress, under which ICAM-1 promotes IEs rolling and binding [START_REF] Ochola | Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome[END_REF]). Moreover, it has been shown that P. falciparum isolates, including those from CM, bind to CD36 10-fold higher than to ICAM-1, and that 80 % of ICAM-1-selected IEs bind to CD36, suggesting an association of CD36 with disease pathogenesis as it facilitates the binding of a high proportion of parasites throughout the body [START_REF] Udomsangpetch | Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1[END_REF]. In addition, under flow conditions, CD36 offers greater and more efficient adherence property than ICAM-1 [START_REF] Cooke | Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin[END_REF]). These results are in agreement with ours as a higher binding level of IEs to CD36 than ICAM-1 has been observed.

Despite the fact that CD36 is poorly expressed in brain vascular endothelia, Turner et al. reported low but significant association between CD36 expression and IEs sequestration in brain vessels (Turner G. D. et al. 1994). Moreover, IEs binding to cerebral endothelial cells may take place indirectly through platelets accumulated in the brain vasculature of CM patients [START_REF] Wassmer | Platelets reorient Plasmodium falciparum-infected erythrocyte cytoadhesion to activated endothelial cells[END_REF]). Platelets express a high level of CD36; this may promote IEs binding and sequestration in the brain, despite the low CD36 expression on brain microvascular endothelium. These cells act as a bridge between brain endothelium and IEs via CD36-binding. This mechanism of sequestration possibly reorients the binding properties of parasites, contributing to CM pathogenesis [START_REF] Wassmer | Platelets reorient Plasmodium falciparum-infected erythrocyte cytoadhesion to activated endothelial cells[END_REF]).

PfEMP-1 variants expressed on the surface of IEs determine the cytoadherence properties of parasite populations [START_REF] Baruch | Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1[END_REF]. These proteins are well-described ligands, and studies have shown that they recognize and interact with more than one receptor independently of IEs subpopulations. One PfEMP-1 variant may allow the cytoadherence to more than one host receptor through its semi-conserved N-terminal head structure DBL1α and CIDR1α [START_REF] Chen | The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors[END_REF]. Likewise, DBL C2 and -1 [START_REF] Bengtsson | A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies[END_REF][START_REF] Oleinikov | High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies[END_REF], the tandem NTS--CIDR varO is essential for IEs binding and rosette formation [START_REF] Vigan-Womas | Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting[END_REF]). CIDRα1 of DC8 and DC13 var genes is a potential ligand of endothelial protein C receptor [START_REF] Turner | Severe malaria is associated with parasite binding to endothelial protein C receptor[END_REF]).

In our study, the transcription of Pfemp-1 variants including groups A, B, var3, DC8, and DC13 was clearly up-regulated in CM parasites compared to UM ones, in agreement with previous studies [START_REF] Bertin | Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin[END_REF], Doritchamou et al. 2012[START_REF] Kyriacou | Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia[END_REF][START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF][START_REF] Rottmann | Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children[END_REF], and supports the implication of these genes in CM pathogenesis. Var2csa, the gene encoding VAR2CSA, is transcribed to a much higher level in isolates from pregnant than from non-pregnant women [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF][START_REF] Ndam | High level of var2csa transcription by Plasmodium falciparum isolated from the placenta[END_REF]. Some CM isolates expressed var2csa at low but higher level than UM isolates, but binding to CSPG was consistently very weak and similar in both groups, indicating that VAR2CSA does not take part in children's malaria pathogenesis. The higher var2csa expression level in selected CM isolates may due to the higher parasitemia and higher MOI of children presenting with CM compared to those with UM.

The transcription level of group B genes positively correlated with the binding level of IEs to CD36, confirming the earlier findings that parasite ligands for CD36 are PfEMP-1 variants encoded by groups B and C var genes through their CIDR1α domain [START_REF] Robinson | Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome[END_REF][START_REF] Smith | Analysis of adhesive domains from the A4VAR Plasmodium falciparum erythrocyte membrane protein-1 identifies a CD36 binding domain[END_REF]). Functional analysis using mutagenesis modifications in the M2 region of CIDR1α linked CD36 binding ability to specific CIDR1α residues [START_REF] Gamain | Modifications in the CD36 binding domain of the Plasmodium falciparum variant antigen are responsible for the inability of chondroitin sulfate A adherent parasites to bind CD36[END_REF]. Recombinant protein of CIDRα1.1 of DC8 also inhibits the binding of IEs to brain endothelium cells, suggesting a role of this domain in binding phenotypes involved in cerebral malaria [START_REF] Avril | A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells[END_REF].

The transcription levels of DC8 and DC13 were higher in CM than in UM isolates, and were not correlated with the binding levels to CD36, ICAM-1, nor CSPG. DC8, DC13 are part of few genes from groups B and A, respectively. It is likely that our study did not involve enough of isolates expressing DC8 and DC13, and lacked of power to detect such correlation between the binding phenotypes and the transcription patterns.

In conclusion, CM isolates bind more to CD36 than UM isolates, and no difference was observed in binding levels to ICAM-1 and CSPG. CM isolates transcribed groups A, B, var2, var3, DC8 and DC13 at higher levels than UM isolates, and the high transcription level of group B var genes correlated with increased binding level to CD36. These results support the role of these PfEMP1 variants in CM. For its survival, the parasite develops sophisticated mechanism to bind host receptors and establish chronic infections. Hence, identifying parasites ligands involved in IEs sequestration during cerebral malaria could be targeted to prevent the disease. PFMC2TM) and unknown hypothetical proteins. The variable expression profiles of VSA families in parasite isolates is probably due to different gene transcriptions between isolates, to the high number of multiclonal infections in field isolates (1-7) as well as to the fact that six isolates were pooled to obtain sufficient quantity of RNA for hybridization. It is also likely that these represent vatiants that doe not exist in 3D7.

Notes

9.3 [1-64] 1 [1-2.6] 0.001 var2 1 [1-4.4] 1 [0-1] 0.001 var3 17.1 [1-34] 1.4 [1-10.4] 0.008 CIDRα1.1 (DC8) 2.11 [1-11.8] 1 [1-2.3] 0.02 DBLβ12 (DC8) 2.8 [1-13.9] 1 [1-4.2] 0.03 DBLγ4/6 (DC8) 1 [0-1] 1 [0-1] 0.4 DBLαCIDRα (DC8) 0 [0-1] 0 [0-1 ] 0.4 DBLα1.7 (DC13) 1.1 [1-8.1] 1 [ 0-1.5] 0.01 CIDRα1.4 (DC13) 1 [1-2.2] 1 [ 0-1] 0.002 0
Furthermore, we compared our transcriptomic outcomes of parasite isolates and 3D7 at ring and late trophozoite stages to transcriptomic data obtained by RNA sequencing of 3D7 at corresponding stages [START_REF] Bartfai | H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3[END_REF]) and observed significant correlation (p = 2.2 e-16 , Kendall test). These outcomes support the efficiency of the array hybridization assay in providing coherent data that are comparable to RNA sequencing method and demonstrate the conserved nature of fundamental biological processes such as metabolic pathways and cell cycle, within in vivo and in vitro conditions. This higher correlation is concordant with the predominance of ring stage parasites in the peripheral blood circulation of infected individuals, and with the changes in gene transcription profiles during the progression to the late trophozoite stage. Moreover, differentially transcribed genes in CM isolates compared to AM parasites suggests an association with parasite-virulence factors. Our arrays detected 99 up-regulated genes in CM compared to AM parasites in late trophozoite stage. Twenty eight genes code for exported proteins with a majority of VSA encoding genes such as var, rif, phist, and surfin. These genes are potentially implicated in parasite cytoadherence and immune avoidance. Interestingly, group A and group B var genes such as Pf11_0521, Pf13_0003, PFI1820w, PfD1235w, PfF1595c were over expressed in CM isolates and in 3D7-Lib line, whereas PfB0020c and PfD0020c were up-regulated only in CM isolates. Pf11_0521 displays DBLα-1.7 and CIDRα1.4, while PFD0020c displays CIDRα-1.1, DBLβ12 and DBL-γ6. These architectural domains characterize Domain Cassettes DC13 and DC8, respectively, and were found to be transcribed at tremendous levels in cerebral malaria isolates from Tanzanian [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]) and Beninese children (Gwladys et al. 2012) compared to mild cases isolates. Likewise, DC5-containing genes such as Pf13_0003 and PfD1235w were also up-regulated in CM and 3D7-Lib. It has been shown that DC5 is associated with severe malaria [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF], and it is implicated in parasites adherence to the human receptor PECAM1 [START_REF] Berger | Plasmodium falciparum expressing domain cassette 5 type PfEMP1 (DC5-PfEMP1) bind PECAM1[END_REF]).

The transcription level of PfD0020c and that of its orthologs IT4var19, HB3var3, and IT4var7 were remarkably elevated in parasite lines selected for binding to human brain endothelial cells (HBEC) [START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF][START_REF] Avril | A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells[END_REF]). These genes code for DC8 and DC13-containing PfEMP-1s and directed antibodies against the NTS-DBLα1 and the NTS-DBLα1-CIDRα1 domains of HB3var3 inhibit the binding of infected erythrocytes to HBEC, supporting their role in cytoadherence [START_REF] Avril | A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells[END_REF]. Recently, endothelial protein C receptor (EPCR) has been identified as a new receptor candidate for PfEMP-1s through CIDRα1.1 of DC8, suggesting the implication of EPCR-binding in malaria pathogenesis [START_REF] Turner | Severe malaria is associated with parasite binding to endothelial protein C receptor[END_REF]. Interestingly, anti-PfEMP-1 IgG antibodies targeting recombinant proteins representing DBLγ6 and CIDRα1.1 of PfD0020c, DBLβ9 of PF13_0003, DBLβ3, DBLγ13, CIDRα1.6 of PfD1235w and CIDRα1.4 of Pf11_0521, with regard to serological recognition, showed that children in malaria endemic regions are exposed to parasites expressing these domains at an early age [START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]. This exposure promotes the induction of the immune system and the synthesis of suitable antibodies against these variants. Moreover, DBL1α1 of 3D7 PF13_0003, which is shared with other genes, such as PaloAlto89F5 VarO, IT4/I29, and IT4var60, found to be implicated in rosetting [START_REF] Albrecht | var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2[END_REF]Vigan-Womas et al. 2008). This gene was over expressed in CM isolates and in the 3D7-Lib. The N-terminal region of DBL1α1 plays a role in pseudo-rosettes formation on the surface of a fibroblast-like cell line COS and baculovirus-infected cells expressing individual PfEMP-1 domains [START_REF] Rowe | P. falciparum rosetting mediated by a parasitevariant erythrocyte membrane protein and complement-receptor 1[END_REF]Vigan-Womas et al. 2008).

