Paris

Rndr Jaroslava

Joniová Pavol

Jozef Šafárik

prof. RNDr Pavol

Miškovský, DrSc. Franck Sureau

PhD. RNDr Zuzana Naďová

Rndr Jaroslava Joniová

PAVOL JOZEF

ACKNOWLEDGEMENT

I would like to thank: Prof. Pavol Miskovsky for making my PhD study unique by giving me the opportunity to be a part of co-tutelle and to have a chance to work in Paris. For his guidance, consultations and help during my study.

Dr. Franck Sureau for the fact that anytime I could come for consultation, for his help, suggestions, guidance, advices, patience and taking care of me during my stays in Paris. Merci, Franck! Dr. Zuzana Nadova, supervisor specialist, for her guidance, support and friendly help, during my study and her suggestions during writing this thesis.

Dr. Alexandra Zahradnikova and Dr. Patrice Petit for their comments and suggestions which enriched this thesis.

My thanks belong as well to Dr. Daniel Jancura for his help and consultations.

Dr. Veronika Huntosova for her friendship, help, sharing her experiences and knowledge, her visits in Paris, great time at the conferences in Brixen and Cordoba and long conversations until morning.

Jana "Kuba" Kubackova for her friendship and "craziness" which made me laugh anytime we talked together. Even when we were chatting and I was smiling at the screen of my computer and trying not to laugh out loud in the office. Barbora Profantova, my fellow in Paris, for her friendship, making the stays in Paris more pleasant and our exploring the beauties of Paris together (and of course introducing me to the geocaching).

Of course, I want to thank to all members of Department of Biophysics in Kosice (especially to Marienka, the soul of the Department of Biophysics) and Laboratoire Jean Perrin in Paris for making very comfortable working atmosphere.

My big thank you belongs to Stanko for his support and patience during my study and to my mom, FOR EVERYTHING… ABSTRACT Photodynamic therapy (PDT), non-invasive method for cancer treatment requires three necessary components: photosensitizer (pts), light of wavelength equal to the wavelength of pts excitation and oxygen. Activation of pts by irradiation results in a formation of reactive oxygen species at the place of light application and starts destructive mechanism. Low density lipoproteins (LDL) are considered as suitable natural in vivo delivery system for hydrophobic pts such as Hypericin (Hyp) and it was shown that over expression of LDL-receptors in tumor cells can be used for specific targeting. However, in spite of extensive tumor accumulation observed in vivo, Hyp, loaded in LDL, can be redistributed towards others biological macromolecules from plasma before cell delivery process. In using cholesterol modified dextran (D ch ) coating of LDL-Hyp complexes we succeed to decrease such redistribution process. We show that this D Ch coating has negligible impact on the cellular uptake of Hyp from [LDL-Hyp]/D Ch complexes. Like LDL, high density lipoproteins (HDL) are able to deliver Hyp to cancer cells as well and their interaction was studied. Because isolation of LDL or HDL is difficult and isolated lipoproteins vary in size and composition, synthetic lipid-based nano-particles (sLNP) were prepared and their interaction with Hyp was analysed by fluorescence spectroscopy. These synthetized sLNP are suitable carriers of Hyp and can enter cells by endocytosis. The main aim of our transport system is to selectively accumulate Hyp in the cancer cells. Subsequently, after Hyp accumulation and irradiation, cell death will occur. In the present work we have focused our attention on some regulation processes involved in Hyp photo-induced cell death pathway. In using specific siRNA, the consequence of gene silencing of protein kinase C PKCα or PKCδ was examined. Post-transcriptional silencing of pkcα gene expression did not significantly modify cell death level but the balance between apoptosis and necrosis id seeply sffected. Our results show that Hyp photo-induced ROS production is significantly increased in PKCα -cells. Such increase of intracellular ROS has been related to the decrease of Bcl-2 phosphorylation and the level of mitochondrial GSH binding. Post-transcriptional silencing of pkcδ gene expression did not affect type of the cell death when we compared it with the effect of pkcα gene silencing, reduced ROS production and effected Bak and Bax translocation in the cells.

RESUME

La photo thérapie dynamique (PDT) est une méthode non invasive de traitement de certains cancers qui requiert la combinaison de trois éléments: un photosensibilisateur (pts), une source lumineuse de longueur d'onde adaptée à l'excitation du pts et la présence d'oxygène. La photo activation du pts induit la production locale d'espèces réactives de l'oxygène (ROS) à l'origine des mécanismes cytotoxiques. Les lipoprotéines de faible densité (LDL) sont considérées comme des vecteurs naturels adaptés au transport des pts hydrophobes tel que l'hypéricine (Hyp) et il a été montré que la surexpression des récepteurs aux LDL dans les cellules tumorales peut être utilisée pour un ciblage spécifique. Cependant, malgré l'accumulation du pts, observée in vivo, dans les cellules tumorales, celle-ci reste limitée du fait de la redistribution de l'Hyp incorporée dans les LDL vers d'autres macromolécules du plasma avant d'atteindre sa cible. Nous avons pu réduire ce processus de redistribution en associant au complexe LDL-Hyp des molécules de Dextran sur lesquelles ont été greffées du cholestérol (DCh). Nous avons pu montrer que l'interaction de ces DCh avait un impact négligeable sur les processus de reconnaissance du complexe [LDL-Hyp]/DCh par les récepteurs au LDL et sur son incorporation cellulaire. Du fait de la grande variabilité de taille et de composition des lipoprotéines et de la complexité d'isolation de fractions pures de LDL ou de HDL, l'utilisation alternative de nanoparticules lipidiques synthétiques (sLNP) pour le transport de l'Hyp a également été étudiée. Nos résultats indiquent que ces sLNP sont des vecteurs appropriés de l'Hyp susceptibles de pénétrer dans les cellules par voie d'endocytose. Dans la perspective d'utilisation de ce type de vecteurs pour une application en PDT, nous nous sommes ensuite intéressés au processus de régulation des voies de mort cellulaire photo-induite par l'Hyp. En utilisant des si-RNA spécifiques, les conséquences de l'extinction de l'expression des gènes de la protéine kinase C α ou δ ont été étudié. Nous avons pu montrer que l'inhibition post-transcriptionelle de ces protéines ne modifie pas significativement le taux global de mort cellulaire mais que cela affecte profondément l'équilibre nécrose/apoptose des voies photo-induites. Nos résultats indiquent, entre autre, une importante augmentation de la production de ROS. Cette augmentation des ROS a pu être corrélée à une réduction de la phosphorylation de Bcl-2 ainsi qu'à une baisse du taux de fixation de la glutathion S-transférase (GSH) au niveau des mitochondries. L'ensemble des ces résultats suggèrent que l'inhibition posttranscriptionelle du gène de la PKC et la réduction consécutive de la concentration intracellulaire en PKC affecte considérablement les propriétés anti-apoptotiques et notamment la fonction anti-oxydante de Bcl-2. Cela signifie que la PKC, en tant que kinase de Bcl-2, protège indirectement les cellules U-87 MG contre le stress oxydatif et la mort cellulaire photo-induite par l'Hyp.

ABSTRAKT

Fotodynamická terapia (PDT) je neinvazívny spôsob liečenia rakoviny, pri ktorom sú potrebné tri veci: fotosenzitívna látka (pts), svetlo excitačnej vlnovej dĺžky pts a kyslík. Po aktivácii pts sa v mieste ožiarenia vytvárajú reaktívne formy kyslíka. V druhej časti tejto práce sme sa zamerali na študovanie procesu regulácie bunkovej smrti vyvolanej fotoaktivovaným Hyp. Použitím siRNA sme študovali vplyv vyblokovanej proteín kinázy C (PKC) α a δ. Post-transkripčná úprava expresie pkcα génu neovplyvnila stupeň bunkovej smrti, ale mala výrazný vplyv na dráhu bunkovej smrti (rovnováha medzi apoptózou/nekroózou). Z našich výsledkov vyplýva, ze pri vyblokovaných PKCα -bunkách, produkcia ROS je po ožiarení Hyp značne zvýšená. Chemotherapy: traditional chemotherapeutic agents act by killing cells. This means that chemotherapy also harms cells that divide rapidly under normal circumstances: cells in the bone marrow, digestive tract, and hair follicles. This results in the most common side-effects of chemotherapy: myelosuppression (decreased production of blood cells, hence also immunosuppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss). Because some drugs work better together than alone, two or more drugs are often given at the same time. This is called "combination chemotherapy"; most chemotherapy regimens are given in a combination [START_REF] Takimoto | Cancer Management: A Multidisciplinary Approach[END_REF].

CHARACTERIZATION OF THE INTERACTION OF LDL-HYP

LIST OF FIGURES

LIST OF TABLES

Targeted therapy: uses drugs or other substances to more precisely identify and attack cancer cells. These drugs tend to have different (and often less severe) side effects than standard chemotherapy drugs [5]. In this category one of the treatments is photodynamic therapy (see Chapter 1.2)

Immunotherapy: uses immune system to reject cancer. The main premise is stimulating the patient's immune system to attack the malignant tumor cells that are responsible for the disease. This can be either through immunization of the patient (e.g., by administering a cancer vaccine), in which case the patient's own immune system is trained to recognize tumor cells as targets to be destroyed, or through the administration of therapeutic antibodies as drugs, in which case the patient's immune system is recruited to destroy tumor cells by the therapeutic antibodies [6].

Hormonal therapy: involves the manipulation of the endocrine system through exogenous administration of specific hormones, particularly steroid hormones, or drugs which inhibit the production or activity of such hormones (hormone antagonists).

Because steroid hormones are powerful drivers of gene expression in certain cancer cells, changing the levels or activity of certain hormones can cause certain cancers to cease growing, or even undergo cell death [START_REF] Jones | A Review of Adjuvant Hormonal Therapy in Breast Cancer[END_REF].

The goal of the treatment is complete removal of the cancer without damage to the rest of the body. Sometimes this can be accomplished by surgery, but the fact that cancers can invade tissue or to spread to distant sites by microscopic metastasis often limits its effectiveness; chemotherapy and radiotherapy can unfortunately have a negative effect on normal cells [START_REF] Enger | Concepts in Biology[END_REF]. To enhance the effect and to get rid of the cancer two or three types of therapy are often used simultaneously.

PHOTODYNAMIC THERAPY

Photodynamic therapy is an entirely new treatment modality and its development can be likened to that of the discovery of antibiotics. This is just beginning, and its possible For more than three thousand years light has been used as therapy. Ancient

Egyptian, Indian and Chinese civilizations used light to treat various diseases, including psoriasis, rickets, vitiligo and skin cancer [START_REF] Daniell | A History of Photodynamic Therapy[END_REF]. At the end of the nineteenth century in Denmark, Niels Finsen further developed 'phototherapy' or the use of light to treat diseases. He found that red-light exposure prevents the formation and discharge of smallpox pustules and can be used to treat this disease. He also used ultraviolet light from the sun to treat cutaneous tuberculosis. This was the beginning of the modern light therapy. More than 100 years ago researchers also observed that a combination of light and certain chemicals could induce cell death. In 1900 German medical student Oscar

Raab reported that certain wavelengths were lethal to infusoria including a species of Paramecium in the presence of acridine. In the same year a neurologist in France named J. Prime found that epilepsy patients who were treated with oral eosin developed dermatitis in sun exposed areas. Later, Herman Von Tappeiner and A. Jesionek treated skin tumors with topically applied eosin and white light. In 1903 they described this phenomenon as 'photodynamic action' [START_REF] Dolmans | Photodynamic Therapy for Cancer[END_REF].

Photodynamic therapy (PDT) can be defined as the administration of a nontoxic drug or even dye known as photosensitizer (pts) to a patient's lesion followed after some time by the illumination of the lesion with visible light, which, in the presence of oxygen, leads to generation of cytotoxic species-reactive oxygen species (ROS) and consequently to cell death and tissue destruction.

Concept of dual selectivity is one of the main attractions of PDT as a therapy. By increasing the selective accumulation of pts in the tumor or other diseased tissue and by delivering the light in a spatially confines and focused manner collateral damage to normal tissue can be minimized. Another advantages of PDT are minimal toxicity, high efficiency, it can be proceeded as diagnostic and therapeutic procedure at the same time, shorter recovery after PDT treatment-typically hours or days when compared to other methods of treatments. Nevertheless PDT can have also side effects including longlasting skin photosensitivity and excessive tissue destruction at the treated site. It is hoped that advances in mechanistic understanding of PDT will minimize these side effects [START_REF] Castano | Mechanisms in Photodynamic Therapy: Part One-Photosensitizers, Photochemistry and Cellular Localization[END_REF].

PHOTOPHYSICS AND PHOTOCHEMISTRY OF PDT

As mentioned above, the effect of PDT consists in combination of three nontoxic agents.

Photosensitizers

A large number of photosensitizing drugs have been tested in vitro and in vivo during last 20 years. Table 1 presents most commonly used pts and precursors in PDT.

The physico-chemical properties of the pts are very important for the efficacy of photosensitization. Chemical purity, capability to localize specifically in neoplastic tissue, short time interval between the administration of the drug and its maximal accumulation in hyper proliferating tissue, rapid clearance from normal tissues, activation at wavelength with optimal tissue penetration, high quantum yields for the generation of singlet oxygen, and lack of dark toxicity are desirable features of ideal pts.

The fundamental prerequisite for optimal response to photosensitization is a sufficient amount of drug localized in the target tissue. Initially, pts are taken up by most normal and hyper proliferating cells, but are retained longer in the hyper proliferating cells. The mechanisms of this selective prolonged retention are not understood in detail yet.

Increased blood vessel permeability as well as poor lymphatic drainage in neoplastic tissues may contribute to the retention of the drug in neoplastic lesions [START_REF] Luksiene | Photodynamic therapy: mechanism of action and ways to improve the efficiency of treatment[END_REF].

Light source

The first light sources used in PDT were conventional lamps where the output wavelengths were defined by the use of filters. A drawback with this was that there was often a significant thermal component, and also calculating the dose of delivered light dose was difficult. These non-coherent light sources are attractive because they are easy to use and relatively cheap. The introduction of lasers equipped with optical fibers revolutionized photosensitization and expanded its applicability in medicine, enabling the endoscopic delivery of light to almost every site of the human body [START_REF] Fisher | Clinical and Preclinical Photodynamic Therapy[END_REF]. They are useful because they produce monochromatic light, and the light dosimetry is relatively easy. The used laser system can be chosen according to the pts used. The most commonly used lasers in PDT are pumped dye lasers and semiconductor lasers.

Oxygen

Numerous investigations supported the idea that the efficacy of photosensitization is directly related to the yield of singlet oxygen ( 1 O 2 ) in the tumor environment and the yield of 1 O 2 depends on the concentration of oxygen in the tissue. The initiating step of the photosensitizing mechanism is the absorption of a light by pts, the unstable excited singlet state with a half life in range of 10 -6 to 10 -9 seconds.

The singlet excited pts either decays back to the ground state, resulting in the fluorescence or undergoes intersystem crossover to the longer lived tripled excited state (10 -3 second). The interaction of the triplet state of the pts with surrounding molecules results in two types of photo-oxidative reaction (Figure 1). [source [START_REF] Luksiene | Photodynamic therapy: mechanism of action and ways to improve the efficiency of treatment[END_REF]]

Type I pathway involves electron or hydrogen atom transfer, producing radical forms of the pts or the substrate. These intermediates may react with oxygen to form peroxides, superoxide ions, and hydroxyl radicals, which initiate free radical chain reactions. Type II mechanism is mediated by the energy transfer process from the triplet state of the pts to the oxygen ( ¹ O 2 ) ground state. The in situ generation of singlet oxygen via type II pathway appears to play the central role in photodynamic cytotoxicity because of the highly efficient interaction of the singlet oxygen species with different biomolecules [START_REF] Luksiene | Photodynamic therapy: mechanism of action and ways to improve the efficiency of treatment[END_REF], [START_REF] Prasad | Introduction to biophotonics[END_REF].

HYPERICIN AS THE MODEL MOLECULE OF PTS FOR PDT

Hypericin (Hyp, Figure 2) is naturally occurring perylene quinone pigment that has stimulated great interest because of its broad spectrum of light induced biological activities. It can be extracted from the plants of Hypericum widely distributed around the world, most common of which is Hypericum perforatum. It is known that Hyp exhibits antiviral, anticancer and antiangiogenic activities against infectious, neoplastic and ophtalmologic diseases [START_REF] Ho | Lipid-Mediated Preferential Localization of Hypericin in Lipid Membranes[END_REF]. Hyp possesses powerful phototoxicity on different cell lines [START_REF] Kamuhabwa | Photodynamic Activity of Hypericin in Human Urinary Bladder Carcinoma Cells[END_REF], displays light induced virucidal activity in vitro against several types of viruses [(herpes, murine cytomegalovirus, sindbis, hepatitis B, equine anemia including the human immunodeficiency virus (HIV)]. Hyp was also used in clinical treatment of AIDS patients [START_REF] Miskovsky | Hypericin -a New Antiviral and Antitumor Photosensitizer: Mechanism of Action and Interaction with Biological Macromolecules[END_REF]. proteins, membrane fragments and cellular compounds) [START_REF] Miskovsky | Hypericin -a New Antiviral and Antitumor Photosensitizer: Mechanism of Action and Interaction with Biological Macromolecules[END_REF]. Fluorescence emission maximum of Hyp is about 600 nm (Figure 3). Depending on different factors like solvent, aggregation, production process, moisture etc, the molar extinction coefficient of Hyp vary from 27 000 to 52 000 at 590 nm. In water insoluble aggregates pellets of Hyp are formed. These aggregates are non-fluorescent, and show very weak visible absorption spectra in the same region as free Hyp (Figure 3), but have much lower extinction coefficients [START_REF] Kubin | Hypericin -the Facts About a Controversial Agent[END_REF]. Hyp monomer is the only biologically active form [START_REF] Miskovsky | Hypericin -a New Antiviral and Antitumor Photosensitizer: Mechanism of Action and Interaction with Biological Macromolecules[END_REF]. With minimal dark toxicity, high quantum yield of singlet oxygen, differential uptake in normal and tumor cells resulting in high tumor selectivity and rapid clearance from the body, Hyp is a suitable choice for PDT application [START_REF] Miskovsky | Hypericin -a New Antiviral and Antitumor Photosensitizer: Mechanism of Action and Interaction with Biological Macromolecules[END_REF]. After photoactivation, Hyp can inhibit protein kinase C (PKC) and other growth factor stimulated protein kinases, it induces peroxidation of membrane lipids, increases the activity of superoxide dismutase and decreases levels of cellular gluthatione as well as other enzymes such as monoamine oxidase and mitochondrial succinoxidase, possibly via generation of singlet oxygen [START_REF] Agostinis | Hypericin in Cancer Treatment: More Light on the Way[END_REF], [START_REF] Fox | Photoactivated Hypericin Is an Anti-Proliferative Agent That Induces a High Rate of Apoptotic Death of Normal, Transformed, and Malignant T Lymphocytes: Implications for the Treatment of Cutaneous Lymphoproliferative and Inflammatory Disorders[END_REF].

The standard for photodynamic activity is the 'quantum yield' [Φ  ] of 1 O 2 production. Quantum yield depends on the reaction media, temperature, pH-value and various other physical and chemical influences. Hyp bound to liposomes shows a singlet oxygen quantum yield of: Φ∆ = 0,4 [START_REF] Ehrenberg | Kinetics and Yield of Singlet Oxygen Photosensitized by Hypericin in Organic and Biological Media[END_REF]. Several mechanisms have been proposed for the photodynamic activity of Hyp, but the type II mechanism (singlet OXY generation is a predominant one) [START_REF] Agostinis | Hypericin in Cancer Treatment: More Light on the Way[END_REF], [START_REF] Wynn | Spectroscopic Properties of Hypericin in Solution and at Surfaces[END_REF].

CELL DEATH

Cell death plays a key role during the physiological processes especially during embryogenesis and metamorphosis. The homeostatic control of cell number is the result of the dynamic balance between cell proliferation and cell death. The death of a cell can be defined as an irreversible loss of plasma membrane integrity. In mammalian cells, three types of cell death have been distinguished by morphological criteria. Autophagy is characterized by a massive accumulation of two-membrane autophagic vacuoles in the cytoplasm [START_REF] Klionsky | Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy[END_REF]. Apoptosis is defined by characteristic changes in the nuclear morphology. All of the changes occur before plasma membrane integrity is lost [START_REF] Hengartner | The Biochemistry of Apoptosis[END_REF].

Necrosis is often defined in a negative manner as death lacking the characteristics of the apoptosis and autophagy processes. The distinction between cell death types is important [26][27].

PDT can activate many of the cell signaling pathways in order to activate the cell death. Concentration, physico-chemical properties of pts, concentration of oxygen, wavelength or the cell type can have important influence at the cell survival and extensity of the cell death [START_REF] Robertson | Photodynamic Therapy (Pdt): A Short Review on Cellular Mechanisms and Cancer Research Applications for Pdt[END_REF].

AUTOPHAGIC CELL DEATH

Macroautophagy (also referred to as autophagy) is a homeostatic and evolutionarily conserved dynamic muti-step process that can be modulated at several steps both positively and negatively [START_REF] Klionsky | Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy[END_REF]. Autophagy is a catabolic cellular mechanism that allows the cell to maintain a balance between the synthesis, degradation, and recycling of cellular products [START_REF] Mathew | Role of autophagy in cancer[END_REF]. A variety of autophagic processes exist, all of which involve the lysosomal degradation of the cellular organelles and proteins. The most well-known mechanism proceeds in the following manner: A double membrane structure called autophagosome surrounds the target region, creating a vesicle that separates its contents from the rest of the cytoplasm. This vesicle is then transported and fused to the lysosome, forming a structure called the autophagolysosome, the contents of which are subsequently degraded by lysosomal hydrolases [START_REF] Kroemer | Lysosomes and autophagy in cell death control[END_REF]. Besides facilitating the disposal of unwanted proteins, organelles, and invading microorganisms, autophagy also allows a cell to reallocate its nutrients from unnecessary processes to life-essential ones in times of starvation or stress. Autophagy is important in all cells for the removal of damaged or long-lived proteins and organelles. Although autophagy is a mechanism of tumor suppression, it also confers stress tolerance that enables tumor cells to survive under adverse conditions. Autophagy is induced in tumor cells within hypoxic tumor regions. Stress-induced autophagy in tumor cells can lead to treatment resistance and tumor dormancy, with eventual tumor regrowth and progression. Understanding the role of autophagy in cancer treatment is critical, because many anticancer therapies have been shown to activate autophagy [START_REF] Yang | The Role of Autophagy in Cancer: Therapeutic Implications[END_REF]. Autophagy and apoptosis often occur in the same cell, mostly in a sequence in which autophagy precedes apoptosis [START_REF] Maiuri | Self-Eating and Self-Killing: Crosstalk between Autophagy and Apoptosis[END_REF]. It often happens that stress stimulates an autophagic response, especially if the level of stress is not lethal. Apoptotic and non-apoptotic lethal programmes are activated when stress exceeds a critical duration or an intensity threshold. In many cases, autophagy constitutes a strategy to adapt to and cope with stress [START_REF] Kroemer | Autophagy and the Integrated Stress Response[END_REF]. Recently Hyp became the first PDT paradigm characterized to be capable of inducing the cell death through formation of autophagy. It should be noted, however, that other pts might also be capable of inducing autophagic cell death but their properties need to be thoroughly and more comprehensively tested [START_REF] Garg | ER stress, autophagy and immunogenic cell death in photodynamic therapy-induced anti-cancer immune responses[END_REF]. The complex role of autophagy in cancer continues to emerge, and it is important to elucidate the mechanisms by which autophagy influences tumorigenesis as well as treatment response. Analysis of autophagic signaling may identify novel therapeutic targets for modulation and therapeutic advantage [START_REF] Yang | The Role of Autophagy in Cancer: Therapeutic Implications[END_REF].

