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Abstract ACCURATE METHODOLOGY FOR MONITORING BIOMEMBRANE EVENTS By Christine A. Winschel, Ph.D. A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in Chemistry at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Director: Dr. Vladimir A. Sidorov ASSOCIATE PROFESSOR, DEPARTMENT OF CHEMISTRY This study describes the synthesis and characterization of a new receptor (cyclen 1) capable of strong selective binding of pyrene-based anionic dyes under near-physiological conditions. This receptor comprises four naphthylthiourea groups tethered to a cyclen core via an ester linkage. The most important finding was the ability of cyclen 1 to bind efficiently to a pH-sensitive pyranine dye, a dye that is commonly used in various biomembrane assays. The high affinity of cyclen 1 to pyranine, its impermeability to the lipid bilayer membrane, fast kinetics of binding, and ability to quench pyranine’s fluorescence were used as a basis for a new membrane leakage assay. This membrane leakage assay is fully compatible with the commonly applied pH-stat transport assay, and therefore it allows for differentiation of ion transport and nonselective leakage mechanisms within a single set of experiments. In the second part of this study a new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of a newly synthesized cascade-blue-labeled lipid through complex formation with cyclen 1. This receptor-dye complexation also has high affinity for binding at micromolar concentrations and can be reversed by either competitive displacement of the lipid probe or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. This new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments, and is less invasive to the model and cell membranes than the commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Lastly, new pH-sensitive lipids were synthesized and utilized in the formulation of liposomes suitable for controlled drug release. These liposomes contain various amounts of internal NaCl and undergo internal acidification upon the exogenous addition of an HCl co-transporter in a physiologically relevant NaCl solution. Therefore, acidification ultimately leads to the hydrolysis of the pH-sensitive lipids and subsequent contents release. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be non-toxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug release model potentially suitable for in vivo applications.

<p>From January, 1988, to December, 1993, 113 patients with FIGO stage IA-IIC epithelial ovarian carcinoma were treated with postoperative radiotherapy. The median follow-up period was 74 months. Tumor recurrences were recorded in 33 cases (30%). The cancer-specific survival rate was 72%. Tumor grade was a significant (P = 0.007) and independent prognostic factor in the multivariate analysis. In a smaller series of 106 patients, a number of prognostic factors (age, FIGO stage, histopathological type, and tumor grade) were studied in relation to regulators of apoptosis (p53, bcl-2, and bax) and growth factor receptors (HER-2/neu and EGFR). Immunohistochemical techniques were used. In a separate series of 103 patients, the DNA content (flow cytometry) and p53 status of the tumors were also studied and related to the same clinicopathological factors. P53 was associated with tumor grade (P = 0.007) and survival status (P = 0.046). In a Cox multivariate analysis, tumor grade (P = 0.0006), bax status (P = 0.020), and EGFR status (P = 0.018) were significant and independent prognostic factors. DNA ploidy of the tumors was strongly associated with tumor grade. </p><p>From January, 1994, to December, 1998, a series of 109 patients with ovarian carcinomas (FIGO IA-IIC) were treated with postoperative adjuvant chemotherapy. The same prognostic factors were studied in this series. The median follow-up was 48 months and the cancer-specific survival rate was 75%. Twenty-five (25%) tumor recurrences were recorded. The most favorable survival rate was seen in patients with tumors negative for p53 and positive for bcl-2 or bax. In a multivariate analysis, tumor grade (P = 0.014) and p53 status (P = 0.020) were independent prognostic factors.</p><p>Clinical, histopathological and biological prognostic factors should be combined in prognostic models to render patient-tailored therapy possible and to define different prognostic groups for future clinical studies of adjuvant therapy in early stage ovarian carcinomas.</p>

From January, 1988, to December, 1993, 113 patients with FIGO stage IA-IIC epithelial ovarian carcinoma were treated with postoperative radiotherapy. The median follow-up period was 74 months. Tumor recurrences were recorded in 33 cases (30%). The cancer-specific survival rate was 72%. Tumor grade was a significant (P = 0.007) and independent prognostic factor in the multivariate analysis. In a smaller series of 106 patients, a number of prognostic factors (age, FIGO stage, histopathological type, and tumor grade) were studied in relation to regulators of apoptosis (p53, bcl-2, and bax) and growth factor receptors (HER-2/neu and EGFR). Immunohistochemical techniques were used. In a separate series of 103 patients, the DNA content (flow cytometry) and p53 status of the tumors were also studied and related to the same clinicopathological factors. P53 was associated with tumor grade (P = 0.007) and survival status (P = 0.046). In a Cox multivariate analysis, tumor grade (P = 0.0006), bax status (P = 0.020), and EGFR status (P = 0.018) were significant and independent prognostic factors. DNA ploidy of the tumors was strongly associated with tumor grade. From January, 1994, to December, 1998, a series of 109 patients with ovarian carcinomas (FIGO IA-IIC) were treated with postoperative adjuvant chemotherapy. The same prognostic factors were studied in this series. The median follow-up was 48 months and the cancer-specific survival rate was 75%. Twenty-five (25%) tumor recurrences were recorded. The most favorable survival rate was seen in patients with tumors negative for p53 and positive for bcl-2 or bax. In a multivariate analysis, tumor grade (P = 0.014) and p53 status (P = 0.020) were independent prognostic factors. Clinical, histopathological and biological prognostic factors should be combined in prognostic models to render patient-tailored therapy possible and to define different prognostic groups for future clinical studies of adjuvant therapy in early stage ovarian carcinomas.