In the present study, the up-regulation of UPS A and UPS B var groups in parasites from children with CM is consistent with findings of the other studies [START_REF] Jensen | Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes[END_REF][START_REF] Kirchgatter | Association of severe noncerebral Plasmodium falciparum malaria in Brazil with expressed PfEMP1 DBL1 alpha sequences lacking cysteine residues[END_REF][START_REF] Kyriacou | Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia[END_REF][START_REF] Rottmann | Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children[END_REF][START_REF] Warimwe | Prognostic indicators of lifethreatening malaria are associated with distinct parasite variant antigen profiles[END_REF], strengthening the relationship between specific PfEMP-1 protein expression and the development of CM.

Other VSA antigens found to be up-regulated in 3D7-Lib line and/or in parasites from CM children are rif genes PfF1545w, Pf10_0403, PfC1100w, Pf11_00520, PfD1230c, Pf11_0009, phist a, b, c genes Pf14_0752, PfL2565w, MAL7P1.7, PfI1785w, MAL8P1.4, PfB0900c. The phist a Pf14_0752 was previously shown to be over expressed in parasite lines selected for binding to HBEC [START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF]. The rif family consists of ~150 clonally variant genes classified into two groups A-and B-type rifins, basing on their sub-cellular localization and the presence of a 25-amino acid motif within the semi-conserved region (C1) of type-A rifin [START_REF] Joannin | Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family[END_REF]. The A-type RIFINs are transported to the surface of the infected erythrocyte [START_REF] Khattab | Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion[END_REF], whereas specific B-type RIFINs have been associated with the extracellular stages; merozoites and gametes [START_REF] Mwakalinga | Expression of a type B RIFIN in Plasmodium falciparum merozoites and gametes[END_REF]. Most of rifin family members are situated close to var genes within the sub-telomeric regions of the chromosomes. These regions are subjected to recombination, which increases the diversity of these genes. Our results showed that in 3D7-Lib, rif Pf11_00520, Pf11_0009, PfD1230c, Pf13_0004, were concomitantly up-regulated with their neighboring var genes Pf11_0521, Pf11_0008, PfD1235w, Pf13_0003 suggesting a unique promoter controls the expression of these genes.

Furthermore, non-VSA genes such as Pf13_0010, Pf14_0010, and PfE0065w, were also found to be highly up-regulated in CM isolates. Pf13_0010 and Pf14_0010 code for GBPH2 and GBPH, respectively. These antigens are members of Maurer's cleft proteins [START_REF] Lanzer | Maurer's clefts: a novel multifunctional organelle in the cytoplasm of Plasmodium falciparum-infected erythrocytes[END_REF], either temporarily during their trafficking to the infected erythrocyte surface with PfEMP-1, RIFIN [START_REF] Petter | Variant proteins of the Plasmodium falciparum RIFIN family show distinct subcellular localization and developmental expression patterns[END_REF], SURFIN [START_REF] Winter | SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes[END_REF], STEVOR [START_REF] Kaviratne | Small variant STEVOR antigen is uniquely located within Maurer's clefts in Plasmodium falciparum-infected red blood cells[END_REF]), PfMC-2TM (Sam-Yellowe et al. 2004), or as constitutive proteins of the Maurer's cleft itself, as Plasmodium falciparum skeleton-binding protein 1 (PfSBP1) which is encoded by PfE0065w (Blisnick et al. 2000). This protein is shown to be involved in PfEMP-1 transporting to the erythrocyte surface (Cook, BM et al. 2006). Pf14_0752 encodes MAHRP1 (Phist a), a Maurer's cleft resident protein that is also necessary for PfEMP-1 trafficking to the surface of infected red blood cells [START_REF] Spycher | Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes[END_REF][START_REF] Spycher | The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes[END_REF]. Our findings demonstrated that the last two genes were transcribed at higher levels in the field isolates comparing to 3D7. In addition, MAHRP1 was among the 15 non-VSA up-regulated genes in parasite lines selected to adhere on HBEC [START_REF] Claessens | A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells[END_REF].

Other differentially transcribed genes in CM isolates are those which code for antigenic proteins, such as P. falciparum sporozoite surface threonine-and asparagine-rich protein (STARP) Pf07_0006, P. falciparum ring-infected erythrocyte surface antigen (RESA) Pf11_0509. These genes were found to be up-regulated in clinical isolates comparing with laboratory-adapted strains [START_REF] Mackinnon | Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates[END_REF]. Some genes implicated in the transcription process, including PfC0805w DNA-directed RNA polymerase II, PfL1525c pre-mRNA splicing factors were highly transcribed in CM isolates as compared to AM and 3D7 parasites.

Our study presented some regulated genes t h a t were also differentially transcribed in other study [START_REF] Mackinnon | Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates[END_REF]). These outcomes suggest a variable expression pattern of these genes between field isolates and 3D7 clone. In addition, these findings support the powerful of transcriptomic analysis by microarray hybridization to obtain reproducible data although different techniques had been used between laboratories. Of these genes, ring-exported protein 1 (REX1) PfI1735c; ring-exported protein 2 (REX2) PfI1740; gametocytogenesis-implicated protein (GIG) PfI1720w; sporozoite threonine-and asparagine-rich protein (STARP) Pf07_0006; lysophospholipase, putative Pf07_0005, conserved Plasmodium protein with unknown function Pf07_0007, (Pfg27) gamete antigen 27/25 Pf13_0011 were all up-regulated in AM and CM isolates used in our study, and were also among the top 20 over expressed genes in clinical isolates of another study [START_REF] Mackinnon | Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates[END_REF]). REX1, REX2 and GIG genes are found in close distance in chromosome 9 whereas STARP, putative lysophospholipase and a conserved Plasmodium gene are localized within close proximity in chromosome 7, proposing a co-regulation of gene transcription due to their proximity. Likewise, three to 12 up-regulated genes in parasites from CM patients compared to AM parasites were harbored on the same chromosome at a distance ranging from 5 to 100 kb where the variability of gene transcription is likely dominated by a single promoter or the same epigenetic mechanism.

On the other hand, cytoadherence of IEs to cerebral microvasculature beds is considered as a major factor in the pathogenesis [START_REF] Macpherson | Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration[END_REF][START_REF] Raventos-Suarez | Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes[END_REF]. In vitro experiments have identified several endothelial receptors that may participate in the adherence of IEs to microvasculature endothelia, and few studies attempted to identify a specific binding phenotype of parasites causing severe malaria which is unlikely or rarely shown in uncomplicated malaria isolates. As yet, no definitive candidate has been incriminated, apart from chondroitin sulfate proteoglycan the main receptor of VAR2CSA involved in placental sequestration during malaria in pregnant women [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF]. Of these receptors, CD36 and ICAM-1 are the most well investigated receptors and the most commonly used by patient parasites [START_REF] Chaiyaroj | Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor[END_REF]. Our knowledge of the role of these receptors in cytoadherence of IE comes mainly from binding assays under static or flow conditions.

However, cytoadherence studies require specific experimental platform as well as particular biological material like "P. falciparum clinical isolates, parasite strains, and mammalian cells expressing the desired receptor or receptors as recombinant proteins" which make the assays difficult to perform resulting in scarce data particularly those obtained from patients with distinct clinical malaria features.

In the second part of my PhD thesis, we aimed to elucidate the potential implication of var genes variants which were found to be up-regulated in CM isolates in our microarrays analysis and may be involved in the pathogenesis of cerebral malaria by promoting increased cytoadherence to specific receptors host such as CD36, ICAM-1. To investigate whether the expression of these PfEMP-1 variants is related to the binding phenotypes to CD36, ICAM-1 or CSPG of clinical parasites isolates, we examined the binding patterns of fresh parasite isolates from Beninese children with cerebral or uncomplicated malaria to ICAM-1, CD36 and CSPG using static binding assay and analyzed the transcripts abundance of groups A, B, var2, var3, DC8 and DC13 for these isolates.

Our results show that CD36 is the major receptor used by clinical isolates regardless the degree of disease severity, consistent with earlier findings demonstrated a strong implication of CD36 in the cytoadherence of IEs [START_REF] Chaiyaroj | Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor[END_REF][START_REF] Ho | Rosette formation of Plasmodium falciparum-infected erythrocytes from patients with acute malaria[END_REF][START_REF] Newbold | Receptor-specific adhesion and clinical disease in Plasmodium falciparum[END_REF]). In contrary to previous studies, we report for the first time that CD36binding levels were higher for parasites from CM that for those freom UM isolates, suggesting a potential implication of CD36 in CM pathogenesis.

Furthermore, we did not observe any difference in binding level to ICAM-1 or CSPG between CM and UM parasites isolates. This result is in agreement with previous investigations where static binding assays were used [START_REF] Heddini | Binding of Plasmodium falciparuminfected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates[END_REF][START_REF] Rogerson | Cytoadherence characteristics of Plasmodium falciparum-infected erythrocytes from Malawian children with severe and uncomplicated malaria[END_REF]. However, the difference in binding levels to ICAM-1 between CM and UM isolates was only detected under flow conditions, which may simulate physiological wall shear stress, under which ICAM-1 promotes IEs rolling, reducing blood flow and providing cytoadherence [START_REF] Ochola | Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome[END_REF].

Further studies are needed to strengthen this observation.

Moreover, It has been found that malaria parasites, including those from CM, bind to CD36 10 times higher than to ICAM-1, and that 80 % of IEs selected for ICAM-1-binding, adhered also to CD36, proposing an association of CD36-binding with disease pathogenesis as it assists the binding of a high proportion of parasites throughout the body, promoting systemic infection that contributes to the severity of the disease [START_REF] Udomsangpetch | Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1[END_REF]. In addition, it has been shown that under flow conditions, CD36 provides higher and more efficient adherence property than ICAM-1 [START_REF] Cooke | Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin[END_REF]). These results are in agreement with ours as a high binding level of IEs to CD36 than ICAM-1 has been observed.

Despite the fact that CD36 is expressed at restricted level in vascular endothelia of the brain, a low but significant association between CD36 expression and IEs sequestration in brain vessels has been observed [START_REF] Turner | An immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration[END_REF]. Further, IEs binding to cerebral endothelial cells probably takes place indirectly by the mean of platelets accumulated in brain vasculature of CM patients (Wassmer et al. 2003). The platelets are known to express high level of CD36 at their surfaces, thus they would promote parasite binding even in the absence of CD36 expression on brain microvascular endothelium. Therefore, these cells would act as a bridge between brain endothelium and IEs through CD36-binding. This mechanism of cytoadherence possibly reorients the adherence properties of parasites and contributes to CM pathogenesis (Wassme et al. 2004).

