Vincent Rincheval
email: vincent.rincheval@uvsq.fr

Marie Bergeaud

Lise Mathieu

Jacqueline Leroy

Arnaud Guillaume

Bernard Mignotte

Le Floch

J.-L Vayssière

Keywords: Reactive oxygen species, cell death, Mitochondria, Bcl-2, caspases, antioxidants mitochondrial membrane potential, eto, etoposide, E/TNF, emetine plus TNF-, MMP, mitochondrial membranes permeabilization, PI, propidium iodide, ROS, reactive oxygen species, t-BHP, tert-butyl hydroperoxide, TNF, tumor necrosis factor, Vit E, vitamin

Differential effects of Bcl-2 and caspases on mitochondrial permeabilization during endogenous or exogenous reactive oxygen species-induced cell death

Introduction

Reactive oxygen species (ROS) are a family of highly reactive molecules that includes hydroxyl radical (HO . ), superoxide anion (O 2 .-), hydrogen peroxide (H 2 O 2 ) and organic peroxide radicals. Under physiological conditions, the mitochondrial respiratory chain is the major site for ROS production in cells [START_REF] Fleury | Mitochondrial reactive oxygen species in cell death signaling[END_REF]. ROS are extremely transient species due to their high chemical reactivity that is responsible for their destructiveness on DNA, proteins, carbohydrates and lipids. It is now admitted that ROS are important mediators of several types of cell death such as apoptosis and necrosis. Apoptosis is mediated by a family of cystein proteases known as caspases. In mammals, there are two main pathways by which caspase activation is triggered: the intrinsic and extrinsic apoptotic pathways. Various signals that trigger the intrinsic pathway (such as oxidative or genotoxic stress) are mainly transduced to the mitochondria which then undergo a series of biochemical events resulting in the mitochondrial membranes permeabilization (MMP) [START_REF] Jourdain | Mitochondrial outer-membrane permeabilization and remodelling in apoptosis[END_REF][START_REF] Kroemer | Mitochondrial membrane permeabilization in cell death[END_REF]) and the release of pro-apoptotic molecules from mitochondria, such as cytochrome c [START_REF] Li | Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade[END_REF]. The extrinsic pathway is activated by the binding of ligands, such as FasL or TNF- to their receptors on the cell surface that can directly activate caspases [START_REF] Thorburn | Death receptor-induced cell killing[END_REF]). This pathway may also require the involvement of mitochondria, notably through the caspase-dependent production of the pro-apoptotic protein tBid [START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF].

The mitochondrial pathway is regulated by members of the Bcl-2 family, which includes both anti-apoptotic proteins (such as Bcl-2) and pro-apoptotic proteins (such as Bax) which respectively repress or stimulate MMP [START_REF] Desagher | Mitochondria as the central control point of apoptosis[END_REF][START_REF] Wang | The role of mitochondria in apoptosis[END_REF].

A ROS-dependent cell death can be triggered by classical apoptosis inducers such as etoposide and TNF-, which respectively trigger the intrinsic and the extrinsic pathways of cell death. We have previously shown in HeLa cells that TNF- and etoposide-induced apoptosis involves early mitochondrial ROS production that strongly accelerates the process of cell death [START_REF] Dumay | The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition[END_REF][START_REF] Sidoti-De Fraisse | TNF-alpha activates at least two apoptotic signaling cascades[END_REF]. Etoposide (eto) is a topoisomerase II inhibitor, which triggers the intrinsic caspase activation pathway, involving mitochondrial perturbations [START_REF] Karpinich | The course of etoposideinduced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c[END_REF], whereas TNF- is a cytokine that triggers the extrinsic caspase activation pathway but does not necessarily involve mitochondria [START_REF] Thorburn | Death receptor-induced cell killing[END_REF]. We have also used exogenous ROS or ROS generators to induce cell death in HeLa cells by incubating them with hydrogen peroxide (H 2 O 2 ), paraquat and tert-butyl hydroperoxide (t-BHP). Among ROS, H 2 O 2 represents a particularly important molecule because it is generated under nearly all oxidative stress conditions and it can participate in several fundamental intracellular processes. Paraquat is a non-selective herbicide which undergoes redox cycling in vivo, being reduced by an electron donor such as NADPH, before being oxidized by an electron receptor such as O 2 to produce superoxide. Next, t-BHP is a short chain analog of lipid hydroperoxides which mimics the toxic effect of peroxidized fatty acids.

In this study, we compared several features of cell death triggered by etoposide and TNF- versus the pro-oxidant molecules H 2 O 2 , t-BHP and paraquat. Our aim was to characterize the relationships that exist between ROS, mitochondrial perturbations, Bcl-2 and caspases, depending on the origin and identity of ROS.

Materials and methods

Reagents

-tocopherol (vitamin E), Butylated hydroxyanisole (BHA), catalase, diphenylene iodonium chloride (DPI), emetine (E), etoposide (eto), hydrogen peroxide solution (H 2 O 2 ), methyl viologen dichloride hydrate (paraquat), N-acetyl-cysteine (NAC), Noridihydroguaiaretic acid (NDGA), propidium iodide (PI), 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron), mouse tumour necrosis factor- (TNF-) and tert-butyl hydroperoxide solution (t-BHP) were from Sigma. 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and 3,3'diethyloxacarbocyanine (DiOC 6 (3)) were from Invitrogen. z-Val-Ala-DL-Asp-Fluoromethylketone (zVAD-fmk) and N-Ac-Asp-Glu-Val-Asp-CHO (DEVD-CHO) were from Bachem. Stock solutions were prepared as follows: NAC (50 mM), PI (1 mg/ml) and tiron (1 M) in water; catalase (10 5 U/ml), emetine (1 mg/ml), paraquat (100 mM), and TNF-g/ml) in serum free medium; BHA (200 mM), DCFH-DA (20 mM), DiOC 6 (3) (1 mM), NDGA (40 mM) and Vit E (100 mM) in ethanol; DPI (3 mM) and etoposide (25 mg/ml) in dimethyl sulfoxide (DMSO); zVAD-fmk (50 mM) and DEVD-CHO (100 mM) in methanol.

All stock solutions were stored at -20°C.

Cell lines, cell culture and induction of cell death HeLa and HeLa-Bcl-2 cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM/F12) supplemented with 10% fetal bovine serum together with penicillin (100 g/ml), streptomycin (100 U/ml) and glutamax (1% v/v) from Invitrogen. The HeLa-Bcl-2 cell line contains the human bcl-2 cDNA under control of the Tet Off inducible expression system (Clontech). These cells were initially cultured in the presence of tetracycline (2 g/ml) to inhibit the expression of exogenous Bcl-2 and then propagated for 10 days without tetracycline to allow the accumulation of the Bcl-2 protein prior the experiments. Cell death was induced by the addition on exponentially growing adherent cells of H 2 O 2 (200 M), paraquat (5 mM), t-BHP (200 M), etoposide (20 g/ml) or TNF- (2.5 ng/ml) plus emetine (1 g/ml). After a 24 h incubation, cells were analyzed by flow cytometry. Antioxidants (BHA (300 M), catalase (1000 units/ml), DPI (2.5 M), NAC (5 mM), NDGA (40 M), tiron (1 mM), Vit E (100 M)) or caspase inhibitors: DEVD-CHO (300 M), zVAD-fmk (50 M), were added at the same time than cell death inducers.

Flow cytometry

Flow cytometric measurements were performed using a XL3C flow cytometer (Beckman-Coulter). Fluorescence was induced by the blue line of an argon ion laser (488 nm) at 15 mW.

Green and red fluorescence were respectively collected with 525 nm and 620 nm band pass filters. Analyses were performed on 10 4 cells. The mitochondrial membrane potential (m) was assessed by the retention of the green fluorescent DiOC 6 (3). This cationic lipophilic fluorochrome is a cell permeable marker that, at low doses, specifically accumulates in mitochondria depending on the mitochondrial membrane potential [START_REF] Petit | Analysis of the membrane potential of ratand mouse-liver mitochondria by flow cytometry and possible applications[END_REF]).

Intracellular ROS level was measured by the production of dichlorofluorescein (DCF) derived from oxidization of DCFH. The non fluorescent DCFH-DA is a freely permeable tracer which is deacylated by intracellular esterases to the nonfluorescent compound DCFH and oxidized to the green fluorescent compound DCF by a variety of peroxides, including hydrogen peroxide [START_REF] Ubezio | Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells[END_REF]. The red fluorescent PI specifically penetrates in cells which have lost their plasma membrane integrity and was therefore used to characterize necrotic cells [START_REF] Darzynkiewicz | Assays of cell viability: discrimination of cells dying by apoptosis[END_REF]. Finally, the decrease in forward scatter that reflects cell shrinkage during the last steps of cell death was also assessed [START_REF] Dive | Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry[END_REF]). The percentage of non necrotic cell death was calculated as the percentage of cell shrinkage in the PI negative population. After drug treatment, the media from the culture dishes (containing late apoptotic cells) were kept in centrifuge tubes. The adherent cells (containing living and early apoptotic cells) were detached using trypsine and pooled with the corresponding media, centrifuged and resuspended in complete medium at a concentration of 1x10 6 cells/ml. Cells were then loaded with 0.1 M DiOC 6 (3) or 20 M DCFH-DA for 30 min at 37°C and incubated with 10 g/ml PI, 5 min prior analysis.

Statistical analysis

Statistical analysis was determined using Student's t-test. P<0.05 was accepted as significant.

Results

The apoptosis inducers etoposide and TNF-, the exogenous ROS H 2 O 2 and t-BHP, as well as the ROS generator paraquat all trigger intracellular ROS accumulation, mitochondrial membrane permeabilization and cell death

We have previously shown that etoposide and TNF- trigger endogenous mitochondrial ROS production in HeLa cells [START_REF] Dumay | The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition[END_REF][START_REF] Sidoti-De Fraisse | TNF-alpha activates at least two apoptotic signaling cascades[END_REF]) whereas H 2 O 2 , paraquat and t-BHP are often used as inducers of oxidative stress and cell death. TNF- used alone is generally not sufficient to kill cultured cells since the interaction between TNF- and its receptors induces antagonistic signals: the formation of the Death Inducing Signaling Complex (DISC), which rapidly activates caspases, and the expression of endogenous caspase inhibitors (IAPs) through the activation of NF-B [START_REF] Nagata | Apoptosis by death factor[END_REF]). Thus, TNF- is currently used in vitro in combination with a protein synthesis inhibitor such as emetine (E) which is able to inhibit the NF-B-dependent expression of caspase inhibitors, without interfering with DISC formation.

Since we wanted to test the influence of ROS on mitochondrial perturbations, we started to determine concentrations of the different compounds (etoposide (eto), emetine plus TNF- (E/TNF), H 2 O 2 , paraquat and t-BHP) that induce a significant drop in m. Fig. 1A shows that after a 24h incubation (concentrations indicated in figure legend), approximately one half of the cells exhibits a low m, indicating that they have undergone a permeabilization of their mitochondrial membranes (MMP). These conditions were considered as ideal to study inhibitory or promoting effects on MMP. Next, we have measured the percentage of cells harboring high ROS levels, corresponding to the cells undergoing oxidative stress. Fig. 1B indicates that etoposide, E/TNF and paraquat can trigger the accumulation of endogenous ROS and that the exogenous H 2 O 2 and t-BHP also provoke an efficient, but probably more direct, oxidative stress. The accumulation of ROS was measured by using DCFH-DA, a probe which becomes fluorescent after reacting with peroxides. Since peroxides are classical by-products of oxidant molecules, DCFH-DA is a good probe to detect oxidative stress from different origins. However, it must be kept in mind that its modification requires a deacetylation process prior oxidation, which cannot be performed in late dead cells with inactive esterases, such as in population (c) (see Fig. 1B). Thus, cells undergoing oxidative stress are always underestimated with this probe when cell death occurs. Next, we were also interested in the events that could occur downstream of mitochondrial perturbations (such as cell death) and we have characterized two additional parameters by flow cytometry.