La photochimiothérapie extracorporelle (PCE) est une immunothérapie autologue, basée sur la réinjection de cellules photochimiquement modifiées. La PCE a démontré son efficacité dans le traitement des formes avancées du CTCL, dans la GvHD ainsi que dans d’autres pathologies médiées par la présence de lymphocytes T pathogènes (rejets de greffes d’organes, pathologies auto-immunes), sans provoquer d’immunossupression généralisée. Bien qu’utilisée depuis de nombreuses années, les mécanismes d’action de la PCE restent peu connus. L’objectif de ma thèse était de les étudier et de mettre en place un modèle chez la souris pour permettre une optimisation des protocoles cliniques. Deux hypothèses ont été testées pour expliquer l’efficacité de la PCE : la mort « tolérogène » où l’effet obtenu serait lié à la génération de lymphocytes T régulateurs et la mort « immunogène » où l’effet pourrait s’expliquer par la génération de lymphocytes T cytotoxiques.L’utilisation de cellules mononucléées humaines obtenues chez des donneurs sains nous a permis de générer des lymphocytes T alloréactifs activés et de montrer que les lymphocytes T traités PUVA émettaient une partie des molécules dites de danger (DAMPs), décrites comme immunogènes, telles que la Calréticuline ou encore le HMGB1. Ces cellules sont phagocytées par les macrophages et les moDCs, mais ne sont pas capables d’induire leur maturation, ce qui n’engendre donc pas de stimulation du système immunitaire.L’utilisation d’un modèle murin de polyarthrite rhumatoïde (CIA) nous a permis de montrer l’efficacité de la PCE dans le traitement de cette pathologie. De plus, nos résultats montrent que l’efficacité du traitement repose sur la présence de lymphocytes T issus de souris « arthritique » dans l’échantillon traité.En conclusion, les mécanismes d’action de la PCE ne semblent pas être associés au concept de mort immunogène. Le modèle de polyarthrite rhumatoïde induite par le collagène chez la souris permettra de poursuivre l’étude des mécanismes d’action et d’optimisation de la PCE. / Extracorporeal photopheresis (ECP) is an autologous immunotherapy based on the reinfusion of photochemical modified mononuclear cells. ECP efficacy is demonstrated in treatment of CTCL, in GvHD and in others T-cell mediated diseases (organ transplant rejection and auto-immune diseases). ECP does not generate generalized immunosuppression. Although it has been used for years, mechanisms of action of ECP are not totally understood. The objective of my thesis was to study these mechanisms of action and set up a murine model to allow an optimization of the clinical protocols. Two hypotheses have been tested to explain the effectiveness of ECP: “Tolerogenic” cell death where the effect would be related to the generation of regulatory T lymphocytes and "immunogenic" death where the effect could be explained by the generation of cytotoxic T lymphocytes.The use of human mononuclear cells obtained from healthy donors enabled us to generate activated alloreactive T cells and to show that the PUVA-treated T-cells emitted a part of the danger molecules (DAMPs), described as immunogenic, such as Calreticulin or HMGB1. These cells are phagocytosed by macrophages and moDCs but do not induce their maturation, which does not therefore generate any stimulation of the immune system.The use of a murine model of rheumatoid arthritis (CIA) allowed us to show the effectiveness of PCE in the treatment of this pathology. Moreover, our results show that the effectiveness of the treatment is based on the presence of T lymphocytes derived from "arthritic" mice in the treated sample.In conclusion, the mechanisms of action of PCE do not seem to be associated with the concept of immunogenic death. The model of collagen-induced rheumatoid arthritis in mice will allow further study of the mechanisms of action and optimization of PCE.

