Le cancer de la prostate (CaP) est actuellement le cancer le plus fréquent en France et constitue l'une des principales causes de décès par cancer chez l'homme dans les pays industrialisés. Un tiers des patients avec un CaP à priori localisé auront déjà des micro-métastases au moment du traitement local. Ces patients qui répondent dans un premier temps à la castration (hormonothérapie) seront cependant en échappement hormonal dans les 2 ans qui suivent. Récemment, plusieurs essais cliniques de phase III ont rapporté un gain de survie avec la chimiothérapie à base de docétaxel dans les CaPs métastatiques résistants à la castration. Néanmoins, la survie n'est prolongée que de 2 ou 3 mois et de nouvelles approches thérapeutiques ciblant des voies de signalisation spécifiques sont donc nécessaires. Les travaux réalisés au cours de cette Thèse ont permis tout d'abord de montrer, grâce à l'utilisation de TMAs, que la surexpression de TP53INP1, une protéine de réponse au stress, était un facteur de mauvais pronostic dans le CaP, prédictif notamment du risque de rechute biologique. Nous avons ensuite pu montrer grâce à des xénogreffes de cellules tumorales (LNCaP) que les taux d'ARNm de TP53INP1 diminuaient durant l'hormonothérapie et que TP53INP1 était de nouveau significativement surexprimée dans les tumeurs résistantes à la castration. Nous avons développé et déposé un brevet pour un oligonucléotide antisens (ASO) inhibant TP53INP1. Le traitement in vitro des lignées cellulaires hormonosensibles LNcaP et hormono-résistantes C4-2 par l'ASO induit une diminution d'expression de la protéine TP53INP1, inhibe la prolifération cellulaire et induit une augmentation de l'apoptose. / Prostate cancer (PC) is the most common malignancy in France and one of the most frequent leading causes of cancer-related death in men in industrialized countries. Even with aggressive screening, approximately one-third of patients believed to have localized PC will already have micro-metastatic disease at the time of definitive local therapy. These patients initially respond to androgen ablative therapy, but with time, their tumors ultimately become unresponsive and recur within 2 years as castration-resistant prostate cancer (CRPC). Recently, docetaxel-based regimens have shown improved survival in men with CRPC in phase III studies. However, the median overall survival was prolonged for only 2-3 months, and thus development of new therapeutic approaches that target relevant signaling pathways are essential to restore the androgen-sensitivity of CRPC. We showed, using tissue micro-array (TMA) analysis, that over-expression of Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1), a cell stress response protein, is a worse prognostic factor in PC, particularly predictive of biological cancer relapse. We also we found that TP53INP1 protein expression decreases during castration therapy (CT) and significantly increases in human CRPC. TP53INP1 mRNA was also significantly increased in castration-resistant (CR) tumors of LNCaP xenograft compared to the castration-sensitive (CS) taken before CT. We developed and world-wide patented one antisense oligonucleotide (ASO) targeting TP53INP1 (PCT/IB2011/054555). Treatment of LNCaP and C4-2 cells in vitro with TP53INP1 ASO downregulates TP53INP1 protein level, inhibits proliferation and induces apoptosis.

In the post genomic era, proteomics has enormous potential in biology and medicine. Among the various bioanalytical tools developed, protein microarray is one of the recent advancements which offer high throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. Fundamentally, the protein microarray involves the immobilization of interacting elements, proteins, on a few square microns of a solid support and in principle, it is capable of detecting analytes with a higher sensitivity than conventional macroscopic immunoassays. Here in the present report we delineates the design, fabrication and functional analysis of protein microarray using semi-synthetic ssDNA tagged-proteins as capturing moiety as well as address on a solid support. Optimization of the platform has been carried out by investigating various parameters such as surface chemistry, signal amplification, and conditions for homogenous liguid phase protein-protein interaction. / Singapore-MIT Alliance (SMA)

Sutherlandia frutescens is a South African herbal remedy traditionally used for various ailments and lately to improve the overall health in cancer and HIV/AIDS patients. Relatively little is known about the mechanisms of action of the constituents present in S. frutescens. The aim of this project was to examine the in vitro influence of crude ethanolic S. frutescens extracts in human breast adenocarcinoma (MCF-7) and non-tumorigenic breast epithelial (MCF-12A) cells after 48 h of exposure. Dose-dependent studies were conducted on cell numbers and metabolic activity by means of spectrophotometry. Morphological changes were determined with light-, fluorescent- and transmission electron microscopy (TEM). Cell cycle progression and apoptosis were analyzed using flow cytometry. The differential effects of S. frutescens extracts on gene expression levels in both the MCF-7 and MCF-12A cells were conducted utilizing micro array analysis. mTOR kinase activity was measured with an ELISA assay. S. frutescens reduced cell proliferation