The first parameter is the percentage of propidium iodide (PI) positive / necrotic cells (Fig. 1C, population (c)). The second one is the percentage of PI negative cells with reduced size (cell shrinkage) which indicates the percentage of late apoptotic (or autophagic) cell death (Fig 1C,population (b)). Our results show that all the inducers trigger both necrotic and non necrotic cell death. The fact that specific inducers of apoptosis (etoposide, E/TNF) also trigger necrosis may be surprising. However, it is well known that a secondary necrotic process provoked by the lack of engulfment of dying cells can occur in vitro.

Etoposide, E/TNF, H 2 O 2 , paraquat and t-BHP all induce a ROS dependent cell death which can be inhibited by several antioxidants Although all inducers seemed to trigger an oxidative stress, MMP and cell death (see Fig. 1), the pathways involved could be different from one inducer to another. Thus, we wanted to determine, in each case, what is the causal link between ROS accumulation and these events.

For this purpose, we have incubated cells with cell death inducers and antioxidants at the same time. Here, we have used 7 antioxidants with distinct properties (see materials and methods for a complete name of the compounds): BHA is a food additive (E320) which has also been shown to inhibit mitochondrial respiration [START_REF] Aldunate | Effects of 2(3)tert-butyl-4-hydroxyanisole (BHA) on in situ mitochondria of Trypanosoma cruzi[END_REF]; catalase is a well known enzyme which converts H 2 O 2 into H 2 O and O 2 ; DPI is a NADPH oxidase inhibitor [START_REF] Meier | Identification of a superoxidegenerating NADPH oxidase system in human fibroblasts[END_REF]; NAC is a precursor to glutathione, a natural antioxidant protein; NDGA is a cyclooxygenase and lipoxygenase inhibitor (Van Wauwe and Goossens 1983); tiron is a superoxide scavenger [START_REF] Greenstock | The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution in chloroplasts[END_REF] and vitamin E is an inhibitor of lipid peroxidation [START_REF] Tappel | Biological antioxidant protection against lipid peroxidation damage[END_REF]. We have quantified cell death (as the percentage of cell shrinkage) in presence or in absence of the antioxidant, and calculated the percentage of cell death inhibition. The results presented in Fig. 2 show that the effect of each inducer is inhibited by at least three different antioxidants, showing that cell death is tightly linked to oxidative stress. Some results were expected: catalase strongly inhibits H 2 O 2 -induced cell death; DPI strongly inhibits the effect of paraquat (mediated by NADPH oxidases); vitamin E inhibits toxicity induced by t-BHP (an analog of lipid hydroperoxides) and BHA inhibits the mitochondrial-dependent production of ROS induced by etoposide and E/TNF. Other results are more surprising and would deserve additional investigations. For instance, it is astonishing that tiron does not inhibit the effect of paraquat which is known to generate superoxide, whereas the effect of t-BHP, a molecule unrelated to superoxide, is strongly inhibited by tiron.

Further studies using other ROS-sensitive probes should help to clarify these results.

Influence of Bcl-2 on MMP, ROS accumulation and cell death

In order to test the effect of the anti-apoptotic protein Bcl-2 on the phenotypes triggered by the five inducers, we have used the HeLa-Bcl-2 cell line which can overexpress the human bcl-2 gene under control of tetracycline (Tet Off system from Clontech). With this system, the absence of tetracycline in the culture medium leads to the accumulation of Bcl-2. The results presented in Fig. 3A show that MMP is significantly inhibited in all cases except for paraquat, demonstrating that the mechanism of MMP triggered by paraquat is different from the other inducers. Next, we can see in Fig. 3B that Bcl-2 significantly inhibits the accumulation of cells with high ROS levels with H 2 O 2 , t-BHP, etoposide and E/TNF. This result is interesting for several reasons: first, since etoposide and E/TNF trigger the production of endogenous mitochondrial ROS (we have previously shown that this production is severely reduced in HeLa  0 cells, demonstrating their mitochondrial origin (Sidoti-de Fraisse et al. 1998)), it indicates that Bcl-2 can act on the mitochondrial production of ROS. In this case, Bcl-2 may directly or indirectly regulate the mitochondrial respiration chain. Second, the fact that Bcl-2 inhibits oxidative stress after exposition to exogenous ROS suggests that Bcl-2 can also act as an antioxidant. Such functions for Bcl-2 have already been described but are still poorly understood [START_REF] Hockenbery | Bcl-2 functions in an antioxidant pathway to prevent apoptosis[END_REF][START_REF] Vrbacky | Inhibitory effects of Bcl-2 on mitochondrial respiration[END_REF]. Finally, Bcl-2 does not seem to inhibit ROS accumulation induced by paraquat. Thus, it is possible that Bcl-2 negatively regulates mitochondrial superoxide production and/or exogenous peroxides accumulation while being unable to act on cytosolic superoxide, produced by enzymes such as NADPH oxidase. Concerning the regulation of cell death, it appears from Fig. 3C that Bcl-2 is both an inhibitor of necrotic and non necrotic cell death, except for paraquat-induced cell death which is apparently totally disconnected from any Bcl-2 regulation. We can hypothesize that Bcl-2 protects against H 2 O 2 , t-BHP, etoposide and E/TNF-induced necrosis because it is a secondary necrotic process (due to a lack of engulfment of dying cells), while necrosis observed during paraquat-induced cell death would be primary necrosis.

Influence of caspase inhibition on MMP, ROS accumulation and cell death

Here we have used two chemical inhibitors to determine the role of initiator and executive caspases: zVAD (Z) which is an inhibitor of both initiator and executive caspases as well as DEVD (D) which is a specific inhibitor of the executive caspases -3 and -7. Fig. 4A shows that zVAD significantly decreases MMP during H 2 O 2 , etoposide and E/TNF-induced cell death, indicating that caspases regulate MMP, acting upstream of or at mitochondria. The effect of DEVD on etoposide and E/TNF is significant (p<0.05), whereas it is nearly significant for H 2 O 2 (p=0.059). Concerning paraquat and t-BHP-induced cell death, it appears that MMP is not influenced by the presence of zVAD or DEVD, demonstrating that this process is caspase-independent and necessarily distinct from the process observed for H 2 O 2 , etoposide and E/TNF. Caspase-inhibitors were also found to influence the accumulation of cells with high ROS levels (Fig. 4B) for H 2 O 2 , etoposide and E/TNF. zVAD totally inhibits ROS accumulation with etoposide and E/TNF, suggesting that endogenous ROS production first requires the activation of caspases. The milder effect of DEVD was found to be significant for etoposide but not for E/TNF (p=0.067). The fact that zVAD inhibits endogenous ROS production in E/TNF cell death was expected since DISC formation and activation of initiator caspases is one on the first events following the interaction between TNF- and its receptors. The results that were found for etoposide also suggest that a caspase, acting upstream of or at mitochondria, regulates ROS accumulation and MMP. Concerning H 2 O 2 , the fact that zVAD and DEVD significantly increase the percentage of cells with high ROS levels is even more surprising. One possibility is that caspase-inhibition would increase the resistance of the cells to (exogenous) H 2 O 2 , therefore delaying their disappearance. This result would necessarily be different from etoposide and TNF- where zVAD and DEVD also inhibit (endogenous) ROS accumulation. In contrast to H 2 O 2 , etoposide and E/TNF, caspase inhibitors do not modify MMP or ROS accumulation during paraquat or t-BHP treatments, indicating that these processes are caspase-independent. Next, Fig. 4C shows that caspase inhibitors attenuate necrotic cell death for H 2 O 2 , etoposide and E/TNF (but not for paraquat and t-BHP), suggesting that secondary necrosis occurs with these three inducers. The caspase inhibitors also diminish non necrotic cell death for all the inducers except paraquat. In conclusion, these results indicate that paraquat-induced cell death is caspase-independent, and that t-BHP-induced necrosis is caspase-independent whereas it was Bcl-2-regulated. Since some studies report that Bcl-2 can inhibit necrosis [START_REF] Guenal | Bcl-2 and Hsp27 act at different levels to suppress programmed cell death[END_REF][START_REF] Kane | Expression of bcl-2 inhibits necrotic neural cell death[END_REF], t-BHP may also induce primary necrosis.

Discussion

Here we have shown that H 2 O 2 , paraquat, t-BHP, etoposide and E/TNF are all able to induce a ROS-dependent MMP and cell death, but that the mechanisms underlying these effects can be different from one inducer to another. Etoposide and E/TNF induce the most similar phenotypes despite the fact that they have quite different targets. For both inducers, our results suggest that a caspase activity is involved upstream of mitochondrial ROS production and MMP since zVAD totally inhibits these processes (Fig. 4). While this is not surprising for E/TNF, because caspases are activated very early during the intrinsic pathway of apoptosis, this result was unexpected for etoposide. However, we have previously shown that zVAD inhibits cytochrome c release in HeLa cells treated with E/TNF whereas it is not the case for etoposide [START_REF] Dumay | The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition[END_REF]. This suggests that contrary to E/TNF, a first wave of cytochrome c release would occur before ROS overproduction and MMP during etoposideinduced cell death. A feedback loop linking caspases to mitochondria would then amplify mitochondrial perturbations [START_REF] Chen | Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis[END_REF]. This feedback loop can be evidenced for both E/TNF and etoposide by the effect of DEVD on MMP and ROS accumulation (Fig. 4).

H 2 O 2 is the inducer that triggers the closest phenotype to etoposide and E/TNF, suggesting that this molecule could be an important mediator of etoposide and E/TNF-induced cell death. Interestingly, it can be noticed that contrary to etoposide and E/TNF, zVAD cannot totally inhibit the drop of m induced by H 2 O 2 (Fig. 4), suggesting that H 2 O 2 can trigger MMP prior caspase activation. In this case, the effect of zVAD would be associated with the inhibition of the caspase-dependent feedback loop on mitochondria. Next, t-BHP, which is also a peroxide, mainly differs from H 2 O 2 by the fact that it triggers a completely caspaseindependent m decrease (Fig. 4). The mechanisms involved in mitochondrial membranes permeabilization is still controversial and two main models have been proposed, which may both exist, depending on death signals and the cellular context [START_REF] Desagher | Mitochondria as the central control point of apoptosis[END_REF][START_REF] Kroemer | Mitochondrial membrane permeabilization in cell death[END_REF]). The first type of models relies on the formation in the outer mitochondrial membrane of large channels constituted of pro-apoptotic members of the Bcl-2 family (such as Bax) which leads to the release of intermembrane space proteins including cytochrome c whose depletion leads to m loss. Alternatively, a deregulation of the permeability transition pore complex (PTP) located at the outer/inner membrane junctions and constituted of the porin VDAC and the adenine nucleotide translocase (ANT), can also lead to m loss and outer membrane permeabilization. Thus it is possible that the regulation of MMP between H 2 O 2 (Bcl-2 and caspase-dependent), t-BHP (Bcl-2-dependent and caspaseindependent) and paraquat (Bcl-2 and caspase-independent) involves the formation of distinct pores having different sensitivities to caspases and Bcl-2. Interestingly, it has been shown in HepG2 cells that t-BHP triggers an hyperpolarization preceding a loss of m, a result which is reminiscent of a model that involves VDAC closure and swelling of mitochondria [START_REF] Desagher | Mitochondria as the central control point of apoptosis[END_REF]. Little is known about the influence of paraquat on MMP [START_REF] Costantini | On the effects of paraquat on isolated mitochondria. Evidence that paraquat causes opening of the cyclosporin A-sensitive permeability transition pore synergistically with nitric oxide[END_REF][START_REF] Palmeira | Mitochondrial bioenergetics is affected by the herbicide paraquat[END_REF]) whereas etoposide-and E/TNF-induced MMP seems to involve pro-apoptotic members of the Bcl-2 family [START_REF] Dumay | The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition[END_REF][START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF].