The thesis entitled “The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer Nanomedicine” elucidates the preparation of various liposomal formulations of cationic monomeric and gemini lipids where hydrophobic domains were consisted of tocopherol, cholesterol and pseudoglyceryl backbone for the cellular transport of nucleic acids. The thesis continues while elucidating the role of various pH sensitive molecules and gold nanoparticles in liposomes to improve the delivery efficacy levels. This thesis also elucidates the role of gold nanoparticles stabilized with natural pH sensitive molecules for efficacious drug delivery applications. Additionally, the role of such pH sensitive gold nanoparticles in association with liposomes for the co-delivery of drug and gene has been discussed. The work has been divided into six chapters. Chapter 1A: Dimeric Lipids Derived from α-Tocopherol as Efficient Gene Transfection Agents. Mechanistic Insights into Lipoplex Internalization and Therapeutic Induction of Apoptotic Activity In this chapter, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. The six gemini lipids possess α-tocopherol as their hydrophobic backbone and differ from each other in terms of their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid 1, 2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA in to nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes which counted significantly better than one well known commercial formulation lipofectamine 2000 (L2K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay. Figure 1. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of p53-EGFP-C3 plasmid DNA that induces significant apoptotic response. Chapter 1B: Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by Cationic Gemini Tocopheryl Lipids Non-viral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding the unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We discuss in this chapter about six tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient co-liposomal formulations derived from each of these geminis and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE) were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized co-liposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz. HEK 293T, HeLa and Caco-2 significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and presence of serum (FBS). Notably, the knockdown activity of co-liposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR and western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines. Figure 2. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of siRNA against survivin that induces significant apoptotic response. Chapter 2: Efficacious in Vitro EGFP Expression and Silencing in Serum by Cationic Pseudoglyceryl Gemini Lipids To elicit the desirable efficacy levels in cationic liposome mediated nucleic acid therapeutics has been part of extensive scientific efforts. This chapter describes three cationic gemini lipids and application of their co-liposomes with DOPE as potent pDNA (plasmid DNA) and siRNA (small interfering RNA) cytofectins for remarkably advanced efficacy levels in numerous cell lines in the presence of serum. The hydrophobic structural lineament of cationic gemini lipids is made up of pseudoglyceryl backbone linked to the hydrocarbon chains via oligo-oxyethylene units. The stable aqueous co-liposomal suspensions of gemini lipids showed an efficient binding to pDNA or siRNA and their significant intracellular delivery in various cell lines. The transfection capabilities of different co-liposomal formulations were profiled based on EGFP expression (pEGFP-C3 pDNA transfection) and EGFP knockdown (anti-GFP siRNA transfections) in EGFP expressing cell lines. The cellular EGFP expression levels and intracellular delivery of labeled nucleic acids were thoroughly studied using flow cytometry (FACS), fluorescence and confocal microscopy. The MTT based cell viability assay revealed no loss in cell viabilities for all of the transfection optimized lipoplexes of siRNA or pDNA. The transfection profile of gemini co-liposomes was noted to be significantly much better than a commercial lipofection reagent, Lipofectamine 2000 used for pDNA and siRNA applications in each of the cell lines studied. The co-liposomes and their transfection optimized lipoplexes were physiochemically characterized extensively by means of zeta potential, dynamic light scattering (DLS) and atomic force microscopy (AFM). In brief, these new gemini co-liposomal formulations seem to offer a great opportunity for