APOPTOSIS

Programmed cell death (PCD), also termed apoptosis, is the necessary mechanism complementary to the proliferation that ensures homeostasis of all tissues. It is used to describe the type of cell death that possesses two major morphological features: cell shrinkage and nuclear condensation [START_REF] Kroemer | The Biochemistry of Programmed Cell-Death[END_REF]. It has been estimated that 50 to 70 billion cells perish each day in the average adult because of PCD a process by which, in a year, each individual will produce and eradicate a mass of cells equal to its entire body weight. This process needs to be highly regulated since defects in the apoptotic machinery will lead to extended cell survival and may contribute to neoplastic cell expansion. Extended cell survival also creates a permissive environment for genetic instability and accumulation of mutations. Furthermore, defects in apoptotic pathways confer resistance to chemotherapy, radiation, and immune-mediated cell destruction [START_REF] Bast | Holland-Frei Cancer Medicine[END_REF]. Although understanding of the detailed signaling pathways that trigger apoptosis is incomplete, this process is controlled by a number of complex proteins, which are activated by various triggers and arranged in sequential signaling modules. Apoptosis occurs through two main pathways. The first, referred to as the extrinsic or cytoplasmic pathway, is triggered through the Fas death receptor, a member of the tumor necrosis factor (TNF) receptor superfamily. The second pathway is the intrinsic or mitochondrial pathway which leads to the release of cytochrome c from the mitochondria and activation of the death signal, when stimulated. Both pathways converge to a final common pathway involving the activation of a cascade of proteases called caspases that cleave regulatory and structural molecules, culminating in the death of the cell (Figure 4) [START_REF] Ghobrial | Targeting Apoptosis Pathways in Cancer Therapy[END_REF].

The Extrinsic Pathway: Fas

This pathway comprises several protein members including the death receptors, the membrane-bound Fas ligand, the Fas complexes, the Fas-associated death domain, and caspases 8 and 10, which ultimately activate the rest of the downstream caspases leading to apoptosis. Activation of the extrinsic pathway is initiated with the ligation of cell surface receptors called death receptors. Fas is a member of the tumor necrosis factor receptor superfamily and is also called Apo-1 or CD95. When a death stimulus triggers the pathway, the membrane-bound Fas interacts with the inactive Fas complexes and forms the death-inducing signaling complex. The Fas death-inducing signaling complex contains the adaptor protein Fas-associated death domain protein and caspases 8 and 10 and leads to activation of caspase 8, which in turn can activate the rest of the downstream caspases. In some cells, the activation of caspase 8 may be the only requirement to execute death, while in other cell types, caspase 8 interacts with the intrinsic apoptotic pathway by cleaving Bid (a pro-apoptotic member of the Bcl-2 family), leading to the subsequent release of cytochrome c. Several pathways and proteins regulate the activation of the extrinsic pathway. Dysregulation of these modulators may also lead to malignant transformation, as mutations or deletions of the Fas gene have been found in some hematologic malignancies [START_REF] Ghobrial | Targeting Apoptosis Pathways in Cancer Therapy[END_REF].

The Intrinsic Pathway

One of the most important regulators of this pathway is the Bcl-2 family of

The Final Pathway: Caspases

The final pathway that leads to execution of the death signal is the activation of a series of proteases termed caspases. Not all caspases are involved in apoptosis. The caspases that have been well described are caspases-3, -6, -7, -8, and -9. The intrinsic and extrinsic apoptotic pathways converge to caspase-3, which cleaves the inhibitor of the caspase-activated deoxyribonuclease, and the caspase-activated deoxyribonuclease becomes active leading to nuclear apoptosis. The upstream caspases that converge to caspase-3 are caspases-9 and -8 in the intrinsic and extrinsic pathways, respectively.

The downstream caspases induce cleavage of protein kinases, cytoskeletal proteins, DNA repair proteins, inhibitory subunits of endonucleaes (CIDE family), and finally, destruction of "housekeeping" cellular functions. Caspases also affect cytoskeletal structure, cell cycle regulation, and signaling pathways, ultimately leading to the morphologic manifestations of apoptosis, such as DNA condensation and fragmentation, and membrane blebbing [START_REF] Ghobrial | Targeting Apoptosis Pathways in Cancer Therapy[END_REF].

CROSS TALK BETWEEN AUTPHAGY AND APOPTOSIS

The turnover of organelles and proteins within cells, and of cells within Therefore, the disruption of the relationship between autophagy and apoptosis has important pathophysiological consequences. The stimuli which cause cell death trigger also autophagy. In these cases, autophagy usually manifests well before apoptosis dismantles the cell [START_REF] Marino | Self-Consumption: The Interplay of Autophagy and Apoptosis[END_REF].

If apoptosis is suppressed, autophagy induction is exacerbated. for instance, by removing pro-apoptotic proteins such as Bcl-2-associated X protein (BAX) and Bcl-2 antagonist or killer (BAK) or by adding caspase inhibitors, including those encoded by viruses [39][40]. By induction of autophagy the cell reflects the instinct to adapt to stress. This is followed by the activation of the cell death pathways in response to multiple external signals such as ionizing radiation, chemotherapeutic anticancer agents, the inhibition of growth factor receptors or a scarcity of essential nutrients [40][41]. The fact that many signal transduction pathways that are elicited by cell-intrinsic stress regulate both autophagy and apoptosis might explain the sequential activation of both processes [START_REF] Marino | Self-Consumption: The Interplay of Autophagy and Apoptosis[END_REF].

Although autophagy and apoptosis are under the control of multiple common upstream signals, these processes also cross-regulate each other, mostly in an inhibitory manner. Thus, autophagy reduces the propensity of cells to undergo apoptosis, and activation of the apoptotic programme is coupled to the suppression of autophagy. The mutual relationship between autophagy and apoptosis is highly context-dependent.

Beyond rare cases of autophagy cell death, in which autophagy truly mediates cell death, there are a few examples in which autophagy or elements of the autophagic machinery may favour the activation of apoptotic or necrotic cell death programmes.

However, in the majority of cases, it seems that apoptosis and autophagy are mutually inhibitory. In most instances, autophagy suppresses apoptosis (or at least raises the threshold of stress required to induce it), which means that autophagy tends to be antiapoptotic rather than pro-apoptotic. The mutually negative regulation of autophagy and apoptosis can be expected to increase the contrast between both processes, hence generating a steeper gradient that differentiates nonlethal from lethal stress levels [START_REF] Marino | Self-Consumption: The Interplay of Autophagy and Apoptosis[END_REF].

NECROSIS

Necrosis is signaled by irreversible changes in the nucleus and in the cytoplasm (condensation and intense eosinophilia, loss of structure, and fragmentation). In contrast to apoptosis, necrosis is accompanied by swelling of the entire cytoplasm (oncosis) and of the mitochondrial matrix, which occur shortly before the cell membrane ruptures [START_REF] Kroemer | The Mitochondrial Death/Life Regulator in Apoptosis and Necrosis[END_REF], [START_REF] Majno | Apoptosis, Oncosis, and Necrosis -an Overview of Cell-Death[END_REF].

Until recently, apoptosis was considered as the only form of regulated cell death and for a longtime necrosis generated limited interest within the scientific community. [START_REF] Galluzzi | Molecular mechanisms of regulated necrosis[END_REF]. Conversely, necrosis was viewed as a merely accidental subroutine of cell death, mostly resulting from very harsh stimuli including steep changes in temperature, osmotic pressure or pH [START_REF] Golstein | Cell Death by Necrosis: Towards a Molecular Definition[END_REF]. Nowdays necrosis is no longer considered as a purely accidental, and hence completely uncontrollable, cell death subroutine [START_REF] Kroemer | Autophagic Cell Death: The Story of a Misnomer[END_REF].

The possibility that necrosis (similar to apoptosis) might also occur in a regulated fashion continued to gather momentum through out the 1990s [44][46], and was definitively confirmed in 2005, when the team of Junying Yuan discovered a groups of molecules that inhibit several instances of necrotic cell death, namely, necrostatins [47][48].

Regulated necrosis plays a major role in both physiologal scenarios (e.g., embryonic development) and pathological settings (e.g., ischemic disorders), consistend efforts have been made throughout the last decade toward the characterization of the molecular mechanisms that underline this cell death modality. Nowdays, various types of the regulated necrosis have been characterized, including (but not limited) necroptosis, mitochondrial permeability transition and parthanatos. The inhibition of onlu one of these modules generally exerts limited cytoprotective effects in vivo, underscoring the degree og interconnectivity that characterized regulated necrosis [START_REF] Galluzzi | Molecular mechanisms of regulated necrosis[END_REF].

CELL DEATH PATHWAYS IN PDT OF CANCER

After PDT treatment there is an acute stress response that leads to changes in calcium and lipid metabolism and causes the production of cytokines and stress response mediators in the cells. Enzymes (particularly protein kinases) are activated and transcription factors are expressed. Many of the cellular responses center on mitochondria and frequently lead to induction of apoptosis by the mitochondrial pathway involving caspase activation and release of cytochrome c. Certain specific proteins (such as Bcl-2) are damaged by PDT-induced oxidation thereby increasing apoptosis, and a build-up of oxidized proteins leads to an ER-stress response that may be increased by proteasome inhibition. Autophagy plays a role in either inhibiting or enhancing cell death after PDT [START_REF] Mroz | Cell Death Pathways in Photodynamic Therapy of Cancer[END_REF].

PDT and autophagy

It is still unclear exactly how autophagy affects the outcome of PDT [50][51]. In general, mammalian cells use autophagy as a defense against ROS mediated damage by clearing the cell of damaged organelles [START_REF] Scherz-Shouval | Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4[END_REF]. Depending on the type of ROS and degree of oxidative injury, PDT may stimulate autophagy [START_REF] Sasnauskiene | Apoptosis, autophagy and cell cycle arrest following photodamage to mitochondrial interior[END_REF] that either acts in a cytoprotective manner or induces autophagic cell death [START_REF] Scherz-Shouval | ROS, mitochondria and the regulation of autophagy[END_REF]. Autophagy may play a role in PDT induced apoptosis, but the two processes can also occur independently of one another [START_REF] Kessel | Initiation of autophagy by photodynamic therapy[END_REF]. A study done on murine leukemia L1210 cells found that a wave of autophagy occurs right before apoptosis [START_REF] Kessel | Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage[END_REF]. It was also found that prevention of autophagy by silencing the Agt7 gene allowed photo-killing to occur at lower light doses. This observation is consistent with the theory that autophagy is a defense mechanism against PDT induced ROS [START_REF] Kessel | Apoptosis and autophagy after mitochondrial or endoplasmic reticulum photodamage[END_REF]. In general, the induction of autophagy in PDT treated cells occurs independently of an apoptotic outcome. While autophagy seems to play a pro-survival role in tumor cells that are capable of apoptosis, it has been shown to promote death in cells that are apoptosis-deficient. In order to understand how PDT affects autophagy it is important to take note of the PDT affected proteins that are involved in this mechanism. Many proteins, some directly involved in the autophagic process, are damaged by PDT induced ROS [START_REF] Mroz | Cell Death Pathways in Photodynamic Therapy of Cancer[END_REF]. Although many proteins involved in the autophagic process are photodamaged by PDT, it appears that those involved in the formation of autophagosomes remain active [START_REF] Kessel | Initiation of autophagy by photodynamic therapy[END_REF]. PDT can also affect autophagy by damaging organelles that are directly involved in the process. Several photosensitizers target autophagy-related organelles such as lysosomes and endosomes. In this type of PDT, lysosomal enzymes are inactivated before the membrane ruptures, which allows for specific targeting of this organelle without causing damage to the rest of the cell.

This treatment can be used to selectively enrich autophagosomes [START_REF] Stromhaug | A novel method for the study of autophagy: Destruction of hepatocytic lysosomes, but not autophagosomes, by the photosensitizing porphyrin tetra(4-sulphonatophenyl)porphine[END_REF].

It can be concluded that the effect of PDT on the autophagy of a tumor cell depends on the type of photosensitizer used. Based on studies with various cancer lines and photosensitizers it can be concluded that PDT directly induces autophagy. This is independent of photosensitizer target, as autophagy was observed with photosensitizers that localize in the ER, mitochondria, lysosomes, and endosomes. A second conclusion that can be drawn is that apoptosis often occurs in cells that are already undergoing autophagy and is also a result of PDT. The rates of autophagy and apoptosis depend on the cancer cell type, photosensitizer, and light dosage. Depending on cell type, autophagy will either promote or prevent cell death from PDT. In cells that are able to undergo apoptosis autophagy alleviates the destructive effects of PDT by recycling damaged organelles [START_REF] Apel | Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy[END_REF]. This opens up a possibility that PDT of these cancer cells can be enhanced by suppressing pro-autophagic proteins. Autophagy has the opposite effect on cells that are apoptosis-deficient, promoting cell death through necrosis. The last conclusion that can be made is that autophagic processes are compromised, but not prevented, in PDT protocols that employ lysosome targeting photosensitizers. In these procedures, autophagy is initiated by formation of autophagosomes but cannot be completed by fusing with the lysosome. This is because the proteins involved in autophagosome formation are not photodamaged by PDT [START_REF] Mroz | Cell Death Pathways in Photodynamic Therapy of Cancer[END_REF].

PDT and necrosis

Studying the factors and parameters that cause cellular necrosis after PDT is not as easy as studying those factors which lead to apoptosis. The crucial factors in determining the type of cell death, e.g., apoptosis or necrosis following PDT are: the cell type, the presence of an intact set of apoptosis machinery, the subcellular localization of the pts, the light dose applied to activate it locally, and the oxygen partial pressure [START_REF] Lavie | A photodynamic pathway to apoptosis and necrosis induced by dimethyl tetrahydroxyhelianthrone and hypericin in leukaemic cells: Possible relevance to photodynamic therapy[END_REF]. The high dose PDT (either a high photosensitizer concentration or a high light fluence or both) tends to cause cell death by necrosis, while PDT administered at lower doses tend to predispose cells towards apoptotic cell death [START_REF] Mroz | Cell Death Pathways in Photodynamic Therapy of Cancer[END_REF]. A common feature of the apoptotic program initiated by PDT is the rapid release of mitochondrial cytochrome c into the cytosol followed by activation of the apoptosome and procaspase-3. With pts localized in the plasma membrane the photosensitization process can rapidly switch the balance towards necrotic cell death likely due to loss of plasma membrane integrity and rapid depletion of intracellular ATP [START_REF] Kessel | Sites of photodamage induced by photodynamic therapy with a chlorin e6 triacetoxymethyl ester (CAME)[END_REF]. It is also possible that high doses of PDT can photochemically inactivate essential enzymes and other components of the apoptotic cascade such as caspases [START_REF] Mroz | Cell Death Pathways in Photodynamic Therapy of Cancer[END_REF].

FAMILY OF PROTEIN KINASE C

Protein kinase C also known as PKC is a family of protein kinase enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. PKC was one of the very first protein kinases to be identified [START_REF] Mellor | The Extended Protein Kinase C Superfamily[END_REF].

In humans the PKC family consists of fifteen isozymes [START_REF] Mellor | The Extended Protein Kinase C Superfamily[END_REF]. They are divided into three subfamilies, based on their second messenger requirements: Novel PKC include the δ, ε, η, and θ isoforms, and require DAG, but do not require Ca 2+ for activation. Thus, conventional and novel PKC are activated through the same signal transduction pathway as phospholipase C. On the other hand, atypical PKC (including protein kinase Mζ and ι / λ isoforms) require neither Ca 2+ nor DAG for activation. The term "protein kinase C" usually refers to the entire family of isoforms [START_REF] Nishizuka | Protein Kinases .5. Protein-Kinase-C and Lipid Signaling for Sustained Cellular-Responses[END_REF].

PKC plays a fundamental signaling role in many physiological processes, including modulating membrane structure, mediating the immune response, and regulating the cell proliferation and differentiation via phosphorylation of various transcription factors. Generally, inactive PKC are considered to be primarily cytoplasmic. However, upon activation by different signals, PKC translocate to the plasma membrane, other membrane organelles and to the nucleus. PKC activity has been reported to be increased in gliomas and glioma cell lines as compared with astrocytes, and PKC inhibitors markedly reduced glioma cell proliferation. Moreover, differential expression of specific PKC isoforms has been reported in gliomas and other malignant cells [START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF].

PKC: ANTI-APOPTOTIC PROTEIN

Protein kinase C alpha (PKCα) is a specific member of the protein kinase family. These enzymes are characterized by their ability to add a phosphate group to other proteins, thus changing their function. PKCα (Figure 5) has been widely studied in the tissues of many organisms including drosophila, xenopus, cow, dog, chicken, human, monkey, mouse, pig, and rabbit [START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF]. There is an extensive literature relating to the role of PKC in suppressing or stimulating apoptosis in various cell types [START_REF] Do Carmo | Pkc Signaling in Glioblastoma[END_REF], [START_REF] Whelan | Loss of Protein Kinase C Function Induces an Apoptotic Response[END_REF]. At the mitochondrial level, key players are members of Bcl-2 family proteins [START_REF] Merino | The Bcl-2 family in autoimmune and degenerative disorders[END_REF]. One of the PKCα roles is phosphorylation of Bcl-2 proteins in several cancerous cell lines including U-87 MG.

Members of the family of Bcl-2 proteins are key regulators of apoptosis by acting either as promoters or as suppressors of the cell death process. Recent studies have shown that phosphorylation of Bcl-2 is required for the anti-apoptotic function of Bcl-2 and that PKC activates Bcl-2 phosphorylation [START_REF] Ruvolo | A Functional Role for Mitochondrial Protein Kinase C Alpha in Bcl2 Phosphorylation and Suppression of Apoptosis[END_REF]. Pro-apoptotic proteins of the Bcl-2 family (e.g. Bax, Bak) can translocate from cytosol to mitochondria and participate in outer membrane destabilization. Anti-apoptotic family members (e.g. Bcl-2) protect the integrity of the outer mitochondrial membrane via inhibitory interactions with proapoptotic Bax and Bak [82][83]. In this context, Bcl-2 contains at least three sites that have been identified as phosphorylation sites in a flexible loop region (Thr69, Ser70 and Ser87). Phosphorylation that occurs at Ser70, the PKC phosphorylation site, is required for full anti-apoptotic function of Bcl-2 [START_REF] Ruvolo | A Functional Role for Mitochondrial Protein Kinase C Alpha in Bcl2 Phosphorylation and Suppression of Apoptosis[END_REF] and can affect the ability to associate with Bax [START_REF] Blagosklonny | Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death[END_REF].

Beyond its classical anti-apoptotic role, Bcl-2 is also known to have a critical anti-oxidant like function which has been linked experimentally to the regulation of cellular glutathione (GSH) content [START_REF] Voehringer | BCL-2 and glutathione: Alterations in cellular redox state that regulate apoptosis sensitivity[END_REF]. GSH is an endogenous antioxidant and key player averting mitochondrial oxidative stress and evading apoptosis [START_REF] Franco | Apoptosis and glutathione: beyond an antioxidant[END_REF] Hyp influenced intracellular localization of PKC reflecting its activity, and this influence differed from that observed for PMA [START_REF] Kocanova | Characterization of the Interaction of Hypericin with Protein Kinase C in U-87 Mg Human Glioma Cells[END_REF]. It was also demonstrated that the majority of PKCα present in U-87 MG cells is already in a catalytically competent form phosphorylated at Thr638, and that it is a Bcl-2 kinase [START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF]. In U-87 MG cells, Hyp localizes in subcellular compartments including ER and mitochondria [START_REF] Huntosova | Interaction Dynamics of Hypericin with Low-Density Lipoproteins and U87-Mg Cells[END_REF]. Photoactivated Hyp affects mitochondrial functions [START_REF] Petrovajova | Monitoring of Singlet Oxygen Luminescence and Mitochondrial Autofluorescence after Illumination of Hypericin/Mitochondria Complex: A Time-Resolved Study[END_REF] and induces predominantly apoptosis with the participation of Bcl-2 family proteins [START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF] [133] [START_REF] Buriankova | Synchrotron based Fourier-transform infrared microspectroscopy as sensitive technique for the detection of early apoptosis in U-87 MG cells[END_REF]. dependent manner [START_REF] Sarkar | Reduction of Protein Kinase C Delta Attenuates Tenascin-C Stimulated Glioma Invasion in Three-Dimensional Matrix[END_REF]. Activation of PKCδ following cerebral ischemia led to the release of cytochrome c from the mitochondria via bad pathway [START_REF] Dave | Activation of Protein Kinase C Delta Following Cerebral Ischemia Leads to Release of Cytochrome C from the Mitochondria Via Bad Pathway[END_REF]. PKCδ plays an important role in osteoclast bone resorption function which thereby opens up new avenues for pharmaceutical intervention in pathological osteolytic conditions [START_REF] Khor | Loss of Protein Kinase C-Delta Protects against Lps-Induced Osteolysis Owing to an Intrinsic Defect in Osteoclastic Bone Resorption[END_REF]. It is known that kinase activity is required for the downregulation and degradation of the kinses [START_REF] Lu | Tumor Promotion by Depleting Cells of Protein Kinase C Delta[END_REF]. Furthermore, PKCδ may serve as an attractive therapeutic target in Parkinson-related neurological diseases [START_REF] Anantharam | Blockade of Pkc Delta Proteolytic Activation by Loss of Function Mutants Rescues Mesencephalic Dopaminergic Neurons from Methylcyclopentadienyl Manganese Tricarbonyl (Mmt)-Induced Apoptotic Cell Death[END_REF]. Several kinase inhibitors are now approved for the treatment of cancer, and many more are advancing through clinical trials.

It was shown that in human malignant glioma cells Hyp inhibits the cell growth, motility and cell invasion and inhibits the tyrosine kinase activity, the activity of succinoxidase, and selectively inhibits the capability of PKC [START_REF] Zhang | Enhancement of radiosensitivity in human malignant glioma cells by hypericin in vitro[END_REF].

TARGETED DRUG DELIVERY

A holy grail in cancer therapy is to deliver high doses of drug molecules to tumor sites for maximum treatment efficacy while minimizing side effects to normal organs.

Drug targeting means an increased accumulation of drug in the required area of the body compared to other tissues and organs mediated by a spontaneous or external force or targeting moiety. For the majority of pharmaceuticals currently in use, the specificity and activity of pharmaceuticals towards disease sites is usually based on the drug's ability to interfere with local pathological processes or with defective biological pathways, but not on its selective accumulation in the specific intracellular compartment or in the target cell, organ or tissue. Usually, pharmaceutical agents are distributed within the body rather evenly, proportionally to the regional blood flow [112].

Moreover, to reach the site of action, the drug has to cross many biological barriers, such as other organs, cells and intracellular compartments, where it can be inactivated or express undesirable effects on organs and tissues that are not involved in the pathological process. As a result, to achieve a required therapeutic concentration of a drug in a certain body compartment or certain tissue, one has to administer the drug in large quantities the great part of which, even in the best case scenario, is just wasted in normal tissues. Drug targeting can bring a solution to all these problems. In a very general sense, drug targeting is the ability of the drug to accumulate in the target organ or tissue selectively and quantitatively, independent of the site and method of its administration. Ideally, under such conditions, the local concentration of the agent at the disease site(s) should be high, while its concentration in other nontarget organs and tissues should be below certain minimal levels to prevent any negative side reactions [START_REF] Cross | Pkc-Delta Is an Apoptotic Lamin Kinase[END_REF]. The following advantages of drug targeting are evident: drug administration protocols may be simplified, the drug quantity required to achieve a therapeutic effect may be greatly reduced, as well as the cost of therapy and drug concentration in the required sites can be sharply increased without negative effects on nontarget compartments [START_REF] Torchilin | Passive and Active Drug Targeting: Drug Delivery to Tumors as an Example[END_REF].

To improve the biodistribution of anticancer drugs, nanoparticles have been designed for optimal size and surface characteristics to increase their circulation time in the bloodstream.Various types of nanoparticles are used in biomedical research and drug delivery (Figure 7): Inorganic nanoparticles: typically composed of inorganic compounds such as silica or alumina. However, the nanoparticle core is not limited to just these two materials; rather, metals, metal oxides and metal sulfides can be used to produce a myriad of nanostructures with varying size, shape, and porosity [START_REF] Faraji | Nanoparticles in Cellular Drug Delivery[END_REF].