Finally, we have shown that H 2 O 2 , paraquat, t-BHP, etoposide and E/TNF all trigger necrotic and non necrotic cell death. While it seems that H 2 O 2 , etoposide and E/TNF trigger secondary necrosis, it would rather be a primary necrosis for t-BHP and paraquat. Surprisingly, paraquat is able to induce a caspase-independent and non necrotic cell death in approximately half of the treated cells, as assessed by the percentage of non necrotic cells undergoing cell shrinkage (Fig. 1). It would be interesting to understand what type of alternative cell death is induced by paraquat and how its execution can be managed independently from caspases. The absence of a bar for several cell death inducer / antioxidant combinations indicates that no protection was found in these particular cases.

Legends to figures

Title:

  Differential effects of Bcl-2 and caspases on mitochondrial permeabilization during endogenous or exogenous reactive oxygen species induced cell death Subtitle: A comparative study of H 2 O 2 , paraquat, t-BHP, etoposide and TNF- induced cell death Vincent Rincheval * , Marie Bergeaud, Lise Mathieu, Jacqueline Leroy, Arnaud Guillaume, Bernard Mignotte, Nathalie Le Floch and Jean-Luc Vayssière Université de Versailles St Quentin-en-Yvelines, EA4589, Laboratoire de génétique et biologie cellulaire / Ecole Pratique des Hautes Etudes : Laboratoire de génétique moléculaire et physiologique, 45 avenue des Etats-Unis, 78035 Versailles cedex, France.

Fig. 1

 1 Fig. 1 Effects of apoptosis inducers and exogenous ROS on (A) mitochondrial membrane

Fig. 2

 2 Fig. 2 Inhibition of etoposide, E/TNF, H 2 O 2 , paraquat and t-BHP-induced cell death by

Fig. 3

 3 Fig. 3 Regulation of etoposide, E/TNF, H 2 O 2 , paraquat and t-BHP-induced cell death by Bcl-

Fig. 4

 4 Fig. 4 Inhibition of etoposide, E/TNF, H 2 O 2 , paraquat and t-BHP-induced cell death by

  Figure 1

Acknowledgments

This work was supported in part by grants from the Ligue Nationale Contre le Cancer (comité des Yvelines).


Cécile Milet
email: cecile.milet@uvsq.fr

Aurore Rincheval-Arnold

Bernard Mignotte
email: bernard.mignotte@uvsq.fr

Isabelle Guénal
email: isabelle.guenal@uvsq.fr

The Drosophila retinoblastoma protein induces apoptosis in proliferating but not in postmitotic cells

Keywords: Retinoblastoma protein, RBF, Drosophila, apoptosis, cell cycle, cell-type specific 3

HAL is

Introduction

The retinoblastoma protein (pRb) was the first tumor suppressor identified in human cells, and pRB loss of function has been linked to the development of many cancers. pRb is a major element in cell cycle regulation: it exerts its functions principally by interacting with the E2F transcription factor family members, which regulate key cellular events, including DNA synthesis and cell cycle progression (for a review see 1). Binding and subsequent inactivation of E2Fs factors by pRb promotes G1 arrest. Cyclin-dependent kinase (CDK)-mediated phosphorylation of pRb disrupts pRb/E2Fs interactions, which allows E2Fs activation and subsequent GI/S progression. pRb is conserved between mammals and flies. A Drosophila pRb homologue, RBF, has been identified [START_REF] Du | RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila[END_REF] and it has been shown that, like its mammalian counterpart, RBF interacts with Drosophila E2F family members and can be regulated during cell cycle progression by CDKs. [START_REF] Du | RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila[END_REF][START_REF] Stevaux | Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2[END_REF][START_REF] Xin | The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth[END_REF] Although pRb function in cell cycle regulation is well established, its effects on apoptosis remain poorly understood. Indeed, numerous studies using different cellular contexts have led to conflicting results. Knock-out mouse embryos for the Rb gene die before day 16 of development, and show ectopic proliferation and extended apoptosis in many tissues. [START_REF] Lee | Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis[END_REF][START_REF] Jacks | Effects of an Rb mutation in the mouse[END_REF] However, it is now clear that most apoptosis observed in Rb -/-embryos is due to abnormal formation of extraembryonic tissues, [START_REF] De Bruin | Rb function in extraembryonic lineages suppresses apoptosis in the CNS of Rbdeficient mice[END_REF] and is therefore an indirect consequence of Rb loss of function. More recent studies in mice have also shown that tissue-specific knock-outs of the Rb gene induce ectopic proliferation but do not have any effect on apoptosis in many proliferative tissues (lung, skin, intestine). [START_REF] Mason-Richie | Retinoblastoma function is essential for establishing lung epithelial quiescence after injury[END_REF][9][START_REF] Wang | The retinoblastoma protein, RB, is required for gastrointestinal endocrine cells to exit the cell cycle, but not for hormone expression[END_REF] Thus, in proliferating tissues in vivo, pRb does not appear to regulate apoptosis. In other cellular contexts, as in cultured mouse embryonic fibroblasts, [START_REF] Almasan | Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis[END_REF][START_REF] Knudsen | RB-dependent S-phase response to DNA damage[END_REF] or in differentiating cells like lens [START_REF] De Bruin | Rb function in extraembryonic lineages suppresses apoptosis in the CNS of Rbdeficient mice[END_REF] and myoblasts, [START_REF] Huh | Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation[END_REF] Rb loss of function induces apoptosis cell-autonomously. This observed apoptosis is thought to be an indirect consequence of inappropriate progression of the cell cycle. In the case of differentiating cells, absence of cell cycle arrest impairs proper differentiation and leads to cell death. Like in mammals, Drosophila RBF loss of function is lethal during development. [START_REF] Du | The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis[END_REF] RBF loss of function clones in proliferative tissues are prone to apoptosis and contain some ectopic S phase cells, both in wing [START_REF] Duman-Scheel | Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin[END_REF][START_REF] Moon | Drosophila E2F1 has context-specific pro-and antiapoptotic properties during development[END_REF] and in eye imaginal discs, [START_REF] Moon | A gradient of epidermal growth factor receptor signaling determines the sensitivity of rbf1 mutant cells to E2F-dependent apoptosis[END_REF][START_REF] Firth | Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye[END_REF] the larval tissues giving rise to adult structures. These results support the view that, as in mammals, Drosophila RBF is required for cell cycle arrest in G1 phase and that the apoptosis observed could be due to cell cycle deregulation.

In spite of these corroborating results concerning pRb anti-apoptotic properties, numerous studies carried out on human cancer cell lines have raised the possibility that pRb can also be pro-apoptotic in many cellular contexts, [START_REF] Bowen | Radiation-induced apoptosis mediated by retinoblastoma protein[END_REF][START_REF] Araki | Retinoblastoma RB94 enhances radiation treatment of head and neck squamous cell carcinoma[END_REF][START_REF] Hsieh | RB regulates the stability and the apoptotic function of p53 via MDM2[END_REF][START_REF] Knudsen | The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death[END_REF][START_REF] Zhao | RB activation and repression of C-MYC transcription precede apoptosis of human prostate epithelial cells[END_REF][START_REF] Lemaire | Caspase-9 can antagonize p53-induced apoptosis by generating a p76(Rb) truncated form of Rb[END_REF] which is in agreement with its described role as a tumor suppressor.

Furthermore, it has been shown recently that pRb can associate together with E2F1 and bind promoters of genes encoding pro-apoptotic factors that are transcriptionally activated in response to genotoxic or oncogenic stress. [START_REF] Ianari | Proapoptotic function of the retinoblastoma tumor suppressor protein[END_REF] In Drosophila, even if loss of function experiments suggest an anti-apoptotic role for RBF, the effects of RBF expression on apoptosis has not been studied in detail. Indeed, in many cases, like in some mammalian cells, observed tissue size reduction resulting from RBF expression could also be explained by induction of apoptosis, and not only by cell cycle inhibition as has been the preferred interpretation until now. [START_REF] Xin | The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth[END_REF][START_REF] Datar | The Drosophila cyclin D-Cdk4 complex promotes cellular growth[END_REF][START_REF] Weng | Critical role of active repression by E2F and Rb proteins in endoreplication during Drosophila development[END_REF] The variety of results concerning pRb pro-or anti-apoptotic effects could be a consequence of the complex role of this protein in many cellular events, such as cell cycle, differentiation and apoptosis.

Therefore, the cellular response to pRb gain or loss of function could depend to a great extent on the proliferation and differentiation context of the cells. We took advantage of the powerful genetic tools developed in Drosophila to better characterize the effects of Drosophila RBF on apoptosis in vivo, taking into account the proliferation and differentiation status of the cells. Our results provide evidence for a dual role for RBF in apoptotic processes. Indeed, in proliferative cells of wing and eye imaginal discs, RBF induces apoptosis in a caspase-dependent manner, while it does not in differentiated post-mitotic cells. In contrast, we show that in these latter cells, RBF is able to suppress apoptosis induced by Dmp53, the drosophila homolog of p53.

Results

RBF is pro-apoptotic only in proliferative tissues

In order to test the effects of RBF expression in different cellular contexts, we took advantage of an inducible transgenic UAS Drosophila strain to precisely choose the expression domain of RBF. To induce UAS-RBF expression in a proliferating tissue, we used the vg-Gal4 driver, which is expressed in wing imaginal discs of third instar larvae, a period of intensive growth. When RBF is expressed in this tissue, resulting adult wings present notches along their margins (Fig . This particular zone corresponds to the zone of non-proliferating cells (ZNC), where cells are arrested in G1 and G2 phases of the cell cycle. Furthermore, RBF expression in this zone using the C96-Gal4 driver did not induce apoptosis (data not shown). These observations suggest that non-cycling cells are more resistant to RBF-induced apoptosis. Thus, RBF expression induces apoptosis, preferentially in proliferating cells.

In addition to apoptosis, the reduction of the size of the tissues could also result from a reduction of cell size. We observed that RBF expression did not reduce cell size neither in wing and eye imaginal discs nor in adult wing (Suppl. Fig. 1). Thus this hypothesis can be excluded. In addition, it has been shown that RBF expression in the wing disc slows cell cycle progression. [START_REF] Neufeld | Coordination of growth and cell division in the Drosophila wing[END_REF] We cannot exclude that, in proliferative tissues, this cell cycle lengthening could have a cumulative effect with apoptosis on the observed deficit in cells in larval and adult structures.