successful nucleic acid (DNA and siRNA) delivery in a practical scenario. Figure 3. Efficacious EGFP expression (pDNA transfection) and EGFP silencing (anti GFP siRNA transfection) mediated by co-liposomes of pseudoglyceryl gemini lipids and DOPE. Chapter 3: Efficient Elicitation of Liposomal Nucleic acid delivery through the Eminence of Gold Nanoparticles Stabilized with pH Responsive Short Tripeptide Derived from Tyrosine Kinase NGF Receptors The prerequisite in the area of gene therapy today is to serve transfection efficient formulations nullifying the enduring key issues. To this end, we discuss in this chapter, the role of hybrid liposomal formulations derived from structurally distinct cationic lipids, a neutral lipid (DOPE) and pH responsive short tripeptide (KFG, Lys-Phe-Gly) capped gold nanoparticles (PAuNPs). The hybrid liposomes are presented to be efficient enough to transfect pDNA leading to remarkably high gene expression levels in various cell lines of different origins in the presence of serum (FBS). Hybrid liposomes could deliver pDNA more effectively than the native liposomes and commercial standard lipofectamine 2000 (L2K) across the entire range of N/P ratios studied under the influence of intracellular pH response and gold nanoparticles prominence. The gene transfection capabilities are profiled based on transfections performed using two different plasmids (pGL3, luciferase activity and p-EGFP-C3, green fluorescent protein expression). pDNA cellular internalization and subsequent gene expression levels are studied using flow cytometry, fluorescence microscopy and confocal microscopic studies. The extensive physiochemical characterization of hybrid liposomal formulation and their complexes with pDNA in comparison with respective native liposomes was performed using AFM, TEM, Zeta, DLS, gel retardation assay, U.V. and fluorescence emission measurements. The hybrid liposomes are shown to possess significantly higher fusion activity at lowered pH of intracellular compartments. These hybrid liposomes are fairly biocompatible across the concentration range used in transfection experiments. Precisely, introduction of these pH responsive tripeptide capped gold nanoparticles in to liposomal formulations straightforwardly must be more advantageous for a practical application in biomedical scenario to achieve therapeutic levels. Figure 4. The hybrid of liposomes and tri-peptide capped gold nanoparticles for significantly improved gene expression levels. Chapter 4: RNA Aptamer Decorated pH Sensitive Liposomes for Active Transport of Nucleic Acids in Specific Cancer Cells This chapter describes the target specific transport of pH sensitive liposomes loaded with a RNA aptamer for promising nucleic acid therapeutics. The pH sensitive liposomes are constructed from a cationic cholesteryl gemini lipid (CGL), neutral helper lipid (DOPE) and gemini analog of a pH sensitive lipid, palmitoyl homocysteine (GPHC). The liposomes are shown to be significantly fusogenic that deliver the cargoes upon lowerin the pH (6.0). The fusogenic behaviour of the liposomes was thoroughly studied by means of dynamic light scattering (DLS), zeta potential, lipid mixing, calcein dequenching and atomic force microscopy (AFM). The facile integration of cholesterol conjugated RNA aptamer in liposomes derived from cholesteryl gemini lipids was exploited for their delivery to specific cancer cells. The RNA aptamer specifically binds to epithelial cell adhesion molecule (EpCAM) with high affinity which is a cell surface marker in various solid cancers such as colorectal and breast carcinoma. These aptamer decorated pH sensitive liposomes could efficiently enter the EpCAM expressing COLO-205, Caco-2, MCF-7 and MDA-MB-231 cell lines while no such noticeable liposome transport was observed in EpCAM negative HEK 293T cells as evidenced by flow cytometry and confocal microscopy. Additionally, the liposomes are shown to be actively transported inside the cells, i.e., receptor mediated endocytosis. These liposomes could complex the nucleic acids (pDNA) in an efficient manner. The MTT based cell viability assay accounted no noticeable loss in cell viabilities for liposome treatments. Concisely, we have formulated RNA aptamer loaded pH sensitive liposomes that would certainly be promising tool in target based cancer nanomedicine. Figure 5. (A) Cellular internalization of DY-647 labeled aptamer loaded pH sensitive liposomes. (B) The liposomes were actively internalized through receptor mediated endocytosis. Each panel (A and B) represents (from left to right) bright field image, aptamer fluorescence, DAPI stained nuclei and merge of previous three impressions. Chapter 5: Natural Tri-peptide Capped Gold Nanoparticles for Efficacious Doxorubicin Delivery in Vitro and in Vivo Nanotechnology has gained ever increasing interest for the successful implementation of chemotherapy based treatment of cancer. This chapter describes the role of gold nanoparticles (AuNPs) capped with a natural pH responsive short tri-peptide (Lys-Phe–Gly or KFG) for significant intracellular delivery of an anti-cancer drug, doxorubicin (DOX). A significantly increased apoptotic response was noted for DOX treatments mediated by KFG-AuNPs in comparison with drug alone treatments in various cell lines (BT-474, HeLa, HEK 293T and U251) in vitro. Furthermore, KFG-AuNPs mediated DOX treatment significantly decreased cell proliferation and tumor growth in BT-474 cell xenograft model in nude mice. In addition, KFG-AuNPs showed efficacious drug delivery in DOX-resistant HeLa cells (HeLa-DOXR) in comparison with drug alone treatments. Figure 6. Representative images of excised tumors after doxorubicin treatment mediated by pH responsive tri-peptide capped gold nanoparticles (DOX-KFG-AuNPs) (C) in comparison with doxorubicin alone treatments (B) and untreated tumors (A). Extensive cell death as observed under Hematoxylin/eosin (H&E) (D) and TUNEL (E) staining of DOX-KFG-AuNPs treated tumor sections. Chapter 6: Significant Apoptotic Activity Induction by Efficacious Co-delivery of p53 Gene and Doxorubicin Mediated by the Combination of Co-liposomes of Cationic Gemini lipid and pH Responsive Tri-peptide Combining chemotherapy with gene therapy has appeared as an efficient tool to treat complex biological disorder like cancer. Herein, we show efficient co-delivery of DNA and an anti-cancer drug, doxorubicin (DOX) by means of gemini cationic liposome (GCL) based lipoplex nanoaggregates that are coated with DOX encapsulated pH responsive tripeptide nanovesicles. The lipoplex, tripeptide vesicles and their association was thoroughly studied using dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM). Flow cytometry, fluorescence and confocal microscopic analysis revealed that the GCL-tripeptide association could significantly co-deliver the p53 expression plasmid (p53-EGFP-C3) and DOX in HeLa and HEK 293T cells in the presence of serum. A synergistic increase in gene expression level and DOX internalization was observed in co-delivery which was even substantially higher than individual lipoplex transfection and DOX treatment. The apoptosis induced due to p53 expression and DOX was profiled with the help of annexin-V positivity analysis under flow cytometry and nuclear damage analysis by DAPI nuclei counterstaining under confocal microscopy which noted to be significantly higher in cells during co-delivery. The MTT based cell viability assay revealed a significantly increased loss in cell viability counts for co-delivery treatments. Such a system delivering synergistically increased significant efficacy levels in combinatorial drug and nucleic acid therapeutics would be certainly advantageous for practical biomedical applications. Figure 7. The co-delivery of pDNA and drug (doxorubicin) mediated by GCL-tripeptide association as observed under (A) confocal microscopy (pDNA; green and doxorubicin; red) and (B) flow cytometry.

Thesis submitted in fulfillment of the requirements for the Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness At the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY 2014 / Diabetes mellitus (DM) results in severe metabolic imbalances and pathological changes in many tissues. Chronic inflammation and oxidative stress have been implicated in the pathophysiology of diabetes mellitus. Garcinia kola (Family: Guttiferae) is a plant well known for its ample medicinal values. The seed of the plant also known as ‘bitter kola’ due to its bitter taste is used as a masticatory agent in traditional hospitality, cultural and social ceremonies in Africa. Kolaviron (KV) is a defatted ethanol extract from the seeds of Garcinia kola (GK). Kolaviron has been shown in experimental models of diseases to have numerous beneficial effects due to the presence of flavonoids (mainly Garcinia biflavonoid (GB)-1, GB-2 and kolaflavanone). However, there is paucity of information regarding the possible effect of kolaviron on inflammatory mediators and oxidative stress in diabetes mellitus. Therefore, this study was carried out to investigate the potential beneficial effects of