Polymeric nanoparticles: most are biodegradable, biocompatible, and have been adopted as a preferred method for nanomaterial drug delivery. They also exhibit a good potential for surface modification via chemical transformations, provide excellent pharmacokinetic control, and are suitable for the entrapment and delivery of a wide range of therapeutic agents [START_REF] Faraji | Nanoparticles in Cellular Drug Delivery[END_REF].

Solid lipid nanoparticles: lipid-based submicron colloidal carriers. They were initially designed in the early 1990s as a pharmaceutical alternative to liposomes and emulsions. In general, they are more stable than liposomes in biological systems due to their relatively rigid core consisting of hydrophobic lipids that are solid at room and body temperatures, surrounded by a monolayer of phospholipids. These aggregates are further stabilized by the inclusion of high levels of surfactants. Because of their ease of biodegradation, they are less toxic than polymer or ceramic nanoparticles [START_REF] Faraji | Nanoparticles in Cellular Drug Delivery[END_REF].

Liposomes: spherical vesicles of phospholipid bilayer membrane. They are related to micelles which are generally composed of a monolayer of lipids. The amphiphilic nature of liposomes, their ease of surface modification, and a good biocompatibility profile make them an appealing solution for increasing the circulating half-life of proteins and peptides. They may contain hydrophilic compounds, which remain encapsulated in the aqueous interior, or hydrophobic compounds, which may escape encapsulation through diffusion out of the phospholipid membrane. Liposomes can be designed to adhere to cellular membranes to deliver a drug payload or simply transfer drugs following endocytosis [START_REF] Faraji | Nanoparticles in Cellular Drug Delivery[END_REF].

Nanocrystals: aggregates of molecules that can be combined into a crystalline form of the drug surrounded by a thin coating of surfactant. They have extensive uses in materials research, chemical engineering, and as quantum dots for biological imaging, but less so in nanomedicine for drug delivery [START_REF] Faraji | Nanoparticles in Cellular Drug Delivery[END_REF].

Nanotubes: self-assembling sheets of atoms arranged in tubes. They may be organic or inorganic in composition and can be produced as single-or multi-walled structures. A popular version of a nanotube involves the use of soluble fullerene derivatives, such as C60. Nanotubes have large internal volumes and the external surface can be easily functionalized. While they are potentially promising for pharmaceutical applications, human tolerance of these compounds remains unknown, and toxicity reports are conflicting [START_REF] Faraji | Nanoparticles in Cellular Drug Delivery[END_REF].

Dendrimers: polymer-based macromolecules formed from monomeric or oligomeric units, such that each layer of branching units doubles or triples the number of peripheral groups. Dendrimers generally have a symmetrical structure, with the potential to create an isolated 'active site' core area through chemical functionalization.

Chylomicrons: transport exogenous TAGs and cholesterol from the intestines to the tissues.

Very Low Density Lipoproteins (VLDL): are synthesized in the liver and function to export TAGs to peripheral tissues.

Low Density Lipoproteins (LDL): are the major vehicles to transport cholesteryl esters to peripheral cells.

High density lipoproteins (HDL): transport endogenous cholesterol from the tissues to the liver [START_REF] Voet | Biochemistry[END_REF]. High density lipoprotein (7-13 nm) is a heterogenous class of plasma lipoproteins that have a density between 1,063 g/ml and 1,21 g/ml. HDL is composed of a hydrophobic core of TAG and cholesterol esters covered in a monolayer of phospholipids into which apolipoprotein A-1 (Apo-I) is embedded (Figure 9) [START_REF] Zhang | Nanostructured Lipid Carriers Constituted from High-Density Lipoprotein Components for Delivery of a Lipophilic Cardiovascular Drug[END_REF].

Chylomicrons
ApoA-I, which is comprised of 10 amphipathic alpha helices each with a hydrophobic domain and a negatively charged hydrophobic domain, is the main protein component of HDL and defines the structure and physiology of HDL in vivo.

HDL is a dynamic serum protective against the development of atherosclerosis and resultant illnesses such as heart disease and stroke [START_REF] Thaxton | Templated Spherical High Density Lipoprotein Nanoparticles[END_REF].

HDL molecules transport cholesterol from the tissues to the liver and have essentially the opposite function of LDL: they remove cholesterol from the tissues.

Circulating HDL acquires its cholesterol by extracting it from cell-surface membranes and converts it to cholesteryl esters. HDL therefore functions as a cholesterol scavenger.

The life spans in the body of HDL is much longer (5 to 6 days) than other lipoproteins [START_REF] Forti | High-density lipoproteins: metabolic, clinical, epidemiological and therapeutic intervention aspects[END_REF].

LOW DENSITY LIPOPROTEIN

This spheroidal particle (~ 22 nm in diameter) consists of 1500 cholesteryl ester molecules surrounded by an amphiphilic coat of 800 phospholipid molecules, 500 cholesterol molecules, and a single 4536-residue molecule of apolipoprotein B-100 (ApoB) that has 550kDa (Figure 10) [START_REF] Voet | Biochemistry[END_REF]. The ApoB are highly insoluble in aqueous solutions and, thus, remain with the lipoprotein particle throughout its metabolism [START_REF] Jairam | Pathophysiology of Lipoprotein Oxidation, Lipoproteins-Role in Health and Diseases[END_REF]. There are three structurally different regions in LDL particle: the outer surface layer, the core region and an interfacial region between these two [START_REF] Forti | High-density lipoproteins: metabolic, clinical, epidemiological and therapeutic intervention aspects[END_REF]. Triglyceride and cholesteryl ester molecules are in the core of LDL molecule and the surface monolayer consists of about phospholipid molecules and a single copy of ApoB-100. In addition, the particles contain molecules of unesterified cholesterol, of which about 1/3 is located in the core and 2/3 in the surface. Toward the surface penetrate a few percent of the triglyceride and cholesteryl ester molecules [START_REF] Segrest | Structure of Apolipoprotein B-100 in Low Density Lipoproteins[END_REF].

Cells take up cholesterol through receptor-mediated endocytosis of LDL. Cells obtain exogenous cholesterol mainly through the endocytosis of LDL in complex with LDL receptor (LDLR), a cell-surface transmembrane glycoprotein that specifically binds ApoB-100. Upon binding to the LDLR, LDL moves into the endosome where a drop of pH causes the receptor to dissociate from LDL. The receptor is recycled back to the surface of the cell while the LDL is moved to lysosome where the particle is degraded. [START_REF] Voet | Biochemistry[END_REF].

LDL and HDL for targeted delivery

In the recent years an important attention has been dedicated to the use of lipoproteins for targeted drug delivery [START_REF] Ng | Lipoprotein-Inspired Nanoparticles for Cancer Theranostics[END_REF]. This fact is based on the establishing of a direct relationship between the relative number of lipoprotein receptors in various tumors and the uptake of drugs by malignant cells [START_REF] Damiano | Templated High Density Lipoprotein Nanoparticles as Potential Therapies and for Molecular Delivery[END_REF]. Of the lipoproteins the most important in terms of drug delivery are LDL, however, the importance of HDL is also recognized and in many cases seems to be even higher than LDL [START_REF] Damiano | Templated High Density Lipoprotein Nanoparticles as Potential Therapies and for Molecular Delivery[END_REF], [START_REF] Davis | Nanoparticle Therapeutics: An Emerging Treatment Modality for Cancer[END_REF]. The capacity of both types of the lipoproteins, LDL as well as HDL, to bind some drugs and their functionality as drug carriers have been examined in several studies [126][131]. It has been also shown that mixing of anticancer drugs with LDL or HDL before administration led to an increase of the cytotoxic effects of the drugs in the comparison when the drugs are administered alone [START_REF] Davis | Nanoparticle Therapeutics: An Emerging Treatment Modality for Cancer[END_REF], [START_REF] Jori | The Role of Lipoproteins in the Delivery of Tumor-Targeting Photosensitizers[END_REF].

LDL are recognized by and internalized into the cells through specific membrane receptors that interact with the ApoB of the LDL particle [START_REF] Hevonoja | Structure of Low Density Lipoprotein (Ldl) Particles: Basis for Understanding Molecular Changes in Modified Ldl[END_REF]. Certain tumor cells and tumor vascular endothelial cells express the LDL receptors in higher number due to either their increased proliferation or increased membrane turnover [START_REF] Vitols | Uptake of Low-Density Lipoproteins by Human Leukemic-Cells Invivo -Relation to Plasma-Lipoprotein Levels and Possible Relevance for Selective Chemotherapy[END_REF]. HDL have been implicated in cholesterol delivery in some malignancies including breast cancer, ovarian cancer, adrenocortical tumors and prostate cancer. The mechanism for HDL mediated delivery appears to be SR-B1 receptor dependent [START_REF] Damiano | Templated High Density Lipoprotein Nanoparticles as Potential Therapies and for Molecular Delivery[END_REF].

It is known that this receptor is broadly expressed among a variety of cancer cells. The above mentioned facts make LDL and HDL particles attractive natural occurring vehicles for drug delivery and targeting to cancer tissues.

Both types of the lipoproteins particles assume a globular shape with an average particle diameter 20-25 nm and 7-13 nm for LDL and HDL, respectively. LDL and HDL have important advantages in comparison to other drug nano-delivery systems: i) as natural molecules both lipoproteins escape recognition by mononuclear phagocytic system, which favors their long circulation time in the plasma, ii) they are not immunogenic, iii) their hydrophobic core and phospholipid shell favor binding of hydrophobic and amphiphilic drugs, respectively [START_REF] Zhang | Nanostructured Lipid Carriers Constituted from High-Density Lipoprotein Components for Delivery of a Lipophilic Cardiovascular Drug[END_REF][124] [START_REF] Lacko | High Density Lipoprotein Complexes as Delivery Vehicles for Anticancer Drugs[END_REF]. The importance of the study of the physicochemical properties of complexes of drugs with lipoproteins is confirmed by the fact that the US Food and Drug Administration encouraged the inclusion of lipoprotein-drug interaction studies as part of any investigational new drug application that contains a hydrophobic compound [START_REF] Wasan | Impact of Lipoproteins on the Biological Activity and Disposition of Hydrophobic Drugs: Implications for Drug Discovery[END_REF].

STATE OF ART AND CONTRIBUTION OF OUR GROUP

PDT is for a long time the main research subject in which our groups

(

ABSORPTION SPECTROSCOPY

Absorbance spectroscopy is frequently used to measure concentration. According to the Beer-Lambert law (Eq. ( 1)), measurements of absorbance for molecules of known ε allow calculation of concentration.

Eq. ( 1) ε = molar absorptivity, a constant for a particular species at a particular wavelength, related to the probability of transition, l = length of cell or cuvette, c = molar concentration.

Alternatively, if the concentration of the absorbing species is known, the ε can be determined [START_REF] Brandt | Introduction to Absorbance Spectroscopy[END_REF].

In this study absorbance spectra were recorded by double beam UV-VIS spectrofotometer Shimadzu UV-2401 PC in the range 400-700 nm which schema is presented on Figure 11. The instrument is equipped with monochromators to select both the excitation and emission wavelengths. Both monochromators are motorized to allow automatic scanning of wavelength. The fluorescence is detected with photomultiplier tubes and quantified with the appropriate electronic devices [140].

Fluorescence spectra in this work were recorded at room temperature by SHIMADZU RF-5301 spectrofluorimeter (Kyoto, Japan). The excitation wavelength was 560 nm and the emission was collected in the range 570-700 nm for all measurements unless it is written differently.

FRET

Förster Resonant Energy Transfer (or Fluorescent Resonant Energy Transfer), FRET, is the energy transfer mechanism between two fluorescent dyes through long range dipole-dipole interactions. For efficient FRET to occur there must be a substantial overlap between the donor fluorescence emission spectra and acceptor fluorescence excitation (or absorption) spectra. The donor is excited at its specific excitation wavelength and this excited state energy is transferred non-radiatively to the acceptor dye which becomes excited, while the donor returns to the ground state. The acceptor dye rapidly loses some energy through vibrational and rotational modes, and thus the energy match with the donor is lost, meaning that this energy cannot be returned to the donor. The acceptor dye eventually returns to the ground state, this time through a radiative process whereby a photon will be emitted.

FRET can only happen when the two fluorescent dyes are in close proximity usually less than 10 nm. The probability of energy transfer is strongly dependent on the inter-dye distance (Figure 13). Another factor which plays a role in the efficiency of the FRET as well is the orientation of the molecules electric dipoles relative to each other [START_REF] Mitchelson | Perspectives in Bioanalysis[END_REF]. Förster, demonstrated that the efficiency of the process (E) depends on the inverse sixth-distance between donor and acceptor (Eq. 2).

Eq. (2)

Ro = Förster distance at which half the energy is transferred r = the actual distance between donor and acceptor.

The distance at which energy transfer is 50% efficient is referred to as the Förster radius (Ro). The magnitude of the Ro is dependent on the spectral properties of the donor and the acceptor. Förster distances ranging from 20 to 90 Å are most useful for studies of biological macromolecules. These distances are comparable to the diameters of many proteins, the thickness of biological membranes, and the distances between sites on multisubunit proteins [START_REF] Forster | *Zwischenmolekulare Energiewanderung Und Fluoreszenz[END_REF].

FLUORESCENCE LIFETIME

The fluorescence lifetime is a measure of the time a fluorophore spends in the excited state before returning to the ground state by emitting a photon. The lifetimes of fluorophores can range from picoseconds to nanoseconds [140]. In general, the behavior of an excited population of fluorophores is described by a familiar rate equation:

Eq. ( 3)

were: n* is the number of excited elements at time t, is the rate constant of emission (dimensions of are sec -1 ), f(t) is an arbitrary function of the time, describing the time course of the excitation.

If excitation occurs at t = 0, the equation Eq. (3) takes form:

= -n* Eq. ( 4
)
and describes the decrease in excited molecules at all further times. Integration gives:

Eq. ( 5)

The lifetime  is equal to -1 .

The lifetime and quantum yield for a given fluorophore is often dramatically affected by its environment. Examples of this fact would be NADH, which in water has a lifetime of ~0,4 ns but bound to dehydrogenases can be as long as 9 ns [START_REF] Gonnella | Fluorescence Lifetime Analysis and Effect of Magnesium Ions on Binding of Nadh to Human Aldehyde Dehydrogenase 1[END_REF].

Excited state lifetimes have traditionally been measured using either the impulse response or the harmonic response method. In principle both methods have the same information content. These methods are also referred to as either the "time domain" method or the "frequency domain" method. In the time domain method, the sample is illuminated with a short pulse of light and the intensity of the emission versus time is recorded. As shown in the Figure 14, the fluorescence lifetime t, is equal to the time at which the intensity has decayed to of the original value. The relation for single exponential decay is given:

were: I t is the intensity at time t  is a normalization term (the pre-exponential factor)

 is the lifetime.

It is more common to plot the fluorescence decay data using a logarithmic scale as shown on Figure 15. If the decay is a single exponential and if the lifetime is long compared to the exciting light then the lifetime can be determined directly from the slope of the curve. If the lifetime and the excitation pulse width are comparable, some type of deconvolution method must be used to extract the lifetime. Great effort has been expended on developing mathematical methods to "deconvolve" the effect of the  Eq. ( 6)

exciting pulse shape on the observed fluorescence decay. With the advent of very fast laser pulses these deconvolution procedures became less important for most lifetime determinations, although they are still required whenever the lifetime is of comparable duration to the light pulse.

If the decay is multiexponential, the relation between the intensity and time after excitation is given by:  Eq. ( 7)

In the frequency domain method a continuous light source is utilized, such as a laser or xenon arc, and the intensity of this light source is modulated sinusoidally at high frequency as shown on Figure 16. Typically, an electro-optic device, such as a Pockels cell is used to modulate a continuous light source. Alternatively, LEDs or laser diodes can be directly modulated.

In such a case, the excitation frequency is described by:

Eq. ( 8) It can be demonstrated that:

E(t)
Eq. ( 9
)
This relationship signifies that measurement of the phase delay φ forms the basis of one measurement of the lifetime τ. In particular it can be demonstrated as:

Eq. ( 10
)
The modulations of the excitation (M E ) and the emission (M F ) are given by: M E = (AC Exc /DC Exc) and M F = (AC Em /DC Em )

Eq. [START_REF] Castano | Mechanisms in Photodynamic Therapy: Part One-Photosensitizers, Photochemistry and Cellular Localization[END_REF] where: AC Exc and AC Em are ratio of amplitude oscillation DC Exc and DC Em are offsets of AC Exc and AC Em

The relative modulation M of the emission is then:

Eq. ( 12
)
 can also be determined from M to the relation:

 Eq. ( 13
)
Using the phase shift and relative modulation one can thus determine a phase lifetime (τ P ) and a modulation lifetime (τ M ). If the fluorescence decay is a single exponential, then τ P and τ M will be equal at all modulation frequencies. If, however, the fluorescence decay is multiexponential then  P < τ M and, moreover, the values of both  P and τ M will depend upon the modulation frequency.

Figure 18 presents multifrequency phase and modulation data [START_REF] Jameson | Introduction to Fluorescence[END_REF].

CONFOCAL FLUORESCENCE MICROSCOPY

In recent years, the confocal scanning laser microscope (CSLM) has become widely established as a research instrument. In a conventional light microscope, object to image transformation takes place simultaneously and parallel for all object points. By contrast, the specimen in a confocal CSLM is irradiated in a pointwise fashion and the physical interaction between the laser light and the specimen detail irradiated is measured point by point as well. To obtain information about the entire specimen, it is necessary to guide the laser beam across the specimen, or to move the specimen relative to the laser beam, a process known as scanning. CSLM uses the confocal aperture (usually called pinhole) arranged in a plane conjugate to the intermediate image plane and, thus, to the object plane of the microscope. As a result, the detector (PMT) can only detect light that has passed the pinhole. The pinhole diameter is variable; ideally, it is infinitely small, and thus the detector looks at a point. The laser beam is focused to a diffraction-limited spot which illuminates only a point of the object at a time; the point illuminated and the point observed lie on conjugate planes. This is called confocal ray path (Figure 19). The pinhole more or less obstructs light coming from object points outside the focal plane and excludes it from detection. Object areas thus obstructed are invisible in the image. Therefore, the confocal microscope is an inherently depthdiscriminating optical system. By varying the pinhole diameter, the degree of confocality can be adapted to practical requirements. With the aperture fully open, the image is non-confocal [START_REF] Wilhelm | Confocal Laser Scanning Microscopy-Principles[END_REF]. Emissions were recorded in single-track configuration with a long-pass filter of 560 nm.

Fluorescence signals were analyzed by the Zen 2011 software (Zeiss, Germany).

FLOW CYTOMETRY

By flow cytometry the physical and chemical properties of live cells or other biological particles can be measured as they pass in a fluid single-cell stream through a measuring apparatus. In the most common scenario, one or more lasers interrogate each particle and, at a minimum, the system measures the degree and direction of scattered Flow cytometry measurements were performed using a MACSQuant®Analyzer (Miltenyi, GER). The obtained data were analyzed by MACSQuantify™ Software (Miltenyi, GER).

DYNAMIC LIGHT SCATTERING

Dynamic light scattering (DLS) is an important experimental technique, one of the most popular methods used to determine the size of particles. It measures the fluctuation in the intensity of the scattered light of the sample due to Brownian motion of the particle in the solution. DLS is applicable in range from about 0.001 to several microns, which is difficult to achieve with other techniques. This is because the dimensions are too small for optical spectroscopy and too large for electron microscopy.

A typical set-up for the scattering experiment consist of a laser beam illuminating a sample and a detector set up at scattering angle  measuring the intensity I(,t) of the scattered light. Typically, the incident and scattered beams are shaped by apertures, slits, or by optics such as lenses. Usually, the incident beam is vertically polarized as the detector moves in a horizontal plane and by this is "catching" the strongest signal. The region of the sample which is illuminated by both incident and outgoing beam, and "seen" by the detector is the "scattering volume". From the collected data the diffusion coefficient of the particle in the sample can be determined, and this can be related to the hydrodynamic radius (R H ) of the particle by the Stokes-Einstein equation:

 Eq. ( 14
)
where: k  is the Boltzmann constant, T is the temperature in degrees Kelvin,   is the viscosity of the solution in which particle is diffusing D is diffusion coefficient [START_REF] Arzenšek | Dynamic light scattering and application to proteins in solutions[END_REF].

Dynamic light scattering for size characterizations of the samples was performed by Malvern Instruments Zetasizer Nano-ZS instrument (Malvern, Worcs. UK) equipped with a 4mW He-Ne laser (633 nm) operating at 90°angle.

CRYO-ELECTRON MICROSCOPY

Cryo-electron microscopy (cryo-EM) is a form of transmission electron microscopy where the sample is studied at cryogenic temperature (generally liquid nitrogen temperature). Cryo-EM is developing popularity in structural biology since it allows the observation of specimen that have not been stained or fixed in any way,

showing them in their native environment. In this technique, the samples are embedded in amorphous ice and images of fully hydrated macromolecules can be obtained ("frozen-hydrated"). The vitrification process enables the structure of macromolecules to be studied in a frozen-hydrated form near their native state. The method involves ultra-rapid cooling of small samples preventing ice crystal to form or to grow sufficiently large to damage the specimen's structure. Then, a beam of voltageaccelerated electrons is transmitted in vacuum through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electron transmitted through the specimen; the image is magnified and focused by means of magnetic lenses onto an imaging device, such as a charge-coupled device camera [START_REF] Diaz | Membrane activities of proapoptotic BAK and antiapoptotoic MCL-1[END_REF].

PROTOCOLS

SYNTHESIS OF DEXTRAN DERIVATES WITH CHOLESTEROL MOIETY

Synthesis of cholesterol-modified dextran (D Ch ) is a two step process. In the first step cholesterol 3-hemisuccinyl chloride was synthetized (cholesterol + linker, Figure Cholesterol 3-hemisuccinate (2 g) was dissolved in 50 ml anhydrous chloroform, and an excess of SOCl 2 (10-fold) in 20 ml anhydrous chloroform was added drop wise under nitrogen protecting. The reaction mixture was stirred vigorously at 30 °C for 14 h, then the solution was evaporated under vacuum to remove solvent and the remained SOCl 2 , the residue was used to further steps as a solution in anhydrous chloroform airproofly.

In the second step cholesterol-modified dextran was synthetised (dextran + linker + cholesterol, Figure 21): Dextran (T 1 with MW 1000, 100 mg) and 0,1 ml dried triethylamine was dissolved in 10 ml anhydrous DMSO. After the mixture was heated up to 50°C, different amount of Chol-succ-COCl in 5 ml CHCl 3 was added drop wise.

The reaction was performed for 18 h under argon protecting atmosphere. Gemini 300 NMR spectrometer). The substituted degree determined from 1 H NMR spectrum and defined as percent content of cholesterol per glucopyranosidic units, was controlled by varying the amount of cholesterol 3-hemisuccinyl chloride, and the mole ratio of glucopyranosidic units of dextran to cholesterol are listed in Table 3. Three different substituted degree of D Ch were synthesized.

PREPARATION OF HDL-HYP AND LDL-HYP COMPLEXES

The stock solution of Hyp was prepared by dissolving Hyp in 100% DMSO.

PREPARATION OF [LDL-HYP]/DEXTRAN COMPLEXES

Unmodified dextran T 1 or D Ch were used for preparation of LDL-Hyp complexes with dextran. Dextran powder T 1 was dissolved in PBS and D Ch in 100% DMSO. Such prepared dextrans were carefully (100 µl by 100 µl) added to LDL-Hyp complex and solution was gently mixed. Prepared complex was stabilized for 1 hour at the room temperature.