This observed cell death could seem contradictory with the prevalent view that RBF has anti-apoptotic effects, according to loss of function experiments described elsewhere. [START_REF] Duman-Scheel | Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin[END_REF][START_REF] Moon | Drosophila E2F1 has context-specific pro-and antiapoptotic properties during development[END_REF][START_REF] Moon | A gradient of epidermal growth factor receptor signaling determines the sensitivity of rbf1 mutant cells to E2F-dependent apoptosis[END_REF][START_REF] Firth | Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye[END_REF] According to the existing literature, the apoptotic properties of pRb depend on the cellular type, and probably on the cycling and differentiation states of the cells. We thus tested if RBF expression could also induce apoptosis in post-mitotic cells. To do so, we used the pan-neural elav-Gal4 driver. To specifically observe neurons we focused on the adult eye, an organ principally composed of photoreceptors which are easy to ). We increased the breeding temperature of the elav-Gal/+;UAS-RBF/+ flies to 29°C to enhance the UAS-RBF expression rate. Even under such extreme conditions, eye and photoreceptor phenotypes were wild type (data not shown). The elav-Gal4/+;UAS-RBF/+ eye imaginal discs were also stained by TUNEL and they showed a similar low amount of apoptosis as that in control elav-Gal4/+ discs (Fig 1 K,N). Thus, RBF expression does not exert any pro-apoptotic activity in these post-mitotic cells, which supports the view that RBF-induced cell death is linked to cell cycle deregulation in proliferative tissues that does not occur in post-mitotic cells.

Caspase activity, is required for RBF-induced apoptosis

In order to test if apoptosis observed when RBF is expressed in proliferating imaginal disc cells depends on caspase activity, the baculovirus p35 caspase inhibitor protein [START_REF] Hay | Expression of baculovirus P35 prevents cell death in Drosophila[END_REF] was induced in wing discs expressing UAS-RBF under control of vg-Gal4. In the wing, the number of notches present at the margin is correlated with the amount of apoptosis. [START_REF] Gaumer | Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila[END_REF] We classified the wing phenotypes into four The apical caspase Dronc plays an important role in developmental and stress-induced apoptosis in Drosophila, [START_REF] Quinn | An essential role for the caspase dronc in developmentally programmed cell death in Drosophila[END_REF] and this caspase is not inhibited by p35. [START_REF] Meier | The Drosophila caspase DRONC is regulated by DIAP1[END_REF] Thus, to test if Dronc is necessary for RBFinduced apoptosis, we used a dronc null mutant allele, dronc I29 , [START_REF] Xu | The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila[END_REF] to assess the strength of the RBF-induced notch phenotype in a dronc heterozygous mutant context (Fig 2 C). As in the UAS-p35 expression context, the phenotype induced by RBF in the wings was significantly weaker in a dronc mutant context (Wilcoxon test, =4.7E-07, n=254). Thus, both effector caspases inhibited by p35 and the apical caspase Dronc are required for RBF-induced apoptosis in the wing.

dE2F1 does not cooperate with RBF to induce apoptosis

The dE2F1 transcription factor is the best characterized target of RBF. When bound to its co-factor dDP, the principal function of dE2F1 is to activate the transcription of major cell-cycle related genes, such as cyclin E, and this activity is inhibited by RBF binding. [START_REF] Van Den Heuvel | Conserved functions of the pRB and E2F families[END_REF] Therefore, RBF and dE2F1 have antagonistic activities in the regulation of the cell cycle. Moreover, dE2F1 overexpression induces apoptosis in Drosophila imaginal discs. [START_REF] Asano | Ectopic E2F expression induces S phase and apoptosis in Drosophila imaginal discs[END_REF][START_REF] Du | Ectopic expression of dE2F and dDP induces cell proliferation and death in the Drosophila eye[END_REF] We thus wondered if dE2F1 cooperates with RBF to induce apoptosis as it has been reported in mammalian cells, [START_REF] Ianari | Proapoptotic function of the retinoblastoma tumor suppressor protein[END_REF] or on the contrary, if it counteracts the effects of RBF as it has been observed in cell cycle regulation. To answer this question, we compared These results therefore show that RBF and dE2F1 have opposite activities and that dE2F1 does not cooperate with RBF to induce cell death in Drosophila wing imaginal discs.

RBF has an anti-apoptotic effect on Dmp53-induced cell death in neurons

As presented above, our results indicate that despite of its pro-apoptotic effects in proliferating cells, RBF alone is not able to induce apoptosis in differentiated cells. We wondered if RBF did not have any influence on apoptotic processes in post-mitotic cells, or if it could enhance apoptosis induced by expression of other pro-apoptotic genes in these cells. We used the UAS-Dmp53 transgene which is known to induce apoptosis when driven in the developing eye disc. [START_REF] Jin | Identification and characterization of a p53 homologue in Drosophila melanogaster[END_REF] Altogether, our results clearly show that Drosophila RBF can be either pro-or anti-apoptotic in vivo, and that the balance between these opposite effects strongly depends on the proliferation and differentiation status of the cells.

Discussion

In this study, we show that RBF expression can be pro-apoptotic in Drosophila in a caspase-dependent manner. The capacity of RBF to be pro-apoptotic depends on the proliferation status of the cells in which it is expressed, as it is observed in proliferating tissues but not in differentiated post-mitotic cells. Non-cell-autonomous mechanisms could explain these results. In this case, RBF-induced effects could be in conflict with proliferating signals sent by surrounding cells. Indeed, in cells of proliferating imaginal discs, RBF expression leads to a lengthening of cell cycle progression, [START_REF] Neufeld | Coordination of growth and cell division in the Drosophila wing[END_REF] whereas, at the same time, these cells receive proliferation signals from the surrounding tissue. These conflicting signals could lead to cell death. In addition, it has been shown that cells with a proliferation disadvantage compared to their neighbors can be eliminated from a tissue by a mechanism known as cell competition. [START_REF] Morata | Minutes: mutants of drosophila autonomously affecting cell division rate[END_REF] It is possible that this process contributes to RBF-induced cell death, even if the effects of RBF expression have never been shown yet to conform to a stringent definition of cell competition. Indeed, according to Moreno, 38 cells carrying a mutation that triggers cell competition are viable when surrounded by cells of the same genotype.

On the other hand, RBF-induced apoptosis could also result from a direct effect of RBF on the cell cycle. In this respect, we have shown that dE2F1/dDP complex has an antagonist effect on RBFinduced cell death. Indeed, our results indicate that dE2F1/dDP overexpression prevents RBF-induced apoptosis, while dE2F1 loss of function enhances this phenotype. These results corroborate with previous reports concerning dE2F1/dDP and RBF interactions. Indeed, dE2F1/dDP and RBF have opposite effects in cell cycle regulation. Taken together, these data support the view that RBF-induced cell death in proliferating tissues is probably a consequence of cell cycle deregulation. In this case, dE2F1/dDP counters the effects of RBF overexpression on cell cycle control, and therefore decreases subsequent apoptosis. However, a recent study performed in mouse has shown that dE2F and pRb can cooperate to induce the transcription of pro-apoptotic factors, [START_REF] Ianari | Proapoptotic function of the retinoblastoma tumor suppressor protein[END_REF] which seems contradictory to our results in Drosophila. Nevertheless, this cooperative effect is observed in response to oncogenic and genotoxic stresses, and not when apoptosis is induced by pRb expression. Thus, the experimental settings are very different from those used in our work, and we cannot exclude that such mechanism may exist in Drosophila as well. Thus, the RBF partner implicated in induction of apoptosis in our model is probably not dE2F1. It could be that the RBF pro-apoptotic effect depends on interactions of the RBF protein with other partner proteins regulating the cell cycle and/or apoptosis in a more direct way. As we do not observe RBF-induced apoptosis in post-mitotic cells, these partners could be absent or inactive in these cells.

Furthermore, we also show in this study that RBF expression can have anti-apoptotic activity in Drosophila neurons when other pro-apoptotic genes are overexpressed. Indeed, when UAS-Dmp53 is overexpressed in neurons to induce apoptosis, we observed an anti-apoptotic effect of RBF toward Dmp53-induced cell death. In mammals, pRb can antagonize p53 apoptotic activity in different ways. [START_REF] Godefroy | p53 and Retinoblastoma protein (pRb): a complex network of interactions[END_REF] Mostly, pRb is able to promote p53 degradation by MDM2 and therefore directly suppresses p53-induced apoptosis. [START_REF] Hsieh | RB regulates the stability and the apoptotic function of p53 via MDM2[END_REF] However, since no Drosophila MDM2 homologue has been discovered yet, this mode of regulation is not likely to be conserved in flies. Furthermore, using the eye-specific GMR-Gal4 driver, we observed that RBF also counteracted the cell death induced by UAS-Debcl, a

Drosophila pro-apoptotic Bcl-2 family member [START_REF] Brachmann | The Drosophila bcl-2 family member dBorg-1 functions in the apoptotic response to UV-irradiation[END_REF][START_REF] Colussi | Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery[END_REF][START_REF] Igaki | Drob-1, a Drosophila member of the Bcl-2/CED-9 family that promotes cell death[END_REF][START_REF] Zhang | Evolutionarily conserved Bok proteins in the Bcl-2 family[END_REF][START_REF] Zhang | Drosophila proapoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell death mechanisms[END_REF] (data not shown). Since there is no data suggesting that Debcl and Dmp53 cooperate to induce apoptosis in Drosophila, one could hypothesize that the anti-apoptotic effect of RBF is not specific to Dmp53-induced cell death and occurs downstream in the apoptotic cascade. As is the case in mammals for pRb, [START_REF] Decary | The retinoblastoma protein binds the promoter of the survival gene bcl-2 and regulates its transcription in epithelial cells through transcription factor AP-2[END_REF][START_REF] Haupt | p53-mediated apoptosis in HeLa cells can be overcome by excess pRB[END_REF][START_REF] Lipinski | Cellautonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system[END_REF] RBF could act downstream of p53 in regulating the expression of survival factors or anti-apoptotic genes, as well as pro-apoptotic genes. This type of anti-apoptotic regulation by RBF has been described in a very restricted area of the wing imaginal disc. Indeed, at the dorso-ventral boundary of wing imaginal discs, RBF and dE2F1 can cooperate to inhibit the expression of the pro-apoptotic gene hid induced by irradiation. [START_REF] Moon | Drosophila E2F1 has context-specific pro-and antiapoptotic properties during development[END_REF] It is possible that a similar mechanism of pro-apoptotic gene repression by RBF could occur in neurons and would be enhanced when RBF is overexpressed. Furthermore, dE2F1 expression is maintained in post-mitotic cells of the eye imaginal disc. [START_REF] Brook | Requirements for dE2F function in proliferating cells and in post-mitotic differentiating cells[END_REF] It would be interesting therefore to test if RBF is also anti-apoptotic in neurons in a dE2F1 loss of function context.

In conclusion, our results show that, like in mammalian cells, Drosophila RBF can have pro-or antiapoptotic properties depending on the proliferative status of the cells. What are the key elements that determine RBF activity on apoptosis? To answer this question, it will be necessary to identify specific RBF partners in mitotic and post-mitotic cells. This type of information would certainly allow a better understanding of the mechanisms by which RBF controls apoptosis and cellular fate.

Materials and methods

Fly stocks

Flies were raised at 25°C on standard medium. The dronc I29 FRT80/TM3 strain was generated by Xu et al. [START_REF] Xu | The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila[END_REF] The UAS-Dmp53 strain was generated by Ollmann et al. [START_REF] Ollmann | Drosophila p53 is a structural and functional homolog of the tumor suppressor p53[END_REF] The UAS-RBF and vg-Gal4 strains were generous gifts from J. Silber. The ey-Gal4/Cyo strain was generous gift from B. Limbourg-Bouchon. The UAS-p35, elav-Gal4, UAS-dE2F1, UAS-dDP/TM6 B and dE2F1 07172 /TM3 strains come from the Bloomington stock center (stock numbers 6298, 458, 4770 and 11717, respectively). The w 1118 strain was used as the reference strain.