in both the non-tumorigenic MCF-12A and the tumorigenic MCF-7 cell line in a dose-dependent manner. The tumorigenic MCF-7 cells were more susceptible to S. frutescens treatment compared to the non-tumorigenic MCF-12A cells. Morphological characteristics of apoptosis and autophagy, including cytoplasmic shrinking, membrane blebbing and an increase in autophagic vacuoles were observed in both cell lines with the MCF-7 cells being more susceptible to autophagy and the MCF-12A cells less susceptible to autophagy and apoptotic cell death. TEM confirmed ultrastructural characteristics of autophagy in both cell lines. Flow cytometry revealed a G2/M arrest with no increase in apoptosis in MCF-7 cells and a G2/M arrest with an increase in apoptosis in MCF-12A cells treated with 1.5mg/ml S. frutescens extract. Microarray analyses revealed 325 statistically significantly differentially expressed genes in MCF-7 cells and 1467 genes in MCF-12A cells. The majority of S. frutescens-treated genes were down-regulated when compared to the vehicle-treated control in both cell lines. Several genes involved in DNA replication and repair were differentially expressed in response to S. frutescens exposure. These include Poly (ADP-ribose) polymerase family, member 2 (PARP-2) (down-regulated in both cell lines), PCNA (down-regulated in MCF-7 cells) and growth arrest and DNA-damage-inducible beta (GADD45B) (up¬regulated in MCF-12A cells). This suggests that abrogated expression of genes involved in DNA replication and repair play a role in inducing a G2/M cell cycle arrest in S. frutescens-treated cells. ELISA analysis of the mTOR kinase revealed a decrease in mTOR kinase activity in both cell lines after S. frutescens exposure. Therefore, attenuated mTOR kinase activity as a result of S. frutescens treatment in both cell lines is regarded as a central mediator in inducing autophagy suppressing gene expression and inhibiting ribosome biogenesis. Understanding of in vitro molecular mechanisms of S. frutescens enables researchers to focus on affected cellular mechanisms and identify active compounds with subsequent evaluation as possible candidates for use in anticancer therapy. The current study contributes to the unraveling of the in vitro molecular mechanisms and signal transduction associated with 70% ethanolic S. frutescens extracts, providing a basis for further research on this multi-purpose medicinal plant in Southern Africa. / Dissertation (MSc)--University of Pretoria, 2007. / Physiology / unrestricted

Une pléthore de processus biologiquement pertinents dépend directement de la sécrétion de biomolécules dans le milieu extracellulaire, aux fonctions régulatrices ou composants structurels. L’analyse de processus biologiques complexes nécessite ainsi la mise au point de nouveaux outils de biodétection. Par conséquent, le but de cette thèse est de fournir des stratégies polyvalentes pour la génération de biocapteurs et de biopuces innovants basés sur la Résonance des Plasmons de Surface (SPR). À l’issue de ces travaux, une méthode de photofonctionnalisation indirecte a été mise au point. Ce procédé a permis de générer des micro-réseaux de protéines dans des conditions entièrement aqueuses, et ainsi de préserver la fonctionnalité des protéines greffées. De plus, nous avons créé et évalué une nouvelle biopuce SPR microstructurée pour le suivi en temps réel des sécrétions cellulaires. Cette biopuce microstructurée présente deux phénomènes optiques différents qui peuvent être utilisés pour la détection cellulaire et le suivi de leurs sécrétions. Enfin, de multiples stratégies de fonctionnalisation ont été évaluées pour la conception d’une biopuce SPR nanostructurée à faisceau de fibres optiques. Parmi ces approches, la génération de monocouches photoréactives auto-assemblées a été la plus adaptée à ce système et est en cours d’optimisation. Une fois réalisée, cette biopuce nanostructurée pourrait ouvrir la voie à la poursuite du développement de systèmes prometteurs de biodétection in vivo. / A plethora of biologically relevant processes depends directly on the effective secretion of biomolecules, from regulatory molecules to structural components. Thus, the analysis of complex biological processes requires the development of novel biosensing tools. Therefore, the aim of this thesis is to provide versatile strategies for the generation of innovative biosensors and biochips based on Surface Plasmon Resonance (SPR). As a result from this research, an indirect photofunctionalization method was developed. This procedure allowed the generation of protein microarrays in fully aqueous conditions while preserving the functionality of the grafted proteins. Furthermore, we created and evaluated a novel microstructured SPR biochip for real-time monitoring of cellular secretions. This microstructured biochip presents two different optical phenomena which could be used for cell detection and the monitoring of their secretions. Finally, multiple functionalization strategies were evaluated for the conception of a nanostructured fiber-bundle SPR biochip. Among the approaches, the generation of photoreactive self-assembled monolayers was the most adapted to this system and currently is being optimized. Once achieved, this nanostructured biochip could pave the way for further development of promising in vivo biosensing systems.