kolaviron on antioxidant status, inflammatory mediators and apoptosis. Other biochemical and histological alterations in the blood, liver and kidney of streptozotocin-induced diabetic rats were also evaluated. A single intraperitoneal injection of freshly prepared solution of streptozotocin (50 mg/kg.b.wt.) in citrate buffer (0.1M, pH 4.5) was administered to overnight fasted rats for diabetes induction. Diabetes was confirmed by stable hyperglycemia (>18 mmol/l) in the tail blood glucose after 5 days of streptozotocin injection. Kolaviron (100 mg/kg b.wt.) was administered to diabetic rats (by gastric gavage) on the 6th day after the induction of diabetes and treatment continued for 6 weeks (5 times weekly). The effects on blood glucose, body weight, organ (liver and kidney) weight, serum biochemical parameters, oxidative status, inflammatory mediators and histology of the liver, kidney and pancreas were assessed. Kolaviron (KV) treatment lowered blood glucose in diabetic and normoglycemic rats and reduced glycated haemoglobin [HbA1C (%)]. Plasma insulin level was raised in diabetic rats treated with KV. Histomorphometric analysis of the pancreas revealed increased β-cell area of pancreatic islets of kolaviron-treated diabetic group. The indices of organ (liver and kidney) damage were increased in diabetic rats. However, KV treatment protected against liver and kidney damage. The characteristic features of diabetic dyslipidemia such as elevated serum triglyceride and cholesterol concentration which are major risk factors for cardiovascular disease were also significantly reduced in KV-treated diabetic rats. Alteration in antioxidant enzymes status was observed in the liver, kidney and blood (erythrocyte, plasma and serum) of diabetic rats. Lowered catalase (CAT) activity was observed in the liver and kidney of diabetic rats while KV treatment significantly (p < 0.05) elevated catalase activity in the liver and kidney. There was no significant change (p > 0.05) in erythrocyte catalase activity among all treatment groups. Erythrocyte of diabetic rats showed a marked reduction in the activity of superoxide dismutase (SOD) with no significant changes in liver and kidney SOD activity of diabetic rats compared to control whereas KV administration to rats markedly increased SOD activity. Glutathione peroxidase (GPX) activity was elevated in the erythrocyte and kidney of STZ-induced diabetic rats with no significant effect on liver GPX activity. KV treatment reversed the alteration in GPX activity in the kidney and erythrocyte. Level of reduced glutathione (GSH), a non-enzymatic antioxidant was decreased in the both liver and kidney of diabetic rats and treatment of diabetic rats with KV elevated GSH concentration in both tissues. Also, malondialdehyde (MDA), a marker of lipid peroxidation was elevated in the liver, kidney and plasma of diabetic rats and significantly (p < 0.05) lowered following KV treatment. Diabetes induction reduced the capacity of liver and kidney to absorb oxygen radicals as demonstrated by lowered oxygen radical absorbance capacity (ORAC) values. KV administration to normal and diabetic rats significantly increased ORAC values. Increased rate of apoptosis, a major cellular response to high glucose induced stress was observed in the renal and hepatic tissues of diabetic control rats. Kolaviron treatment of diabetic rats protected the liver and kidney against hyperglycemia-induced apoptosis and decreased the number of TUNEL positive cells A significant (p < 0.05) elevation of pro-inflammatory cytokines; monocyte chemoattractant protein (MCP-1), Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF)-𝛂 was observed in the liver of diabetes rats. KV treatment lowered these inflammatory biomarkers. On the other hand, the kidney of diabetic rats showed elevated concentration of pro-inflammatory IL-1β with no significant effect on kidney TNF-𝛂. An increase in the serum concentration of MCP-1 and IL-1β was observed in the untreated diabetic rats while kolaviron treatment normalized the alteration in serum concentration of MCP-1, IL-1β and vascular endothelial growth factor (VEGF). In conclusion, persistent and chronic hyperglycemia promotes the generation of free radicals and inflammatory molecules which contributes to progressive development of micro- and macro vascular complications and multi-organ damage. Kolaviron demonstrated beneficial effects on markers of oxidative stress and inflammation in the diabetic rats and also promoted the survival and functional integrity of the liver and kidney.