Test of phenotype suppression in the wing

To test caspase implication in RBF-induced apoptosis, the severity of the notch phenotype induced by UAS-RBF expression was assayed in a UAS-p35 co-expression context and in a dronc I29 heterozygous loss of function context. UAS-RBF Drosophila males were crossed with either vg-Gal4 or UAS-p35;vg-Gal4/Cyo females, and vg-Gal4;UAS-RBF/TM3Sb Drosophila females were crossed with either w 1118 or dronc I29 /TM3Sb males. Concerning the interaction between dE2F1 and RBF, wing phenotype was assayed in a UAS-dE2F1, UAS-dDP co-expression context and in a dE2F1 07172 heterozygous loss of function context. vg-Gal4;UAS-RBF Drosophila males were crossed with either w 1118 or dE2F1 07172 /TM6B females, vg-Gal4 Drosophila males were crossed with UAS-RBF females, and vg-Gal4;UAS-RBF males were crossed with UAS-dE2F1, UAS-dDP/TM6B females. The progenies of all crosses were classified according to the number of notches on their wings. The data were analyzed by the Wilcoxon test as described previously. 50

TUNEL staining of imaginal discs

vg-Gal4, ey-Gal4 and elav-Gal4 female flies were crossed either with w1118 males for a control or with UAS-RBF males. Third instar larvae of the progeny were dissected in PBS pH 7.6, fixed in PBS/formaldehyde 3.7%, washed three times for 20 min in PBT (1XPBS, 0,5% Triton). Discs were then dissected, TUNEL staining was performed following manufacturer's instructions (ApopTag® Red in situ apoptose detection kit, Chemicon), then discs were observed with a conventional Leica DMRHC research microscope using the N2.1 filter.

Western blot analysis

vg-Gal4 and elav-Gal4 female flies were crossed either with w1118 males for a control or with UAS-RBF males. 60 wing and 60 eye imaginal discs of the progeny were dissected in PBS pH 7.6 and homogenized in 80 l of NP40 ice cold buffer (50 mM Tris-Cl pH=8, 150mM NaCl, 1% NP40, 1mM DTT, AEBSF SC ). Proteins were separated in NuPAGE 4-12 % Bis-Tris polyacrylamide gels according to the manufacturer's instructions (Invitrogen) and transferred onto PVDF membrane (Millipore).

Blots were incubated with primary antibodies (rabbit polyclonal anti-RBF,1:500, Custom antibody, Proteogenix and rabbit polyclonal anti-Actin, 1:500, Sigma) overnight at 4°C and were then incubated for 1 h with corresponding peroxidase-conjugated antibody (anti-rabbit immunoglobulin, Biosystem).

Immunoreactive bands were detected by Immobilon TM Western Chemiluminescent HRP Substrate (Millipore).

7.

larvae imaginal discs by western blotting. Endogenous RBF is detected in control wing discs (first lane) and in control eye discs (third lane). UAS-RBF overexpression is clearly visible in wing discs under control of vg-Gal4 (second lane), and in eye discs under control of elav-Gal4 (fourth lane).

Actin is used as a loading control.

  observe. Eyes of adult elav-Gal4/+;UAS-RBF/+ raised at 25°C have a wild type structure similar to control elav-Gal4/+ eyes (Fig 1 C, G), and there are 7 photoreceptors visible in each ommatidia (Fig 1 D, H). Since we did not observe any pro-apoptotic effects of RBF in photoreceptors, we verified that the UAS-RBF transgene was correctly driven by elav-Gal4. Protein detection by western blotting experiments showed that elav-Gal4 indeed induces UAS-RBF expression under these conditions (Fig 1 O

  categories (wild type, weak, intermediate and strong) according to the number of notches (Fig2 A, asterisks). We assayed for the strength of the notch phenotype in wings of vg-Gal4;UAS-RBF flies in presence or absence of UAS-p35 (Fig2). UAS-p35 expression driven by vg-Gal4 does not induce any notch phenotype. When p35 was co-expressed together with RBF, distribution of the phenotypes shifted to weaker phenotypes when compared to the expression of RBF alone (Fig2 B) and the difference is statistically significant (Wilcoxon test, =7.7E-6, n=493). To ensure that change in phenotype is not due to dilution of Gal4 activity over two UAS , we co-expressed UAS-RBF and UAS-EGFP transgenes under control of the vg-Gal4 driver. We did not observe any significant change in phenotype distribution between vg-Gal4; UAS-RBF flies and vg-Gal4; UAS-RBF/UAS-EGFP flies (data not shown). Thus, these results clearly show that p35 suppresses the notch phenotype induced by RBF in the wing, and therefore that this apoptosis is caspase dependent.

  the wing phenotypes induced by UAS-RBF expression under control of the vg-Gal4 driver, either in a dE2F1 07172 heterozygous loss of function context, or together with UAS-dE2F1,UAS-dDP (Fig 3). UAS-dE2F1, UAS-dDP expression driven by vg-Gal4 does not induce any notch phenotype. The distribution of the RBF-induced notch phenotypes shifted to stronger phenotypes in a dE2F1 07172 partial loss of function context (Wilcoxon test, =4.5E-07 n=462) (Fig 3 A), whereas it shifted to weaker phenotypes in the dE2F1/dDP overexpression context (Wilcoxon test, =0 n=1069) (Fig 3 B).

  When we overexpressed UAS-Dmp53 in neurons in elav-Gal4/+;UAS-Dmp53/+ flies, we observed apoptosis induction in eye imaginal discs (TUNEL staining, data not shown) and we obtained very strong adult phenotypes: about 85% of the flies died before eclosion and escapers could hardly move and had small altered eyes (Fig 4 B). Strikingly, co-expression of UAS-RBF together with UAS-Dmp53 under control of elav-Gal4 restored viability significantly (about 43% of the pupae hatched) and reduced the Dmp53induced eye phenotype (Fig 4 C). As for the vg-Gal4 driver, expressing UAS-EGFP together with UAS-Dmp53 did not modify UAS-Dmp53-induced phenotype using the elav-Gal4 driver (data not shown) excluding an effect due to potential Gal4 dilution. Therefore, RBF expression has an inhibitory effect on Dmp53-induced apoptotic phenotype in neurons. Finally, we wondered if RBF could have an anti-apoptotic effect toward Dmp53-induced cell death in proliferating wing disc cells as well. When UAS-RBF and UAS-Dmp53 were co-expressed in the wing disc under control of the vg-Gal4 driver, apoptosis is enhanced compared to apoptosis induced by Dmp53 alone in vg-Gal4/UAS-Dmp53 flies (data not shown), indicating an additive effect of RBF on Dmp53-induced apoptosis. Therefore, the observed anti-apoptotic property of RBF is not specific to Dmp53-induced apoptosis, but depends on the cellular context, and most probably on the differentiation status of the cells.

Fig 2 :

 2 Fig 2: RBF-induced apoptosis in the wing is caspase dependent. (A) Phenotypes are grouped into four

Fig 3 :

 3 Fig 3: RBF-induced apoptosis in the wing does not require dE2F1. (A) dE2F1 07172 heterozygous flies

Fig 4 : 1 :

 41 Fig 4: RBF suppresses Dmp53-induced apoptosis in neurons. (A) Wild type elav-Gal4/+ control eye.

Acknowledgements

We are grateful to S Gaumer for helpful discussions and to C Lasbleiz and AM Prêt for critical reading of the manuscript. We wish to thank MN Soler, for expert support with confocal microscopy. The Imaging and Cell Biology facility of the IFR87 (FR-W2251) "La plante et son environnement" is supported by Action de Soutien à la Technologie et la Recherche en Essonne, Conseil de l'Essonne. We thank B. Limbourg-Bouchon and J. Silber for gifts of flies and the Bloomington Drosophila Stock Center for fly stocks. This work was supported in part by grants from the Association pour la Recherche contre le Cancer (#3819) and the Ligue Nationale Contre le Cancer. Cécile Milet held successive fellowships from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche and the Ecole Pratique des Hautes Etudes.

8.


J Castillo

J Escareno

I Boussaada

O Labbani

K Camarillo

Modeling and Control of an Aerial Multi-Cargo System: Robust Acquiring and Transport Operations

In this paper, we introduce the concept of a multilink unmanned aerial system (ML-UAS) designed for multicargo transportation tasks. Such a system features three links who are actuated by four flying robots. We present in detail the dynamics modeling based on the Euler-Lagrange formulation. A preliminary control scheme based on robust Adaptive Integral Sliding Mode Control (AISMC) is applied considering the ML-UAS model uncertainties and external disturbances. The control objective is to follow/track avian-inspired acquiring patterns for acquiring operations. The effectiveness of the proposed strategy is validated by numerical simulations.

I. INTRODUCTION

In recent years, Unmanned Aerial Vehicles (UAVs) have been used for different tasks in the industrial or the scientific field. Recently, UAVs applications have been evolving specially those involving aerial environment-interactivity (manipulation, assembling, picking, transport) it is also the case for swarm-based cooperation which demands a high degree of dexterity [START_REF] Hou | A survey on the formation control of multiple quadrotors[END_REF]. Aerial robots have been extensively used for natural disaster assessment. In this mission context, the contribution of micro aerial vehicles (MAVs) remains restricted to the collection of images either in hovering or in-motion flight. Thus, the vehicles capable of picking, transporting and placing represent an ideal solution for survival-kit delivering, rescue operation, sensor deployment/acquiring. The impact of these capabilities might be amplified whether we multiply the aerial robots and develop efficient interaction algorithms.

A. Related work

There exist different approaches and strategies as well as a variety of configurations to address the the multi-vehicles interaction problem. Inspired by parallel manipulators, [START_REF] Six | The kinematics, dynamics and control of a flying parallel robot with three quadrotors[END_REF] presents a flying robot composed of three off-the-shelf quadcopters rigidly attached via an articulated structure. The implementation aerial system, which comprises three links and a platform, yielded to interesting results showing the system's dexterity as well as enhanced cargo capacity. The necessity to increase efficiency flight raises the interest in transformable/morphing flying robots. In [START_REF] Anzai | Multilinked multirotor with internal communication system for multiple objects transportation based on form optimization method[END_REF] the problems of flight stability and center of gravity shifting due to the payloads motion are studied. In this regard, [START_REF] Zhao | Transformable multirotor with two-dimensional multilinks: modeling, control, and motion planning for aerial transformation[END_REF] exposes a multi-rotor aerial vehicle with two-dimensional multi-links and demonstrates stable aerial transformation for high mobility in three-dimensional environments. The DRAGON transformable aerial robot is introduced in [START_REF] Zhao | Design, modeling, and control of an aerial robot dragon: A dual-rotorembedded multilink robot with the ability of multi-degree-of-freedom aerial transformation[END_REF]. This flying machine is a dual-rotor-embedded multi-link robot with the ability of multi-degree of freedom aerial transformation and the full pose control regarding the center of gravity of multi-links.

