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ABSTRACT Renal cell carcinoma (RCC), the most common type of kidney cancer, is a highly metastatic disease. Late stage metastatic RCC is essentially incurable and lethal. The average survival of patients, following metastatic RCC, is about 4 months and only 10% of patients survive for one year. None of the currently available chemotherapy, radiotherapy, hormonal or biological therapies have a significant impact on the progression of the disease. Novel chemotherapeutics are urgently required for the treatment of this deadly disease. The mechanisms that pose the greatest challenges to chemotherapeutics are resistance of tumour cells to apoptosis, tumour angiogenesis and multi-drug resistance. Resistance to apoptosis may be mediated by the up-regulation of anti-apoptotic proteins, especially Bcl-2 and Bcl-XL, and/or by the down- regulation of pro-apoptotic proteins, particularly Bax. Angiogenesis is pivotal for tumour growth and metastasis. Of all identified pro-angiogenic molecules, vascular endothelial growth factor (VEGF) is considered to be a key molecule. Drug resistance is thought to be mediated by the up-regulation of multi-drug resistance molecules such as MDR-1 and MRP-1. Up-regulation of Bcl-2 also confers drug resistance to cancer cells. The main hypothesis of this thesis was that treatment targets of metastatic RCCs are likely to multifactorial and that inhibition of molecules that regulate the processes of apoptosis, angiogenesis and multidrug resistance are likely to be better targets than those that regulate only one of these processes. In this regard, the transcription factor nuclear factor kappaB (NF-kB) meets the criterion, regulating the apoptotic, angiogenic and multi-drug resistance pathways of cancer cells. Its inhibition appeared to be an attractive strategy for the treatment of metastatic RCC. Many studies have demonstrated an association between the over-expression NF-kB and RCC. Thus, the major aim of this thesis was to explore the anti-cancer effect of pyrrolidine dithiocarbamate (PDTC), a potent NF-kB inhibitor on human metastatic RCC cell lines. The thesis is divided into seven Chapters. In Chapter 1, the literature on RCC, NF-B and the role of NF-kB in RCC development and progression are reviewed. The rationale for the inhibition of NF-kB as a potential anti-RCC strategy using PDTC is established. During the course of this research, the use of PDTC as an anti-cancer agent has risen to prominence. Chapter 2 describes the materials and methods used in the project. In Chapter 3, the expression of NF-kB in human kidney and the RCC cell lines, ACHN and SN12K1, was established. The proof of hypothesis that NF-kB inhibition using PDTC is an effective anti-cancer strategy was demonstrated. PDTC was selectively toxic to the RCC cell lines, but not to normal human kidney cells. PDTC induced apoptosis and inhibited proliferation of the RCC cells. PDTC also inhibited NF-kB, its upstream regulatory molecules such as the inhibitory protein family of the IkBs, and the kinase IKK complex. PDTC also inhibited anti-apoptotic Bcl-2 and Bcl-XL, but not pro-apoptotic Bax. Chapter 4 demonstrated the in vitro and ex vivo anti-angiogenic and anti-metastatic effects of PDTC. Protein microarrays for angiogenic factors produced controversial results. PDTC inhibited epidermal growth factor (EGF) produced in endothelial cells. VEGF had neutral effect on angiogenesis under the experimental conditions used. In the RCC cell lines, several pro-angiogenic molecules were modulated. Interestingly, the pro-angiogenic molecule interleukin (IL)-8 was up-regulated in both RCC cell lines. The monocyte chemoattractant protein-1 (MCP-1) was decreased in ACHN cells, but increased in SN12K1 cells. The implications of these controversial findings are discussed. Chapter 5 demonstrated the ability of PDTC to overcome drug resistance in a synergism with cisplatin. Individual non-toxic concentrations of PDTC and cisplatin, when combined, induced significant toxicity of RCC cell lines. The synergistic effect was not mediated by the inhibition of NF-kB, but rather through the inhibition of transcriptional activation of NF-kB. Bcl-2 rather than MDR-1 or the regulatory protein MRP-1 may be important in overcoming drug resistance in RCC. Chapter 6 showed the anti-cancer effect of PDTC in an animal model of RCC. PDTC significantly decreased the growth of RCC implanted in the kidney of severe combined immunodeficiency (SCID) mice. PDTC inhibited NF-kB and was not toxic to normal cells. The expression of Bcl-2, Bcl-XL and Bax were contradictory to the in vitro findings and a theory about the spread of RCC based on these findings is discussed. In Chapter 7, the findings are summarised. A case for PDTC as a potential therapeutic agent for RCC is established. Under the experimental conditions used, PDTC was demonstrated to be an effective anti-RCC agent by targeting the three most important characteristics of RCC that pose the greatest challenges to chemotherapeutics: resistance of tumour cells to apoptosis, tumour angiogenesis and multi-drug resistance. PDTC was selectively toxic to RCC, but not to normal renal cells. Thus PDTC appears to be a promising anti-cancer agent. This is supported by the current increase in interest, and in the number of publications, on the use of PDTC in several cancers. Some future directions are also discussed in this Chapter. These include, but are not limited to, an investigation of what is protecting normal cells from the toxicity of PDTC, the creation of an Australian database on RCC, and the characterisation of RCC based on NF-kB expression.