Chez les mammifères, les cellules apoptotiques peuvent être complètement dégradées par des cellules phagocytaires spécialisées ou servir de vecteur d'ADN Le transfert d'oncogènes via les cellules apoptotiques aboutit à la transformation des cellules receveuses uniquement si celles-ci sont déficientes en p53. Sachant que Fniicogène E6 des papillomavirus humains (HPV) à haut risque induit la dégradation de p53, il est concevable que son transfert par la cellule apoptotique soit à l'origine d'un mécanisme alternatif de carcinogenèse associée aux HPV. Afin de confirmer cette hypothèse, l'apoptose de cellules dérivées de cancer du col de l'utérus, abriiam ou non des séquences d'HPV, a été induite. En collaboration avec l'équipe de Patrick Sandoz du laboratoire d'optique de FEMTO-ST, nous avons adapté leur inicr;système référencé en position à l'observation automatisée de Finternalisation des cellules apoptotiques. Nous avons aussi déterminé que les cellules apoptotiques son! phagocytées par les fîbroblastes quel que soit leur statut virologique. Seules les cellules apoptotiques dérivées de cellules abritant de l'ADN d'HPV transforment les cellules receveuses. L'expression de l'ADN viral, dont E6, dans les fîbroblastes transformés ainsi que la perte d'expression des protéines p53 et p21 suggère que les oncogènes d'HPV pourraient être à l'origine de la transformation. Les résultats présentés dans ces travaux mettent en évidence un nouveau mécanisme de carcinogenèse associée aux HPV via la phagocytose des cellules apoptotiques, potentiellement impliqué dans la transformation de cellules primaires et la progression des tumeurs associées aux HPV. / Apoptotic cells in mammals may be completely degraded by specialized phagocytic cells or serve as a DNA vector. The oncogene transfer via apoptotic cells leads to the transformation of récipient cells but only when they are p53 deficient. As the E6 oncogene of high-risk human papillomavirus (HPV) leads to p53 dégradation, its transfer from apoptotic cells may be the cause of an alternative mechanism of HPV-associated carcinogenesis. To confirm this hypothesis, we induced apoptosis of cervical cancer cells that may harbor HPV sequences. In collaboration with Patrick Sandoz's team at the FEMTO-ST optical laboratory, we used their position-referenced microsystem for the automated observation of apoptotic cell internalization. We also found that apoptotic cells are phagocytized by fibroblasts regardless of their virological status. Only apoptotic cells from cells harboring HPV DNA transform recipient cells. Viral DNA expression, including E6, in transformed fibroblasts and the loss of p53 and p21 protein expression suggest that HPV oncogènes may cause transformation. These results highlight a new mechanism of HPV-associated carcinogenesis via apoptotic cell phagocytosis. This mechanism may be involved in thé transformation of primary cells and the progression of HPV-associated tumors.

L’ischémie/reperfusion (I/R) occurrente à toutes transplantations d’organe solide, constitue un stimulus pro-autophagique/pro-apoptotique pour les cellules endothéliales. Nous avons récemment démontré que les cellules endothéliales apoptotiques (CEapo) sécrètent des vésicules apoptotiques exosome like (ApoExo) qui induisent une réponse auto-immune et accélèrent le rejet vasculaire dans un modèle de greffe aortique chez la souris. Ces ApoExo qui diffèrent des corps apoptotiques classiques par leur structure et leur contenu protéique contiennent le fragment C-terminal du perlécan, LG3. Le LG3, un auto-antigène d’importance en transplantation, favorise le remodelage vasculaire et est augmenté en circulation chez les patients greffés rénaux atteints d’un rejet vasculaire. De plus, la présence d’anticorps anti-LG3 avant la transplantation est associée à un risque plus élevé de développer un rejet vasculaire chez des patients greffés du rein et à la perte du greffon à long terme. Il est connu que la génération du fragment LG3 implique la protéolyse du perlécan par la cathepsine L, une protéase lysosomiale, mais le mécanisme de transport de ce fragment au sein des ApoExo est encore inconnu. Nous avons émis l’hypothèse que l’activité lysosomiale et l’autophagie jouent un rôle important dans la maturation et le chargement du LG3 dans les vésicules ApoExo sécrétées par les CEapo. Une étude longitudinale de microscopie électronique chez les cellules endothéliales humaines carencées en sérum pendant 1h, 2h et 3h, nous a révélé la présence du perlécan / fragment LG3 au sein de compartiments autophagiques (autophagosomes et autophagolysosomes) à différentes étapes du processus autophagique. Après 3 heures de carence en sérum, nous avons identifié le fragment LG3 dans des vésicules membranaires situées au sein de grands réseaux vacuolaires s’apparentant à des autophagolysosomes. L’inhibition de la cathepsine L, de l’acidification du lysosome et de l’autophagie diminuent la présence du fragment LG3 dans les vésicules ApoExo sans affecter la sécrétion de vésicules démontrant donc le rôle de l’autophagie dans l’intégration