The task of manipulation several cargos implies the understanding of the phenomena present in transporting a single object which is a case widely studied in the literature. Hereof, the implementation of Kalman Filter to estimate the payloads disturbances [START_REF] Escareno | Generalized disturbance estimation via eslkf for the motion control of rotorcraft having a rod-suspended load[END_REF] and the trajectory path picking generation in windy environments [START_REF] Escareño | Navigation strategy in-flight retrieving and transportation operations for a rotorcraft mav[END_REF] can be cited, along with the use of learning automatas [START_REF] Cabral | Design of model predictive control via learning automata for a single uav load transportation[END_REF] and of fuzzy logic for computing the effects of the cargos in several quadrotors [START_REF] Barawkar | Fuzzy logic based variable damping admittance control for multi-uav collaborative transportation[END_REF]. Nonetheless, the control by the means of the H ∞ control theory has been adopted due to its robustness [START_REF] Jafar | H infinity controller for unmanned aerial vehicle against atmospheric turbulence[END_REF]. By combining the H 2 and H ∞ controls, [START_REF] Raffo | A load transportation nonlinear control strategy using a tilt-rotor uav[END_REF] solves the trajectory tracking problem of a tilt-rotor UAV when transporting a suspended load. The Sliding Mode Control (SMC) is another robust strategy control applied to aerial vehicles as in [START_REF] Escareño | Navigation strategy in-flight retrieving and transportation operations for a rotorcraft mav[END_REF], [START_REF] Huang | Adaptive sliding-mode control for nonlinear systems with uncertain parameters[END_REF] or [START_REF] Pan | Integral sliding mode control: performance, modification and improvement[END_REF].

B. Paper contribution

This paper presents the detailed longitudinal modeling and robust control of a novel multi-link aerial interactive system that is studied for multi-object acquisition and transport tasks. The Euler-Lagrage approach is used to deduce the equations of motion and couplings of the ML-UAS. The resulting expressions are rewritten for the sake of the control design. In terms of control, a Sliding Mode Controller alongside an adaptive gain with an integral term of the tracking error added to the sliding surface is designed (for details [START_REF] Pan | Integral sliding mode control: performance, modification and improvement[END_REF], [START_REF] Zhao | Design, modeling, and control of an aerial robot dragon: A dual-rotorembedded multilink robot with the ability of multi-degree-of-freedom aerial transformation[END_REF], [START_REF] Escareño | Navigation strategy in-flight retrieving and transportation operations for a rotorcraft mav[END_REF]). Furthermore, an extension to the multilink case of the avian-inspired trajectory generation scheme for aerial grasping is presented [START_REF] Escareño | Navigation strategy in-flight retrieving and transportation operations for a rotorcraft mav[END_REF]. The latter is endowed with a links-dependant trajectory dynamics to generate a smooth pattern to achieve multi-object acquisition/placing.

C. Problem Formulation

In the regard of aerial picking and transportation, a single aerial rotorcraft is limited in dexterity and payload-carrying capacity. The employment of several rotorcrafts and the theory of multi-agent systems is often the emergent solution to the aforementioned issues despite the interaction difficulties and constrains [START_REF] Hou | A survey on the formation control of multiple quadrotors[END_REF], [START_REF] Zhao | Transformable multirotor with two-dimensional multilinks: modeling, control, and motion planning for aerial transformation[END_REF].

I z I x (x,z) l l Q 1 q 2 f 2 f 4 q 4 Q 3 b 3 f 3 q 3 Q 2 l 3 d 4 p 3 d 3 l 2 b 1 q 1 f 1 lp 1 b 2 p 2 d 2 d 1 p 1 l 1
Fig. 1. Multi-linked Unmanned Aerial System description A flying kinematic chain inspired by the capabilities of aerial vehicles, the dexterity of arm manipulators and a train-like transporting operation is introduced herein to overcome the established problematic.

D. Paper Outline

The paper is organized as follows: Section I corresponds to the general description of the paper. Section II comprises the equations describing the kinematics and the dynamics of the chain. The mathematical motion description of the system is used in Section III to generate a robust control law to overcome the aforementioned problematic while the trajectory generation is fully described in Section IV. We validate our proposal by numerical simulations whose explanation and results are given in Section V. Finally, the conclusions and future work remarks are written in Section VI.

II. MATHEMATICAL MODELLING

Let us consider the 3 links and 4 rotorcrafts system as a flying kinematic chain and its lateral dynamics only as shown in Fig. 1.

The links are considered to be attached to the center of gravity of the corresponding vehicles and to have a frictionless angular motion since the due-to disturbances can be mitigated by the robust controller [START_REF] Six | The kinematics, dynamics and control of a flying parallel robot with three quadrotors[END_REF] [10] [START_REF] Pan | Integral sliding mode control: performance, modification and improvement[END_REF]. In addition, we assume that the links have the same length l l ∈ IR and mass m l ∈ IR and, as a consequence, the same moment of inertia I l ∈ IR. The attitude of the j-th link is described by the angle Θ j ∈ IR where j = 1, 2, 3. The attitude of the i-th rotorcraft (with i = 1, 2, 3, 4) is given by the angle θ i ∈ IR and measured as shown in Fig. 1. The masses of the vehicles are given as m r ∈ IR and their moments of inertia as I r ∈ IR. The pose of j-th pendulum-like load is described by β j ∈ IR. Furthermore, we consider their masses and lengths to be m p j ∈ IR and l p j ∈ IR.

A. Kinematic equations

Based on the position of the chain ξ = [x z] T ∈ IR 2 and the angles Θ j and β j , the positions of the rotorcrafts ξ r i , the links ξ l j and the pendulums ξ p j w.r.t. the inertial frame can be computed as follows:

ξ r i = x r i z r i = ξ + Q i l l 2 C Θ 2 +V i C Θ D i -S Θ 2 -V i S Θ D i ∈ IR 2 (1) ξ l j = x l j z l j = ξ + E i l l 2 C Θ 2 +C Θ j -S Θ 2 -S Θ j ∈ IR 2 (2)
ξ p j = x p j z p j = ξ l j -l p j S β j C β j ∈ IR 2 (3)
where

S • = sin (•), C • = cos (•), Q k = sign (k -2.5), D k = (2k + 1) /3, E k = k -2 and V k = k 2 -5k + 6.
By time differentiating the Eqs. ( 1)-( 3), the velocity of each component are obtained, hence:

ξr i = ξ - Q i l l 2 S Θ 2 Θ2 +V i S Θ D i ΘD i C Θ 2 Θ2 +V i C Θ D i ΘD i ξl j = ξ - E i l l 2 S Θ 2 Θ2 + S Θ j Θ j C Θ 2 Θ2 +C Θ j Θ j ξp j = ξl j -l p j C β j β j -S β j β j
The dynamics of the flying multi-link robot is then modelled by the means of the Euler-Lagrange formalism.

B. Dynamics of the ML-UAS

Let the Lagrangian of the system be expressed as L = K -U ∈ IR such that K and U stand for the total kinetic and potential energies, respectively. Both terms are defined based on the energies of the quadrotors (K r and U r ), the links (K l and U l ) and the pendulums (K p and U p ), in the manner:

K = K r + K l + K p ∈ IR ; U = U r +U l +U p ∈ IR with K r = 4 ∑ k=1 m r 2 ẋ2 r k + ż2 r k + I r 2 θ 2 k U r = gm r 4 ∑ k=1 z r k K l = 3 ∑ k=1 m l 2 ẋ2 l k + ż2 l k + I l 2 Θ2 k U l = gm l 3 ∑ k=1 z l k K p = 3 ∑ k=1 m p k 2 ẋ2 p k + ż2 p k U p = g 3 ∑ k=1 m p k z p k
where g ∈ IR is the constant of gravity acceleration.

Let the vector of generalized coordinates be defined as q = [q 1 q 2 ... 5 . By applying the equation provided by the Euler-Lagrange formalism, i.e.

q 5 ] T = [x z Θ 1 Θ 2 Θ 3 ] T ∈ IR
d dt ∂ ∂ qa L - ∂ ∂ q a L = τ a with a = 1, 2, ..., 5 (4)
to the Lagrangian, the dynamics of the ML-UAS is provided.

It must be noticed that the attitudes of the vehicles are not considered as generalized coordinates since these are used as control inputs.

The equations of motion for the translation of the chain can be expressed as

µ ẍ - 3 ∑ k=1 R k α k S Θ k Θk +C Θ k Θ2 k - m p k l p k C β k βk -S β k β 2 k = τ x (5) µ (z + g) - 3 ∑ k=1 R k α k C Θ k Θk -S Θ k Θ2 k + m p k l p k S β k βk +C β k β 2 k = τ z (6)
where R k = k 2 -3k + 1 and µ, α 1 , α 2 and α 1 ∈ IR are constants defined as

µ = 4m r + 3m l + 3 ∑ j=1 m p j α 1 = l l m r + m l 2 + m p 1 2 α 2 = l l 2 m p 1 -m p 3 α 3 = l l m r + m l 2 + m p 3 2
Meanwhile, the equations of motion of the links follow the general form:

ι j Θ j -R j α j S Θ j ẍ +C Θ j (z + g) + E 2 j l l α j 2 C (Θj-Θ2) Θ2 + S (Θj-Θ2) Θ2 2 + E j η j S (Θj-βj) β j -C (Θj-βj) β 2 j + 1 -E 2 j 3 ∑ k=1 E k η k S (Θ 2 -β k ) βk -C (Θ 2 -β k ) β 2 k + l l α k 2 C (Θ k -Θ 2 ) Θk -S (Θ k -Θ 2 ) Θ2 k = τ l j (7)
By applying Eq. ( 4) and considering the β j instead of the generalized coordinates q a , the dynamics of the payloads can be represented in the general form:

m p j l 2 p j β j -m p j l p j C β j ẍ -S β j (z + g) + E j η j S (Θ2-βj) Θ2 +C (Θ2-βj) Θ2 2 + S (Θj-βj) Θ j +C (Θj-βj) Θ2 j = τ p j (8)
Along the Eqs. ( 7) and ( 8), new constants were introduced to reduce the expression, these are:

η j = m p j l p j l l 2 ∈ IR ι 1 = l 2 l m r + m l 4 + m p 1 4 + I l ∈ IR ι 2 = l 2 l m r + m l 2 + m p 1 4 + m p 3 4 + I l ∈ IR ι 3 = l 2 l m r + m l 4 + m p 3 4 + I l ∈ IR
As previously mentioned, the aerial vehicles are the actuators of the system, thus we consider their rotational dynamics

I r θi = u d i
where u d i ∈ IR is the control input of the i-th vehicle.

C. Disturbed system

The system itself is composed by the actuators (MAVs) and the rigid links, in this regard the effects of the payloads can be treated as disturbances, which leads to a nonlinear representation of the system in the form:

M (q) q C (q, q) q + G (q) = u + ρ (9)
such that

M (q) =       m 11 0 αS Θ 1 0 -αS Θ 3 0 m 22 αC Θ 1 0 -αC Θ 3 αS Θ 1 αC Θ 1 m 33 m 34 0 0 0 m 43 m 44 m 45 -αS Θ 3 -αC Θ 3 0 m 54 m 55       ∈ IR 5×5
with the elements of the matrix being

m 11 = m 22 = 4m r + 3m l m 33 = m 55 = l 2 l (m r + 0.25m l ) + I l m 34 = m 43 = 0.5l l α c C Θ 1 -Θ 2 m 44 = l 2 l (m r + 0.5m l ) + I l m 45 = m 54 = 0.5l l α c C Θ 2 -Θ 3 α = l l (m r + 0.5m l )
The term C (q, q) q in Eq. ( 9) corresponds to the Coriolis and centripetal effects and it is given as:

C (q, q) q =       α C Θ 1 Θ2 1 -C Θ 3 Θ2 3 -α S Θ 1 Θ2 1 -S Θ 3 Θ2 3 l l 2 αS Θ 1 -Θ 2 Θ2 2 -l l 2 α S Θ 1 -Θ 2 Θ2 1 -S Θ 2 -Θ 3 Θ2 3 -l l 2 αS Θ 2 -Θ 3 Θ2 2       ∈ IR 5
The gravitational effects are comprised in the vector

G (q) = 0 m 22 g αgC Θ 1 0 -αgC Θ 3 T ∈ IR 5
At the right part of Eq. ( 9), the control input vector u = u x u z u Θ 1 u Θ 2 u Θ 3 T ∈ IR 5 and the disturbances vector ρ ∈ IR 5 are found.