PREPARATION OF LUV, LUV WITH CHOLESTEROL AND LUV STAINED

WITH RHODAMINE B

Large unilamellar vesicles (LUV) were prepared as followed: stock solution of DOPC with concentration 5x10 -2 M was prepared by dissolving DOPC in chloroform and methanol (1:1m v/v). 100 µl of DOPC was added to the round bottom glass tube and the solution was evaporated under the stream of N 2 forming a thin film of lipids visible at the bottom of the glass tube. 1 ml of PBS was added (making the final concentration of lipids = 5x10 -3 M) to the lipid film, then the solution was vortexed for 1 minute at the highest settings. The emulsion was then extruded using Avanti Polar Lipids extruder with 100 nm filter. The particles were extruded 8-10 times each.

The LUV with cholesterol (LUV-Chol) were prepared the same way as LUV without cholesterol. Three different ratios of LUV-Chol were prepared (8:2, 7:3 and

PRODUCTION OF SYNTHETIC LDL

Egg yolk phosphatidyl choline (PC) = 11,25 mg, triolein (TO) = 8,73 mg, and cholesteryl oleate (CO) = 3,17 mg were combined in a 3:2:1 molar mixture: in chloroform and methanol (1:1, v/v) in the volume of 1ml. The solvent was evaporated under the stream of N 2 . Tris-saline buffer (6 ml, 20 mM, pH 7,2) was used to reconstitute the lipids, and the solution was then vortexed for 1 min on the high setting.

Butylated hydroxytoulene (20 µM) was added to the solution to protect against oxidation during the processing of the particles. The solution was sonificated for 1 hour on ice, under nitrogen, to break up larger lipid complexes. The particles were spun for 20 min at 4000 rpm to remove particulates. The emulsions were then extruded using Avanti Polar Lipids extruder to form smaller particles at 55°C. The particles extruded 4

times each first through a 200 nm, 100 nm filter and then through a 50 nm filter. The final emulsion was filter sterilized using a 0,22 µm filter. Emulsion was combined with the synthetic peptide at 2,15 µMm 10 -3 M). The mixture was lightly vortexed for 1 min.

The solution was allowed to stand at room temperature for 30 min before the start of dialysis to facilitate the binding of peptide to lipid emulsion. Unbound peptide was removed by two days of dialysis against Tris-saline buffer at 4°C with two changes.

Ultracentifugation was utilized to separate lipid particles by density in a Beckman

Ultracentrifuge using a TL100.2 rotor. Samples were adjusted to d = 1,063 g/ml by addition of sodium bromide and spun for 24 hrs at 40000 rpm (4°C) in 1 ml tubes. At the conclusion of the spin, the top 167 µl of sample (d<1,063 g/ml fraction) was collected by aspiration. An additional 167 µl was collected by aspiration and discarded.

Samples were adjusted to d = 1,21 g/ml by addition of 334 µl of sodium bromide solution (d = 1,4744 g/ml) and spun for 48 hrs at 40000 rpm (4°C) in 1 ml tubes. At the conclusion of this spin, the top 167 µl of the sample (d = 1,063 -1,21 g/ml fraction) was collected by aspiration. An additional 167 µl was collected by aspiration and discarded. The remaining infranatant was retained. All three fractions (d<1,063 g/ml, d = 1,064-1,21 g/ml, d>1,21 g/ml) were dialyzed against Tris-saline buffer with two buffer changes.

Protein concentration of the sLDL was determined according to the Modified Lowry protein kit protocol.

Synthesis of synthetic lipid-based nano-particles

sLNP were synthesized similarly to sLDL skipping adding the synthetic peptide and ultracentrifugation process.

sLNP stained with DiO18

The stained sLNP were the same as those previously described for synthesis of sLNP.

CELL CULTURE AND HYP PHOTOACTIVATION

U-87 MG human glioma cells (Cell Lines Services, Germany) were routinely maintained in Dulbecco's modified Eagle medium (D-MEM) containing L-glutamine (862 mg.l -1 ), sodium pyruvate (110 mg.l -1 ), glucose (4500 mg.l -1 ), streptomycin (50 µg.ml -1 ), penicilin (50 µg.ml -1 ) and supplemented with 10% FBS or serum substitute 2% Ultroser G, in the presence of 5% CO 2 humidified atmosphere at 37°C.

Cells were incubated for 1 hour in dark condition in culture medium containing 2% Ultroser G and Hyp. The final concentration of Hyp was either 100 nM or 500 nM.

For all experiments final content of DMSO was less than 0,1%. After incubation with Hyp, medium was removed and normal D-MEM containing 10% FBS was added. Cells were irradiated by monochromatic homemade diode illuminator at 590 nm wavelength and light dose of 4 J.cm -2 . Cellular response was observed 0, 5 and 24 hours after irradiation. Misuth). Control siRNA (FITC Conjugate)-A was used as a negative control to avoid false positive results according to manufacturer's recommendation.

SI

IMMUNOCYTOCHEMISTRY PROTOCOL

Cells (2x10 5 cells) were seeded onto 35 mm culture dishes with integral No.0 glass cover slip bottoms (MatTek, USA). Cells were incubated with 5x10 -6 M CellROX ® Green or Orange Reagent for 15 min, Hoechst 33258 for 30 min at room temperature, 4x10 -7 M MitoTracker® Orange [START_REF] Balogova | Relationship between Bax and Bak Distribution and Apoptosis Onset in U-87 Mg Cells Upon Hyppdt[END_REF] and/or with 0.2x10 -7 M ThiolTracker TM Violet [START_REF] Mandavilli | Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy[END_REF] for 10 min at 37°C. For followed indirect immunofluorescence staining the cells were washed three times with 0.2 % BSA/PBS and fixed with 3.7 % formaldehyde for 15 min. Fixed cells were permeabilized with 0.2 % Triton X -100 for 5 min at room temperature and washed 3 times with 0.

CO-LOCALIZATION ANALYSIS

Co-localization analysis was performed on fluorescence images obtained above using the ZEN 2011 image processing. Mander's coefficients, defined as follow, were used for co-localization analysis:

Eq. ( 15)

Eq. ( 16)

Eq. ( 17)

where R i and G i are the signal intensity of the pixel number "i"obtained for the red and the green channel, respectively. M-values are thus ranging from 0 to 1. Absolute co-localization corresponds to M = 1. The M 1 correlation coefficient, displays how well the red pixels co-localize with some green ones, and the M 2 correlation coefficient displays how well the green pixels co-localize with some red ones. Overlap coefficient does not depend on the relative strengths of each channel, but can be affected by the background signal [START_REF] Huntosova | Cell Death Response of U87 Glioma Cells on Hypericin Photoactivation Is Mediated by Dynamics of Hypericin Subcellular Distribution and Its Aggregation in Cellular Organelles[END_REF]. The attempt for preparing the complexes without using DMSO was not successful resulting in irreproducible results.

PREPARATION OF COMPLEXES WITH DMSO

Because the attempt to exclude DMSO from the preparation process was not successful DMSO was used for solubilization of Hyp and D Ch . The amount of DMSO in all experiments was not higher than 0,5%. Two different ways of preparation were tested:

First protocol:

[LDL-Hyp] was incubated overnight. The next day, D Ch was added for 1 hour and fluorescence intensity was measured. Then the complex was dialyzed for ~ 17 hours at 4°C and fluorescence intensity was measured again and compared with the intensity measured before dialysis.

Figure 26 shows the comparison of fluorescence intensity of Hyp in complex [LDL-Hyp]/D Ch before and after dialysis. The difference of intensity is due to the fact that during 17 hour dialysis Hyp went from LDL through the dialysis membrane to the PBS against which the complex was dialyzed. There is equilibrium between Hyp in LDL and Hyp in PBS. We came to conclusion that dialysis with Hyp cannot be performed when the complex was prepared. incubation. The exact method is described in the Chapter 2.9.3.

For future experiments we suggest that complexes should be prepared with ~ 17 hours dialysis of [LDL-D Ch ] followed by exact determinination of LDL concentration by protein kit. Appropriate amount of Hyp could then be added to the complex.

EXAMINATION OF THREE SYNTHESIZED DEXTRANS

As mentioned in Chapter 2.9.

Efficiency of covering [%] ratio [LDL-dextran] D

CHARACTERIZATION OF THE INTERACTION OF LDL-HYP WITH DCH 3

We already have known that with the increasing concentration of Hyp inside LDL Hyp starts to form aggregates at the ratio ~ 50 molecules of Hyp per one molecule of LDL [START_REF] Kascakova | Fluorescence Spectroscopic Study of Hypericin-Photosensitized Oxidation of Low-Density Lipoproteins[END_REF]. But how does Hyp behave in the presence of D Ch 3? Figure 28 FRET [START_REF] Huntosova | Interaction Dynamics of Hypericin with Low-Density Lipoproteins and U87-Mg Cells[END_REF]) and the other 50% of the intensity is characterized by a lifetime of about 3,9 to 4 nsec (it corresponds to some Hyp molecules that are partially quenched by homo-FRET (FRET from monomer toward aggregates). Anyhow, the situation is the same in both samples.

In another experiment, interaction of Hyp in the presence of D Ch 3 was monitored (Figure 31). The presence of D Ch 3 has no impact on the behavior of Hyp inside LDL and slightly reduces the size of LDL.

DETERMINATION OF THE BEST RATIO BETWEEN [LDL-HYP] COMPLEX AND DCH 3

In order to find the best ratio that would decrease the redistribution process of As mentioned in Chapter 1.7.2, LDL particles are recognized by cells through the LDL-receptors on the surface of the cells. This receptor-mediated endocytosis is a general mechanism by which cells take up LDL particles [START_REF] Jairam | Pathophysiology of Lipoprotein Oxidation, Lipoproteins-Role in Health and Diseases[END_REF]. It was already shown that presence of D Ch 3 decreases/minimizes the redistribution process of Hyp to free LDL particles (Figure 33). However, the modification of the LDL surface could possibly modify and inhibit the recognition of LDL particles through LDLR. In order to find out if LDL molecules covered by D Ch can enter glioma cells the monitoring of cellular uptake of Hyp by U87-MG cells was carried out. Figure 34 For 37°C the process of uptake of LDL by endocytosis is almost identical when D Ch is or is not present. As control uptake of Hyp at 4°C is presented when the endocytosis process is almost completely blocked for both samples (uncoated and D Ch coated). Thus, D Ch coating of LDL does not preclude the binding of ApoB moiety to the LDL specific receptors for cells.

In order to further study the interaction of [LDL-Hyp]/D Ch 3 transport system with U87-MG cells, fluorescence imaging microscopy was made. [LDL-Hyp] complex with and without D Ch 3 was added to the 10% FBS medium containing U-87 MG cells and was incubated for 1 hour. U-87 MG cells were first grown in 2% UG medium which does not contain any lipids. In this medium the proliferation of the LDLR in cells was increased [START_REF] Kascakova | High Level of Low-Density Lipoprotein Receptors Enhance Hypericin Uptake by U-87 Mg Cells in the Presence of Ldl[END_REF]. It is known that upon administration into the bloodstream Hyp associates with serum proteins [START_REF] Chen | Efficacy of Antitumoral Photodynamic Therapy with Hypericin: Relationship between Biodistribution and Photodynamic Effects in the Rif-1 Mouse Tumor Model[END_REF]. 10% FBS medium contains lipids and therefore after adding Hyp in LDL that was or was not covered by D Ch 3 the redistribution process occurred.

[LDL-Hyp] [LDL-Hyp]/D Ch 3

S SY YN NT TH HE ET TI IC C L LD DL L 3.5 SYNTHESIS OF SYNTHETIC LDL/SLNP

As it was already described in Chapter 1.7.2.1, LDL are ideal for the delivery of cancer drug and imaging agents since they are able to circulate in the blood stream for a significant amount of time, the hydrophobic core facilitates the incorporation of poorly soluble drugs or imaging agents, and they are highly amenable to bioconjugation [START_REF] Ng | Lipoprotein-Inspired Nanoparticles for Cancer Theranostics[END_REF].

Despite all of this advantages of LDL as carrier for hydrophobic drugs, it is less than ideal as targeting agent since it is difficult to isolate LDL in large quantities. Also, each LDL is variable in composition and size. Another approach used reconstituted LDL consisting of a lipid emulsion containing drugs stabilized by purified ApoB [START_REF] Song | Naphthalocyanine-Reconstituted Ldl Nanoparticles for in Vivo Cancer Imaging and Treatment[END_REF]. The ApoB protein, however, is difficult to isolate due to its large size and propensity to aggregate and is therefore not useful for generation of large batches of reconstituted LDL.

A part of my study was to construct synthetic nano-particles with the most possible similarity in composition and properties to natural LDL. We decided to prepare synthetic LDL (sLDL) according to the way Nikanjam et al. have prepared [START_REF] Nikanjam | Synthetic Nano-Low Density Lipoprotein as Targeted Drug Delivery Vehicle for Glioblastoma Multiforme[END_REF]. sLDL particles were constructed by combining a synthetic peptide containing a lipid binding motif and the LDL receptor binding domain of ApoB-100 with a lipid emulsion of phosphatidyl choline, triolein and cholesteryl oleate in a molar ratio 3:2:1. The exact preparation is described in the Chapter 2.9.5. Modified Lowry kit was used for defining the peptide concentration of prepared sLDL particles. In general, the concentration was ~ 10 -4 M. The size of sLDL was determined by DLS to be 89 nm (Figure 37). 6. The ratio 1:1 decreased the size of sLDL from 89 nm to 68,43 nm.

[sLDL-D Ch 3] ratio size [nm]

1:1 After studying the interaction of D Ch 3 with sLDL we wanted to define the steady state curve of Hyp in sLDL. We supposed that Hyp would behave inside sLDL similarly as it does in LDL (Figure 28)-when in excess of sLDL, Hyp would be in monomer form and with increasing concentration of Hyp it would start to form aggregates and the fluorescence intensity would slowly decrease. Different amount of Hyp was mixed with sLDL and the fluorescence intensity was measured (Figure 38). We found out that even after 90 min of incubation of Hyp with sLDL in ratios from [sLDL-Hyp] = 1:1 to 1:1000, the fluorescence intensity did not significantly change. For comparison fluorescence intensity of Hyp in LDL in ratio [LDL-Hyp] = 1:1 and 1:30 was measured as well. We came to the conclusion that synthetic peptide which was used for the synthesis of sLDL and was responsible for forming the LDLR binding domain of ApoB-100 caused insolubility of Hyp inside sLDL so when Hyp was mixed with sLDL, Hyp did not enter sLDL and stayed in the solution.

We decided to eliminate synthetic peptide from the protocol of synthesis of sLDL. We are aware of the fact that sLDL particles would not enter cells through the receptor mediated pathway. This fact is not important because in the future plans the Department of Biophysics in Koisce is going to work with DARPins-designed ankyrin repeat proteins which are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. These DARPins will be attaches to the surface of sLDL and this way much higher targeting of the nanoparticles which will be loaded with photosensitizer to the desired cancer cells will be reached. For now, we assume that sLDL without the LDLR binding domain of ApoB-100 will enter cells through the endocytosis process. After several different tries of preparing sLDL where we compared the size and characterized the interaction of such prepared particles with Hyp, we came to the conclusion that ultracentrifugation is not necessary for the synthesis either and we skipped this step at the preparation of sLDL. Because there is a difference in the preparation of sLDL according to Nikanjam The size our sLNP was measured by DLS (Figure 39) and cryo-electron microscopy (Figure 40) and was the same as size of sLDL which were prepared according to the protocol by Nikanjam et al. It was determined to be 89 nm.

CHARACTERIZATION OF INTERACTION OF SLNP PARTICLES

WITH HYP

We were not able to define the ratio between sLNP and Hyp or sLNP and D Ch 3 anymore since we eliminated synthetic peptide and were not able to determine protein concentration of sLNP. We wanted to know the cholesterol content in sLNP. It was defined through cholesterol kit and was generally ~ 0,58 µg/µl.

To confirm the theory that synthetic peptide was responsible for Hyp insolubility in the presence of sLDL particles, the interaction of Hyp into sLNP was studied.

Different concentrations of Hyp were mixed with sLNP and incorporation of Hyp into sLNP was measured in time (Figure 41). As it can be seen on this figure, Hyp is able to incorporate to sLNP.

B [sLDL-Hyp] complex can not be obtained either because of the unknown problem

in the preparation of sLDL or because the synthetic peptide preclude Hyp incorporation in sLDL. This result supports our suggestion that because of synthetic peptide which was present in sLNP, Hyp did not incorporate to sLDL and interacted with these particles very poorly. The fluorescence intensity of different concentration of Hyp after 150 min incubation with sLNP was taken from incorporation graph (Figure 41) and steady state curve was made (Figure 42).

REDISTRIBUTION OF HYP FROM [SLNP -HYP]/DCH 3 TOWARDS FREE NATURAL LDL

To know if D Ch 3 has similar effect of decreasing/minimalizing of the redistribution of Hyp towards free lipid particles as it does when Hyp is loaded in LDL particles, redistribution of Hyp from sLNP covered by D Ch 3 to free natural LDL was studied.

sLNP were synthesized, incubated with Hyp and covered by D Ch 3. The fluorescence intensity was measured after adding free LDL particles (Figure 43). Three different concentrations of Hyp in sLNP were measured (10 -7 M, 5x10 -7 M and 10 -6 M)

with one concentration of D Ch 3 (1.5x10 -7 M).

Hyp is able to incorporate into sLNP particles and the steady state curve of Hyp

in the presence of sLNP resembles the steady state curve in the presence of natural LDL. From the redistribution process on Figure 43, covering of sLNP by D Ch 3 was not as effective as it was in the case when Hyp was in LDL. At the concentration of

[Hyp] = 5x10 -7 M and [D Ch 3] = 1.5x10 -7 M the redistribution was 3,65% decreased. It is hard to find the appropriate ratio so D Ch 3 would be as effective as it is with LDL since we were not able to determine the exact ration between sLNP, Hyp and D Ch 3. In the future measurement it is necessary to find the concentration of D Ch 3 which will be as effective as ratio of [LDL-Hyp]/ D Ch 3 = 1:50:75 is.

The first attempts of decreasing/minimalizing of the Hyp redistribution proces towards free LDL molecules by covering the surface of sLNP with D Ch 3 were performed. Yet, the right concentration of D Ch 3 must be determinted in the future experiments.

INTERACTION OF SLNP WITH CELLS

sLNP were stained with DiOC 18 (3) and were incubated with U-87 MG cells to know if they are able to vectorize such hydrophobic compound to glioma cells without LDLR binding domain of ApoB-100. Figure 44 shows that even though there is no Apo-B in sLNP, DiOC 18 (3) is taken up by cells probably through endocytosis process.

sLNP can be used as transport vesicles form Hyp until above mentioned DARPins will be attached to the surface.

S ST TU UD DY Y W WI IT TH H C CH HO OL LE ES ST TE ER RO OL L

Cholesterol is one of the most essential components of natural membranes which can influence the function and structure properties of the membrane as well as the interaction of drugs. The cholesterol molecule (Figure 45) is made up of three groups that have all proven to be essential for their effect on membranes: the fused rigid steroid rings, the hydroxyl group attached to one of the rings and the short flexible hydrocarbon chain [START_REF] Ho | Lipid-Mediated Preferential Localization of Hypericin in Lipid Membranes[END_REF].

INFLUENCE OF CHOLESTEROL ON HYP

In order to investigate how Hyp behaves in the presence of 20% cholesterol ovelap between Rhod B fluorescence emission spectra and Hyp fluorescence excitation (or absorption) spectra was used for FRET between Hyp and (Rhod B). Firstly LUV without cholesterol and with 20% cholesterol content were prepared and incubated with Hyp overnight. Subsequently, LUV-Rhod B which preparation is described in the Chapter 2.9.4was added to the solution and fluorescence intensity of Hyp and Rhod B was measured Figure 46. The interaction of Hyp with model membranes in the absence or in the presence of cholesterol was studied. Uptakes of Hyp monomer within LUV were measured by fluorescence intensity. Large unilamellar vesicles (LUV) containing 0%, 20%, 30% and 50% of cholesterol were prepared. The exact protocol of preparation is described in Chapter 2.9.4.

As previously shown (Figure 28), high accumulation of Hyp in vesicles leads to the formation of non fluorescent aggregates. We wanted to find out how would the fluorescence intensity of Hyp differ in the presence of 20%, 30% and 50% cholesterol.

S ST TU UD DY Y O OF F T TH HE E I IN NT TE ER RA AC CT TI IO ON N O OF F H HD DL L W WI IT TH H H HY YP P

As it was mentioned in Chapter 1.7.2.1 in the recent years the use of lipoproetins as transport veicles for drug delivery has been widely studied [123][126]. We have already studied the behavior of Hyp in the presence of LDL [START_REF] Buriankova | Kinetics of Hypericin Association with Low-Density Lipoproteins[END_REF][131] [START_REF] Huntosova | Interaction Dynamics of Hypericin with Low-Density Lipoproteins and U87-Mg Cells[END_REF]. In this part of the study the emphasis is given on the kinetics of Hyp incorporation into free HDL molecules and the redistribution of Hyp from the complex HDL-Hyp=70:1 to free lipoprotein particles. Similarly to our study of LDL-Hyp complexes [START_REF] Buriankova | Kinetics of Hypericin Association with Low-Density Lipoproteins[END_REF][131], we have utilized the fluorescence spectroscopy approach and the fact that molecules of Hyp form non-fluorescent aggregates in aqueous solutions and the Hyp aggregates can be formed also in lipoprotein particles at high local Hyp concentrations [131][155]. The differences between characteristics of Hyp association with LDL and HDL are also discussed.

INCORPORATION OF HYP INTO HDL

The fluorescence emission spectra of Hyp in the presence of HDL resemble those in the presence of LDL (not shown). The spectral maximum is positioned at 599 nm [START_REF] Kascakova | Fluorescence Spectroscopic Study of Hypericin-Photosensitized Oxidation of Low-Density Lipoproteins[END_REF] and the intensities are also almost identical in the presence of both types of the lipoproteins (at the same Hyp concentrations and molar ratio Hyp/lipoprotein=1:1). The dependence of Hyp fluorescence intensity at its maximum on the molar ratio HDL-Hyp, 300 minutes after mixing of Hyp with HDL solutions (HDL concentration in the mixtures was 10 nM), is presented in Figure 48. nm) 2 . It suggests that the molecules of monomeric Hyp are most probably distributed on the "like sphere surface", likely in phospholipid-cholesterol outer layer of LDL and HDL molecules. Further increase of Hyp concentration (above the ratio 1:8 and 1:30 for HDL-Hyp and LDL-Hyp) leads to a Hyp clustering in lipid media which has been previously proposed [START_REF] Kascakova | Fluorescence Spectroscopic Study of Hypericin-Photosensitized Oxidation of Low-Density Lipoproteins[END_REF][156] and also theoretically supported [START_REF] Jambeck | Molecular Dynamics Studies of Liposomes as Carriers for Photosensitizing Drugs: Development, Validation, and Simulations with a Coarse-Grained Model[END_REF]. This clustering is mostly due to π-stacking interaction between aromatic cores of Hyp molecules. It was proposed by several groups that Hyp is mainly localized close to the polar headgroups of the outer layer of lipid structure (liposomes, biological membranes) [START_REF] Ho | Lipid-Mediated Preferential Localization of Hypericin in Lipid Membranes[END_REF][157] [START_REF] Strejckova | Spatial Orientation and Electric-Field-Driven Transport of Hypericin inside of Bilayer Lipid Membranes[END_REF].

This is very probably also the case for the localization of Hyp inside LDL and HDL molecules, however the presence of cholesterol in outer shell of the lipoproteins molecules can move Hyp a more deeply inside the LDL or HDL particles as it was proposed for Hyp localization in cholesterol enriched biological membranes [START_REF] Ho | Lipid-Mediated Preferential Localization of Hypericin in Lipid Membranes[END_REF].