du fragment LG3 au sein des ApoExo. Toutefois, l’injection de vésicules ApoExo LG3-, provenant de cellules murines aortiques traitées à la bafilomycine, dans un modèle murin de greffe aortique induit une réponse auto-immune anti-LG3 ainsi qu’un remodelage vasculaire à des niveaux similaires aux souris ayant reçu des ApoExo véhicule. Une analyse protéomique de ces vésicules ApoExo LG3-, nous a révélé que la bafilomycine modifie le contenu protéique des ApoExo en induisant une augmentation de la présence de protéines lysosomiales et de la matrice extracellulaire dont le perlécan. Ceci suggère que la présence du motif LG3 au sein du perlécan natif non clivé dans les ApoExo LG3- pourrait être responsable de la mise en place de la réponse anti-LG3 observées chez les souris. Les travaux de ce doctorat ont permis de mettre en évidence que l’autophagie dans les cellules endothéliales productrices d’ApoExo ne régule pas l’immunogénicité des ApoExo. Cependant, elle régule au sein des cellules endothéliales apoptotiques le transport et le clivage du perlécan qui lui est immunogène. En effet, l’autophagie module les différentes formes de perlécan natif et clivé sécrétées dans les ApoExo. L’étude chez la souris greffée nous a donc permis de considérer non plus l’implication du fragment LG3 mais l’implication du motif LG3 présent au sein du perlécan natif non clivé et de ses différentes formes intermédiaires dans la réponse auto-immune anti-LG3 et le rejet vasculaire. La modulation de la sécrétion du perlécan et du LG3 dans les ApoExo constitue une cible thérapeutique potentielle afin de diminuer la réponse auto-immune pouvant augmenter le dommage vasculaire après la greffe / Ischemia/reperfusion (I/R) occurring in all solid organ transplantation, constitute a proautophagic / pro-apoptotic stimulus on endothelial cells. We recently demonstrated that apoptotic endothelial cells (CEapo) secrete apoptotic exosome-like vesicles (ApoExo) that induce autoimmune response and accelerate vascular rejection in a mouse aortic transplant model. These ApoExo, that differ from classical apoptotic bodies in structure and protein content, contain the C-terminal fragment of perlecan, LG3. LG3, an autoantigen of importance in transplantation, promotes vascular remodeling and is increased in circulation in renal transplant patients undergoing vascular rejection. In addition, the presence of anti-LG3 antibodies prior to transplantation is associated with a higher risk of developing vascular rejection in kidney transplant patients and long-term graft loss. It is known that the generation of LG3 fragment involves the proteolysis of perlecan by cathepsin-L, a lysosomal protease, but the mechanism of export of this fragment within ApoExo is still unknown. We hypothesized that lysosomal activity and autophagy play an important role in the maturation and the secretion of LG3 in ApoExo vesicles secreted by CEapo. Longitudinal electron microscopy study after 1h, 2h and 3h in serum starved endothelial human cells revealed the presence of perlecan / LG3 fragment within autophagic compartments (autophagosomes and autophagolysosomes) at different stages of the autophagic process. After 3 hours of serum starvation, we identified LG3 fragment in membrane vesicles located within large vacuolar networks reminiscent of autophagolysosomes. Inhibition of cathepsin-L, of lysosomal acidification and of autophagy decrease the presence of LG3 fragment in ApoExo vesicles without affecting vesicle secretion thus demonstrating the role of autophagy in the secretion of LG3 fragment within ApoExo. However, Injection of ApoExo LG3- vesicles from bafilomycin-treated aortic murine cells into a murine aortic transplant model induces autoimmune anti-LG3 response and vascular remodeling at levels similar to the ApoExo vehicle mice control group. Proteomic analysis of ApoExo from bafilomycin-treated aortic murine cells has demonstrated that bafilomycin modifies ApoExo protein content by inducing an increase of the presence of lysosomal proteins and the extracellular matrix, including perlecan. This suggests that the presence of LG3 motif in uncleaved native perlecan in ApoExo LG3- could be responsible for the establishment of the anti-LG3 response as well as the vascular remodeling observed in mice. Collectively, these results demonstrate that autophagy in endothelial cells producing ApoExo does not regulate the immuogenicity of ApoExo. However, it regulates within apoptotic endothelial cells the processing and cleavage of perlecan who is immunogenic. Indeed, autophagy modulates the different forms of native and cleaved perlecan secreted in ApoExo. Grafted mice study thus allowed us to consider neither the involvement of the LG3 fragment but the implication of the LG3 motif present in the uncleaved native perlecan and its intermediate forms in the anti-LG3 autoimmune response and vascular rejection. Modulating perlecan and LG3 secretion in ApoExo vesicles is a potential therapeutic target to reduce the autoimmune response that can increase vascular damage after transplantation.