The course of microsporidiosis caused by Encephalitozoon cuniculi in immunocompetent BALB/c mice and immunodeficient SCID mice was screened using molecular methods. The site of infection in organs was located using molecular and histology methods. The effectiveness of albendazole treatement and possibility of infection relapse after immunosuppresion (cyclosporine A, tacrolimus, mycofenolate mofetil) was also studied. Moreover, the course of excretion of microsporidial spores in feces was monitored during the whole time of experiment.

OBJECTIVES: Models for the development of new treatment options in vestibular schwannoma (VS) treatment are lacking. The purpose of this study is to establish a quantifiable human VS xenograft model in mice. STUDY DESIGN AND METHODS: Both rat malignant schwannoma cells (KE-F11 and RT4) and human malignant schwannoma (HMS-97) cells were implanted near the sciatic nerve in the thigh of severe combined immunodeficiency (SCID) mice. Additionally, human benign VS specimens were implanted in another set of SCID mice. Three-dimensional tumor volumes were calculated from magnetic resonance images over the next 6 months. RESULTS: Mice implanted with malignant schwannoma cells developed visible tumors within 2 weeks. Imaging using a 4.7-tesla magnetic resonance imaging and immunohistopathologic examination identified solid tumors in all KE-F11 and HMS-97 xenografts, whereas RT4 xenografts consistently developed cystic schwannomas. VS xenografts demonstrated variability in their growth rates similar to human VS. The majority of VS xenografts did not grow but persisted throughout the study, whereas two of 15 xenografts grew significantly. Histopathologic examination and immunohistochemistry confirmed that VS xenografts retained their original microscopic and immunohistochemical characteristics after prolonged implantation. CONCLUSIONS: This study describes the first animal model for cystic schwannomas. Also, we demonstrate the use of high-field magnetic resonance imaging to quantify VS xenograft growth over time. The VS xenografts represent a model complimentary to Nf2 transgenic and knockout mice for translational VS research.