THE KINETICS OF HYP INTERACTION WITH HDL PARTICLES

The The fluorescence intensity of Hyp in HDL-Hyp complexes (until molar ratio 1:8) increases and reaches saturation 10 minutes after mixing. For the complexes with higher HDL-Hyp ratios an increase of Hyp fluorescence observed immediately after mixing is followed by its decrease. This decrease is caused, as it was already mentioned, by the creation of Hyp aggregates and self-quenching of Hyp fluorescence inside HDL particles in situation when continuous flow of Hyp molecules into HDL particles leads to a number of Hyp molecules per one HDL particle above 8. For the HDL-Hyp = 1:1 and 1:2 complexes a major part of Hyp molecules is incorporated into HDL in the slow phase (Table 7). On the other hand, for the complexes with the molar ratios 1:6 and 1:10, the major part of Hyp (about 70%) is incorporated into HDL in the first, rapid, phase (Figure 49,Table 7). This behavior is slightly different than the properties of LDL-Hyp complex, where it was observed that most of Hyp molecules (about 65%) are associated with LDL in the slow phase of the incorporation process until concentration ratio 1:30 [START_REF] Buriankova | Kinetics of Hypericin Association with Low-Density Lipoproteins[END_REF]. For the determination of the half-times of Hyp incorporation into HDL (until ratio HDL-Hyp =1:8) the experimental data were fitted by mono-exponential function. However, we are aware that the process of Hyp incorporation into lipoproteins is complicated and mono-exponential fitting is only a rough approximation of the real situation. The obtained half-times for the internalization of Hyp molecules into HDL are summarized in Table 7. The comparison of these values with those for LDL-Hyp complex reveals that the process of internalization of Hyp molecules into lipoproteins is more rapid in the presence of HDL than LDL. The half-times for Hyp incorporation into HDL is 2-7 min depending on the final HDL-Hyp ratio, meanwhile the half-times for Hyp association with LDL is 14-26 min [START_REF] Buriankova | Kinetics of Hypericin Association with Low-Density Lipoproteins[END_REF]. The reason for this observation should be in a relative higher containing of cholesterol molecules in LDL (11%) in comparison with HDL (7%) [START_REF] Jonas | Lipoprotein structure[END_REF]. There are similar properties of the kinetics of Hyp incorporation into HDL and LDL molecules, however, incorporation of Hyp into HDL is more rapid than to LDL also the relative contribution of the fast phase vs slow phase of Hyp accumulation into HDL particles is higher than into LDL. The concentration of cholesterol molecules in outer shell of the lipoprotein particles is probably the determining factor for the kinetics of Hyp incorporation into lipoproteins.

The major part of Hyp molecules is incorporated into HDL in the slow phase for ratios HDL-Hyp = 1:1 and 1:2. In the ratios 1:6 and 1:10 about 70% of Hyp is incorporated to HDL in the rapid phase.

The appropriate amounts of HDL were added to the stabilized complex HDL-Hyp =1:70 (stabilization for 2 hours at the room temperature) and the evolution of Hyp fluorescence intensity at its maximum (599 nm) was registered. After adding free HDL to the HDL-Hyp =1:70 complex, the increase of Hyp fluorescence for all HDL-Hyp final ratios was observed. The observed redistribution has two phases. First one is represented by the increase of Hyp fluorescence immediately (5-10 sec) after mixing of HDL and HDL-Hyp =1:70 solutions and the second is expressed by the further gradual fluorescence increase (Figure 50). When compared to the redistribution of Hyp from LDL-Hyp to free LDL [START_REF] Buriankova | Kinetics of Hypericin Association with Low-Density Lipoproteins[END_REF] Hyp translocates from the HDL-Hyp complex towards free HDL faster than it does from LDL-Hyp complexes to free LDL. Lower rates for the Hyp translocation from the complex with LDL to free LDL molecules in comparison with those observed in the presence of HDL again suggests the role of cholesterol in incorporation of Hyp to and releasing from lipoprotein particles. The mechanism of redistribution of Hyp molecules from HDL/LDL-Hyp complex to free HDL/LDL particles is not resolved yet. We think that both direct Hyp translocation from saturated HDL-Hyp complex to free HDL molecules as well as translocation through water phase are possible in this redistribution process. The partition coefficient of Hyp in octanol/water mixture has been determined to be log P=3,43 [START_REF] Jurgenliemk | Dissolution, Solubility and Cooperativity of Phenolic Compounds from Hypericum Perforatum L. In Aqueous Systems[END_REF]. This value clearly suggests a strong preference of Hyp to be incorporated in lipid structures in comparison with aqueous phase. As it was previously mentioned, Hyp aggregates formed at high HDL-Hyp ratios can be sticked to the HDL/LDL surface. Under collisions of HDL-Hyp complex with free HDL particles, a part of Hyp molecules can be transferred directly to these free particles. However, certain part of Hyp molecules exists as aggregates in aqueous phase. From this phase, molecules of Hyp can be also incorporated into free HDL molecules. More thorough studies are necessary to be performed to definitely resolve this problem. The role of anti-apoptotic PKCα in response to Hyp photodynamic action in U-87 MG cells has been previously investigated [START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF]. The purpose of study in this part of the thesis was to focus closer on the effect of pkcα gene silencing in U-87 MG cell line after Hyp photo-activation and to study the impact and consequence of such silencing on cell survival and cell death.

When compared to the redistribution of

TRANSFECTION EFFICIENCY

Control siRNA (FITC Conjugate)-A was used to determine the transfection efficiency. This control contains a scrambled sequence that does not lead to a specific degradation of any known cellular RNA and represents a negative control to avoid false positive results.

PKC GENE SILENCING

To silence pkcgene, U-87 MG cells were treated according to siRNA transfection protocol (Chapter 2.9.7). Confocal fluorescence microscopy along with western blot analysis was used to confirm gene silencing.

CELL SURVIVAL AFTER HYP PHOTO-ACTIVATION

It was already shown that cells undergo apoptosis after Hyp photo-activation [75][163]. Thus, taking into account that PKC is an anti-apoptotic protein [START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF], we can expect that silencing of this protein by using of siRNA would have an effect on Hyp photo-induced cell death. Our predictions were that the level of apoptosis could occur Hyp, (2) 5x10 -7 Hyp, (3) DNA Ladder, transfected PKCα -cells treated with (4) 1x10 -7 Hyp, (5) 5x10 -7 Hyp.

DETECTION OF APOPTOSIS/NECROSIS AFTER HYP PHOTO-

ACTIVATION

Because of cell viability tests showed that decreased amount of PKCα did not have expected influence on the cell survival, the effect of pkc gene silencing on the type of cell death was further investigated by flow cytometry (Figure 54).

A significant difference between transfected PKCα -cells was detected when compared with non-transfected cells after Hyp photo-activation. Our results show that cells with blocked pkcα gene undergo necrosis prior to apoptosis which was present in the non-transfected cells and in the next steps we were trying to find out the reason.

This decrease of apoptosis and increase of necrosis was slightly evident right after photo-activation of Hyp and was more pronounced for 24 hours post-irradiation time (Figure 54). pkcα gene silencing does not affect the cell survival after Hyp photo-activation.

REACTIVE OXYGEN SPECIES PRODUCTION

In order to better understand how PKCα silencing can lead to such modification of the cell death pathway, intracellular ROS formation was monitored by confocal fluorescence microscopy and flow cytometry analysis (Figure 55). As it was already shown, the level of ROS formation plays an important homeostatic role regulating signal transduction involved in proliferation and cell survival [START_REF] Finkel | Signal Transduction by Reactive Oxygen Species[END_REF]. Moreover, when ROS formation is deregulated and surpasses antioxidant defenses, oxidative stress takes place, and when oxidative stress exceeds the capacity of the cell to repair biomolecule oxidation (nucleic acids, lipids and proteins), irreversible oxidative damage occurs. Thus, predominance of necrosis prior to apotosis in transfected cells can be considered to be related to such ROS increase.

BCL-2 PHOSPHORYLATION ON SERINE70 (PBCL-2(SER70))

Taking into account that members of the Bcl-2 family of proteins are important regulators of programmed cell death pathways with individual members that can suppress (e.g. Bcl-2, Bcl-XL) or promote (e.g. Bax, Bad) apoptosis [START_REF] Ruvolo | Phosphorylation of Bcl2 and Regulation of Apoptosis[END_REF], flow cytometry experiments focused on the role of Bcl-2 protein in the cell death were also performed. In this view, phosphorylation of Bcl-2 (pBcl-2) was assessed since such modification is needed in order to protect cells from apoptosis [START_REF] Ruvolo | Phosphorylation of Bcl2 and Regulation of Apoptosis[END_REF]. The results are Recent studies have shown that agonist-activated phosphorylation of Bcl-2 at Ser70 (single site phosphorylation), a site within the flexible loop domain is required for Bcl-2's full and potent anti-apoptotic function [START_REF] Ruvolo | Phosphorylation of Bcl2 and Regulation of Apoptosis[END_REF]. Thus observed decrease of Bcl-2 phosphorylation after siRNA gene silencing and Hyp photo-activation can explain decreased amount of apoptotic cells. However, question why we observed such important increase of ROS production and subsequent necrosis in transfected PKCα - cells after Hyp photo-activation remains to be answered.

FLUORESCENCE IMAGING OF PBCL-2(SER70),

MITOCHONDRIA AND GSH

Our hypothesis is that the increased oxidative stress generated during photodynamic action of Hyp in transfected cells as well as during cell death development could be a key factor for the observed shift from apoptosis to necrosis. It was reported previously that mitochondria, ROS, gluthation (GSH) and Bcl-2 are closely related things [START_REF] Esteve | Oxidative Damage to Mitochondrial Dna and Glutathione Oxidation in Apoptosis: Studies in Vivo and in Vitro[END_REF]. Indeed, GSH is considered as a key endogenous anti-oxidant to combat ROS produced by the electron transport chain in mitochondria. The role of Bcl-2 in GSH transport into mitochondria is well described in the work of Wilkins et al. [START_REF] Wilkins | Bcl-2 Is a Novel Interacting Partner for the 2-Oxoglutarate Carrier and a Key Regulator of Mitochondrial Glutathione[END_REF].

We previously reported changes in the mitochondrial NAD(P)H content and possible alteration in the mitochondrial metabolic state after Hyp photo-activation. Observed rise pkcα gene silencing decreases the level of phosphorylation of Bcl-2.

of mitochondrial NAD(P)H fluorescence could be explained with relation to enzymatic elimination of mitochondrial oxidative stress with participation of GSH [START_REF] Benderdour | Decreased Cardiac Mitochondrial Nadp(+)-Isocitrate Dehydrogenase Activity and Expression: A Marker of Oxidative Stress in Hypertrophy Development[END_REF].

It was shown that Bcl-2 is an anti-oxidant like protein [START_REF] Hockenbery | Bcl-2 Functions in an Antioxidant Pathway to Prevent Apoptosis[END_REF][171] which over expression leads to an increase of cellular contents of GSH [START_REF] Voehringer | Redox Aspects of Bcl-2 Function[END_REF]. In contrast, Bcl-2 knockout mice had reduced level of GSH in brain tissue and were more sensitive to neuronal cell death induced by mitochondrial oxidative stress [START_REF] Hochman | Enhanced Oxidative Stress and Altered Antioxidants in Brains of Bcl-2-Deficient Mice[END_REF]. In the context of anti-oxidative protection of mitochondria against oxidative stress we decided to investigate a co-localization of GSH with Bcl-2 and mitochondria. Fluorescence imaging microscopy has been used to assess relative co-localization between mentioned parameters despite the fact that Bcl-2 is localized in outer mitochondrial membrane and GSH within the mitochondrial matrix where its anti-oxidative functions are linked to other enzymatic systems of the mitochondria [START_REF] Benderdour | Decreased Cardiac Mitochondrial Nadp(+)-Isocitrate Dehydrogenase Activity and Expression: A Marker of Oxidative Stress in Hypertrophy Development[END_REF]. siRNA PKCδ(h) specifically blocks the pkcδ gene expression without having any impact on the other isoforms of PKC protein family.

CELL SURVIVAL AFTER PKC GENE SILENCING AND HYP PHOTO-ACTIVATION

The examining of the effect of pkcδ gene silencing on the cell survival after Hyp photo-activation was tested through MTT colorimetric assay (Figure 59). The obtained results were slightly different to those obtained for pkcα gene silencing. MTT tests were assessed with two concentrations of Hyp where, as expected, 5x10 -7 M Hyp was more effective when compared to 1x10 -7 M concentration. From the amount of cell viability (%) point of view for transfected PKCδ -cells the amount of living cells was a higher compared to non-treated cells even though the difference between non-transfected and transfected PKCδ -cells was little. PKCδ generally functions as a pro-apoptotic protein during DNA damage-induced apoptosis [START_REF] Basu | Two Faces of Protein Kinase C Delta: The Contrasting Roles of Pkc Delta in Cell Survival and Cell Death[END_REF], so silencing of pkcδ gene can cause that transfected PKCδ -cells were more viable after Hyp photo-activation.

DETECTION OF APOPTOSIS/NECROSIS AFTER HYP PHOTO-

ACTIVATION

In the next step, determination of the cell death after Hyp photo-activation in the control and transfected PKCδ -cells was performed by flow cytometry. For the detection of apoptosis/necrosis after Hyp photo-activation cells were stained with Annexin V-FITC/PI. Three major populations of cells were observed in the cytotoxicity assay:

Annexin V -/PI -cells were defined as live cells, Annexin V + /PI -cells as apoptotic, and PI + as death cells (late apoptotic or necrotic).

As it was already mentioned cells incubated with Hyp undergo apoptosis after

Hyp photo-activation [START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF][163] and our results discussed in the previous chapter (Chapter 3.16) where the cell death after Hyp photo-activation in the cells treated with siRNA PKCα was examined showed that in this case cell death pathway is different and cells undergo necrosis prior to apoptosis after irradiation. Blocking of expression of proapoptotic protein PKC did not influence the type of cell death (apoptosis/necrosis).

Figure 60 shows the percentage of Hyp photo-induced apoptotic and necrotic cells in the control cells and cells treated with siRNA after irradiationPost-transcriptional silencing of pkcδ gene in U87-MG cells after Hyp photo-activation does not have any impact on the way of the cell death pathway. Here again the effect on the cell surviving of the transfected PKCδ -cells after Hyp photo-activation can be observed where amount of the dead transfected PKCδ -cells was lower when compared to non-transfected cells.

This result is in agreement with MTT test described above (Figure 59).

pkcδ gene silencing has a little impact on the cell survival after Hyp photoactivation. Transfected PKCδ -cells are more viable after Hyp photo-activation.

EXAMINATION OF THE ROS PRODUCTION

As it was already mentioned one of the key regulators of the cell survival is level of ROS formation. Because decreasing of PKCδ proliferation in the glioma cells did not have any impact on the cell death pathway, ROS production was investigated in nontransfected and transfected PKCδ -cells.

For the set of flow cytometry experiments CellROX Deep Red™ was used for ROS production in the non-treated and treated PKCδ -cells 0, 1 and 3 hours after Hyp photo-activation. A substantial difference between transfected PKCδ -and non-pkcδ gene silencing does not have any impact on the type of Hyp photo-induced cell death.

transfected cells was observed (Figure 61). When control cells were compared to cells with transfected PKCδ -cells we found out that the level of the ROS production after Hyp photo-activation in the cells without siRNA treatment was immediately increased.

On the other side, in the transfected PKCδ -cells ROS formation after Hyp photoactivation was significantly decreased. This phenomenon can be observed for both concentrations of Hyp (1x10 -7 M and 5x10 -7 M). This result from flow cytometry can explain why the amount of cell death was slightly lower in the transfected PKCδ -cells. Transfected PKCδ -cells did not produce as much ROS as control cells and they were a little bit more viable after Hyp photoactivation. PKCδ is known for its apoptotic properties and is involved in the cellular response to DNA damage and oxidative stress [START_REF] Marengo | Pkc Delta Sensitizes Neuroblastoma Cells to L-Buthionine-Sulfoximine and Etoposide Inducing Reactive Oxygen Species Overproduction and Dna Damage[END_REF]. Our results show that blocking of pkcδ gene blocks the induction of oxidative stress (ROS production) which results in higher cell viability of transfected PKCδ -cells after Hyp photo-activation compared to control cells.

pkcδ gene silencing decreases the intracellular photo-induced ROS production.

LOCALIZATION OF BAK AND BAX

From the literature [75][163] as well as from our results (Figure 62) it was observed that apoptosis is the type of cell death pathway which is followed after Hyp photo-activation. In most cases, cells undergo oxidative stress related apoptosis via an intrinsic death pathway, in which the key role play mitochondria and Bcl-2 family of proteins [START_REF] Hsiao | Morphine Induces Apoptosis of Human Endothelial Cells through Nitric Oxide and Reactive Oxygen Species Pathways[END_REF]. Bcl-2 proteins can act as pro-apoptotic or anti-apoptotic regulators [START_REF] Kroemer | The Proto-Oncogene Bcl-2 and Its Role in Regulating Apoptosis[END_REF][177] and the outer mitochondrial membrane (OMM) is their main site of action.

However, except of mitochondria there have been reports of other cell localization of Bcl-2 proteins such as endoplasmic reticulum and in the nucleus [START_REF] Annis | There Is More to Life and Death Than Mitochondria: Bcl-2 Proteins at the Endoplasmic Reticulum[END_REF]- [179][180]. Most anti-apoptotic Bcl-2 proteins can be constitutively found in the OMM and endoplasmic reticulum in normal healthy cells. Bak, pro-apoptotic protein, is localized on the OMM and on the other side Bax can be predominantly found in the cytosol [START_REF] Gilmore | Integrin-Mediated Survival Signals Regulate the Apoptotic Function of Bax through Its Conformation and Subcellular Localization[END_REF].

In through the phosphorylation of 14-3-3ζ [START_REF] Park | Pkc Delta in Preeclamptic Placentas Promotes Bax Dissociation from 14-3-3 Zeta through 14-3-3 Zeta Phosphorylation[END_REF]. Other interaction between PKCδ and Bak/Bax proteins was not described. Taking in to account that similar to other PKCs, activation of PKCδ stimulates its translocation to cellular membrane and increases its serine/threonine kinase activity silencing of pkcδ gene can cause translocation of Bak and Bax to the mitochondria near the plasma membrane as tendency of the Bak and Bax protein to interact and "find" PKCδ throughout the cell.

CONCLUSION AND SUMMARY

Two main problems were studied in this thesis. One was focused on the developing of efficient nano-delivery transport system which would help to keep Hyp inside LDL.

In this topic, not only interaction of Hyp with LDL was studied, but other possible lipid particles were examined as well. Incorporation and redistribution of Hyp from HDL was monitored from kinetics point of view and synthetic lipid-based nano-particles able to transport Hyp were produced. Our nano-delivery transport system will be used in the PDT treatment where after Hyp photo-activation cell death will occur. During the cell death PKC isoforms (α and δ) play among other processes important role. In the second part of the study mechanism of the cell death pathway after gene silencing of PKCα and PKCδ was studied. In the beginning of my thesis two main objectives were given and the summarized results are presented in these conclusions: protocol turned out to be not suitable carriers for Hyp since Hyp was not able to incorporate to these particles. sLNP were alternatively synthetized and their ability of Hyp incorporation as well as similar behavior of Hyp inside sLNP like Hyp inside LDL was showed. First attempts to find the right ratio (concentration) between sLNP, Hyp and D Ch in order to minimalize/decrease Hyp redistribution from these particles towards free lipids was done, but further investigation to find the best ratio is required in the future. It was also showed that even if sLNP does not have LDL receptor binding domain of ApoB-100 these particles can enter cells probably through endocytosis. In the future plans DARPins will be attached to the surface of sLNP which will make this delivery system highly tumor targeted. The influence of cholesterol on the Hyp incorporation was studied and it was shown that the presence of cholesterol in LUV leads probably to a higher local Hyp concentration around cholesterol rich domains. As consequence of this fact, a formation of non-fluorescent Hyp aggregates around these domains is reached at lower total Hyp concentration than in the case when cholesterol is not present in LUV. Incorporation of Hyp into HDL particles was studied and it was showed that until molar ratio HDL-Hyp = 1:8 Hyp is incorporated into HDL as monomers. At higher ratios Hyp starts to form non-fluorescent aggregates. The number of Hyp molecules which can exist in the fully monomeric state in the HDL particles is different from that in the case of LDL molecules (8 vs 30). This difference is mainly related to the smaller size of HDL molecule in comparison with LDL molecule (11 nm vs 22 nm). The kinetics of the association of Hyp molecules with free HDL particles, HDL-Hyp = 1:10 complex as well as the redistribution of Hyp molecules from overloaded HDL-Hyp = 1:70 complex to free HDL molecules reveal a qualitative similar characteristics of these processes with those obtained for the association of Hyp with LDL. However, some quantitative differences have been detected. In the presence of LDL, the major part of Hyp is incorporated into lipoprotein particles in the slow phase (ca 65 %). On the other hand, in the presence of HDL this statement is valid only for the very low HDL-Hyp ratios (1:1 and 1:2). Higher HDL-Hyp ratios lead to a situation when most of Hyp molecules is incorporated into lipoprotein in the rapid phase. The lower concentration of cholesterol molecules in outer shell of HDL particles is probably the determining factor for the more rapid kinetics of Hyp incorporation to and redistribution from these molecules when comparing with LDL particles.

  Nízkohustotné lipoproteíny (LDL) sú považované za vhodné in vivo transportné systémy pre hydrofóbne pts akým je hypericín (Hyp). Bolo preukázané, že LDL môžu byť používané pre cielenú terapiu vďaka zvýšenej expresii LDL-receptorov v nádorových bunkách,. Aj keď napriek pozorovanej zvýšenej akumulácii Hyp in vivo, Hyp nachádzajúci sa v LDL môže byť redistribuovaný k ďalším voľným biologickým makromolekulám nachádzajúcich sa v plazme. Pomocou cholesterol-modifikovaného dextránu (D ch ) sa nám podarilo znížiť proces redistribúcie. Ukázali sme, že pokrytie LDL-Hyp komplexu pomocou D ch nemá nežiaduci účinok na vychytávanie [LDL-Hyp]/D Ch bunkami. Tak ako LDL, aj vysokohustotné lipoproteíny (HDL) sú schopné transportovať Hyp k nádorovým bunkám a ich interakcia s Hyp bola študovaná. Proces izolácie LDL aj HDL je veľmi zložitý pričom izolované lipoproteíny sa odlišujú veľkosťou a zložením. Preto boli vytvorené syntetické nano-častice na báze lipidov (sLNP) a pomocou fluorescenčnej spektroskopie bola študovaná ich interakcia s Hyp. Hlavným cieľom nášho transportného systému je selektívna akumulácia Hyp v nádorových bunkách. Následne, po nahromadení a ožiarení Hyp nastáva bunková smrť.

2 1. 3

 23 Toto zvýšenie vnútrobunkových ROS súvisí s poklesom fosforylácie Bcl-2 ako aj s nižším stupňom mitochondriálneho GSH. Pri porovnaní post-transkripčnej úpravy expresie pkcδ génu s post-transkripčnou úpravou pkcα génu, vlpyv na druh bunkovej smrti, produkciu ROS nebol vyrazný, ale ovplyvnil translokáciu Bak a Bax v bunkách s vyblokovanou PKCδ -. Ph ho ot to op ph hy ys si ic cs s a an nd d p ph ho ot to oc ch he em mi is st tr ry y o of f P PD DT T . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . HYPERICIN AS THE MODEL MOLECULE OF PTS FOR PDT .................

1 1 . .3 3 . .1 1 P 2 1. 4 3 1 1 . .4 4 . .2 2 A 3 1 1 . .4 4 . .4 4 N 3 1 1 . .4 4 . .5 5 C 40 1. 5 FAMILY 4 1 1 .