PfEMP-1 variants trafficked to the surface of IEs determine the cytoadherence patterns of parasite populations. These proteins are well-investigated ligands, and it has been demonstrated that these antigens have the ability to recognize and bind more than one receptor independently of IEs subpopulations [START_REF] Mccormick | Intercellular adhesion molecule-1 and CD36 synergize to mediate adherence of Plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells[END_REF]. One PfEMP-1 variant promotes the cytoadherence to more than one host receptor through its semi-conserved Nterminal head structure DBL1α and CIDR1α [START_REF] Chen | The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors[END_REF]. Simirarily, DBLβ-C2 of groups B, C and DBLβ3 of group A bind ICAM-1 [START_REF] Bengtsson | A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies[END_REF][START_REF] Howell | Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors[END_REF][START_REF] Oleinikov | High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies[END_REF]), the tandem NTS-DBLα-CIDRϒ of varO is essential for IE binding and rosettes formation [START_REF] Vigan-Womas | Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting[END_REF], and CIDRα1 of DC8 and DC13 var genes is a potential ligand of endothelial protein C receptor [START_REF] Turner | Severe malaria is associated with parasite binding to endothelial protein C receptor[END_REF].

In this study, we found a noticeable up-regulation in the transcription of Pfemp-1 variants including groups A, B, var3, DC8, and DC13 in CM parasites compared to UM ones which is in agreement with previous studies [START_REF] Bertin | Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin[END_REF][START_REF] Doritchamou | First-trimester Plasmodium falciparum infections display a typical "placental" phenotype[END_REF][START_REF] Kyriacou | Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia[END_REF][START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF][START_REF] Rottmann | Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children[END_REF]) and supports their implications in CM pathogenesis.

It is believed that the transcription level of var2csa the coding gene of VAR2CSA is higher in isolates from pregnant than non-pregnant women [START_REF] Salanti | Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria[END_REF][START_REF] Ndam | High level of var2csa transcription by Plasmodium falciparum isolated from the placenta[END_REF]. Surprisingly, we found that some CM parasites transcribed var2csa at higher levels than UM parasites which might be due to the higher parasite density and higher multiplicity of infection of CM parasites comparing to UM isolates. However, the binding level to CSPG was low and no significant difference was detected between the two groups pointing out the non-important role of VAR2CSA in children malaria pathogenesis.

More interestingly, we show a correlation between the transcription level of group B and the binding level of IEs to CD36, confirming the earlier findings reporting that parasite ligands for CD36 are PfEMP-1 variants encoded by group, B and C var genes through CIDR1α domain [START_REF] Robinson | Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome[END_REF][START_REF] Smith | Analysis of adhesive domains from the A4VAR Plasmodium falciparum erythrocyte membrane protein-1 identifies a CD36 binding domain[END_REF]. Furthermore, functional analysis using mutagenesis modifications in the M2 region of CIDR1α have linked the binding activity to CD36 to specific residues within CIDR1α [START_REF] Gamain | Modifications in the CD36 binding domain of the Plasmodium falciparum variant antigen are responsible for the inability of chondroitin sulfate A adherent parasites to bind CD36[END_REF]. CIDRα1.1 of DC8 was also found to inhibit the binding of IEs to brain endothelium cells suggesting a role of this domain in binding phenotypes involved in cerebral malaria [START_REF] Avril | A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells[END_REF].

The transcription levels of DC8 and DC13 were higher in CM than UM isolates in agreement with several findings [START_REF] Bertin | Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin[END_REF]Cleassens et al. 2012;[START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]). However, the transcription levels of these DC were not correlated with the binding levels to CD36, ICAM-1 or CSPG. DC8, DC13 are part of few genes of group B and A respectively, probably more isolates are needed to detect such correlation between the binding phenotypes and the transcription patterns.

In conclusion, the transcriptomic analysis using DNA microarrays that we achieved is a powerful technique. It allowed us to explore the majority of genetic variations between isolates from cerebral malaria, uncomplicated malaria and asymptomatic carriers at the same time. Thus a distinct gene expression profile has been identified in cerebral malaria isolates comparing to asymptomatic carriers. Of these genes, groups A and B var genes containing DC4, DC5, DC8 and DC13 are of clinical interest. These genes are probably implicated in the pathogenesis of cerebral malaria. The implication of the proteins encoded by these genes in virulence, namely PfEMP-1, arises from their ability to mediate the cytoadherence of IEs to post capillary endothelial cells.

Therefore, we applied static cytoadherence test to study the role of var group genes, which were found to be up-regulated in cerebral malaria isolates by the microarrays. We show that parasites from CM isolates bind more to CD36 than those from UM isolates and that they transcribed groups A, B, var2, var3, DC8 and DC13 higher than those rom UM isolates. The high transcription level of group B var genes correlated with increased binding level to CD36. These results support the role of these PfEMP1 variants in CM pathogenesis.

However, as for any research investigation, our study has certain scientific and technical limitations. Indeed, we did not find out any differences in the expression patterns between cerebral malaria and uncomplicated malaria isolates when DNA microarray has been used. This may due to the fact that fewer numbers of isolates from each group had been used (only 18 isolates from each group, CM and UM), which might be insufficient to detect the distinct genetic variation between these two groups. Moreover, although DNA microarray approach is high throughput and cost effective, it still suffers lack of perfect processing methods and acceptable sensitivity [START_REF] Tu | A method for detecting and correcting feature misidentification on expression microarrays[END_REF]).

On the other hand, studying the cytoadhesion phenotypes of P. falciparum isolates from CM and UM children using static binding assay did not show any significant difference in binding levels to ICAM-1 between these two groups. This might be due to several reasons: first, it is possible that there is a degree of uncontrolled variation in clinical definition of malaria, resulting in over or under estimation of the binding properties of parasites to their cognate receptor, if any exists. This is due to the fact that we classified the clinical groups based on signs and symptoms presented by children at the moment of admission to hospital. Thus, cerebral malaria isolates drawn from peripheral blood at the time of enrollment, may not represent those sequestering and causing damages of host tissues. Likewise, uncomplicated malaria in children may have evolved towards severe malaria. In both cases, this may interfere with the relationship between isolate characteristics and disease classification.

Moreover, the multiplicity of infection (MOI), where each parasite population may exhibit a specific binding phenotype, is an additional confounding factor. In our cytoadherence study, the MOI for CM isolates was 3.4 clones while it was 2.2 clones for UM. If one clone displays a specific adhesion property, and sequesters in brain microvasculature endothelia leading to CM, while another clone circulates in the peripheral blood representing different binding phenotype at the moment of sampling, then the observed phenotype would not match the real clone causing cerebral malaria.

Finally, static binding system employed in our study does not necessarily reflect the intensity of ligand-receptor that occurs in vivo. This model may not confer the appropriate receptor conformation, accessibility or mobility thereby the number of IEs bound in vitro may not represent the real proportion of parasites that sequester in vivo. The development of new binding assays closely mimicking physiological ligand-receptor interaction will provide relevant knowledge on receptor affinity to IEs.

As perspectives, it would be interesting to pursue the exploring of the role of VSA and non-VSA genes, found to be upregulated in CM isolates by our transcriptomic analysis, in the pathogenesis of cerebral malaria. This could be achieved by the production of recombinant proteins and their corresponding antibodies, which would allow as to:

-Evaluate the capacity of these proteins to induce a specific antibody response (ELISA). -Conduct binding assays (static and flow) to analyse the cytoadherence phenotypes of these proteins to endothelial receptors such as CD36 and ICAM-1. -Carry out inhibition binding assays to test the ability of specific antibodies to abolish the binding of these proteins to the tested receptors.

Inhibition the binding of parasite ligand to its preferred/specific receptor by specific antibodies or suitable agonist molecules could be an efficient strategy to prevent severe complication of P. falciparum infections, such as cerebral malaria.

  Malaria History at a glance Malaria or mal' aria "spoiled air" is caused by parasites which have existed with us since the dawn of time. The earliest Plasmodium falciparum DNA has been detected in two Pharaonic mummies aged more than 3200 years B.C.

Fig. 1 :

 1 Fig. 1: Rawhide Bag of Cinchona bark, namely "Seron" used to store and transport Cinchona barks, brought from Peru in 1777 by Hipolito Ruiz Lopez and Antonio Pavon y Jimienz who had been sent by Spanish monarch Charles III to discover the region. Source: Science and society, 2014. (http://www.scienceandsociety.co.uk)

Fig. 2 :

 2 Fig. 2: P. falciparum malaria risk defined by Annual Parasite Incidence in 2010. Carmine color refers to stabletransmission geographical zone where annual parasite incidence PfAPI ≥ 0.1%, whereas pink color represents unstable-transmission zone with PfAPI < 0.1%. Light grey mentions to free malarial areas. Source: Malaria Atlas Projects (Map), 2010.

Fig. 3 :

 3 Fig. 3: Global map showing the localization of Benin in west Africa (Top-left), its neighbors and the two capitals, Cotonou and Porto-Novo. Source: Modified from President's Malaria Initiative (PMI). (http://www.pmi.gov/countries/profiles/benin.html)

Fig. 3 :

 3 Fig.3: Life cycle of Plasmodium falciparum.(1) Sporozoites injected by mosquito, migrate through the circulatory system to the liver, where they invade hepatocytes, multiply then released to blood stream as merozoites which are able to invade erythrocytes. Parasites enter asexual intraerythrocytic multiplications (2), which take 48 hours for P. falciparum. Each schizont gives birth to 30 daughter merozoites. Once erythrocyte ruptures, these merozoites infect other erythrocytes. Some merozoites differentiate to male and female gametocytes, which are taken by mosquito with blood meal, to be fertilized, differentiate and replicate resulting in sporozoites stages ready to infect human host. Source: Modified from (Bousema and Drakeley

Fig. 5 :

 5 Fig. 5: Sequestration phenomenon of infected erythrocyte. Schematic cytoadherence mechanisms of P. falciparum infected erythrocytes (a). Cytoadherence of infected erythrocytes to endothelial cells (b), Rosetting (c) and Clumpling (d). Source: (Rowe et al. 2009).

Fig. 6 :

 6 Fig. 6: The crystal structure of N-Terminal Ig-like domain of human ICAM-1 shown with fibrinogen, LFA-1 and P. falciparum binding sites. The binding site of Rhinovirus includes residues dispersed throughout Ig-like domain.The strands of the β-barrel are named from A to G. Some residues have been numbered for wild type ICAM-1. Source:[START_REF] Tse | Divergent binding sites on intercellular adhesion molecule-1 (ICAM-1) for variant Plasmodium falciparum isolates[END_REF]).

Fig. 7 :

 7 Fig. 7: Schematic organization of STEVOR, type-A and type-B RIFIN proteins. (Indel): insertion/deletion of 25 aminoacid conecnsus located in typ-A RIFIN, but not in type-B; (SP?): Potential signal; (TM): transmembrane domain; (V, V1, V2) are variable regions and (C1 and C2) are conserved regions. Source: (Joannin et al. 2011).

Fig. 8 :

 8 Fig. 8: General characteristics of var genes. All members of var gene family consist of two exons separated by conserved intron. The variable extra cellular domain encoded by exon 1 and involves: N-terminal segment (NTS); Duffy binding like domains (DBL); cysteine rich inter domain regions (CIDR) and transmembrane domain (TM). The intracellular domain (exon 2) encodes a semi conserved amino acid terminal segment (ATS) Source: Modified from (Sherf, 2008).