The perturbations affecting the translational motion are set based on Eqs ( 5) and ( 6) as

ρ x = 3 ∑ k=1 m p k ẍ + m p k l p k C β k βk -S β k β 2 k ρ z = 3 ∑ k=1 m p k (z + g) -m p k l p k S β k βk +C β k β 2 k
For each link motion disturbance, the friction between the corresponding link and the pendulous payload is considered by the addition of the term γ j β j -Θ j where γ j ∈ IR is the friction coefficient. This last consideration implies that the friction effect must be equally considered in the dynamics of the cargos (Eq. ( 8)) in the term τ p j .

ρ Θ 1 = -l 2 l m p 1 4 Θ1 -l l m p 1 2 S Θ 1 ẍ +C Θ 1 z - l 2 l m p 1 4 C (Θ 1 -Θ 2 ) Θ2 + S (Θ 1 -Θ 2 ) Θ2 2 + η 1 S (Θ 1 -β 1 ) β1 -C (Θ 1 -β 1 ) β 2 1 -l l m p 1 2 gC Θ 1 + γ 1 β1 -Θ1 ρ Θ 2 = -l 2 l m p 1 4 + m p 3 4 Θ2 - l l 2 m p 1 -m p 3 S Θ 2 ẍ +C Θ 2 z - l 2 l m p 1 4 C (Θ 1 -Θ 2 ) Θ1 -S (Θ 1 -Θ 2 ) Θ2 1 - l 2 l m p 3 4 C (Θ 3 -Θ 2 ) Θ3 -S (Θ 3 -Θ 2 ) Θ2 3 + η 1 S (Θ 2 -β 1 ) β1 -C (Θ 2 -β 1 ) β 2 1 - η 3 S (Θ 2 -β 3 ) β3 -C (Θ 2 -β 3 ) β 2 3 - l l 2 m p 1 -m p 3 gC Θ 2 + γ 2 β2 -Θ2 ρ Θ 3 = -l 2 l m p 3 4 Θ3 + l l m p 3 2 S Θ 3 ẍ +C Θ 3 z - l 2 l m p 3 4 C (Θ 3 -Θ 2 ) Θ2 + S (Θ 3 -Θ 2 ) Θ2 2 - η 3 S (Θ 3 -β 3 ) β3 -C (Θ 3 -β 3 ) β 2 3 + l l m p 3 2 gC Θ 3 + γ 3 β3 -Θ3
The well defined model in Eq. ( 9) is used in the upcoming section in order to design the AISM controller.

III. CONTROL

For control purposes, let the dynamics of the system, based on Eq. ( 9), be expressed as: q = f (q, q) + B (q) u + w [START_REF] Jafar | H infinity controller for unmanned aerial vehicle against atmospheric turbulence[END_REF] where f (q, q) = f o (q, q)+∆ f is the dynamic state-dependent function of the system defined by the nominal dynamics f o (q, q) = -M -1 (q) (C (q, q) q + G (q)) and the unmodeled uncertainties ∆ f ∈ IR 5 . The control matrix B (q) ∈ IR 5×5 is composed by the nominal control matrix B o (q) = M -1 (q) and the uncertainties in the control matrix ∆B ∈ IR 5×5 resulting in B (q) = B o (q) + ∆B. The term w -M -1 (q) ρ ∈ IR 5 comprises the external disturbances [START_REF] Huang | Adaptive sliding-mode control for nonlinear systems with uncertain parameters[END_REF].

According to [START_REF] Huang | Adaptive sliding-mode control for nonlinear systems with uncertain parameters[END_REF] and [START_REF] Pan | Integral sliding mode control: performance, modification and improvement[END_REF], we must provide a control input u = u o + u w such that u o ∈ IR 5 mitigates the nominal dynamics and u w ∈ IR 5 compensates the parametric and environmental uncertainties. Defining the tracking error vector as e = qq d ∈ IR 5 where the vector q d defines the desired trajectories, we can propose a sliding surface as follows

σ = ė + λ e + ε ∈ IR 5 ( 11
)
where ε is a vector containing the integral terms of the error [START_REF] Escareño | Navigation strategy in-flight retrieving and transportation operations for a rotorcraft mav[END_REF] and

Λ = diag λ x λ z λ Θ 1 λ Θ 2 λ Θ 3 ∈ IR 5×5 is a diagonal
matrix of control gains such that each λ -gain satisfies λ > 0.

The control input must approach σ to zero and sustain it there (σ σ < 0). Then the nominal control input shall provide stability once the system has reached the sliding surface, i, e. σ = 0 ∈ IR 5 (with 0 the zero vector), i.e:

0 = ë + Λ ė + e
By the substitution of the error and its time derivatives, we obtain that

u o = C (q, q) q + G (q) + M (q) qd -Λ ė -e (12)
To mitigate the plant parameter variations and the external disturbances, a viable solution is to consider the control input u w according to the SMC theory as

u w = -M (q) HT (σ ) (13)
such that

H = diag η x η z η Θ 1 η Θ 2 η Θ 3 ∈ IR 5×5
corresponds to a matrix of adaptive control gains, all subjected to the restriction η > 0 ∈ IR and T = sign σ x sign σ z ...

sign σ Θ 3 T ∈ IR 5 .
Let us recall Eq. ( 11) and express its time derivative based on the model in Eq. ( 10) as:

σ = f o (q, q) + B o (q) u -qd + Λ ė + e +W L (14)
with the term W L = ∆ f + ∆Bu + w ∈ IR 5 being the Lumped uncertainties vector, bounded in the manner

||W L || < H d (15)
where

H d = diag η d x η d z η d Θ 1 η d Θ 2 η d Θ 3 ∈ IR 5×5
is the terminal value of H. In order to achieve H d , the dynamics of H is defined as

Ḣ = A -1 S A ∈ IR 5×5 (16) with S A = diag |σ x | |σ z | σ Θ 1 σ Θ 2 σ Θ 3 ∈ IR 5×5 and A = diag α x α z α Θ 1 α Θ 2 α Θ 3 ∈ IR 5×5
which defines the adaptation speed of the η-gains.

A. Lyapunov stability analysis

Let us consider the following Lyanpunov candidate function

V = 1 2 σ T σ + 1 2 1 T HT A H1 ∈ IR ( 17
)
with H = H -H d being the adaptation error and 1 ∈ IR 5 the ones vector. By time differentiating Eq. ( 17) and considering Eqs. ( 12), ( 13) and ( 14), the time derivative of the Lyapunov candidate function can be expressed as

V = σ T W L -H d T (σ )
and since σ T H d T (σ ) ≥ 0 by definition, and considering the boundness property of W L (Eq. ( 15)), it follows that V ≤ 0 x , z , , ,

d d d d d Q Q Q 1 2 3 f , t q d q, q . real u f ,u i di r
Fig. 2. Control strategy for the ML-UAS ∀t > 0 which ensures the asymptotic stability of the system subjected to the proposed control law.

B. MAVs control

In order to control the attitude of the vehicles, we choose a PD controller given as

u d i = -K p i e p i -K v i e v i
where the constants K p i , K v i ∈ IR correspond to the proportional and derivative gains, respectively, the position and velocity errors are defined as e p i = θ iθ d i ∈ IR and e v i = θiθ d i ∈ IR in which the index d stands for the desired position and velocity. The stability of this controller has already been proved in [START_REF] Castillo-Zamora | Comparison of pd, pid and sliding-mode position controllers for vtail quadcopter stability[END_REF]. The desired orientation of the vehicles and the forces to be exerted by each aerial vehicle are computed based on the control inputs generated by the AISM controller. Let us recall the control input vector in Eq. ( 9) and the description of the system in Fig. 1, thus

      u x u z u Θ 1 u Θ 2 u Θ 3       =       S θ 1 f 1 + S θ 2 f 2 + S θ 3 f 3 + S θ 4 f 4 C θ 1 f 1 +C θ 2 f 2 +C θ 3 f 3 +C θ 4 f 4 l l 2 C Θ 1 -θ 1 f 1 -C Θ 1 -θ 2 f 2 l l 2 C Θ 2 -θ 2 f 2 -C Θ 2 -θ 3 f 3 l l 2 C Θ 3 -θ 3 f 3 -C Θ 3 -θ 4 f 4      
To avoid the under actuation of the system, let us establish ∀t > 0 that

θ d = θ d 1 = θ d 2 = θ d 3 = θ d 4 ∈ IR which leads to θ d = tan -1 u x u z
Therefore the force that each MAV should exert over the system can be computed according to the expression

    f 1 f 2 f 3 f 4     = 1 4     1 3 2 1 1 -1 2 1 1 -1 -2 1 1 -1 -2 -3            u 2 x + u 2 z 2u Θ 1 / l l C Θ 1 -θ d 2u Θ 2 / l l C Θ 2 -θ d 2u Θ 3 / l l C Θ 3 -θ d        l 3 l 2 l 1 (x,z) Q 3 d Q 2 d Q 1 d Desired trajectory l /2 l l /2 l (x ,z ) d d z -(l +z ) ref p o z -(l +z ) ref p o
Fig. 3. Desired Θ angles relations

The control section is summarized in Fig. 2 which shows the control algorithm in a graphical representation for a better understanding. Additionally, the block of trajectory planning can be appreciated thus it is described in the following section.

IV. TRAJECTORY PLANNING AND PICKING STRATEGY

The trajectory for the altitude of the overall system is based on a co-sinusoidal function whose parameters (frequency and amplitude) are updated based on the object location (x o , z o ) [START_REF] Escareño | Navigation strategy in-flight retrieving and transportation operations for a rotorcraft mav[END_REF]: The desired trajectory in I x is given in order to keep a constant velocity v h ∈ IR, i. e.

x d (t) = v h t ∈ IR Based on this speed pattern, and considering the position in I x of the object (x o ∈ IR), we define the grasping time t g ∈ IR as

t g = v h x o
For the motion in I z, the desired trajectory is defined based on t g , the altitude of the chain during the operation z re f ∈ IR, the length of the pendulums l p = l p 1 = l p 2 = l p 3 and the position of the object in the corresponding axis z o ∈ IR.

z d (t) = z re f + z re f -(l p + z o ) cos π t g t (18)
The desired attitude of the links is computed based in a tangent relation w.r.t. the translational pattern. By time differentiation of Eq. ( 18) and adopting a sign change in the sin function due to the measurement convention, we compute Θ d 2 as:

Θ d 2 = tan -1 z re f -(l p + z o ) π t g sin π t g t ∈ IR
which, alongside the geometric relations described in Fig. 3, leads to define

Θ d 1 = π -2 tan -1 2 z re f -(l p + z o ) l l + Θ d 2 ∈ IR Θ d 3 = 2 tan -1 2 z re f -(l p + z o ) l l + Θ d 2 -π ∈ IR V.

NUMERICAL SIMULATIONS AND RESULTS

To validate our control strategy proposal, we developed a numerical simulation with the parameters presented in Table I in which we compared the performance of the system under the command of a PD controller and the AISM controller. The PD control law was defined as u = -K P e -K V ė + M q d qd + C q d , qd qd + G q d where K P , K V ∈ IR 5×5 are the diagonal control gain matrices of the PD controller to command the overall system. The initial conditions of the flying chain were all set to zero as well as the initial attitude of the vehicles. Moreover, the simulation was divided in 3 different phases described in Table II. The results of the simulation are exposed in Figs. 456789, where the vertical dashed lines in black represents the time marks described in Table II. The three different colors at the background indicates the moment when a payload is picked and transported. Fig. 4 shows the translational motion of the flying kinematic chain and the corresponding errors. It can be inferred from the I x plot that both controllers drive the system to reach the desired horizontal velocity. It is clear that the motion in I z is successfully tracked by the two controllers until the picking operation starts, in this regard, we can appreciate the robustness of the AISMC. The motion of the payloads is described in Fig. 6 where the amplitude of the oscillation reaches a larger value in the case of the AISM control than in the application of the PD control.