 1312431423144314540541 Ph hy ys si ic co o--c ch he em mi ic ca al l a an nd d p ph ho ot to od dy yn na am mi ic c p pr ro op pe er rt ti ie es s o of f H Hy yp p . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . CELL DEATH .......................................................................... 1 1. .4 4. .1 1 A Au ut to op ph ha ag gi ic c c ce el ll l d de ea at th h . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Ap po op pt to os si is s. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 3 1.4.2.1 The Extrinsic Pathway: Fas ................................................................................ 1.4.2.2 The Intrinsic Pathway ........................................................................................ 1.4.2.3 The Final Pathway: Caspases ............................................................................ 1 1. .4 4. .3 3 C Cr ro os ss s t ta al lk k b be et tw we ee en n a au ut tp ph ha ag gy y a an nd d a ap po op pt to os si is s . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Ne ec cr ro os si is s . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Ce el ll l D De ea at th h P Pa at th hw wa ay ys s i in n P PD DT T o of f C Ca an nc ce er r . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 3 1.4.5.1 PDT and autophagy ............................................................................................ 1.4.5.2 PDT and apoptosis: involvement of Bcl-2 family in the PDT response .......... 1.4.5.3 PDT and necrosis ...............................................................................................OF PROTEIN KINASE C ................................................... : : a an nt ti i--a ap po op pt to ot ti ic c p pr ro ot te ei in n . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..5 5. .2 2 P PK KC C : :p pr ro o--a ap po op pt to ot ti ic c p pr ro ot te ei in n . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 4 1.6 TARGETED DRUG DELIVERY ...................................................... 1.7 LIPOPROTEINS .......................................................................

2. 1 2 . 3 FLUORESCENCE

 123 CHEMICALS ........................................................................... 2.2 ABSORPTION SPECTROSCOPY .................................................... SPECTROSCOPY ................................................

2 2 . .3 3 . .1 1 F 5 2. 4 FLUORESCENCE

 23154 FR RE ET T . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . LIFETIME ........................................................ 2.5 CONFOCAL FLUORESCENCE MICROSCOPY .................................... 2.6 FLOW CYTOMETRY .................................................................. 2.7 DYNAMIC LIGHT SCATTERING ................................................... 2.8 CRYO-ELECTRON MICROSCOPY ................................................. 2.9 PROTOCOLS ...........................................................................

2 2 . .9 9 . .1 1 S 6 2 2 . .9 9 . .2 2 P 7 2 2 . .9 9 . .3 3 P 7 2 2 . .9 9 . .4 4 P 7 2 2 . .9 9 . .5 5 P 7 2. 9 . 5 . 1

 29162927293729472957951 Sy yn nt th he es si is s o of f d de ex xt tr ra an n d de er ri iv va at te es s w wi it th h c ch ho ol le es st te er ro ol l m mo oi ie et ty y . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Pr re ep pa ar ra at ti io on n o of f H HD DL L--H Hy yp p a an nd d L LD DL L--H Hy yp p c co om mp pl le ex xe es s. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Pr re ep pa ar ra at ti io on n o of f [ [L LD DL L--H Hy yp p] ]/ /d de ex xt tr ra an n c co om mp pl le ex xe es s . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Pr re ep pa ar ra at ti io on n o of f L LU UV V, , L LU UV V w wi it th h c ch ho ol le es st te er ro ol l a an nd d L LU UV V s st ta ai in ne ed d w wi it th h R Rh ho od da am mi in ne e B B . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Pr ro od du uc ct ti io on n o of f s sy yn nt th he et ti ic c L LD DL L . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Synthesis of synthetic lipid-based nano-particles .......................................

WITH DCH 3 86 3 . 4

 334 DETERMINATION OF THE BEST RATIO BETWEEN [LDL-HYP] COMPLEX AND DCH 3 .................................................................................... SYNTHETIC LDL ................................................................. 3.5 SYNTHESIS OF SYNTHETIC LDL/SLNP ....................................OF HYP FROM [SLNP -HYP]/DCH 3 TOWARDS FREE NATURAL LDL ............................................................................. 3.8 INTERACTION OF SLNP WITH CELLS ........................................ STUDY WITH CHOLESTEROL ................................................. 3.9 INFLUENCE OF CHOLESTEROL ON HYP ...................................... STUDY OF THE INTERACTION OF HDL WITH HYP .................... 3.10 INCORPORATION OF HYP INTO HDL ....................................... 3.11 THE KINETICS OF HYP INTERACTION WITH HDL PARTICLES ........ 3.12 KINETICS OF HYP REDISTRIBUTION FROM HDL-HYP COMPLEXES EFFECT OF PKC EXPRESSION ON BCL-2 PHOSPHORYLATION AND CELL DEATH BY HYPERICIN ................................................... 3.13 TRANSFECTION EFFICIENCY ................................................... 3.14 PKC GENE SILENCING ......................................................... 3.15 CELL SURVIVAL AFTER HYP PHOTO-ACTIVATION....................PKC ON HYPERICIN PHOTO-INDUCED CELL DEATH .......................................................................................

Figure 1 :

 1 Figure 1: Jablonski diagram and Scheme of Type I and Type II mechanism of PDT. .......................................................................................................

Figure 2 :

 2 Figure 2: A-Hypericum perforatum (blossom), B-chemical structure of Hyp, C-Hypericum perforatum (blatt). .........................................................

Figure 3 :

 3 Figure 3: Absorbance and fluorescence spectrum of Hyp in PBS and DMSO ....

Figure 4 :

 4 Figure 4: Extrinsic and intrinsic pathway of apoptosis ......................................

Figure 5 :

 5 Figure 5: Crystal Structure Analysis of PKC (alpha)-C2 domain complexed with Ca2+ and PtdIns(4,5)P2 ...............................................................

Figure 6 :

 6 Figure 6: A-C2 domain from protein kinase c-The structure of PKC ......

Figure 7 :

 7 Figure 7: Various types of nanoparticles used in biomedical research and drug delivery ..................................................................................................

Figure 8 :

 8 Figure 8: Lipoprotein classes ...............................................................................

Figure 9 :

 9 Figure 9: Molecule of HDL ..................................................................................

Figure 10 :

 10 Figure 10: LDL particle-the major cholesterol carrier of the bloodstream .........

Figure 11 :

 11 Figure 11: The layout of dual beam spectrofotometer .........................................

Figure 12 :

 12 Figure 12: Schematic diagram of spectrofluorimeter ...........................................

Figure 13 :

 13 Figure 13: Conditions for FRET ...........................................................................

Figure 14 :

 14 Figure 14: Example of the intensity decay ............................................................

Figure 15 :

 15 Figure 15: Fluorescence decay data in logarithmic scale .....................................

Figure 16 :

 16 Figure 16: Sinusoidal modulation of the light source .........................................

Figure 17 :

 17 Figure 17: The phase delay between the excitation and emission ......................

Figure 18 :

 18 Figure 18: The frequency response curve (phase and modulation .....................

Figure 19 :

 19 Figure 19: Ray path in a confocal CSLM..............................................................

Figure 20 :

 20 Figure 20: Scheme of cholesterol moiety (cholesterol + linker) .........................

Figure 21 :

 21 Figure 21: Scheme of dextran derivate with cholesterol moiety .........................

Figure 22 : 1 H

 221 Figure 22: 1 H NMR spectra of dextran and different type of modified dextrans Figure 23: A-Chemical structure of cholesterol-modified dextran-DCh 3 from GPC measurement, B-FT IR spectra of DCh 3 .......................................

Figure 24 :

 24 Figure 24: Overlap of the flow cytometry band pass filter of B1, B2 and B3 channels detection with the florescence spectra of PI, CellROX® Deep Red, Annexin V-FITC and Hyp ...................................................

Figure 25 :

 25 Figure 25: Dependance of the fluorescence intensity of Hyp in different [LDL-Hyp] and [LDL-Hyp]/ DCh ratios prepared without using DMSO ......

Figure 26 :

 26 Figure 26: Fluorescence intensity of Hyp before and after dialysis of complex .

Figure 27 :

 27 Figure 27: Redistribution of Hyp to free LDL from [LDL-Hyp] complexes covered by DCh 1 (A), DCh 2 (B) and DCh 3 (C) with different ratios of dextrans. ................................................................................................

Figure 28 :

 28 Figure 28: Steady state curve-dependance of the fluorescence intensity of Hyp in different LDL:Hyp ratios with and without DCh 3 ....................

Figure 29 :

 29 Figure 29: DLS of LDL with DCh 1, DCh 2 and DCh 3 at the ratio [LDL-DCh 3] = 1:75 ........................................................................................................

Figure 30 :

 30 Figure 30: Fluorescence lifetime of complexes [LDL-Hyp] and [LDL-Hyp]/DCh 3 measured at different intensities ......................................

Figure 31 :

 31 Figure 31: Solubilization of Hyp in DCh 3 ............................................................

Figure 32 :

 32 Figure 32: Scheme of two populations of Hyp in the solution without and in the presence of DCh 3 .............................................................................

Figure 33 :

 33 Figure 33: Redistribution of Hyp from complex [LDL-Hyp]/DCh 3 ....................

Figure 34 :

 34 Figure 34: Flow cytometry fluorescence histogram of uptake of Hyp in U-87 MG cells observed at 4°C and at 37°C by flow cytometry. ...................

Figure 35 :

 35 Figure 35: Fluorescence microscopy images of U-87 MG cells incubated with complex [LDL-Hyp] and [LDL-Hyp]/DCh 3 in 10% FBS ......................

Figure 36 :Figure 37 :

 3637 Figure 36: Confocal fluorescence microscopy images of U-87 MG cells incubated with complex [LDL-Hyp] and [LDL-Hyp]/DCh 3 in 2% UG Figure 37: DLS of sLDL........................................................................................

Figure 38 :

 38 Figure 38: Fluorescence intensity of Hyp in different ratios of [sLDL-Hyp] after 90 min of incubation ....................................................................

Figure 39 :

 39 Figure 39: DLS of sLNP without synthetic peptide and ultracentrifugaion .......

Figure 40 :

 40 Figure 40: Cryo-electron microscopy of sLNP (A) and LDL (B) ......................

Figure 41 :

 41 Figure 41: Graph of incorporation of Hyp into sLNP .........................................

Figure 42 :

 42 Figure 42: Steady state curve of Hyp in different amount of sLNP and LDL ....

Figure 43 :

 43 Figure 43: Redistribution of Hyp from [sLNP -Hyp]/DCh 3 to free LDL ..........

Figure 44 :

 44 Figure 44: Fluorescence microscopy image of U-87 MG cells incubated with sLNP stained with DiO18 (3) ...............................................................

Figure 45 :

 45 Figure 45: Cholesterol structure .........................................................................

Figure 46 :

 46 Figure 46: Fluorescence intensity of Hyp and Rhod B in the LUV without cholesterol and with 20% cholesterol .................................................

Figure 47 :

 47 Figure 47: Steady state of Hyp inside LUV with different amount of cholesterol ............................................................................................

Figure 48 :

 48 Figure 48: Steady state of incorporation of Hyp into LDL or HDL ...................

Figure 49 :

 49 Figure 49: Time dependence of fluorescence intensity ......................................

Figure 50 :

 50 Figure 50: Redistribution of Hyp from complex /HDL-Hyp = 70:1 to free HDL ......................................................................................................

Figure 51 :

 51 Figure 51: Detection of siRNA transfection efficiency in U87-MG cells ............

Figure 52 :

 52 Figure 52: Distribution of PKCα in human U87-MG cells .................................

Figure 53 :

 53 Figure 53: Cell viability test of non-treated and siRNA PKCα(h) treated U87-MG cells before and after Hyp photo-activation. ................................

Figure 54 :

 54 Figure 54: Determination of the cell death (apoptosis/necrosis) induced by photo-activated Hyp ............................................................................

Figure 55 :

 55 Figure 55: ROS production after Hyp photo-activation .....................................

Figure 56 :Figure 57 :

 5657 Figure 56: Level of Bcl-2 phosphorylation on Ser70 after Hyp photo-activaion127 Figure 57: Cellular distribution of pBcl-2(Ser70) and GSH after Hyp photoactivation and its co-localization with mitochondria ..........................

Figure 58 :

 58 Figure 58: Distribution of PKCδ in the human U87-MG cells ...........................

Figure 59 :

 59 Figure 59: Cell viability test of non-treated and siRNA PKCδ(h) treated U87-MG cells before and after Hyp photo-activation. ................................

Figure 60 :

 60 Figure 60: Determination of the cell death (apoptosis/necrosis) induced by photo-activated Hyp within 24 hours after cell irradiation ................

Figure 61 :

 61 Figure 61: ROS production after Hyp photo-activation .....................................

Figure 62 :

 62 Figure 62: Fluorescence imaging of Bak and Bax localization after Hyp photoactivation. ................................................................................... 139

  uses are only limited by the imagination. (McCaughan J. S. Drugs & Aging 1999; 15:49-68.)

Figure 1 :

 1 Figure 1: Jablonski diagram and Scheme of Type I and Type II mechanism of PDT.

Figure 2 :

 2 Figure 2: A-Hypericum perforatum (blossom), B-chemical structure of Hyp, C-Hypericum perforatum (blatt).

Figure 3 :

 3 Figure 3: Absorbance and fluorescence spectrum of Hyp in PBS and DMSO

  proteins. Members of the Bcl-2 family are key regulators of apoptosis and are over expressed in many malignancies. Increased expression of Bcl-2 causes resistance to chemotherapeutic drugs and radiation therapy, while decreasing Bcl-2 expression may promote apoptotic responses to anticancer drugs. In addition, over expression of Bcl-2 may result in accumulation of cells in the G0 phase of cell cycle division and contribute to chemo résistance. The Bcl-2 family includes pro-apoptotic members such as Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim, and Hrk, and anti-apoptotic members such Bcl-2, Bcl-XL, Bcl-W, Bfl-1, and Mcl-1. Anti-apoptotic Bcl-2 members act as repressors of apoptosis by blocking the release of cytochrome c, whereas pro-apoptotic members act as promoters. These effects are more dependent on the balance between Bcl-2 and Bax than on Bcl-2 quantity alone. Following a death signal, pro-apoptotic proteins undergo posttranslational modifications that include dephosphorylation and cleavage resulting in their activation and translocation to the mitochondria leading to apoptosis. All BH3only molecules require multidomain BH3 proteins (Bax, Bak) to exert their intrinsic pro-apoptotic activity. In response to apoptotic stimuli, the outer mitochondrial membrane becomes permeable, leading to the release of cytochrome c and second mitochondria-derived activator of caspase. Cytochrome c, once released in the cytosol, interacts with Apaf-1, leading to the activation of caspase-9 proenzymes. Active caspase-9 then activates caspase-3, which subsequently activates the rest of the caspase cascade and leads to apoptosis. Activated caspases lead to the cleavage of nuclear lamina and breakdown of the nucleus through caspase-3 Figure 4 [32].

Figure 4 :

 4 Figure 4: Extrinsic and intrinsic pathway of apoptosis[source[START_REF] Ghobrial | Targeting Apoptosis Pathways in Cancer Therapy[END_REF]]

  organisms, respectively are controlled by autophagy and apoptosis. Many stress pathways sequentially elicit autophagy, and apoptosis within the same cell. Generally autophagy blocks the induction of apoptosis, and apoptosis-associated caspase activation shuts off the autophagic process. However, in special cases, autophagy or autophagy-relevant proteins may help to induce apoptosis or necrosis, and autophagy has been shown to degrade the cytoplasm excessively, leading to 'autophagic cell death'. The normal clearance of dying cells, as well as immune recognition of the dead cell antigens is influenced by the dialogue between autophagy and cell death pathways.

1. 4 . 5 . 2

 452 PDT and apoptosis: involvement of Bcl-2 family in the PDT responseIn mammals the Bcl-2 family has at least 20 relatives, all of which share at least one conserved Bcl-2 homology domain. The family includes four other anti-apoptotic proteins: BclXL, Bcl-w, A1 and Mcll, and two groups of proteins that promote apoptosis: the Bax and the BH3-only families[START_REF] Oltvai | Bcl-2 heterodimerizes in vivo with the conserved homologue, Bax, that accelerates programmed cell death[END_REF][START_REF] Cory | The Bcl2 family: Regulators of the cellular life-or-death switch[END_REF]. Bcl-2 and other members of the family potently inhibit apoptosis in response to many, but not all cytotoxic insults. Bcl-2 and several other pro-survival molecules associate with the mitochondria outer membrane and the endoplasmic reticulum/nuclear membrane and maintain their integrity[START_REF] Ross | Testicular degeneration in Bcl-w deficient mice[END_REF]. They probably regulate the activation of several other caspases, independently of mitochondrial damage Erreur ! Source du renvoi introuvable.. PDT is thought to induce photodamage of Bcl-2 and related antiapoptotic proteins and activate the proapoptotic members of the family[START_REF] Oleinick | The role of apoptosis in response to photodynamic therapy: What, where, why, and how[END_REF]. Alteration in expression of members of Bcl-2 family proteins following PDT has been reported in various cell lines and tumors[START_REF] He | A study on the mechanism of 5aminolevulinic acid photodynamic therapy in vitro and in vivo in cervical cancer[END_REF]-[START_REF] Amo | Mechanism of cell death by 5-aminolevulinic acid-based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937[END_REF]. Hyp mediated PDT in human breast adenocarcinoma cell line involved downregulation of Bcl-xl and upregulation of Bax[START_REF] Koval | Degradation of HER2 receptor through hypericin-mediated photodynamic therapy[END_REF]. This study indicated a possible involvement of the antiapoptotic Bcl-2 family members in PDT induced apoptotic cell death, but a more definite role for Bcl-2 comes from studies involving antisense treatment and overexpression approaches.Bcl-2 family members seem to spend most of their time simply trying to block each other's next move. Many family members can homodimerize, but more importantly, pro-and anti-apoptotic members can form heterodimers[START_REF] Adams | The Bcl-2 protein family: arbiters of cell survival[END_REF][START_REF] Antonsson | The Bcl-2 protein family[END_REF].

  isoforms α, β I , β II , and γ. These require Ca 2+ , diacylglycerol (DAG), and a phospholipid such as phosphatidylserine for activation.

Figure 5 :

 5 Figure 5: Crystal Structure Analysis of PKC (alpha)-C2 domain complexed with Ca2+ and PtdIns(4,5)P2 Protein chains are colored from the N-terminal to the C-terminal using a rainbow (spectral) color gradient [source [77]]

1. 5 . 2

 52 PKC:PRO-APOPTOTIC PROTEINProtein kinase C delta (PKC) was the first new/novel PKC isoform to be identified by the screening of mammalian cDNA libraries. PKCFigure 6 is expressed ubiquitously among cells and tissues. PKChas catalytic and regulatory domains in the carboxyl-and amino-terminal halves, respectively (Figure6 B). The catalytic domain contains two conserved regions, C3 and C4, in common with other members of the PKC family[START_REF] Kikkawa | Protein Kinase C Delta (Pkc Delta): Activation Mechanisms and Functions[END_REF].

Figure 6 :

 6 Figure 6: A-C2 domain from protein kinase c-The structure of PKC A: Protein chains are colored from the N-terminal to the C-terminal using a rainbow (spectral) color gradient; B: The domain structure of PKC schematically shown, with the phosphorylation sites of serine (S), threonine (T), and tyrosine (Y) residues.[source[START_REF] Kikkawa | Protein Kinase C Delta (Pkc Delta): Activation Mechanisms and Functions[END_REF], [111]]

Figure 7 :

 7 Figure 7: Various types of nanoparticles used in biomedical research and drug delivery [source [115]]

Table 2 :Figure 9 :

 29 Figure 9: Molecule of HDL [source [119]]

Figure 10 :

 10 Figure 10: LDL particle-the major cholesterol carrier of the bloodstream [source [116]]

1 CHEMICALS

 1 photoactivation, it can induce oxidation of certain amino acid residues which leads to irreversible inactivation of PKC[START_REF] Jurgenliemk | Dissolution, Solubility and Cooperativity of Phenolic Compounds from Hypericum Perforatum L. In Aqueous Systems[END_REF].c) The role of anti-apoptotic PKCα in response to Hyp photodynamic therapy in U-87 MG cells was examined as well. It was shown that the presence of Hyp without irradiation does not affect PKCα distribution but after Hyp photo-activation, PKCα is activated and localized along the plasma membrane and partially in the nuclei. The regulation of PKCα activity may be imporant for increasing cancer cells'sensitivity toward anticancer PDT treatments. In particular, dephosphorylation of PKCα and phosphorylated Bcl-2 may play a role[START_REF] Dzurova | The Role of Anti-Apoptotic Protein Kinase Calpha in Response to Hypericin Photodynamic Therapy in U-87 Mg Cells[END_REF].At this stage of knowledge the second objective of my thesis was defined: to study molecular mechanism of the photodynamic induced cell death: understand the impact of PKCα and PKCδ on the cell death after knocking out their expression in U87-MG cells.

Figure 11 :

 11 Figure 11: The layout of dual beam spectrofotometer[source[START_REF] Brandt | Introduction to Absorbance Spectroscopy[END_REF]]

Figure 12 :

 12 Figure 12: Schematic diagram of spectrofluorimeter [source [139]]

Figure 13 :

 13 Figure 13: Conditions for FRET [source [142]]

Figure 14 :

 14 Figure 14: Example of the intensity decay [source [143]]

Figure 15 :

 15 Figure 15: Fluorescence decay data in logarithmic scale [source [143]]

Figure 16 :

 16 Figure 16: Sinusoidal modulation of the light source [source [143]]

  and Eo are the intensities at time t and 0, M E is the modulation factor which is related to the ratio of the AC and DC parts of the signal, is the angular modulation frequency; = 2f where f is the linear modulation frequency.Due to the persistence of the excited state, fluorophores subjected to such an excitation will give rise to a modulated emission which is shifted in phase relative to the exciting light as shown on Figure17which illustrates the phase delay (φ) between the excitation E(t) and the emission F(t). Also shown are the AC and DC levels associated with the excitation and emission waveforms.

Figure 17 :

 17 Figure 17: The phase delay between the excitation and emission [source [143]]

Figure 18 :

 18 Figure 18: The frequency response curve (phase and modulation[source[143]

Figure 19 :

 19 Figure 19: Ray path in a confocal CSLM [source [148]]

  light-indicators of the particle's size, shape and structure. If particles have been stained with one or more fluorescent dyes-known as fluorochromes, the light source excites these dyes to provide additional biological information about each particle, such as metabolic activity, DNA content and the presence of specific surface and intracellular markers. Precise optical and electronic elements collect the fluorescent pulses and scattered light, convert them into digital values and send them to a computer for analysis. The unique power of flow cytometers is that they can rapidly and quantitatively measure multiple simultaneous parameters on individual live cells and then isolate cells of interest [149].

  20): Succinic anhydride (2g) was added to solution of cholesterol (2 g) in 50 ml of pyridine. The mixture was stirred for 3 hours at 70 °C. The crude product was dissolved in a minimum amount of H 2 O/ethanol (1:10, v/v), and cholesterol 3-hemisuccinate was crystallized from H 2 O/methanol (1:10, v/v), then recrystallized from ethanol.

Figure 20 :

 20 Figure 20: Scheme of cholesterol moiety (cholesterol + linker)

Figure 21 :

 21 Figure 21: Scheme of dextran derivate with cholesterol moiety

Figure 22 : 1 HTable 3 : 5 Figure 23 :

 2213523 Figure 22: 1 H NMR spectra of dextran and different type of modified dextrans (* solvent)

  Molar concentration was determined by absorption spectra using Lamber-Beer law and extinction coefficient for Hyp 44 000 cm -1 M -1 for =600 nm. Hyp in DMSO was kept in the dark at 4°C. A stock solution of HDL and LDL was prepared in 150 mM NaCl at pH=7,4 in the presence of 0,1% EDTA. The HDL-Hyp or LDL-Hyp complexes used in experiments were obtained by mixing of appropriate volumes of HDL/LDL and Hyp stock solutions in PBS at pH=7,4. The final quantity of DMSO in all HDL-Hyp and LDL/Hyp solutions was under 0,5%. As revealed by UV-Vis absorption and fluorescence spectroscopy, HDL-Hyp or LDL-Hyp complexes are stable in PBS (pH=7.4) at room temperature (23 o C) for several days (5 days in this case).