Fig. 9 :

 9 Fig. 9: Genomic organization and nuclear position of P. falciparum var genes.Global organization of subtelomeric regions of the 14 linear chromosomes shows that they all share non coding repeats of various sizes called TARE 1-6 (telomere associated repeat elements) situated beside telomere repeats. Subtelomeric site is followed by a member of the group B var genes, usually accompanied by another group A var genes transcribed in the opposite direction. Group C var genes are clustering in the chromosome center and rif genes are often interspersed with var genes. Source: Modified from[START_REF] Scherf | Antigenic variation in Plasmodium falciparum[END_REF]).

Fig. 10 :

 10 Fig. 10: Plasmodium falciparum erythrocyte membrane protein-1 domain architectures. PfEMP-1 antigens display global structure similarity, they all contain: N-terminal segment (NTS); variable number of Duffy binding like domain (DBL), one or two cysteine rich inter domain regions (CIDR), trans-membrane domain (TM) and acidic terminal segment (ATS). The NTS, DBL1α and CIDR1α make up a conserved head structure of exon I which displays multiple cytoadherence properties. Source: (Kraemer and Smith 2006).

Fig. 11 :

 11 Fig. 11: Identification of 23 domain cassettes with their corresponding upstreams of var groups and their structures (tandem) of DBL and CIDR domains. DC4 is absent in this figure. Source: (Rask et al. 2010).

Fig. 12 :

 12 Fig. 12: Different binding phenotypes of var gene groups are potentially correlated with specific clinical outcomes. Source: (Kraemer and Smith 2006).

  of Plasmodium falciparum in children with cerebral malaria and asymptomatic carriers Talleh Almelli 1,2, Grégory Nuel 2, Emmanuel Bischoff 3,4, Agnès Aubouy 5, Mohamed Elati 6, Christian William Wang 7, Marie-Agnès Dillies 8, Jean-Yves Coppée 8, Georges Nko Ayissi 9, Leonardo Kishi Basco 10,11, Christophe, Rogier 12, Nicaise Tuikue Ndam 1,2, Philippe Deloron 1,2, Rachida Tahar 1,2,10 1 Institut de Recherche pour le Développement (IRD), UMR 216 Mère et Enfant Face aux Infections Tropicales, Université Paris-Descartes, Près Sorbonne Paris-Cité, 4, Avenue de l'Observatoire, 75270 Paris 2 PRES Sorbone Paris Cité, Université Paris Descartes, Faculté de Pharmacie, Paris, 75270, France 3 Institut Pasteur, Unit of Molecular Immunology of Parasites, Unit of Insect Vector Genetics and Genomics, Department of Parasitology and Mycology, F-75015 Paris, France 4 Centre National de la Recherche Scientifique (CNRS), URA 3012, F-75015 Paris, France 5 Institut de Recherche pour le Développement (IRD), UMR 152 Pharmacochimie etpharmacologie pour le développement -(PHARMA-DEV), Université Paul Sabatier, Toulouse, France 6 Institute of Systems and Synthetic Biology, CNRS, University of Evry, Genopole, 91030, Evry, France 7 Centre for Medical Parasitology at Department of International Health, Immunology, and Microbiology, University of Copenhagen and at Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark 8 Plate-forme Transcriptome et Epigénome, Departement Génomes et Génétique, Institut Pasteur, Paris, France 9 Hôpital d'Olembé, Yaoundé, Cameroon 10 Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), Laboratoire de Recherche sur le Paludisme, B. P. 288, Yaoundé, Cameroon 11 Institut de Recherche pour le Développement (IRD), UMR 198 Unité de Recherche des Maladies Infectieuses et Tropicales Emergentes, Faculté de Médecine La Timone, Université Aix-Marseille, 13385 Marseille, France 12 Institut Pasteur de Madagascar, B.P. 1274, Ambatofotsikely, 101 Antananarivo, Madagascar Corresponding author: Rachida Tahar Institut de Recherche pour le Développement (IRD), UMR 216 Mère et Enfant Face aux Infections Tropicales, Université Paris-Descartes, Près Sorbonne Paris-Cité, 4, Avenue de l'Observatoire, 75270 Paris email Rachida.Tahar@ird.fr Tel: 331 53 73 99 33 Fax:331 53 73 96 17

  Figure. 1. Mean MA plot associated with the comparison of cerebral malaria with asymptomatic malaria (mean log ratio of expression versus mean log intensity)

Figure 2 :

 2 Figure 2: The expression level measured by quantitative RT-qPCR for arrays data confirmation. PFB0106c and PF13_0003 transcripts were highly up-regulated (fold change>6) by arrays in 3D7-Lib-Ring (R) versus 3D7-R and 3D7-Lib-Late Trophozoïte (LT) versus 3D7-LT, respectively. PFD1235W, PF11_0521, PFA0015c, PFD0065w, and PF11_0454 transcripts were moderately up-regulated (fold change range [2.5-5]) in 3D7-LT versus 3D7Lib-LT, CM-LT versus AM-LT, CM-LT versus 3D7-LT and 3D7-Lib-R versus 3D7-R, respectively. PFE1620C and PFL0030C transcripts were down-regulated (Fold change range [4 to 0]) in CM-LT versus3D7-LT.

Figure 2

 2 Figure 2 SI: Spearman's rank correlation (r = 0.775; P < 0.001) between arrays hybridization and qPCR methods for gene expression analysis.

Figure ( 3

 3 Figure (3). Transcription pattern of UPS A1, A3, and B1, var genes in parasites from CM and UM. The differential in transcription was compared between groups by the Mann-Whitney U test, significant P-values are shown in the figure.

Figure 1 .

 1 Figure 1. Prevalence of high-binder isolates of children presenting cerebral (CM, blue bars) and uncomplicated malaria (UM, yellow bars) on CSPG, ICAM-1 and CD36. The difference in prevalence to a given receptor was tested by the Chi-square test; significant P-value is shown in the figure.

Figure 2 .

 2 Figure 2. Transcript levels of var genes from group A, B, var2 and var3 in P. falciparum isolates from children presenting with either cerebral (CM) or uncomplicated malaria (UM). Transcript patterns of specific var genes were compared between groups by the Mann-Whitney U test, significant P-values are shown in the figure.

Figure 3 .

 3 Figure 3. Transcript abundance of DC8 and DC13 targeted by CIDRα1.1, DBLβ12, DBLα1.7, and CIDRα1.4 primers in in P. falciparum isolates from children presenting with either cerebral (CM) or uncomplicated malaria (UM). Transcript patterns were compared between groups by the Mann-Whitney U test, significant P-values are shown in the figure.

L

  'enfant a-t-il eu des convulsions ? _______________________________ L'enfant a t-il reçu un traitement avant de venir à l'hôpital ? est-il comateux ? ____________________A-t-il des convulsions ?_______________ Calcul du score de Blantyre :

Table 1 . A coma scale for children, Blantyre coma scale Type of stimulus Observation Score

 1

	Eye movements	-Directed (e.g., follows mother's face)	1
		-Not directed	0
	Verbal response	-Appropriate cry	2
		-response Moan or inappropriate cry	1
		-None	0
	Best motor response	-Localizes painful stimulus	2
		-Withdraws limb from pain	1
		-Non-specific or absent response	0
		Total	0 -5

Table 1 .

 1 Clinical and biological characteristics of P. falciparum-infected patients and asymptomatic

	carriers
	Characteristics	cerebral malaria 1	uncomplicated	asymptomatic
				malaria	carriers
	Number of enrolled	18	18	18
	patients
	Mean age ± SD	15.3 ± 6.6 (6-25)	28.9 ± 15.9 (7-60)	65.3 ± 7.4 (48-72)
	(range)(months)
	Sex ratio F/M (n)	0.8 (8/10)	1.0 (9/9)	1.25 (10/8)
	Geometric	mean	30,000 (9,550-	50,100
	parasitemia	(95%	94,000;
	confidence	interval;	300-309,000)
	range)	(asexual
	parasites/µl of blood)

List of down regulated genes in 3D7-LibR vs 3D7R:

			1, PfEMP1
	PF3D7_1000100	PF10_0001	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_1150400	PF11_0521	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_1240300	PFL1950w	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_1240400	PFL1955w	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_1240600	PFL1960w	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_1300300	PF13_0003	erythrocyte membrane protein
	[New Gene ID]	[Previous ID]	[Product Description] 1, PfEMP1 (VAR)
	PF3D7_1300400	PF13_0004	rifin (RIF)
	PF3D7_0100300 PF3D7_1400100	PFA0015c PF14_0001	var-like erythrocyte membrane erythrocyte membrane protein
			protein 1 1 (PfEMP1), truncated,
	PF3D7_0115700	PFA0765c	erythrocyte membrane protein pseudogene
			1, PfEMP1 (VAR)
	PF3D7_0201600	PFB0080c	Plasmodium exported protein
			(PHISTb), unknown function
	PF3D7_0201800	PFB0090c	RESA-like protein with PHIST
	PF3D7_0201900 [New Gene ID]	PFB0095c [Previous ID]	and DnaJ domains erythrocyte membrane protein [Product Description]
	PF3D7_0202000 PF3D7_0102500 PF3D7_0202100 PF3D7_0424200 PF3D7_0202200 PF3D7_0609000 PF3D7_0223300 PF3D7_0324900 PF3D7_1237900 PF3D7_0421300 PF3D7_1417200 PF3D7_0424200	PFB0100c PFA0125c PFB0105c PFD1150c PFB0106c PFF0445w PFB1045w PFC1120c PFL1835w PFD1015c PF14_0170 PFD1150c	3 (EMP3) erythrocyte binding knob-associated histidine-rich antigen-181 (EBA181) protein (KAHRP) reticulocyte binding Plasmodium exported protein protein homologue 4 (PHISTc), unknown function (RH4) Plasmodium exported protein, conserved Plasmodium unknown function protein, unknown erythrocyte membrane protein function 1 (PfEMP1), truncated conserved Plasmodium erythrocyte membrane protein protein, unknown 1, PfEMP1 (VAR) function erythrocyte membrane protein NOT family protein, 1, PfEMP1 (VAR) reticulocyte binding protein putative
			homologue 4 (RH4)
	PF3D7_0425800	PFD1235w	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_0600200	PFF0010w	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_0600400	PFF0020c	erythrocyte membrane protein
			1 (PfEMP1)-like protein (VAR-
			like)
	PF3D7_0713300	MAL7P1.61	erythrocyte membrane protein
			1 (PfEMP1),pseudogene
	PF3D7_0732800	MAL7P1.186	var-like erythrocyte membrane
			protein 1
	PF3D7_0733000	MAL7P1.187	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_0800300	PF08_0140	erythrocyte membrane protein
			1, PfEMP1 (VAR)
	PF3D7_0833300	MAL7P1.214	VAR pseudogene fragment,
			erythrocyte membrane protein
			1
	PF3D7_0937600	PFI1820w	erythrocyte membrane protein