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In this dissertation, first we consider the problem of testing homogeneity and order in a contaminated normal model, when the data is correlated under some known covariance structure. To address this problem, we developed a moment based homogeneity and order test, and design weights for test statistics to increase power for homogeneity test. We applied our test to microarray about Down’s syndrome. This dissertation also studies a singular Bayesian information criterion (sBIC) for a bivariate hierarchical mixture model with varying weights, and develops a new data dependent information criterion (sFLIC).We apply our model and criteria to birth- weight and gestational age data for the same model, whose purposes are to select model complexity from data.

Background: Prostate Cancer (PC) is a common and highly variable disease. Using current diagnostic methods, the prostate specific antigen (PSA) blood test and histological grading of prostate tissue needle biopsies, it is often difficult to evaluate whether the patient has a PC that requires active treatment or not. The absolute majority of all 10,000 cases of PCs diagnosed annually in Sweden have tumours graded as Gleason score (GS) 6-7 and a PSA value in blood below 10. Many of these are harmless and can be left without active treatment and hence spared problematic post-therapy side-effects, others are highly malignant and require early diagnosis and treatment. Better prognostic markers are needed and the aim of this study was to evaluate prognostic markers and to test if these markers could identify patients with indolent tumours. Methods: We have studied tumour material from 419 men consecutively diagnosed with PC at transurethral resection (1975-1990). The majority of these patients (295) had no metastasis at diagnosis and was not given any curative treatment and only hormonal treatment upon symptoms from metastatic progression. Standard histological sections and tissue microarrays (TMA) from these tumours and surrounding normal prostate tissue were stained and evaluated for cell proliferation (Ki67), blood vessels (endoglin and von Willebrand factor, vWf) and the extracellular matrix component hyaluronan (HA). An orthotopic rat PC model was used to explore hyaluronan staining, hyaluronic acid synthase (HAS)-1 mRNA levels and the effect of local HA treatment on tumour growth. Results: Tumour cell proliferation (Ki67) and the density of intra-tumoural endoglin stained blood vessels were independent prognostic markers (i.e. they added prognostic information to the conventional prognostic markers; clinical stage and GS). None of the GS 6 patients with low staining for both Ki67 and endoglin died of PC within 15 years of follow-up. High HA staining in the tumour epithelium and stroma was a negative prognostic marker of cancer specific survival but they were not independent of GS. High HA staining and high vascular density in the stroma of the surrounding morphologically normal prostate were prognostic for short cancer specific survival. Implantation of tumour cells in the normal rat prostate resulted in an increase in HA and HAS-1 mRNA levels in the prostate tissue surrounding prostate tumours. Concurrently intra-prostatic injection of HA also stimulated tumour growth. Conclusions: By evaluating both tumour cell proliferation (Ki67) and vascular density, it is possible to identify patients with very low risk of cancer specific death in the absence of active treatment. Prostate tumours influence the surrounding non-malignant prostate tissue, for example they cause an increased angiogenesis and synthesis of hyaluronan. Such responses can possibly be used to diagnose PC and to evaluate PC aggressiveness.