5: 5 )

 5 . The cholesterol powder was dissolved in chloroform (with [cholesterol] = 5x10 - 2 M) and then according to the ratio of LUV-Chol 80 µl of DOPC and 20 µl of Chol, or 70 µl of DOPC and 30 µl of Chol, or 50 µl of DOPC and 50 µl of cholesterolwere added to the glass bottom tube. All the other preparation of the LUV-Chol was the same as preparation of the SUV without cholesterol. The preparation of LUV stained with Rhodamine B (LUV-Rhod B) consisted of 1% of DOPE-Rhod B which was introduced with DOPC ([DOPC]= 10 -3 M). Organic solvent was added to the DOPC and DOPE-Rhod B solutions (chloroform:ethanol = 3:5, v/v). Thin film was obtained by evaporation of 100 µl of the solution. All the other steps were the same as at the preparation of the pure LUV.

  photostable when incorporated into membranes. It has an extremely high extinction coefficient and short excited-state lifetimes (~1 ns) in lipid environments. Once applied to cells, the dye diffuses laterally within the plasma membrane[152]. Stock solution of DiO 18 was prepared in acetone with final concentration of 10 -3 M. For sLNP staining, 0,1% of DiO 18 was added to the mixture of PC:TO:CO = 3:2:1 in chloroform:methanol before evaporation under the stream of N 2 . Next steps in the preparation of DiOC 18[START_REF] Siegel Rebecca | Cancer Statistics, 2013[END_REF]

  -RNA TRANSFECTION PROTOCOL Cells were seeded onto 35 mm culture dishes (Corning, USA) at density of 2x10 5 cells per well in 2 ml antibiotic free D-MEM supplemented with 10% FBS. Then they were incubated at 37°C in CO 2 incubator until they were 60-80% confluent. For each transfection, 5 µl of siRNA duplex (solution A) was diluted in 100 µl siRNA Transfection Medium and 5 µl of siRNA Transfection Reagent (solution B) was also diluted in 100 µl siRNA Transfection Medium. The siRNA duplex solution (solution A) was added to the Transfection Reagent solution (solution B) and mixture was incubated for 45 minutes at room temperature. Meanwhile, cells were once washed with 2 ml of siRNA Transfection Medium. After 45 minutes of mixture incubation 0,8 ml of siRNA Transfection Medium was added to each tube containing the siRNA Transfection Reagent mixture (Solution A + Solution B). Solution was mixed gently and was overlaid to the washed cells and incubated 5 hours in CO 2 incubator. 1 ml of DMEM containing 20% FBS and 2% of antibiotics was added to cells without removing the transfection mixture. Cells were incubated for an additional 18 hours and were treated according to PDT protocol. The effectiveness of transfection was assessed by confocal microscopy after indirect immunofluorescence staining of PKCα and subsequently verified by western blot analysis (all western bloth analysis were performed by M.

2 . 9 . 10 . 2

 29102 In normal viable cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. However, in the intermediate stages of apoptosis, PS is translocated from the inner to the outer leaflet of the membrane, exposing PS to the external cellular environment where it can be detected. Highly fluorescent annexin V-FITC conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine. Propidium iodide (PI) is a red-fluorescent nuclear and chromosome counterstain. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. Staining protocol: Cells (approximately 0.5 x 10 6 cells per sample) were harvested by low-speed centrifugation, resuspended in 100 μl Annexin-binding buffer and incubated with 5 µl AnnexinV-FITC for 15 min at room temperature. After incubation, 400 μl of Annexinbinding buffer was added to each sample. 2 l Propidium iodide (PI) was added to each sample just before measurements to distinguish the necrotic cells. 10000 cells were counted and analysis was performed using a MACSQuant®Analyzer (Miltenyi, GER). Fluorescence of AnnexinV-FITC was detected in B1 channel (525 ± 50nm), fluorescence of Hyp was detected in B2 channel (585 ± 40nm) and fluorescence of PI was detected in B3 channel (655-730nm). The data were analyzed by MACSQuantify™ Software. Oxidative Stress Detection Oxidative stress results from an imbalance between production of reactive oxygen species (ROS) and the ability of cells to scavenge them. ROS play an important role in the progression of several diseases including inflammation, atherosclerosis, aging, and age-related degenerative disorders as well as in cell death processes.CellROX® Deep Red Reagent is a novel fluorogenic probe for measuring cellular oxidative stress in both live and fixed cell imaging, with absorption/emission maxima at ~644/665 nm. The cell-permeant dye is non-fluorescent while in a reduced state, and exhibits bright fluorescence upon oxidation by reactive oxygen species (ROS).Staining protocol: cells were harvested by low-speed centrifugation and resuspended in PBS (approximately 0.5 x 10 6 cells pre sample) and 5.10 -6 M CellROX® Deep Red was added. After 30 min incubation at room temperature samples were measured using a MACSQuant Analyzer. Flouorescence was excited with an Argon laser 488 nm and emission of CellROX® Deep Red was collected in B3 channnel (655nm-730nm). The data were analyzed by MACSQuantify™ Software.

Figure 24 :

 24 Figure 24: Overlap of the flow cytometry band pass filter of B1, B2 and B3 channels detection with the florescence spectra of PI, CellROX® Deep Red, Annexin V-FITC and Hyp

Figure 25 :

 25 Figure 25: Dependance of the fluorescence intensity of Hyp in different [LDL-Hyp] and [LDL-Hyp]/ D Ch ratios prepared without using DMSO LDL was added to D Ch dried under the stream of N 2 and solution was incubated for 1 hour following ~17 hours dialysis. Consequently, different amount of Hyp was added to [LDL-D Ch ] for overnight incubation.

Figure 26 :

 26 Figure 26: Fluorescence intensity of Hyp before and after dialysis of complex A: Fluorescence spectrum of Hyp in [LDL-Hyp]/D Ch complex before and after dialysis. B: Comparison of the fluorescence intensity of Hyp at the different ratios LDL:Hyp.

1 ,

 1 three dextrans were synthesized with different cholesterol moiety. All three of them were used for the fluorescence measurements of the redistribution of Hyp to free LDL (Figure 27) and compared to the redistribution of Hyp from complexes that were not covered with D Ch . After incubation of [LDL-Hyp] with D Ch , free LDL was added to the complexes in order to reach the final ratio of three In order to find the best protocol for preparation of [LDL-Hyp]/D Ch complexes, three different protocols were tried. Overnight incubation of LDL and Hyp followed by addition of little amounts (100μl) of D Ch and additional 1 hour incubation was selected as the best protocol for complex preparation. Hence, preparation and purification of [LDL-Hyp]/D Ch complexes needs further improvement.Hyp per one LDL. At this ratio all molecules of Hyp are in fluorescent monomer form.After addition of free LDL to the [LDL-Hyp]/D Ch complexes, increase of fluorescence intensity of Hyp was observed. This simulates the situation how our transport system would behave in the blood stream. Hyp would be redistributed to other plasma proteins (in our case to free LDL molecules). The redistribution is expressed with the increase of fluorescence intensity of Hyp because some molecules of Hyp which were incorporated in [LDL-Hyp] complex (where it formed aggregates) were relocated to free LDL. This relocation caused increased amount of Hyp in monomer form and consequently, the increase of fluorescence intensity of Hyp. As Figure27 C, Table4shows, D Ch 3 is the most efficient to slow down the Hyp redistribution process and was subsequently used for all further experiments.

Figure 27 :

 27 Figure 27: Redistribution of Hyp to free LDL from [LDL-Hyp] complexes covered by D Ch 1 (A), D Ch 2 (B) and D Ch 3 (C) with different ratios of dextrans. [Hyp] = 2 x 10 -7 M; initial ratio of complexes were [LDL-Hyp] = 1:50 or [LDL-Hyp]/D Ch = 1:50: different ratios of D Ch , after adding free LDL to complexes final ratios of [LDL-Hyp] was 1:3.

  shows steady state curve-the fluorescence intensity of Hyp as a function of different [LDL-Hyp] and [LDL-Hyp]/D Ch 3 ratios. It is important to notice that the presence of D Ch 3 does not influence the Hyp aggreggation process inside LDL since for LDL-Hyp ratios > 50 aggregates are forms in both cases. D Ch 3 is the most efficient for limitation of Hyp redistribution process.

Figure 28 :

 28 Figure 28: Steady state curve-dependance of the fluorescence intensity of Hyp in different LDL:Hyp ratios with and without D Ch 3 [LDL] was constant = 4 x 10 -9 M; different amount of Hyp was added to LDL and such prepared complexes were stabilized overnight.

Figure 29 :Table 5 : 1 -LDL with D Ch 2 -LDL with D Ch 3 Figure 30 :

 29512330 Figure 29: DLS of LDL with D Ch 1, D Ch 2 and D Ch 3 at the ratio [LDL-D Ch 3] = 1:75 ([LDL]=10 -8 M, [LDL was mixed with D Ch and complexes were incubated for 1h at room temperature following by ~17 hour dialysis against PBS at 4ºC)

  After adding Hyp to the solution of D Ch 3 in PBS the fluorescence intensity can be observed. This fluorescence was slowly decreased in time after Hyp started to form aggregates inside D Ch 3. Thus the higher fluorescence intensity in the Figure 28 at the ratios above 100 Hyp per LDL can be due to the fact that Hyp which was not incorporated in LDL and was in solution was solubilized in D Ch 3. Even though we found out that Hyp is slightly solubilized in D Ch 3 its solubilization is -Hyp] ratio 1:50 • [LDL-Hyp]/DCh 3 ratio 1:50:75 minimal and does not affect our purpose that is to decrease/reduce the redistribution process of Hyp towards other serum proteins.

Figure 31 : 3 A:

 313 Figure 31: Solubilization of Hyp in D Ch 3 A: Fluorescence intensity spectra of Hyp; B: the evolution of fluorescence in time. The spectra were taken 0-120 min after adding of Hyp to solution of D Ch 3 in PBS; [Hyp] = 2x10 -7 M, [D Ch 3] = 3x10 -7 M.

Figure 32 :

 32 Figure 32: Scheme of two populations of Hyp in the solution without and in the presence of D Ch 3

  Hyp the most effectively, fluorescence measurement of the redistribution of Hyp to free LDL from the complex [LDL-Hyp]/D Ch 3 with different ratios of D Ch 3 was performed. The initial ratio of [LDL-Hyp] was 1:50. After adding free LDL the final ratio was [LDL-Hyp] = 1:3. From all the ratios between LDL and D Ch 3, at the ratio [LDL-D Ch 3] = 1:75 the redistribution of Hyp was 27% slower than the redistribution of Hyp toward free LDL from [LDL-Hyp] complex not covered by D Ch (Figure 33). Because of this result along with the result from DLS where at the presence of D Ch 3 with the same ratio the size of LDL was decreased (Figure 29) we used D Ch 3 at the ratio [LDL-D Ch 3] = 1:75 in all the other experiments.

Figure 33 : 3 [

 333 Figure 33: Redistribution of Hyp from complex [LDL-Hyp]/D Ch 3 [Hyp] = 2x10 -7 M; initial ratio of complex[LDL-Hyp] was 1:50, after adding free LDL the ratio [LDL-Hyp] was 1:3.

  shows the uptake of Hyp by U-87 MG cells at 37°C and at 4 °C measured at the FACS MACSQuant®Analyzer where fluorescence of Hyp was detected in B2 channel (Band Pass Filter 585/40nm). The data were analyzed by MACSQuantify™ Software.

Figure 34 :

 34 Figure 34: Flow cytometry fluorescence histogram of uptake of Hyp in U-87 MG cells observed at 4°C and at 37°C by flow cytometry. Cells were incubated with [LDL-Hyp] or [LDL-Hyp]/D Ch 3 for 1 hour either at 37 °C or 4 °C. 10000 cells were counted and fluorescence of Hyp was detected in B2 channel (Band Pass Filter 585 ± 40nm).

Figure 35 :

 35 Figure 35: Fluorescence microscopy images of U-87 MG cells incubated with complex [LDL-Hyp] and [LDL-Hyp]/D Ch 3 in 10% FBS A: U-87 MG cells incubated in the presence of [LDL-Hyp] = 1:50 for 30 min B: U-87 MG cells incubated in the presence of [LDL-Hyp]/D Ch 3 = 1:50:75 for 30 min C: U-87 MG cells incubated in the presence of [LDL-Hyp] = 1:50 for 60 min D: U-87 MG cells incubated in the presence of [LDL-Hyp]/D Ch 3 = 1:50 for 60 min

Figure 36 :

 36 Figure 36: Confocal fluorescence microscopy images of U-87 MG cells incubated with complex [LDL-Hyp] and [LDL-Hyp]/D Ch 3 in 2% UG A: U-87 MG cells incubated in the presence of [LDL-Hyp] = 1:50 for 30 min B: U-87 MG cells incubated in the presence of [LDL-Hyp]/D Ch 3 = 1:50:75 for 30 min

Figure 37 :

 37 Figure 37: DLS of sLDL ([sLDL] = 10 -8 M in PBS)

Figure 38 :

 38 Figure 38: Fluorescence intensity of Hyp in different ratios of [sLDL-Hyp] after 90 min of incubation [sLDL] was constant and different amount of Hyp was added. Complexes were stabilized for ~12 hours and then fluorescence intensity of Hyp was measured.

  et al. (no synthetic peptide and centrifugation), name synthetic lipid-based nanoparticles (sLNP) is more appropriate.

Figure 39 :

 39 Figure 39: DLS of sLNP without synthetic peptide and ultracentrifugaion ([sLNP] = 10 -8 M in PBS)

Figure 40 :

 40 Figure 40: Cryo-electron microscopy of sLNP (A) and LDL (B)

Figure 41 :

 41 Figure 41: Graph of incorporation of Hyp into sLNP Different amount of Hyp was added to sLNP and incorporation of Hyp was measured.

Figure 42 :

 42 Figure 42: Steady state curve of Hyp in different amount of sLNP and LDL

Figure 43 :

 43 Figure 43: Redistribution of Hyp from [sLNP -Hyp]/D Ch 3 to free LDL Hyp at different concentrations was added to sLNP. These complexes were stabilized for ~ 3 hours and then D Ch 3 was added. The whole complexes were incubated for 1 hour at the room temperature. Free LDL was added and fluorescence intensity of Hyp was measured.

Figure 44 :

 44 Figure 44: Fluorescence microscopy image of U-87 MG cells incubated with sLNP stained with DiO 18 (3) U87-MG cells were incubated with sLNP-DiOC 18 (3) complex and fluorescence microscopy was used for determining the localization of DiOC 18 (3) inside the cells. Standard filters system: FITC filter set (λ exc = 450-490 nm, λ em = 520-570 nm) was used for measurement. Exposure time was set to 5 s.

Figure 45 :

 45 Figure 45: Cholesterol structure [source [15]]

Figure 46 :

 46 Figure 46: Fluorescence intensity of Hyp and Rhod B in the LUV without cholesterol and with 20% cholesterol

Figure 47 shows

 47 Figure 47 shows steady state curve of Hyp in LUV particles in the absence and presence of three different amounts of cholesterol. Similarly as in the case of Hyp interaction with natural LDL or with sLNP, the fluorescence measurements revealed that in the presence of LUV the fluorescence intensity linearly increase until certain Hyp concentration ([Hyp] = 4x10 -7 M) and beyond this concentration the quenching of Hyp fluorescence is observed (Figure 47, black line). At high Hyp concentrations > 4x10 -6 M the fluorescence almost disappears. It is obvious that this behavior is common for all known Hyp-lipid structure studies [122][131][133][134]. It means that Hyp can penetrate into lipid structures from aqueous environment and is monomerized in the lipid media. However, an entrance of other Hyp molecules leads to a high local concentration of Hyp resulting in a formation of Hyp aggregates or self-quenching of Hyp fluorescence.

Figure 47 :

 47 Figure 47: Steady state of Hyp inside LUV with different amount of cholesterol Hyp was added to LUV with different amount of cholesterol. Solutions were stabilized for ~ 3 hours and fluorescence intensity was measured.

Figure 48 :

 48 Figure 48: Steady state of incorporation of Hyp into LDL or HDL Dependence of Hyp fluorescence intensity on concentration ratio HDL-Hyp and LDL-Hyp after 120 min of stabilization of the complexes (fluorescence maximum 599 nm, excitation line 515 nm). Concentration of HDL and LDL was kept constant during the measurements (10nM).

  time evolution of the fluorescence intensity of Hyp after mixing of Hyp molecules with free HDL particles is shown in Figure 49. It is evident that there exists two phases of the incorporation of Hyp into HDL molecules. The rapid phase of the incorporation is manifested by a non-zero fluorescence intensity of Hyp immediately (5-10 seconds) after mixing of Hyp and HDL solutions. The second, slow phase of the process lasts several minutes. Hyp is incorporated to HDL molecule in the monomer form until the ratio 1:8. Above this ratio Hyp starts to form non-fluorescent aggregates.

Figure 49 :

 49 Figure 49: Time dependence of fluorescence intensity Time dependence of fluorescence intensity of Hyp after mixing of Hyp and HDL solutions. Concentration of HDL was kept constant (10nM). Fluorescence of Hyp was recorded at 599 nm, the excitation line was 515 nm.

Figure 51 A

 51 Figure 51 A shows confocal fluorescence images of cells transfected with scrambled siRNA. siRNA entered the cells, and green fluorescence originated from Control siRNA(FITC Conjugate)-A was detected in the cytoplasm. Based on nuclear (blue fluorescence originated from Hoechst 33258) and mitochondrial (red fluorescence originated from MitoTracker Orange®) morphology, no significant changes have been detected. The effect of scrambled Control siRNA-A on key investigated parameters (such as cell viability, ROS production and Bcl-2 phosphorylation) is further described within the corresponding paragraphs. Figure 51 B shows histograms of non-transfected control cells (black line) and cells transfected with Control siRNA (FITC Conjugate)-A (red line) obtained by flow cytometry. Flow cytometry clearly demonstrates that after 5 hours the transfection efficiency was about 87%.

Figure 51 :

 51 Figure 51: Detection of siRNA transfection efficiency in U87-MG cells A: Confocal fluorescence image of nuclei (blue) and mitochondria (red) of U87-MG cells entered by FITC-conjugated scrambled siRNA Control-A (green). Scrambled siRNA was transfected into U87-MG cells and nuclei (Hoechst 33258) and mitochondria (MitoTracker Orange) were stained after 5 hours. B: Flow cytometry histograms of control non-transfected cells (black line) and cells entered by FITCconjugated scrambled siRNA Control-A (red line).

Figure 52 A

 52 shows confocal fluorescence image of non-transfected and transfected cells PKC -were fluorescence intensity of cells treated with siRNA is much lower than fluorescence intensity of siRNA transfection has no impact on the nuclear and mitochondrial morphology.After 5 hours the transfection efficiency was ~ 90%. control cells. This result was verified by western blot analysis (Figure52 B) where optical density of transfected PKC -cells was 6,37% when compared to non-transfected cells with optical density 93,62%. β-actin determined in both samples was used as reference protein. This result proves that pkcα gene silencing carried through siRNA efficiently decreased the amount of PKCα protein in transfected U-87 MG cells. The specificity of siRNA PKCα(h) transfection was verified by confocal microscopy (Figure52 C). Nontransfected and transfected PKCα -cells were stained by indirect immunofluorescence, and PKCδ (as a representative member of PKCs) was visualized. Fluorescence intensity and the outer profile of the measured cells (marked with white line) are comparable (Figure52 C) and indicate that silencing of pkcα gene expression is specific and the expression of PKCδ is not affected. The specificity of pkc silencing observed in our work was confirmed also by other groups[START_REF] Adhikary | Pkc-Delta and -Eta, Mekk-1, Mek-6, Mek-3, and P38-Delta Are Essential Mediators of the Response of Normal Human Epidermal Keratinocytes to Differentiating Agents[END_REF].

Figure 52 :

 52 Figure 52: Distribution of PKCα in human U87-MG cells A: The figure shows confocal fluorescence image ofU87-MG non-transfected control cells (left) and cells transfected with siRNA PKCα(h) (right) both stained with primary Ab against PKCα (E195, Novus,

  more easily and that this modulation of PKC expression would result in more effective treatment with lower concentration of Hyp and consequently would reduce amount of survival cells. MTT colorimetric assay showed different cell behavior. Pure silencing of pkcα gene did not show noticeable effect on the cell survival with only little changes between control cells and cells with decreased transcription of PKCSurprisingly, combination of PKCα silencing and Hyp photo-activation did not result in bigger impact on the cell viability either. MTT tests were assessed with two concentrations of Hyp. It is obvious that 5x10 -7 M Hyp is more effective when compared to 1x10 -7 M concentration, but in both cases cell survival was similar for transfected PKCα -cells and siRNA PKCα(h) specifically blocks the pkcα gene expression without having any impact on the other isoforms of PKC protein family. non-transfected one (Figure53 A). The colorimetric MTT assay (Figure53 B) shows that scrambled Control siRNA-A has no influence on cell viability. Figure53 Cshows DNA fragmentation in non-transfected and transfected PKCα -cells after Hyp photoactivation where the DNA ladder is clearly visible in non-transfected cells, but in transfected PKCα -cells DNA fragmentation was not observed.

Figure 53 :

 53 Figure 53: Cell viability test of non-treated and siRNA PKCα(h) treated U87-MG cells before and after Hyp photo-activation.

Figure 54 :

 54 Figure 54: Determination of the cell death (apoptosis/necrosis) induced by photo-activated Hyp A: Representative flow-cytometric dot plot analysis of apoptosis/necrosis after Annexin V-FITC/PI staining in non-transfected cells and cells transfected with siRNA PKCα(h) obtained 0h, 5h or 24 h after Hyp photo-activation. Three major populations of cells can be observed in the cytotoxicity assay: Annexin V -/PI -cells are defined as live cells, Annexin V + /PI -cells as apoptotic cells and PI + as dead cells (late apoptotic or necrotic). B: Histograms showing the comparison ratio of apoptosis and necrosis (live cells are not considered) in the cells non-treated and treated with siRNA PKCα(h)24 hours after Hyp photo-activation. Values are mean ± S.D. of three independent experiments.

B

  pkcα gene silencing shifts Hyp photo-induced cell death from apoptosis towards necrosis.

Figure 55 :

 55 Figure 55: ROS production after Hyp photo-activation

presented in Figure 56 .

 56 After Hyp photo-activation we observed a slight decrease of Bcl-2 phosphorylated on Ser70 with much higher decrease of the phosphorylation of Bcl-2 in the transfected PKCα -cells. This result was verified by western blot analysis (Figure 56 B). It proves that pkcα gene silencing decreases phosphorylation of Bcl-2 protein at Ser70.

Figure 56 :

 56 Figure 56: Level of Bcl-2 phosphorylation on Ser70 after Hyp photo-activaion

BA:

  Flow cytometry histogram of Bcl-2 phosphorylation on Ser70 after indirect immuno-fluorescence staining with rabbit mAb (5H2) (Cell Signaling Technology, USA) against human pBcl-2(Ser70). Level of Bcl-2 phosphorylation on Ser70 after Hyp photo-activation is obtained for control cells (grey line), non-transfected cells (black line) and cells transfected with siRNA PKCα(h) (red line). B: Western blot analysis of endogenous expression of pBcl-2(Ser70) in non-transfected (PKCα) and transfected (PKCα -) U87-MG cells. Cell lysates were prepared according to the Whole cell lysate preparation protocol described in Material and Methods and were subjected to SDS-PAGE and Western blot analysis. The membranes were probed with Ab (ab47473, Abcam, UK), which specifically recognized Bcl-2αphosphorylated at Ser70. As protein reference control, anti β-actin antibody (Abcam, UK) was used.

Figure 57 :

 57 Figure 57: Cellular distribution of pBcl-2(Ser70) and GSH after Hyp photo-activation and its colocalization with mitochondria A: Cellular distribution of pBcl-2(Ser70) and mitochondria, B: cellular distribution of glutathione and pBcl-2(Ser70) and C: cellular distribution of glutathione and mitochondria in in control cells (left), nontransfected (middle) and PKCα -cells transfected with siRNA PKCα after Hyp photo-activation (right).