List of up-regulated genes in 3D7-LibT vs 3D7T:

			1, PfEMP1 (VAR) protein, unknown function thrombospondin repeat protein condensation, putative
	PF3D7_0201700 PF3D7_0315100 PF3D7_0420300	PFB0085c PFC0635c PFD0985w	DnaJ protein, putative translation initiation factor 4E (SPATR) transcription factor with AP2
	PF3D7_0201800 PF3D7_0214200	PFB0090c PFB0645c	RESA-like protein with PHIST (eIF4E) mitochondrial ribosomal domain(s) (ApiAP2)
	PF3D7_0315900	PFC0695w	and DnaJ domains conserved Plasmodium protein L13 precursor, putative
	PF3D7_0202200 PF3D7_0216000	PFB0106c PFB0730w	Plasmodium exported protein, protein, unknown function DEAD/DEAH box helicase,
	PF3D7_0319600	PFC0870w	unknown function elongation factor 1 (EF-1), putative
	PF3D7_0202600.1 PF3D7_0217500	PFB0125c PFB0815w	conserved Plasmodium putative calcium dependent protein
	PF3D7_0320100	PFC0890w	protein, unknown function SNARE protein (SEC22) kinase 1 (CDPK1)
	PF3D7_0208500 PF3D7_0323600 PF3D7_0217600	PFB0385w PFC1055w PFB0820c	acyl carrier protein (ACP) conserved Plasmodium conserved Plasmodium protein,
	PF3D7_0212300	PFB0550w	peptide chain release factor protein, unknown function unknown function
	PF3D7_0324200 PF3D7_0302200	PFC1085c PFC0110w	subunit 1, putative Plasmodium exported protein cytoadherence linked asexual
	PF3D7_0213700	PFB0620w	conserved protein, unknown (hyp4), unknown function protein 3.2 (CLAG3.2)
	PF3D7_0401600.1 PF3D7_0302500	PFD0070c PFC0120w	function rifin (RIF) cytoadherence linked asexual
	PF3D7_0217700 PF3D7_0407400	PFB0826c PFD0365c	conserved Plasmodium conserved Plasmodium protein 3.1 (CLAG3.1)
	PF3D7_0304100	PFC0185w	protein, unknown function protein, unknown function membrane skeletal protein
	PF3D7_0217800 PF3D7_0409300	PFB0830w PFD0460c	40S ribosomal protein S26e, conserved Plasmodium IMC1-related (ALV2)
	PF3D7_0308800	PFC0371w	putative protein, unknown function conserved protein, unknown
	PF3D7_0219200 PF3D7_0412700	PFB0885w PFD0625c	40S ribosomal protein S30, erythrocyte membrane protein function
	PF3D7_0309200	PFC0385c	putative 1, PfEMP1 (VAR) serine/threonine protein
	PF3D7_0219300 PF3D7_0412900	PFB0886c PFD0630c	conserved Plasmodium erythrocyte membrane protein kinase, putative (ARK2)
	PF3D7_0312200	PFC0515c	protein, unknown function 1, PfEMP1 (VAR) TPR domain containing protein
	PF3D7_0219400.1 PF3D7_0413100 PF3D7_0313600	PFB0888w PFD0635c PFC0570c	ribosome associated erythrocyte membrane protein conserved Plasmodium protein,
			membrane protein RAMP4, 1, PfEMP1 (VAR) unknown function
	PF3D7_0313800	PFC0575w	putative conserved Plasmodium protein,
	PF3D7_0219400.2 List of down regulated genes 3D7-LibT vs 3D7T: PFB0888w PF3D7_0315200 PFC0640w	ribosome associated membrane protein RAMP4, unknown function circumsporozoite-and TRAP-
	PF3D7_0219700 [New Gene ID] PF3D7_0315500	PFB0900c [Previous ID] PFC0675c	putative Plasmodium exported protein related protein (CTRP) [Product Description] mitochondrial ribosomal
	[New Gene ID] PF3D7_0100100 PF3D7_0100300 PF3D7_0111500 PF3D7_0113200 PF3D7_0113300 PF3D7_0113900 PF3D7_0115700 PF3D7_0200100 PF3D7_0219900 PF3D7_0220100 PF3D7_0220600 PF3D7_0220700 PF3D7_0221900 PF3D7_0223300 PF3D7_0300100 PF3D7_0302300 PF3D7_0304900 PF3D7_0306300 PF3D7_0306900 PF3D7_0307100 PF3D7_0307400 PF3D7_0310600.1 PF3D7_0314500 PF3D7_0102200 PF3D7_0102500 PF3D7_0315600 PF3D7_0103800 PF3D7_0317200 PF3D7_0105700 PF3D7_0106000 PF3D7_0317500 PF3D7_0319000 PF3D7_0110500 PF3D7_0111000 PF3D7_0320800 PF3D7_0111300 PF3D7_0321300 PF3D7_0114000 PF3D7_0402300 PF3D7_0203100 PF3D7_0203200 PF3D7_0403600 PF3D7_0206800 PF3D7_0407800 PF3D7_0207500 PF3D7_0208100.1 PF3D7_0408500 PF3D7_0410700 PF3D7_0210200 PF3D7_0411900 PF3D7_0415200 PF3D7_0210300 PF3D7_0415800 PF3D7_0212100 PF3D7_0416000 PF3D7_0212600 PF3D7_0418600	[Previous ID] PFA0005w PFA0015c PFA0555c PFA0630c PFA0635c PFA0670c PFA0765c PFB0010w PFB0910w PFB0920w PFB0930w PFB0932w PFB0975c PFB1045w PFC0005w PFC0115c PFC0221c PFC0271c PFC0290w PFC0295c PFC0310c PFC0441c PFC0600w PFA0110w PFA0125c PFC0680w PFA0190c PFC0755c PFA0280w PFA0295c PFC0770c PFC0840w PFA0510w PFA0535c PFC0915w PFA0545c PFC0940c PFA0675w PFD0110w PFB0150c PFB0150c PFD0170c PFB0300c PFD0385w PFB0335c PFB0365w PFD0420c PFD0530c PFB0460c PFD0590c PFD0735c PFB0465c PFD0765w PFB0540w PFD0775c PFB0570w PFD0900w	[Product Description] erythrocyte membrane protein 1, PfEMP1 (VAR) var-like erythrocyte membrane protein 1 UMP-CMP kinase, putative Plasmodium exported protein, unknown function Plasmodium exported protein (hyp1), unknown function Plasmodium exported protein (hyp8), unknown function (GEXP10) erythrocyte membrane protein 1, PfEMP1 (VAR) erythrocyte membrane protein (PHISTc), unknown function (GEXP20) Plasmodium exported protein, unknown function DnaJ protein, putative Plasmodium exported protein (hyp9), unknown function Plasmodium exported protein (hyp9), unknown function erythrocyte membrane protein 1 (PfEMP1), truncated erythrocyte membrane protein 1 (PfEMP1), truncated erythrocyte membrane protein 1, PfEMP1 (VAR) erythrocyte membrane protein 1 (PfEMP1), pseudogene conserved Plasmodium protein, unknown function glutaredoxin 1 (GRX1) 40S ribosomal protein S23, putative 40S ribosomal protein S12, putative ATP-dependent Clp protease proteolytic subunit (ClpP) SAC3/GNAP family-related protein, putative conserved Plasmodium protein L29/L47 precursor, ring-infected erythrocyte putative surface antigen (RESA) conserved Plasmodium protein, erythrocyte binding antigen-unknown function 181 (EBA181) cdc2-related protein kinase 4 actin-related protein (ARP1) (CRK4) asparagine-rich antigen Pfa35-2 kinesin-like protein, putative conserved Plasmodium protein, P-type ATPase, putative unknown function (ATPase7) bromodomain protein, putative ATP-dependent RNA helicase, kinesin, putative putative (DOZI) replication factor c protein, conserved Plasmodium protein, putative unknown function RESA-like protein with DnaJ normocyte binding protein domain, putative (GEXP06) 1,reticulocyte binding protein protein kinase, putative homologue 1 conserved Plasmodium protein, conserved Plasmodium protein, unknown function unknown function merozoite surface protein 2 conserved Plasmodium protein, (MSP2) unknown function serine repeat antigen 6 (SERA6) flap endonuclease 1 (FEN1) conserved Plasmodium protein, GTPase, putative unknown function DNA polymerase alpha conserved Plasmodium protein, conserved Plasmodium protein, unknown function unknown function monocarboxylate transporter, RING zinc finger protein, putative putative conserved Plasmodium protein, RNA binding protein, putative unknown function secreted protein altered regulator of chromosome

List of up-regulated genes in CMT vs AMT:

	PF3D7_0600400 PF3D7_0610400	PFF0020c PFF0510w	erythrocyte membrane protein 2TM) histone H3 (H3)
	PF3D7_0105300 PF3D7_0612700	PFA0260c PFF0615c	1 (PfEMP1)-like protein (VAR-cyclase-associated protein, 6-cysteine protein (P12)
	PF3D7_0613400	PFF0650w	like) putative (C-CAP) apicoplast ribosomal protein L18
	PF3D7_0605900 PF3D7_0109000	PFF0290w PFA0440w	long chain polyunsaturated fatty photosensitized INA-labeled precursor, putative
	PF3D7_0617900	PFF0865w	acid elongation enzyme, protein 1, PhIL1, putative histone H3 variant, putative
	PF3D7_0114100	PFA0680c	putative Pfmc-2TM Maurer's cleft two (H3.3)
	[New Gene ID] PF3D7_0617100 PF3D7_0628300	[Previous ID] PFF0830w PFF1375c	alpha adaptin-like protein, transmembrane protein (MC-choline/ethanolaminephosphotr [Product Description] putative 2TM) ansferase, putative (CEPT)
	PF3D7_0102200 PF3D7_0632800 PF3D7_0206200 PF3D7_0630400 PF3D7_0104300 PF3D7_0701900 PF3D7_0206800 PF3D7_0631400 PF3D7_0113300 PF3D7_0702100 PF3D7_0207600 PF3D7_0210000 PF3D7_0702400 PF3D7_0113600 PF3D7_0702300 PF3D7_0217500 PF3D7_0725400 PF3D7_0200300 PF3D7_0709000 PF3D7_0221500 PF3D7_0730400 PF3D7_0200800 PF3D7_0720700 PF3D7_0808200 PF3D7_0302200 PF3D7_0810300 PF3D7_0201700 PF3D7_0726100 PF3D7_0811600 PF3D7_0202200 PF3D7_0302500 PF3D7_0730900 PF3D7_0817800 PF3D7_0205700.1 PF3D7_0316600 PF3D7_0732100 PF3D7_0819600 PF3D7_0206300 PF3D7_0320900 PF3D7_0801900 PF3D7_0821000 PF3D7_0214100 PF3D7_0321400 PF3D7_0812500 PF3D7_0414900 PF3D7_0821800 PF3D7_0219700 PF3D7_0819900 PF3D7_0423500 PF3D7_0828600 PF3D7_0825500 PF3D7_0828800 PF3D7_0220900 PF3D7_0301800 PF3D7_0318200 PF3D7_0324800 PF3D7_0400400 PF3D7_0400800 PF3D7_0401900 PF3D7_0415300 PF3D7_0424200 PF3D7_0425800 PF3D7_0501400 PF3D7_0502400 PF3D7_0527400 PF3D7_0101300 PF3D7_0609800 [New Gene ID] PF3D7_0525800 PF3D7_0522600 PF3D7_0518300 PF3D7_0935600 PF3D7_0515700 PF3D7_0508000 PF3D7_0933400 PF3D7_0505800 PF3D7_0933300 PF3D7_0503400 PF3D7_0920400 PF3D7_0908600 PF3D7_0502900 PF3D7_0830600 PF3D7_0501600 PF3D7_0830500 PF3D7_0501500 PF3D7_0827000 PF3D7_0423700 PF3D7_0903200	PFA0110w PFF1595c PFB0275w PFF1475c PFA0215w PF07_0004 PFB0300c PFF1525c PFA0635c MAL7P1.7 PFB0340c PFB0450w PF07_0007 PFA0650w PF07_0006 PFB0815w MAL7P1.141 PFB0020c MAL7P1.27 PFB0960c PF07_0127 PFB0045c MAL7P1.109 PF08_0108 PFC0110w MAL8P1.109 PFB0085c PF07_0107 PF08_0091 PFB0106c PFC0120w MAL7P1.171 PF08_0057 PFB0255w PFC0725c MAL7P1.183 MAL8P1.62 PFB0279w PFC0920w MAL8P1.154 MAL8P1.53 PFB0640c PFC0945w PF08_0086 PFD0720w MAL8P1.51 PFB0900c PF08_0049 PFD1110w MAL8P1.13 PF08_0026 PF08_0008 PFB0946c PFC0090w PFC0805w PFC1115w PFD0020c PFD0035c PFD0085c PFD0740w PFD1150c PFD1235w PFE0070w PFE0120c PFE1365w PFA0065w PFF0485c [Previous ID] PFE1285w PFE1130w PFE0915c PFI1720w PFE0785c PFE0395c PFI1615w PFE0285c PFI1615w PFE0165w PFI1000w PFI0415c PFE0140c MAL8P1.4 PFE0080c PF08_0003 PFE0075c MAL8P1.19 PFD1120c PFI0155c	erythrocyte membrane protein metabolite/drug transporter, conserved Plasmodium protein, ring-infected erythrocyte 1, PfEMP1 (VAR) putative unknown function surface antigen (RESA) Plasmodium exported protein, merozoite surface protein 2 Pfmc-2TM Maurer's cleft two ubiquitin carboxyl-terminal unknown function (MSP2) transmembrane protein (MC-hydrolase, putative Plasmodium exported protein serine repeat antigen 5 (SERA5) 2TM) Plasmodium exported protein (PHISTb), unknown function, secretory complex protein 61 conserved Plasmodium protein, (hyp1), unknown function pseudogene gamma subunit (Sec61-gamma) unknown function surface-associated interspersed sporozoite threonine and calcium dependent protein conserved Plasmodium protein, gene (SURFIN), pseudogene asparagine-rich protein (STARP) kinase 1 (CDPK1) unknown function (SURF1.2) chloroquine resistance Pfmc-2TM Maurer's cleft two conserved Plasmodium protein, erythrocyte membrane protein transporter (CRT) transmembrane protein, unknown function 1 (PfEMP1), exon2 phosphoinositide-binding pseudogene plasmepsin X erythrocyte membrane protein protein, putative cytoadherence linked asexual protein phosphatase, putative 1 (PfEMP1), truncated Plasmodium exported protein, protein 3.2 (CLAG3.2) conserved Plasmodium protein, DnaJ protein, putative unknown function cytoadherence linked asexual unknown function Plasmodium exported protein, Plasmodium exported protein, protein 3.1 (CLAG3.1) conserved Plasmodium protein, unknown function unknown function formate-nitrite transporter, unknown function conserved Plasmodium protein, erythrocyte membrane protein putative conserved Plasmodium protein, unknown function 1 (PfEMP1), pseudogene (VAR) histone H2A variant, putative unknown function conserved Plasmodium protein, conserved Plasmodium protein, (H2A.Z) conserved Plasmodium protein, unknown function unknown function protein kinase, putative unknown function protein transport protein sec31 RNA binding protein, putative conserved ARM repeats protein, secretory complex protein 61 (SEC31) U6 snRNA-associated Sm-like unknown function beta subunit (Sec61-beta) Plasmodium exported protein protein LSm3, putative (LSM3) glideosome associated protein folate transporter 1 (FT1) (PHISTc), unknown function conserved protein, unknown with multiple membrane spans 2 GPI-anchored micronemal (GEXP20) function (GAPM2) antigen (GAMA) Plasmodium exported protein, 1,merozoite surface protein 8 conserved Plasmodium protein, unknown function transmembrane protein (MC-zinc finger protein, putative Pfmc-2TM Maurer's cleft two IMC1-related (MSP8) membrane skeletal protein [Product Description] function ring-stage membrane protein conserved protein, unknown (FIRA) putative interspersed repeat antigen proteasome subunit beta type 1, 1, PfEMP1 (VAR) protein (GIG) 40, putative (GAP40) erythrocyte membrane protein gametocytogenesis-implicated glideosome-associated protein homologue 4 (RH4) unknown function 6-cysteine protein (P38) reticulocyte binding protein conserved Plasmodium protein, putative (SUMO) (CRK3) unknown function small ubiquitin-related modifier, cdc2-related protein kinase 3 conserved Plasmodium protein, (ADF1) acyl-CoA synthetase (ACS6) unknown function actin-depolymerizing factor 1 stevor conserved Plasmodium protein, putative 1, PfEMP1 (VAR) methyltransferase, putative TIM10 associated protein, erythrocyte membrane protein ribosomal RNA mitochondrial inner membrane rifin (rifin (3D7-rifT3-7)) (PHISTc), unknown function (RAP2) II, putative Plasmodium exported protein rhoptry-associated protein 2 DNA-directed RNA polymerase antigen (TryThrA) (RAP3) unknown function tryptophan/threonine-rich rhoptry-associated protein 3 Plasmodium exported protein, DBP10, putative (DBP10) protein 4 (ETRAMP4) unknown function, pseudogene ATP-dependent RNA helicase early transcribed membrane Rab GTPase 7 (Rab7)

List of down regulated genes in CMT vs AMT:

List of up-regulated genes in CMT vs 3D7T:

	PF3D7_0220500	PFB0926c	cyclodiphosphate synthase protein L14 precursor, putative Plasmodium exported protein homologue 4 (RH4) putative
	PF3D7_0523500 PF3D7_0424300 PF3D7_1035800	PFE1173c PFD1155w PF10_0350	(IspF) outer arm dynein lc3, putative (hyp2), unknown function erythrocyte binding antigen-probable protein, unknown
	PF3D7_0210000 PF3D7_0600300 PF3D7_0220700	PFB0450w PFF0015c PFB0932w	secretory complex protein 61 rifin (RIF) Plasmodium exported protein 165, pseudogene (EBA165) function
	PF3D7_0600800 PF3D7_0425700 PF3D7_1100800	PFF0040c PFD1230c PF11_0014	gamma subunit (Sec61-rifin, pseudogene (hyp9), unknown function rifin (RIF) Pfmc-2TM Maurer's cleft two
	PF3D7_0605400 PF3D7_0222100 PF3D7_0425800	PFF0265c PFB0985c PFD1235w	gamma) calcium-binding protein, Pfmc-2TM Maurer's cleft two erythrocyte membrane protein transmembrane protein (MC-
	PF3D7_0215700	PFB0715w	DNA-directed RNA polymerase putative transmembrane protein (MC-1, PfEMP1 (VAR) 2TM)
	PF3D7_0607400 PF3D7_0425900 PF3D7_1101700	PFF0365c PFD1240w PF11_0025	II second largest subunit, G-protein associated signal 2TM) rifin (RIF) Pfmc-2TM Maurer's cleft two
	PF3D7_0223300	PFB1045w	putative transduction protein, putative erythrocyte membrane protein transmembrane protein (MC-
	PF3D7_0219400.1 PF3D7_0612000 PF3D7_0300900 List of up-regulated genes in AMT vs 3D7T: PFB0888w PFF0585c PFC0045w PF3D7_1144000 PF11_0454	ribosome associated membrane protein RAMP4, conserved Plasmodium protein, unknown function 1 (PfEMP1), truncated stevor-like 2TM) 40S ribosomal protein S21e,
	PF3D7_0219400.2 PF3D7_0618600 PF3D7_0301400 [New Gene ID] PF3D7_1255200	PFB0888w PFF0900c PFC0070c [Input ID] PFL2665c	putative ribosome associated rhomboid protease ROM10 (ROM10) Plasmodium exported protein, unknown function putative [Product Description] erythrocyte membrane protein
	[New Gene ID] PF3D7_0100900 PF3D7_0107000 PF3D7_0114500 PF3D7_0115700 PF3D7_0207000 PF3D7_0207400 PF3D7_0207700 PF3D7_0207800 PF3D7_0208000 PF3D7_0209300 PF3D7_0303500 PF3D7_0310400 PF3D7_0312100 PF3D7_0320600 PF3D7_0324500 PF3D7_0400600 PF3D7_0400800 PF3D7_0401500 PF3D7_0401800 PF3D7_0401900 PF3D7_0413700 PF3D7_0414200.1 PF3D7_0416700 PF3D7_0417900 PF3D7_0420500 PF3D7_0422600 PF3D7_0500200 PF3D7_0501300 PF3D7_0502000 PF3D7_0506200 PF3D7_0511800 PF3D7_0512100 PF3D7_0519100 PF3D7_0621100 PF3D7_0622700 PF3D7_0624100 PF3D7_0624500 PF3D7_0626100 List of down regulated genes in CMT vs 3D7T: Previous ID] PFA0045c PFA0345w PFA0700c PFA0765c PFB0310c PFB0330c PFB0345c PFB0350c PFB0360c PFB0420w PFC0165w PFC0435w PFC0510w PFC0911c PFC1100w PFD0028w PFD0035c PFD0065w PFD0080c PFD0085c PFD0670c PFD0692c PFD0807c PFD0870w PFD0993c PFD1065c PFE0010c PFE0065w PFE0100w PFE0305w PFE0585c PFE0600c PFE0960w PFF1020c PFF1090c PFF1160w PFF1180w PFF1265w [New Gene ID] [Previous ID] PF3D7_0102500 PFA0125c PF3D7_0105700 PFA0280w PF3D7_0106600 PFA0325w PF3D7_0113100 PFA0625w PF3D7_0200300 PFB0020c PF3D7_0203100 PFB0150c PF3D7_0203200 PFB0150c PF3D7_0206200 PFB0275w PF3D7_0206300 PFB0279w PF3D7_0210600 PFB0475c PF3D7_0212600 PFB0570w PF3D7_0214600 PFB0665w PF3D7_0214800 PFB0675w PF3D7_0220300 PFB0923c PF3D7_0302300 PFC0115c PF3D7_0304000 PFC0180c PF3D7_0304100 PFC0185w PF3D7_0305200 PFC0235w PF3D7_0312900 PFC0540w PF3D7_0315200 PFC0640w PF3D7_0316000 PFC0700c PF3D7_0319700 PFC0875w PF3D7_0321300 PFC0940c PF3D7_0324800 PFC1115w PF3D7_0401700 PFD0075w PF3D7_0402300 PFD0110w PF3D7_0404700 PFD0230c PF3D7_0406000 PFD0300w PF3D7_0408000 PFD0395c PF3D7_0408200 PFD0403w PF3D7_0410000 PFD0495c PF3D7_0418600 PFD0900w PF3D7_0418700 PFD0905w PF3D7_0419700 PFD0955w PF3D7_0422200 PFD1045c PF3D7_0422900 PFD1080w PF3D7_0423800 PFD1130w PF3D7_0424000 PFD1140w PF3D7_0424200 PFD1150c PF3D7_1003500 PF10_0038 PF3D7_1001500 PF10_0019 PF3D7_1000800 PF10_0009 PF3D7_0936900 PFI1785w PF3D7_0936800 PFI1780w PF3D7_0936300 PFI1755c PF3D7_0936200 PFI1750c PF3D7_0936000 PFI1740c PF3D7_0935900 PFI1735c PF3D7_0931200 PFI1515w PF3D7_0902600 PFI0125c PF3D7_0831800 MAL7P1.231 PF3D7_0712800 MAL7P1.55 PF3D7_0702900 MAL7P1.10 PF3D7_0702400 PF07_0007 PF3D7_0114100 PFA0680c PF3D7_1301400 PF13_0073 PF3D7_0114500 PFA0700c PF3D7_1301700 MAL13P1.61 PF3D7_0221500 PFB0960c PF3D7_1353100 PF13_0275 PF3D7_0316600 PFC0725c PF3D7_1353200 PF13_0276 PF3D7_0324500 PFC1100w PF3D7_1370300 MAL13P1.413 PF3D7_0401800 PFD0080c PF3D7_1372200 MAL13P1.480 PF3D7_1425900 PF14_0239 PF3D7_0424900 PFD1185w PF3D7_1445500 PF14_0432 PF3D7_0501300 PFE0065w PF3D7_0532300 PFE1600w PF3D7_1478000 PF14_0752 PF3D7_0631400 PFF1525c	[Product Description] rifin (RIF) centrin-1 (CEN1) Plasmodium exported protein (hyp10), unknown function erythrocyte membrane protein 1, PfEMP1 (VAR) merozoite surface protein 4 (MSP4) serine repeat antigen 7 (SERA7) serine repeat antigen 4 (SERA4) serine repeat antigen 3 (SERA3) serine repeat antigen 1 (SERA1) 2C-methyl-D-erythritol 2,4-membrane protein RAMP4, putative spindle pole body protein, putative parasite-infected erythrocyte surface protein (PIESP1) RING zinc finger protein, putative conserved Plasmodium protein, unknown function rifin (RIF) erythrocyte membrane protein 1 (PfEMP1), exon1, pseudogene stevor stevor, pseudogene Plasmodium exported protein (PHISTb), unknown function (PfD80) acyl-CoA synthetase (ACS6) lysine decarboxylase-like protein, putative calmodulin-like protein CDGSH iron-sulfur domain-containing protein, putative conserved Plasmodium protein, unknown function conserved Plasmodium protein, unknown function conserved Plasmodium protein, unknown function erythrocyte membrane protein 1 (PfEMP1), pseudogene skeleton-binding protein 1 (SBP1) conserved Plasmodium protein, unknown function transcription initiation factor TFiid, TATA-binding protein (TBP) myo-inositol 1-phosphate synthase, putative conserved Plasmodium protein, unknown function mitochondrial ribosomal conserved Plasmodium protein, unknown function conserved Plasmodium membrane protein, unknown function conserved Plasmodium protein, unknown function anaphase promoting complex subunit, putative oxidoreductase, short-chain dehydrogenase family, putative [Product Description] erythrocyte binding antigen-181 (EBA181) asparagine-rich antigen Pfa35-2 conserved Plasmodium protein, unknown function surface-associated interspersed gene 1.1 (SURFIN 1.1) (SURF1.1) erythrocyte membrane protein 1 (PfEMP1), exon2 protein kinase, putative conserved Plasmodium protein, unknown function metabolite/drug transporter, putative conserved Plasmodium protein, unknown function conserved Plasmodium protein, unknown function secreted protein altered thrombospondin repeat protein (SPATR) serine/threonine protein kinase, putative conserved Plasmodium membrane protein, unknown function Plasmodium exported protein, unknown function erythrocyte membrane protein 1 (PfEMP1), pseudogene inner membrane complex protein 1a, putative (IMC1a) membrane skeletal protein IMC1-related (ALV2) conserved Plasmodium protein, unknown function hypothetical protein circumsporozoite-and TRAP-related protein (CTRP) conserved Plasmodium protein, unknown function ABC transporter, putative conserved Plasmodium protein, unknown function rifin (rifin (3D7-rifT3-7)) Plasmodium exported protein, unknown function, pseudogene normocyte binding protein 40S ribosomal protein S20e, reticulocyte binding protein protein 10.1 (ETRAMP10) (PHISTc), unknown function early transcribed membrane Plasmodium exported protein stevor, pseudogene unknown function pseudogene conserved Plasmodium protein, (PHISTb), unknown function, unknown function Plasmodium exported protein conserved Plasmodium protein, (PHISTc), unknown function associated antigen Plasmodium exported protein erythrocyte membrane-ring-exported protein 3 (REX3) apical merozoite protein (Pf34) (hyp11), unknown function unknown function Plasmodium exported protein conserved Plasmodium protein, ring-exported protein 2 (REX2) condensation, putative ring-exported protein 1 (REX1) regulator of chromosome selenoprotein (Sel2) unknown function FIKK family (FIKK9.7) conserved Plasmodium protein, serine/threonine protein kinase, unknown function histidine-rich protein II (HRPII) conserved Plasmodium protein, 1, PfEMP1 (VAR) unknown function erythrocyte membrane protein conserved Plasmodium protein, centrin, putative unknown function unknown function conserved Plasmodium protein, conserved Plasmodium protein, dipeptidyl peptidase 3 (DPAP3) 2TM) homologue1 transmembrane protein (MC-1,reticulocyte binding protein 1, PfEMP1 (VAR) Pfmc-2TM Maurer's cleft two Plasmodium exported protein transmembrane protein (MC-(hyp12), unknown function 2TM) Plasmodium exported protein Plasmodium exported protein (hyp8), unknown function (hyp10), unknown function (GEXP07) Pfmc-2TM Maurer's cleft two Plasmodium exported protein, transmembrane protein, unknown function pseudogene membrane associated histidine-formate-nitrite transporter, rich protein (MAHRP2) putative membrane associated histidine-rifin (RIF) rich protein (MAHRP1) Plasmodium exported protein histidine-rich protein III (HRPIII) (PHISTb), unknown function conserved Plasmodium protein, (PfD80) unknown function Plasmodium exported protein conserved Plasmodium protein, (PHISTa), unknown function unknown function skeleton-binding protein 1 (SBP1) Plasmodium exported protein Plasmodium exported protein (PHISTa), unknown function (PHISTb), unknown function Pfmc-2TM Maurer's cleft two (GEXP17)

List of down regulated genes in AMT vs 3D7T:

	PF3D7_0212600	PFB0570w	secreted protein altered antigen
	PF3D7_0422900	PFD1080w	thrombospondin repeat protein conserved Plasmodium protein,
			(SPATR) unknown function
	PF3D7_0214600 PF3D7_0423800	PFB0665w PFD1130w	serine/threonine protein kinase, conserved Plasmodium protein,
			putative unknown function
	PF3D7_0214800 PF3D7_0424000	PFB0675w PFD1140w	conserved Plasmodium membrane Plasmodium exported protein
			protein, unknown function (PHISTc), unknown function
	PF3D7_0220300 PF3D7_0424200	PFB0923c PFD1150c	Plasmodium exported protein, reticulocyte binding protein
			unknown function homologue 4 (RH4)
	PF3D7_0220500 PF3D7_0424300	PFB0926c PFD1155w	Plasmodium exported protein erythrocyte binding antigen-165,
			(hyp2), unknown function pseudogene (EBA165)
	PF3D7_0220700 PF3D7_0425700	PFB0932w PFD1230c	Plasmodium exported protein rifin (RIF)
	PF3D7_0425800	PFD1235w	(hyp9), unknown function erythrocyte membrane protein 1,
	PF3D7_0222100	PFB0985c	Pfmc-2TM Maurer's cleft two PfEMP1 (VAR)
	PF3D7_0425900	PFD1240w	transmembrane protein (MC-2TM) rifin (RIF)
	PF3D7_0223300	PFB1045w	erythrocyte membrane protein 1
			(PfEMP1), truncated
	PF3D7_0300900	PFC0045w	stevor-like
	PF3D7_0301400	PFC0070c	Plasmodium exported protein,
			unknown function
	PF3D7_0302300	PFC0115c	erythrocyte membrane protein 1
			(PfEMP1), pseudogene
	PF3D7_0304000	PFC0180c	inner membrane complex protein
			1a, putative (IMC1a)
	PF3D7_0304100	PFC0185w	membrane skeletal protein IMC1-
			related (ALV2)
	PF3D7_0305200	PFC0235w	conserved Plasmodium protein,
			unknown function
	PF3D7_0312900	PFC0540w	hypothetical protein
	PF3D7_0315200	PFC0640w	circumsporozoite-and TRAP-
			related protein (CTRP)
	PF3D7_0316000	PFC0700c	conserved Plasmodium protein,
			unknown function
	PF3D7_0319700	PFC0875w	ABC transporter, putative
	PF3D7_0321300	PFC0940c	conserved Plasmodium protein,
			unknown function
	[New Gene ID] PF3D7_0324800 PF3D7_0401700	[Previous ID] PFC1115w PFD0075w	rifin (rifin (3D7-rifT3-7)) [Product Description] Plasmodium exported protein,
	PF3D7_0102500 PF3D7_0402300 PF3D7_0105700 PF3D7_0106600 PF3D7_0404700 PF3D7_0113100 PF3D7_0406000 PF3D7_0200300 PF3D7_0408000 PF3D7_0203100 PF3D7_0408200 PF3D7_0203200 PF3D7_0410000 PF3D7_0206200 PF3D7_0418600 PF3D7_0206300 PF3D7_0418700 PF3D7_0210600 PF3D7_0419700 PF3D7_0422200	PFA0125c PFD0110w PFA0280w PFA0325w PFD0230c PFA0625w PFD0300w PFB0020c PFD0395c PFB0150c PFD0403w PFB0150c PFD0495c PFB0275w PFD0900w PFB0279w PFD0905w PFB0475c PFD0955w PFD1045c	unknown function, pseudogene erythrocyte binding antigen-181 normocyte binding protein (EBA181) 1,reticulocyte binding protein asparagine-rich antigen Pfa35-2 homologue 1 (RH1) conserved Plasmodium protein, dipeptidyl peptidase 3 (DPAP3) unknown function conserved Plasmodium protein, surface-associated interspersed unknown function gene 1.1 (SURFIN 1.1) (SURF1.1) conserved Plasmodium protein, erythrocyte membrane protein 1 unknown function (PfEMP1), exon2 conserved Plasmodium protein, protein kinase, putative unknown function conserved Plasmodium protein, conserved Plasmodium protein, unknown function unknown function metabolite/drug transporter, regulator of chromosome putative condensation, putative conserved Plasmodium protein, conserved Plasmodium protein, unknown function unknown function conserved Plasmodium protein, apical merozoite protein (Pf34) unknown function erythrocyte membrane-associated