The movement of the cargos influences directly the dynamics of the overall system as it adds the disturbances previously described in section II. 8 where the disturbances are observed to follow the same behaviour for both cases, differing only in the peak at t = 70 s presented when applying the AISMC. These peaks imply that the transition is suddenly done and that a modification in the transition strategy needs to be considered in order to have a smoother disturbances influence. It can be inferred from Fig. 9 that the perturbations are mitigated by the MAVs rotational motion. When implementing the AISMC, the magnitude of the disturbances rises nevertheless the picking pattern is successfully tracked due to the actuators response. In the case of the PD control, no considerable oscillations are present in the motion of the aerial vehicles though the picking trajectory is not followed.

An animation of the simulation is available at https://youtu.be/4lIr6Zuocmg.

VI. CONCLUDING REMARKS AND FUTURE WORK

In this paper, we have computed the dynamic model of a novel flying kinematic chain, besides, a robust controller based on sliding mode control and adaptive parameters was conceived. Based on the results obtained from the simulations and the Lyapunov stability analysis, the AISM controller provides stability to the system and disturbances tolerance to accomplish the picking and transport operations. An improvement of the picking strategy shall be proposed based on a moving objective, in addition the motion of the payloads and the velocity of the operation will be analyze to design an strategy for smooth links transition. The dynamics of the actuators play an important roll in the performance of the system since so a robust control law will be applied to these MAVs. The extension to a three dimensional space and the experiments concerning our proposal are conjointly left for future works.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Translational behavior of the ML-UAS comparison

Fig. 6 .Fig. 7 .

 67 Fig. 6. Payloads motion comparison

  Fig 7 shows the magnitude and

Fig. 8 .

 8 Fig. 8. Rotational links motion disturbances

LS2A IPSA, Ivry-sur-Seine, France. jose.castillo@ipsa.fr

L2S, Université Paris Sud-CNRS-CentraleSupelec, Université Paris Saclay, Gif-sur-Yvette, France

XLIM UMR CNRS 7252, Université de Limoges, Limoges, France

Inria Saclay, Equipe DISCO

Mechanical Engineering Department, Tecnológico Nacional de México en Celaya, Celaya, Mexico


Justin Kamga

Louis P Sandjo
email: plsandjo@yahoo.fr

Hervé M Poumale

Bathélémy Ngameni

Yoshihito Shiono

Mehdi Yemloul

Vincent Rincheval

Bonaventure T Ngadjui
email: ngadjuibt@yahoo.fr

Gilbert Kirsch

Politamide, a new constituent from the stem bark of Ficus polita Vahl (Moraceae)

Keywords: Ficus polita, moraceae, cerebroside, cytoxicity

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Introduction

The plants are always a great source of drugs discovery. It is the case of Ficus which is a genus endemic in west, central, north and east Africa. 1,[START_REF] Abdel-Hameed | [END_REF] The leaves of some species are used in Ivory Coast folk medicine to treat worms and abdominal pains. 3 Some previous biological studies have shown that some species have anti-HIV 4 and antimicrobial properties. 5 That is why our attention is focused on Ficus polita (Moraceae) which is a small tree about 10 to 15 meters high. 1 The discovery of bioactive compounds prompted us to investigate this plant. This paper deals with isolation, structural elucidation of a new cerebroside based on the spectrometric methods.

Results and Discussion

The methanolic crude extract was subjected to repeated columns chromatography yielding a new cerebroside 1, 3-O-β-D-glucopyranoside sitosterol 2, 6 betulinic acid 3, 7 stigmasterol 4 8 and lupeol 5. 9 Compound 1 was obtained as a dark amorphous solid from mixture CH 2 Cl 2 /MeOH (9:1). The positive test with Molisch reagent suggested that, 1 is a glycosylated compound. Its positive mode HRESI-MS spectrum showed a peak at m/z 910.6869 (calcd 910.6954) corresponding to the formula [C 50 H 97 O 11 N+Na] + requiring 3 double bond equivalents. The FT-IR spectrum exhibited a broad absorption band for OH groups (3368 cm -1 ), two strong absorption bands for olefinic functions (1649 cm -1 ) and for secondary amide (1637 and 1542 cm -1 ). 10 The NMR spectra of 1 displayed signals corresponding to those of a phytoceramide. 11 In fact, a triplet of 6H was observed at δ H /δ C 0.83 (J = 5.9 Hz)/13.9 and assigned to the two terminal CH 3 groups. A broad singlet observed between δ H /δ C 1.22-1.75/(24.2-29.0) corresponded to the sequence of CH 2 groups. A proton signal linked to a nitrogen (H-N) appeared as doublet at δ H 7.50 (J = 9.2 Hz) and the other linked to azomethine at δ H /δ C 4.11 (brd, J = 8.8 Hz)/49.8. Furthermore this information, a set of signals of β-D-glucopyranose was observed on both spectra which showed an anomeric proton at δ H /δ C 4.12 (d, J = 8.0 Hz)/103.4, four oxymethines at δ H /δ C 3.35 (m)/73. 4, 3.05 (m)/76.4, 3.10 (m)/70.0, 3.16 (m)/76.8 and the oxymethylene at δ H /δ C [3.65 (dd, J = 3.0, 11.8 Hz), 3.42 (dd, J = 5.8, 11.8 Hz)]/61. 12 In addition, the chemical shifts of a double bond having cis configuration 11 were also observed at δ H 5.30 (brd, J = 6.2 Hz)/129.4 and 5.33 (brd, J = 6.2 Hz)/130.1. The foregoing data suggested to a glucophytoceramide derivative with an olefinic bond. The sugar moiety was located using long range correlations exhibited by the HMBC spectrum. This showed correlations from diastereotopic protons H-1a [3.65 (dd, J = 5.8, 10.6 Hz)]/69. 5 and H-1b [3.90 (m) The spatial location of hydroxyl groups in sphingosine was evident by exploitation of NOESY spectrum (Scheme 1) which showed interactions between the proton at δ H 4.11 and those at δ H 3.90, 3.35; both protons at δ H 3.35 and 2.92 interacted with the one at δ H 4.90 (HO-C-3) supporting that all the five protons were in the same spatial orientation. The absolute configurations of carbons C-2, C-3, C-4, and C-2' were determined to be (S), (S), (R) and (R) respectively since the NMR data were close to those of phytoceramide. 11 So the carbon C-5 is in the (S) configuration considering its spatial position. 4 7-25, 4'-7',12'-14' 1.22-1.75 (brs) 24.2-29.0 0.83, 1.90, 2.10 13.9 29, 15' 0.83 (t, 6.0, CH3) 13.9 Trap detector (Thermo Electron, Zellik, Belgium) equipped with an ESI interface run in the positive ion mode. The separation of sample components was achieved on an X-Terra MS C18 (5 µm particle size, 3.9 x 150 mm) (Waters, Overijse, Belgium), equipped with an X-Terra MS C18 pre-column (5 µm particle size, 3.9 x 10 mm) and operated at 37 °C. Injection volume was 15 µL. The mobile phase consisted of a mixture of 5 mM ammonium formate buffer at pH 3.8 (A) and acetonitrile (B). Separation conditions for all compounds were as follows: 0.0-0.50 min, A/B hold at 50/50, v/v; 0.50-9.0 min, eluant B increase to 97%; 9.0-12.0 min, eluant B hold at 97%, 12.0-12.5 min, eluant decrease to 50%, 12.5-13.0 min, A/B hold at 50/50, Before each run the column was equilibrated for 6 min at A/B 50/50. The flow rate for column equilibration and analytical runs was 0.

  ]/69.5 to the anomeric carbon at δ C 103.4. Additional correlations were observed between the proton H-N at δ H 7.50 and the carbonyl at δ C 173.6 as well as other interactions from proton H-2' at δ H /δ C 3.85 (m)/70.8 to carbonyl function (173.6) and both successive CH 2 groups [C-3' (δ C 34.3), C-4' (δ C 24.2)]. Fatty acid moiety is αhydroxylated since the protons of α-OH group [δ H 5.59 (d, J = 6.2 Hz)] and the one of the oxymethine C-2' [δ H 3.85 (m)/70.8] correlated with the carbonyl (173.6).

Scheme 1 .

 1 Scheme 1. Spatial interactions (NOESY) of compound 1.

Scheme 2 .

 2 Scheme 2. Proposal of fragmentation mechanism justifying olefin bond position in fatty acid side.

Table 1 .

 1 The 1 H-NMR (DMSO-d 6 , 400 MHz) and 13 C-NMR (100 MHz) spectral data of compound 1

	Position	δH (J Hz)	δC (APT)	Cosy	HMBC
	ceramide
	NH	7.50 (d, 9.2)	-	4.12	49.8, 173.6
	1	3.65 (dd, 5.8, 10.6, Ha)	69.5	4.12, 4.11	49.8, 103.4
		3.90 (m, Hb)
	2	4.11 (brd, 8.8)	49.8	3.90, 7.50	69.5
	3	3.10 (dd, 8.8, 11.2)	76.8	4.90	49.8, 69.5
	4	3.35 (m)	69.9	4.75
	5	2.92 (dt, 5.9, 8.8, 11.2)	73.4	3.35, 3.10	69.9, 76.8
	6	1.95 (m, Ha), 2.10 (m, Hb) 31.9	1.22-1.75, 3.35 24.2-29.0,
					73.

Table 1 .

 1 Continued

	1.22-1.75	24.2-29.0

  4 ml/min. Ionization of the analytes was carried out as follows: sheath gas flow rate (nitrogen), 47 arbitrary units; auxiliary gas flow rate (helium), 18 arbitrary units; spray voltage, 5.0 kV; capillary temperature, 200 °C; capillary voltage, 36 V; Data acquisition was performed in a time segment between 0.2 and 11.5 min after injection. The full MS-MS spectrum of [M+H] + ions was monitored for all compounds, isolation width was 2.5 m/z, normalized collision energy was 28.0%. Ficus polita (Moraceae) was collected From Yaoundé central region of Cameroon in May 2007. A sample (N° 39955/HNC) has been deposited in the National Herbarium of Yaoundé, Cameroon.Methanolysis and LC-ESI-MS analysis.Compound 1 (1.2 mg) was refluxed (70 °C) for 18 h in 2.5 mL of MeOH containing 1.5 mL of 0.9N HCl under magnetic stirring. The mixture was neutralized with aqueous solution of Na 2 CO 3 and extracted with CHCl 3 . The fatty acid methyl ester was carefully characterized by LC-ESI-MS at m/z 270 (Retention time: 8.87 min).

	Extraction and isolation

© ARKAT USA, Inc.

D

Acknowledgements L. P. Sandjo would like to thank EGIDE and SCAC (Service de Cooperation et d'Actions Culturelles: French Grant Government No. 607832K) for their financial support and Health National Laboratory, Toxicological Division, University of Luxembourg. Dr. H.M. Poumale is thankful to the JSPS (Japan Society for the Promotion of Science) for the Postdoctoral fellowship through financial support (P08430) and a research stay at the University of Yamagata.