Mitochondrial localization of pBcl- 2 (Figure 57 .

 257 Figure 57. Confocal microscopy showed (Figure57 A) that the pBcl-2(Ser70) is distributed through whole cells in control conditions. However, after Hyp photoactivation, strong re-localization of pBcl-2(Ser70) to mitochondria occurred in both, non-transfected and transfected PKCα -cells. There is evidence of co-localization between pBcl-2(Ser70) and mitochondria, but the information from the image is just qualitative. Co-localization coefficients calculated for better understanding and quantification of co-localization are presented in Table8, where M 1 coefficient displays co-localization of the red pixels with green ones, and the M 2 coefficient displays colocalization of the green pixels with red ones. Quantitative results of the co-localization analysis (Table8) describe (i) the presence of pBcl-2(Ser70) in mitochondria, (ii) colocalization of pBcl-2(Ser70) and GSH (iii) the presence of GSH in mitochondria. This means that we have observed a slight decrease of co-localization of pBcl-2(Ser70) and mitochondria 2 hours after Hyp photo-activation (M 2 = 0,91) compared with control

Figure 57 C

 57 photo-activation, redistribution of GSH to cellular membranes was detected, and the association of GSH with mitochondria was still evident (M 2 = 0,98). In transfected PKCα -cells co-localization coefficients suggested that photo-activation of Hyp after pkcα gene silencing (M 2 = 0,88) affects and decreases the presence of GSH in mitochondria.

Figure 58 :

 58 Figure 58: Distribution of PKCδ in the human U87-MG cells A: The figure shows fluorescence image ofU87-MG non-transfected control cells (left) and cells transfected with siRNA PKCδ(h) (right) both stained with primary Ab against PKCδ (Acris antibodies, USA) for visualization of distribution of PKCδ. In both cases images were taken with the same measuring parameters (brightness, contrast and laser power). The scale bar represents 10 µm. B: Western blot analysis of endogenous expression of PKCδ in non-transfected and transfected (PKCδ -) U87-MG cells. The membranes were probed with Ab (Acris antibodies, USA). As protein reference control anti β-actin antibody (Abcam, GB) was used. C: Evaluation of the optical density bands from the western blot analysis by ImageJ software. Values are mean ± S.D. of three independent experiments.

Figure 59 :

 59 Figure 59: Cell viability test of non-treated and siRNA PKCδ(h) treated U87-MG cells before and after Hyp photo-activation. Viability of non-transfected U87-MG cells and cells transfected with siRNAPKCδ(h) after Hyp photoactivation was determined by the MTT test. The cells were plated into 96-well tissue culture plates at the density of 10 000 cells per well and were cultivated until an optimal population density was reached. Cells were incubated with Hyp and Hyp photo-activation protocol was used. Results of adherent cell numbers from three independent experiments are expressed relative to control (100%) and represent mean values.

Figure 60 :

 60 Figure 60: Determination of the cell death (apoptosis/necrosis) induced by photo-activated Hyp within 24 hours after cell irradiation Histograms showing the comparison ratio of apoptosis and necrosis (live cells are not considered) in the cells non-treated and treated with siRNA PKCδ(h) 24 hours after Hyp photo-activation. Values are mean ± S.D. of three independent experiments.

Figure 61 :

 61 Figure 61: ROS production after Hyp photo-activation Histograms of ROS production obtained by flow cytometry after CellROXDeepRed™ staining in nontransfected cells and cells transfected with siRNA PKCδ(h) 1 hour after Hyp photo-activation.

  the Balogova et al. study expression patterns of Bak and Bax in U87-MG cells before and after Hyp photo-activation was studied [182]. The occurrence of two distinct mitochondria populations was observed. One contained both proteins simultaneously and the other contained Bax only. By immunocytochemistry we studied the translocation of Bak and Bax in transfected PKCδ -cells and compared them to the control non-transfected cells. Incubation of cells with siRNAδ without Hyp photoactivation leads to the translocation of both Bak and Bax from cytosol to mitochondria near the plasma membrane (Figure 62 B). It was observed that PKCδ catalytic fragment activated Bax and caused its redistribution and release of cytochrome c, activation of the caspase cascade, and loss of inner mitochondrial membrane integrity [185]. It was shown that PKCδ in preeclamptic platentas promotes Bax dissociation from 14-3-3 ζ

Figure 62 :

 62 Figure 62: Fluorescence imaging of Bak and Bax localization after Hyp photoactivation. The figures show fluorescence image of U87-MG non-transfected control cells (first row) andcells transfected with siRNA PKCδ(h) (second row). All the samples were stained with primary Ab against Bak (green fluorescence) and Bax (red fluorescence) for visualization of distribution of Bak and Bax. The scale bar represents 10 µm.

1 .

 1 ) to optimize properties of [LDL-Hyp]/DCh delivery system, study the cellular uptake of Hyp delivered by complex [LDL-Hyp]/Dch and its intracellular redistribution which included the development of a synthetic LDL based delivery system and beside nanodelivery system based on the LDL molecules to study and characterize the interaction of HDL with Hyp as well. It was showed that cholesterol-modified dextran is a good solution for Hyp anchoring inside LDL particles. Three cholesterol modified dextrans with different cholesterol moiety were synthetized and the most efficient one was chosen. D Ch 3 was proven to be the most effective. The redistribution of Hyp from [LDL-Hyp] complexes covered by D Ch 3 was reduced by 27% when compared to the redistribution of Hyp from complex not covered by D Ch. Covering of the LDL particles by cholesterolmodified dextran was applied for the patent. Modification of LDL by D Ch could possibly modify and inhibit the recognition of LDL particles through LDLR. By flow cytometry analysis and confocal fluorescence microscopy it was showed that modification of the LDL surface does not influence the cellular uptake of [LDL-Hyp]/ D Ch complexes. Flow cytometry analysis showed that for 37°C the process of uptake of LDL by endocytosis was almost identical when D Ch was or was not present. By confocal fluorescence microscopy it was shown that [LDL-Hyp]/ D Ch complex is able to deliver higher amount of Hyp to the cells when cells are incubated with [LDL-Hyp]/ D Ch in the lipid containing medium. Preparation of sLDL according to Nikanjam et al.

2 . 11 

 211 ) to study molecular mechanism of the photodynamic induced cell death: understand the impact of PKCα and PKCδ on the cell death after knocking out their expression in U87-MG cells. The contribution of PKCα in the cell death induced by photo-activated Hyp was investigated. It was shown that blocking of PKCα expression by siRNA results in significant difference of type of cell death after Hyp photo-activation. The level of necrosis was considerably increased in PKCαtransfected cells as the consequence of higher ROS production. It was shown that the Hyp photo-induced decrease of the phosphorylation level of Bcl-2 protein on Ser70 enhanced after silencing of pkcα gene. Such enhancement can explain decreased amount of apoptotic cells and could be considered as another reason of increased necrosis after Hyp photo-activation in transfected PKCα -cells. Moreover, silencing of pkcα gene and subsequent Hyp photoactivation causes significant decrease of co-localization of pBcl-2(Ser70) with mitochondria, pBcl-2(Ser70) with GSH and affects and decreases the presence of GSH in mitochondria. In summary: post-transcriptional silencing of pkcα gene significantly increased necrosis as a consequence of acute oxidative stress in relation to the level of Bcl-2 phosphorylation and reduced anti-oxidant protection of mitochondria. Posttranscriptional silencing of pkcδ gene did not influence the type of cell death (apoptosis or necrosis), however amount of death cells (%) where PKCδ was not blocked was higher compared to cells with blocked PKCδ. This can be due to the fact that production of ROS was notably decreased in cells treated with siRNAδ. It was shown that inhibition of PKCδ leads to translocation of Bak and Bax from cytosol to mitochondria located near the plasma membrane. This translocation can be explained as effort of cells to target possible PKC after reduction of cytosolic PKCδ in cells. Jaroslava Joniova, Matus Misuth, Franck Sureau, Pavol Miskovsky and Zuzana Nadova: The effect of PKC δ on hypericin photo-induced cell death; 12th Greta Pifat Mrzljak International School of Biophysics, September 2014 (poseter presentation) 7 FINANCIAL SUPPORT My thesis was realized in co-tutoring program between Pavol Jozef Safarik University in Kosice and Piere et Marie Curie University in Paris (3 times 6 moths stays were supported by French government fellowship) and was supported by:  Project CELIM funded by 7.FP EU  International Program for Scientific Cooperation (PICS N°5398) from the French National Center of Scientific Research (CNRS)  APVV-0242-The Agency of the Ministry of Education of Slovak Republic for the Structural funds of the European Union: Operational program Research and Development-Doktorand, ITMS cod: 26110230013 NanoBioSens ITMS cod: 26220220107  KVARK  VVGS PF 32/2011/F  the European Society for Photobiology (ESP): -fellowship for attending the 16th International Congress on Photobiology, Cordoba, Argentina, September 2014.

  Hi ig gh h d de en ns si it ty y l li ip po op pr ro ot te ei in n . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 5 1 1. .7 7. .2 2 L Lo ow w d de en ns si it ty y l li ip po op pr ro ot te ei in n . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 5 1.7.2.1 LDL and HDL for targeted delivery ................................................................... . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 5

	1 1. .7 7. .1 1 H
	1 1. .7 7. .3 3 S St ta at te e o of f a ar rt t a an nd d c co on nt tr ri ib bu ut ti io on n o of f o ou ur r g gr ro ou up p .

.2 MATERIAL, METHODS AND PROTOCOLS .............................

  . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 8

	3 3. .1 1. .2 2 P Pr re ep pa ar ra at ti io on n o of f c co om mp pl le ex xe es s w wi it th h D DM MS SO O .
	2 2. .9 9. .6 6 C Ce el ll l c cu ul lt tu ur re e a an nd d H Hy yp p p ph ho ot to oa ac ct ti iv va at ti io on n .
	. 7
	2 2. .9 9. .1 10 0 F Fl lo ow w c cy yt to om me et tr ri ic c a an na al ly ys si is s o of f a ap po op pt to os si is s/ /n ne ec cr ro os si is s a an nd d R RO OS S p pr ro od du uc ct ti io on n . .. .. .. . 7
	8

..... 2.9.5.2 sLNP stained with DiO18 ................................................................................... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 7 2 2. .9 9. .7 7 s si i--R RN NA A t tr ra an ns sf fe ec ct ti io on n p pr ro ot to oc co ol l . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 7 2 2. .9 9. .8 8 I Im mm mu un no oc cy yt to oc ch he em mi is st tr ry y p pr ro ot to oc co ol l . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 7 2 2. .9 9. .9 9 C Co o--l lo oc ca al li iz za at ti io on n a an na al ly ys si is s . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 2.9.10.1 Apoptosis/Necrosis analysis ............................................................................ 2.9.10.2 Oxidative Stress Detection ..............................................................................

3 RESULTS AND DISCUSSION .............................................. STUDY CONCERNING LDL, HYP AND DCH ................................. 3.1 OPTIMALIZATION OF THE PROTOCOL FOR PREPARATION [LDL-HYP]/DCH COMPLEXES ...................................................................

3 3. .1 1. .1 1 P Pr re ep pa ar ra at ti io on n o of f c co om mp pl le ex xe es s w wi it th ho ou ut t D DM MS SO O . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . .3.2 EXAMINATION OF THREE SYNTHESIZED DEXTRANS .......................

Table 1 :

 1 Photosensitizers and their precursors used in experimental and clinical PDT applications ......................................................................

Table 2 :

 2 Characteristics of the major classes of lipoproteins in human plasma

Table 3 :

 3 Percent and number content of cholesterol per gluocopyranosidic units ........................................................................................................

Table 4 :

 4 Efficiency of covering of dextrans with different cholesterol moiety ...

Table 5 :

 5 The size of [LDL-DCh 1, DCh 2 and DCh 3] at the ratio 1:75 ....................

Table 6 :

 6 Size of sLDL particles with different [sLDL-DCh 3] ratios ...................

Table 7 :

 7

		LIST OF ABBREVIATIONS
	1 O 2	singlet oxygen		cholesterol
	Ab	antibody	LUV-Rhod B	large unilamellar vesicles stained
	ALA	aminolevulinic acid		with Rhodamine B
	apoB	apolipoprotein B-100	M E	modulations of the excitation
	BHT	butylated hydroxytoulene	M F	modulations of the emission
	BSA	bovine serum albumin	MTT	3-[4, 5-dimethylthiazol-2-yl]-2, 5-
	CO	cholesteryl oleate		diphenyltetrazolium bromide
	cryo-EM	cryo-electron microscopy	OMM	outer mitochondrial membrane
	CSLM	confocal scanning laser	pBcl-2	phosphorylation of Bcl-2
		microscopy	PBS	phosphate saline buffer
	DAG	diacylglycerol	PC	egg yolk phosphatidyl choline
	D Ch	cholesterol modified dextran	PCD	programmed cell death
	DiOC 18 (3)	3,3′-dioctadecyloxacarbocyanine	PDT	photodynamic therapy
		perchlorate	PI	propidium iodide
	DLS	dynamic light scattering	PKC	protein kinase C
	D-MEM	Dulbecco's modified Eagle	PKCα	protein kinase C alpha
		medium	PKC	protein kinase C delta
	DMSO	dimethyl sulfoxide	PMA	phorbol myristate acetate
	DOPC	1,2-dioleoyl-sn-glycero-3-	PMT	photo-multiplyer (detector)
		phosphocholine	PS	phosphatydylserine
	DOPE	1,2-dioleoyl-sn-glycero-3-	pts	photosensitizer
		phosphoethanolamin	R H	hydrodynamic radius
	DOPE-Rhod B	1,2-dioleoyl-sn-glycero-3-	ROS	reactive
		phosphoethanolamine-N -
		lissamine rhodamine B sulfonyl
	FBS	fetal bovine serum
	FRET	Förster Resonant Energy
		Transfer
	GSH	glutathione
	HDL	high density lipoprotein
	HIV	immunodeficiency virus
	Hyp	hypericin
	LDL	low density lipoprotein
	LDLR	LDL receptor
	LUV	large unilamellar vesicles
	LUV-Chol	large unilamellar vesicles with

Half-times of the slow phase of Hyp incorporation into free HDL molecules .............................................................................................. Table 8: Co-localization analysis of pBcl-2(Ser70) in mitochondria, pBcl-2(Ser70) and GSH, and the presence of GSH in mitochondria. ........

oxygen species sLDL synthetic low density lipoprotein sLNP synthetic lipid-based nano- particles

	1 INTRODUCTION
	1.1 CANCER AND CANCER TREATMENT
	Cancer has been a huge problem for mankind for centuries. The earliest written
	record regarding cancer is from 3000 BC in the Egyptian Edwin Smith Papyrus and
	describes cancer of the breast. Hippocrates (ca. 460 BC-ca. 370 BC) described several
	kinds of cancer, referring to them with the Greek word carcinos (crab or crayfish). The
	Greek, Celsus (25 BC-50 AD) translated carcinos into the Latin cancer, also meaning
	crab and recommended surgery as treatment [1].
	TAG	triacylglycerol
	TNF	tumor necrosis factor
	TO	triolein
	UG	ultroser G
	VLDL	very low density lipoprotein
	τ M	modulation lifetime
	τ P	phase lifetime

Cancer is the world's major public health problem. One in 4 deaths in the United States is due to the cancer. In 2013, total predicted number of cancer deaths in the European Union was 1 314 236 and according to American Cancer Society a total of 1 660 290 new cancer cases and 580,350 cancer deaths were projected to occur in the United States in 2013

[START_REF] Malvezzi | European Cancer Mortality Predictions for the Year 2013[END_REF]

,

[START_REF] Siegel Rebecca | Cancer Statistics, 2013[END_REF]

.

As understanding of the cancer biological processes has increased the treatment of the cancer has undergone evolutional changes. Tumor removal surgeries have been documented in ancient Egypt, hormone therapy was developed in 1896, and radiation therapy was developed in 1899. Chemotherapy, immunotherapy, and newer targeted therapies are the products of the 20 th century. As new information about the biology of cancer emerges, treatments will be developed and modified to increase effectiveness, precision, survivability, and quality of life. Depending on the location, type and stage of cancer as well as the general state of the patient the proper treatment is proposed. There are several types of treatment: Surgery: in theory, non-hematological cancers can be cured if entirely removed by surgery, but this is not always possible. When the cancer has metastasized to other sites in the body prior to surgery, complete surgical excision is usually impossible.

Radiation therapy: use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy injures or destroys cells in the area being treated by damaging their genetic material. Although radiation damages both cancer cells and normal cells, most normal cells can recover from the effects of radiation and function properly. The goal of radiation therapy is to damage as many cancer cells as possible, while limiting harm to nearby healthy tissue. Radiation therapy may be used to treat almost every type of solid tumor or soft tissue sarcomas.

Table 1 : Photosensitizers and their precursors used in experimental and clinical PDT applications

 1

	[source: [12]]

1.3.1 PHYSICO-CHEMICAL AND PHOTODYNAMIC PROPERTIES OF HYP

	solutions, organic bases such as pyridine, and polar organic substances including
	acetone, ethanol, dimethyl sulfoxide (DMSO), methanol, ethyl acetate, ethyl methyl
	ketone and other solvents. In most organic solvents Hyp forms a red solution and green
	solution in basic media. Hyp can be soluble in biological media by formation of
	complex with biological macromolecules (e.g. human serum albumin and other plasma
	Hyp (C 30 H 16 O 8 )-1,3,4,6,8,13-hexahydroxy-10,11-dimethylphenanthro[1,10,9,8-
	opqra]perylene-7,14-dione (Figure 2A) is hydrophobic molecule insoluble in water, oil,
	methylene chloride and most other nonpolar solvents. It is soluble in alkaline aqueous

  The results obtained by Wilkins et al. suggest that Bcl-2 plays a central role in the regulation of mitochondrial GSH transport through its interaction with 2oxoglutarate GSH carrier and maintenance of the mitochondrial glutathione pool[START_REF] Wilkins | Bcl-2 is a novel interacting partner for the 2oxoglutarate carrier and a key regulator of mitochondrial glutathione[END_REF].

	It was shown that Hyp and phorbol 12-myristate-13-acetate (PMA; PKC
	activator) might competitively bind to the regulatory C1B domain of PKC. Furthermore

[START_REF] Circu | Glutathione and apoptosis[END_REF]

. Synthesis of GSH occurs in cytoplasm, and originated GSH must be actively transported into mitochondria.

Table 4 : Efficiency of covering of dextrans with different cholesterol moiety

 4

		Ch 1	D Ch 2	D Ch 3
	1:5	15,8	3	14,4
	1:50	18,5	0,5	21,1
	1:500	6,8	7,6	3,9
	1:1000	19,2	12,8	12.9

Table 6 : Size of sLDL particles with different [sLDL-D Ch 3] ratios

 6

	:15	89,5
	1:50	128,6
	1:100	157,96
	1:500	122,5
	1:1000	148,5

Table 7 : Half-times of the slow phase of Hyp incorporation into free HDL molecules

 7

  Hyp from LDL, Hyp is translocated from HDL-Hyp complexes to free HDL molecules faster than from LDL-Hyp complexes to free LDL. Molecules of LDL contain more cholesterol than molecules of HDL which can cause the difference in the kinetics of the redistribution.

	E EF FF FE EC CT T O OF F P PK KC C  E EX XP PR RE ES SS SI IO ON N O ON N B BC CL L--2 2 P PH HO OS SP PH HO OR RY YL LA AT TI IO ON N
	A AN ND D C CE EL LL L D DE EA AT TH H B BY Y H HY YP PE ER RI IC CI IN N

  The distribution of pBcl-2(Ser70), glutathione and mitochondria was detected by confocal microscopy following vital cell staining with ThiolTracker™ Violet and appropriate MitoTracker (Green or Red) and indirect immunofluorescence staining with 5H2 antibody against Bcl-2 phosphorylated on Ser70.

		pBcl-2(Ser70)	ThiolTracker™Violet	ThiolTracker™Violet_
		MitoTracker®Orange	pBcl-2(Ser70)	MitoTracker®Orange
		overlap	M1	M2	overlap	M1	M2	overlap	M1	M2
	control	0,83	0,62	0,99	0,77	0,17	0,99	0,75	0,37	0,96
	5x10 -7 M Hyp	0,77	0,51	0,91	0,68	0,65	0,88	0,65	0,34	0,98
	5x10 -7 M Hyp/PKCα -0,63	0,55	0,75	0,65	0,41	0,73	0,87	0,65	0,88

Table 8 : Co-localization analysis of pBcl-2(Ser70) in mitochondria, pBcl-2(Ser70) and GSH, and the presence of GSH in mitochondria.

 8 Co-localization analysis was performed on the obtained fluorescence images using the ZEN 2011 image processing (Zeiss, Germany). Co-localization coefficients M 1 ; M 2 represents a relative number of colocalized pixels in channel 1 or 2 as compared to the total number of pixels about thresholds (min. value 0: no co-localization, max value 1: all pixels co-localized). Co-localization was calculated only from the cytoplasmic region of measured cells, while fluorescence signal from the nucleus was disregarded.

Modification of the degree of branching may allow for encapsulation of a molecule within this structure. [START_REF] Faraji | Nanoparticles in Cellular Drug Delivery[END_REF].

LIPOPROTEINS

Lipoproteins consist of noncovalently associated lipids and proteins which function in the blood plasma as transport vehicles for triacylglycerols (TAGs) and cholesterol. Lipids, such as phospholipids, TAG, and cholesterol, are sparingly soluble in aqueous solution. Hence, the globular micelle like particles-droplets of TAGs and cholesteryl esters coated with a monolayer of phospholipids, cholesterol, and apolipoproteins transport these lipids by the circulation as components of lipoproteins.

Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties (Table 2, Figure 8) [START_REF] Voet | Biochemistry[END_REF]. [source [START_REF] Jairam | Pathophysiology of Lipoprotein Oxidation, Lipoproteins-Role in Health and Diseases[END_REF]]

KINETICS OF HYP REDISTRIBUTION FROM HDL-HYP

COMPLEXES

Similarly as for transport of Hyp molecules from saturated complex LDL-Hyp to free LDL molecules [START_REF] Buriankova | Kinetics of Hypericin Association with Low-Density Lipoproteins[END_REF], the translocation of Hyp molecules from saturated HDL-Hyp =1:70 complex to free HDL particles was studied. This investigation is possible due to the fact that this translocation leads to a lower number of Hyp molecules per one lipoprotein particle which consequently leads to appearance of Hyp fluorescence. The

T TH HE E E EF FF FE EC CT T O OF F P PK KC C  O ON N H HY YP PE ER RI IC CI IN N P PH HO OT TO O--I IN ND DU UC CE ED D C CE EL LL L D DE EA AT TH H

From the regulation point of view PKCδ has two faces. It is considered to be pro-apoptotic protein and plays a critical role in the regulation of various cellular processes, including cell proliferation, cell death, and tumor promotion but it is also important to recognize that PKCδ not only induces apoptosis, but can also function as an anti-apoptotic protein during receptor-initiated cell death and confer resistance to anticancer drugs. Furthermore, PKCδ is required for the survival of several cancers. The function of PKCδ depends on various factors, including its localization, tyrosine phosphorylation, and the presence of other pro-and anti-apoptoic signaling molecules.

[90].

In the next chapter the effect of PKCδ on the cell death pathway after pkcδ gene silencing and Hyp photo-activation was explored.

PKC GENE SILENCING

Like in the case of post-transcriptional silencing of pkcα gene in U87-MG cells siRNA PKCδ(h) was used for post-transcriptional silencing of pkcδ gene as well.

Distribution of PKC was monitored by fluorescence microscopy on cells which were not treated with siRNA (control cells) and transfected PKCδ -cells. Fluorescence intensity of primary antibody for PKC (pSer645) stained with Alexa Fluor® 546 is lower in cells where expression of pkcgene was silenced (Figure 58

A AT TT TA AC CH HM ME EN NT TS S