Table 1 . Parasitological and Clinical Characteristics of Children Enrolled in Cotonou, 2012- 2013.

 1 Comparisons between P. falciparum isolates from children presenting with cerebral (CM) and uncomplicated malaria (UM) were achieved by the Mann-Whitney U test

		Cerebral Malaria	Uncomplicated Malaria	P-value
		(N = 52)	(N = 36)
	Sex ratio (female/male)	23/29	18/18	0.2
	Age (months), median [range]	42 [5-72]	33 [8-58]	0.09
	Parasitemia (P/µl), median	75,806 [889-	49,453.5 [1,164-	0.01
	[range]	3,600,000]	246,857]
	Hemoglobin (g/dl), median	5.15 [0.8-12.6]	7.45 [5.2-12.5]	0.0001
	[range]
	Blantyre score, median [range]	2 [0-2]	-	-
	Temperature, median [range]	38°C [35.5°C-40°C]	38.5°C [36.5°C-40°C]	0.15
	Multiplicity of infection, median [range]	3.4 [1-6]	2.2 [1-6]	0.0001
	Number of deaths	19	0	-

Table 2 .

 2 Receptor Binding Data. Values are the mean number of infected erythrocytes bound (range) /mm² to a given receptor minus the number of infected erythrocytes bound to BSA.

		Cerebral malaria	Uncomplicated malaria	All samples
		(n = 38)	(n = 27)	(n = 65)
	CD36	350.5 (0-2552)	85.1 (0-379)	244.0 (0-2552)
	ICAM-1	48.3 (0.6-215)	39.3 (0-294)	45.2 (0-294)
	CSPG	11.3 (0-62)	9.6 (0-72)	10.7 (0-72)

Table 3 . Transcript Abundance of var Genes from Groups A, B, var2, var3, DC8 and DC13.

 3

	Comparisons between P. falciparum isolates from children presenting with cerebral (CM)
	and uncomplicated malaria (UM) were achieved by the Mann-Whitney U test
	Primers ID	T u _CM	T u _UM	P-value
		median [IQR]	median [IQR]
	A1	1.4 [1-6.8]	1 [0-1.7]	0.01
	A2	1.2 [1-5.3]	1 [1-6.3]	0.8
	A3	19.6 [4.3-33]	6.6 [1-14.3]	0.02
	B1

average_control primers ). Transcript units were then determined as Tu = 2 , as described by[START_REF] Lavstsen | Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children[END_REF]).

Diagnostic :.................................................................................................................................

Acknowledgments:

We would like to acknowledge all the medical staff of Olembé Hospital and Nlongkak Dispensary in Yaoundé, Cameroon who actively participated in this work. We also thank Caroline Proux for technical assistance. We thank Dr. Thomas Lavstsen (Centre for Medical Parasitology, University of Copenhagen, Denmark) for providing the 3D7-Lib parasite line. This study was funded by the Direction Générale des Armées (DGA) of France and Institute of Research for Development (IRD), France.

Acknowledgments. We would like to thank all children and their parents for participating in this study. We are grateful to the pediatricians and the nurses in HOMEL, CNHU-HKM and Suru Léré for their help in sample collection. We thank Prof Ayvivi and Dr Layla for facilitating fieldwork. UMR216 is part of the Laboratory of Excellence GR-Ex, reference ANR-

Best GOs

P-value Gene Ontology term description.

CM T versus AM T upregulated genes

GO:0009405 5 x 10 -4 Pathogenesis GO:0020013 9 x 10 -4 modulation by symbiont of host erythrocyte aggregation GO:0022407 9 x 10 -4 regulation of cell-cell adhesion GO:0030155 9 x 10 -4 regulation of cell adhesion GO:0034109 -4 9 x 10 homotypic cell-cell adhesion GO:0034110 9 x 10 -4 regulation of homotypic cell-cell adhesion GO:0034117 9 x 10 -4 erythrocyte aggregation GO:0034118 9 x 10 -4 regulation of erythrocyte aggregation I a 3D7Lib T vs 3D7 upregulated genes GO:0009405 4 x 10 -17 Pathogenesis GO:0044406 6 x 10 -12 adhesion to host GO:0022407 9x10 -12 regulation of cell-cell adhesion GO:0034109 9 x 10 -12 homotypic cell-cell adhesion GO:0034117 9 x 10 -12 erythrocyte aggregation GO:0051701 3 x 10 -09 interaction with host GO:0051805 9 x 10 -05 evasion or tolerance of immune response of other organism involved in symbiotic interaction GO:0020033 2 x 10 -4 antigenic variation 3D7-Lib R vs 3D7 upregulated genes GO:0009405 1 x 10 -22 Pathogenesis GO:0030155 9 x 10 -17 regulation of cell adhesion GO:0034109 9 x 10 -17 homotypic cell-cell adhesion GO:0034117 9 x 10 -17 erythrocyte aggregation GO:0020035 2 x 10 -16 cytoadherence to microvasculature, mediated by symbiont protein GO:0051701 2 x 10 -13 interaction with host GO:0020033 2 x 10 -12 antigenic variation GO:0006950 1 x 10 -09 response to stress

The reported p-values are based on the "OddsRatio score", a measure of gene-enrichment (Falcon et al.2007).

Chapter 3

Cytoadherence phenotype of P. falciparum infected erythrocytes is associated with specific Pfemp-1 expression in parasites from children with cerebral malaria

Talleh Almelli 1,2 , Nicaise Tuikue Ndam 1,2 , Sem Ezimegnon 3 , Maroufou J. Alao 4 , Charles Ahouansou 3 , Gratien Sagbo 5 , Annick Amoussou 6 , Philippe Deloron 1,2 , Rachida Tahar

Discussion and Perspectives

Comparative transcriptomic analysis using microarrays is a robust technique that has improved our knowledge of the relationship between gene expression patterns and their potential roles in biological processes [START_REF] Roch | Discovery of gene function by expression profiling of the malaria parasite life cycle[END_REF][START_REF] Blythe | Plasmodium falciparum STEVOR proteins are highly expressed in patient isolates and located in the surface membranes of infected red blood cells and the apical tips of merozoites[END_REF]. Furthermore, differences in gene expression profiles between P. falciparum isolates and laboratory-adapted lines have been revealed [START_REF] Daily | In vivo transcriptional profiling of Plasmodium falciparum[END_REF]Ndam, et al. 2008). These variations of genes transcriptions between in vitro and in vivo conditions may associate with the biological mechanisms and the adaptation of parasites within the host or with the in vitro conditions. The host-parasite interactions that require gene activation involve different metabolic pathways essential for in vivo parasite survival, sexual differentiation for parasite transmission, parasite cytoadherence and immune evasion.

The overall aim of my PhD thesis was to gain more insights of the parasite genetic factors implicated in cerebral malaria and the potential relation of gene expression profiles of a specific subset of var genes with cytoadherence phenotype to a particular receptor.

In order to attain our goals we first compared the whole transcriptome of P. falciparum parasites isolated from children with distinct clinical manifestations, including uncomplicated malaria (UM) and cerebral malaria (CM) and from asymptomatic carriers (AM) at ring and late trophozoite stages to investigate gene transcription patterns underlying malaria pathology. As controls, gene expression profiles of 3D7-Lib line were compared with 3D7 clone. In addition, transcriptomic patterns of isolates were compared to 3D7 clone as well.

Our results revealed consistent differences in the transcription profiles of parasite isolates compared to 3D7, and between the selected 3D7-Lib compared t o 3D7. Moreover, we detected important differences in gene transcription patterns between parasites from children with CM compared to those from asymptomatic children.

The up-regulated genes that could be implicated in CM pathogenesis are those that code for proteins involved in cytoadherence of IEs to brain endothelial receptors and antigenic variation that would allow the parasites to avoid antibodies responses. This result was supported by gene ontology test of up-regulated genes in CM and 3D7-Lib which showed over representation of pathogenesis, regulation of cell adhesion, cytoadherence to microvasculature, erythrocytes aggregation and antigenic variation.

The majority of 3D7 and 3D7-Lib transcripts that were not detected in our arrays were also found also absent in other studies (Le [START_REF] Roch | Discovery of gene function by expression profiling of the malaria parasite life cycle[END_REF][START_REF] Bozdech | The transcriptome of the Intra erythrocytic developmental cycle of Plasmodium falciparum[END_REF], suggesting untranscribed ORFs in cultured parasites. However, many o f t h o s e transcripts we r e found in our transcriptome data of field isolates and in the transcriptome of other clinical isolates [START_REF] Vignali | NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children[END_REF], suggesting that these genes are not useful for in vitrosurvival of asexual-stage parasites. Of the undetected transcripts in ring and trophozoite stages of 3D7 clone variant surface antigen (VSA) families (PfEMP-1, RIFIN, STEVOR, and

Note d'information

Projet de Recherche sur le paludisme : FPPG Etude des facteurs parasitaires impliqués dans le paludisme grave

Le paludisme, présent particulièrement en Afrique, peut être responsable d'une maladie très grave, le neuropaludisme qui se caractérise par une phase de coma. Les jeunes enfants sont particulièrement vulnérables aux accès palustres car ils ne disposent pas d'une immunité naturelle suffisante. Celle-ci se constitue au fil des années et des infections palustres successives.

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