Le Cancer Colo Rectal (CCR) est la deuxième cause de mortalité par cancer dans le monde. Le risque de récidive après traitement curatif atteint 45% pour les stades 3. Une des hypothèses à l’heure actuelle pouvant expliciter le processus métastatique et les récidives est la présence en son sein de cellules « souches », pouvant « initier » le cancer. Notre réflexion s’inscrit dans la continuité des travaux réalisés au sein de notre Laboratoire, intitulés «Stratégies d’isolement et de caractérisation des cellules initiatrices de cancer colorectal», (Mélin et al, 2012). Dans une première partie, notre travail a porté sur l’analyse in vitro de la sensibilité des fractions enrichies en CIC aux différentes molécules de chimiothérapie les plus couramment utilisées en cancérologie colorectale.Puis, dans une deuxième partie, l’analyse des tumeurs obtenues après greffe a été faite ex ovo sur la Membrane Chorio-Allantoïdienne d’embryon de poulet (CAM), modèle facilement manipulable, peu onéreux et très rapide. Ce modèle étant naturellement immunodéprimé, il permet d’obtenir des informations concernant les phénomènes clés de la tumorigénèse et de la néoangiogénèse. Des analyses de la croissance tumorale, de l’histologie (prolifération, apoptose et vascularisation) et des analyses protéiques ont été menées en parallèle. De cette dernière étude, un marqueur particulier la E cadhérine, a été mis en évidence comme témoin indirect d’agressivité. En effet, au sein des tumeurs obtenues à partir de F1 HCT116, fraction himiosensible, l’expression de la E cadhérine est augmentée contrairement aux tumeurs obtenues à partir de F3 WiDr, fraction chimiorésistante, montrant une diminution d’expression de la E cadhérine.Ainsi, dans une troisième partie, sachant qu’un lien entre cellules initiant le cancer et E cadhérine a été mis en évidence, nous nous sommes focalisés sur son expression. Nous avons alors étudié son expression in vitro sur des cellules résistantes au 5 Fluorouracile. Puis, son expression a été étudiée ex vivo au sein de tissus et de sérums de patients opérés de cancer colorectal. / The ColoRectal Cancer (CCR) is the second leading cause of cancer mortality in the world. The risk of recurrence after curative treatment reaches 45% for stages 3. One of the hypotheses currently able to explain the metastatic process and the recurrences is the presence within it of "stem" cells, which can "initiate" the cancer. Our reflection is in line with the work carried out in our Laboratory, entitled "Strategies for the isolation and characterization of cells that initiate colorectal cancer" (Mélin et al, 2012).In the first part, our work focused on in vitro analysis of the sensitivity of fractions enriched in CIC to different chemotherapy molecules most commonly used in colorectal cancer (CRC). Then, in a second part, the analysis of the tumors obtained after transplantation was made ex ovo on Chicken Embryo Chorio-Allantoid Membrane (CAM), an easily manipulated model, inexpensive and very fast. This model provides information on the key phenomena of tumorigenesis and neoangiogenesis. Analyzes of tumor growth, histology (proliferation, apoptosis and vascularization) and protein analyzes were carried out in parallel. From this last study, a particular marker, E cadherin, was highlighted as an indirect link with witness of aggressiveness. Indeed, within the tumors obtained from F1 HCT116, the chemosensitive fraction, the expression of cadherin E was increased in contrast to the tumors obtained from F3 WiDr, chemoresistant fraction, showing a decrease in expression of E cadherin.Thus, in a third part, knowing that a link between cells initiating cancer and E cadherin was highlighted, we focused on its expression. We first studied its expression in vitro on 5 Fluorouracilresistant cells. Its expression was then studied ex vivo in tissues and sera of operated patients of CRC.

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Gene delivery for therapeutic purposes is quickly emerging as the best potential treatment option for inherited genetic diseases and cancer. Viral gene carriers have been the choice for this purpose due to their high efficiency, but harmful immunogenic and oncogenic host reactions have limited their in vivo use. Cationic polymers provide a safe alternative to viral carriers as they can be engineered to reduce immunogenic and toxic responses and serve therapeutic purposes in the body. Due to their strong positive charge, they are able to compact the negatively charged nucleotides to small nano-sized particles appropriate for cellular uptake. Additionally, they efficiently encapsulate the highly sensitive nucleotides, and protect them against degradation by the nucleases present at the physiological milieu. In this thesis work, I have used a novel approach for gene delivery by combining the critical properties of a cationic polymer (i.e., nucleotide condensing ability) with that of a fatty acid (i.e., lipid membrane compatibility). The resulting lipid modified polymer increased delivery of our gene of interest into target cells and resulted in increased siRNA delivery for cancer gene therapy. / Biomedical Sciences

Utilization of tolerogenic dendritic cells (tolDCs) as a cell-based therapy represents a promising strategy in treatment of autoimmune diseases including type 1 diabetes (T1D). Numerous protocols have been established to generate tolDCs ex vivo and their therapeutic effect has been demonstrated in animal models of autoimmune diseases. In this thesis we compared three different variants of such protocols which are based on the combined treatment of bone marrow- derived DCs with vitamin D and dexamethasone applied at different time points of their maturation towards tolDCs. We assessed the efficiency of these protocols in regards of their effect on the expression of co-stimulatory molecules CD40, CD80, CD86, and MHC II and the chemokine receptor CCR7 on the surface of tolDCs. Then, we evaluated the migration pattern of antigen unloaded tolDCs in vivo as well as their effect on the induction of immune responses and cell proliferation of lymph node cells. This was achieved by labelling of tolDCs with membrane dye PKH26 and by following their migration path by flow cytometry after intraperitoneal (i.p) or subcutaneous (s.c.) injection into either left or right side of the body. On day 1, 3, 5, 7, and 9, the presence of PKH26+ tolDCs was examined in spleen, pancreatic, mesenteric, inguinal and axillary...

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