Le cancer de la prostate est le cancer le plus fréquemment diagnostiqué et la troisième cause de mortalité liée au cancer chez les hommes au Canada. Un quart des patients diagnostiqués développeront une forme plus agressive de ce cancer. Bien que nous possédions plusieurs indices cliniques pronostiques dans les cancers localisés (score de Gleason, taux sérique d’antigène prostatique spécifique (APS), stade, etc.), ceux-ci sont insuffisants pour adéquatement distinguer les patients à faible risque de progression de ceux à haut risque. A ce jour, aucun biomarqueur pronostique n’est encore utilisé en clinique. Les cliniciens ont donc besoin de nouveaux outils plus efficaces pour stratifier ce cancer et pour s’assurer d’adapter au mieux le traitement à chaque patient. En nous basant sur la littérature et sur des études préliminaires (cohortes de moins de 65 patients), notre hypothèse est que les protéines PUMA-NOXA et les récepteurs membranaires de la famille ERBB seraient, lorsqu’utilisés en combinaison, des biomarqueurs prédictifs de la progression du cancer de la prostate. Les objectifs de cette thèse sont : 1) identifier et valider de bons anticorps pour chaque biomarqueur d’intérêt, 2) définir les niveaux d’expression de chaque biomarqueur sur une cohorte de 285 patients, et 3) établir les corrélations entre les niveaux d’expression et les données cliniques des patients. Dans l’optique d’une utilisation en clinique, des anticorps de type monoclonal ont été choisis pour identifier les biomarqueurs d’intérêts. Ces anticorps ont été testés et validés pour leur spécificité par immunobuvardage de type western blot et par immunofluorescence. La localisation de la protéine d’intérêt a été validée sur des échantillons de tissus de patients suivie de l’optimisation du multi-marquage sur les cellules épithéliales et basales. Après perfectionnement de l’analyse d’images, nous avons montré qu’une expression extrême (faible ou forte) de PUMA couplée à une forte expression de NOXA dans les glandes bénignes est associée à la rechute biochimique des patients. La présence de ces biomarqueurs dans les glandes bénignes permet d’envisager d’améliorer l’identification lors des premières biopsies des patients se qualifiant pour la surveillance active. Par ailleurs, le suivi de l’expression des récepteurs de la famille ERBB dans les glandes tumorales permet une stratification des patients atteints d’un cancer de la prostate en fonction des risques de rechute biochimique, de développement de métastases et de mort liée au cancer. Ainsi, les patients présentant la combinaison d’une forte expression de EGFR et d’une faible expression de ERBB3 sont les plus susceptibles de mourir spécifiquement de leur cancer de la prostate, en particulier si les cellules tumorales présentes en plus une faible expression de ERBB2 entrainant un fort risque de développer des métastases. Mon projet de doctorat aura donc permis d’identifier et de valider des biomarqueurs d’intérêt pour prédire l’évolution du cancer de la prostate et démontrer l’intérêt de suivre ces biomarqueurs en combinaison afin d’obtenir une meilleure stratification des patients. Ces résultats devront être validés sur une cohorte indépendante et multicentrique en vue de fournir aux cliniciens un plus grand nombre d’outils pour leur permettre de réaliser une stratification fine des patients atteints d’un cancer de la prostate, et ouvrirait la voie à de nouvelles stratégies thérapeutiques plus ciblées. / Prostate cancer is the most frequently diagnosed cancer and the third leading cause of cancer-related death in men in Canada. A quarter of patients will develop a more aggressive form of this cancer. While there are several clinical prognostic variables for localized prostate cancer (Gleason score, prostate specific antigen (PSA) levels, stage, etc.), these are insufficient to adequately distinguish between low and high-risk of progression cases. As a result, clinicians need new, more effective tools to stratify this cancer and to ensure that treatments are best tailored to each patient. To date, no prognostic biomarker has yet been used clinically. Based on the literature and preliminary studies of small cohorts (less than 65 patients), we hypothesize that the protein expression of PUMA-NOXA and ERBB family members could help with the prediction of prostate cancer progression. The objectives of this thesis are: 1) to identify and validate antibodies for each biomarker of interest, 2) to define the expression levels of each biomarker on a 285 patient cohort, 3) to evaluate the correlation between marker expression levels and patient clinical data. For clinical use, monoclonal-type antibodies were chosen to identify the biomarkers of interest. These antibodies were validated for specificity by western blot and immunofluorescence techniques. The localization of the protein of interest was further identified within samples of patient tissues and additional optimization involving combinatorial staining for epithelial and basal cells. After refining the imaging and statistical analysis of PUMA and NOXA in benign glands, we found that extreme (weak or strong) PUMA expression coupled with high NOXA expression was associated with biochemical relapse. In addition, these proteins have significant potential for predicting disease evolution based on the initial radical prostatectomy sample. The presence of these proteins in benign glands would allow the identification of patients less suitable for active surveillance. Additionally, statistical analysis of ERBB family receptors in tumor glands, when used alone, allow stratification of prostate cancer patients for the prediction of biochemical relapse, development of metastases and also specific death from prostate cancer. Moreover, patients expressing a combination of high EGFR expression and low ERBB3 expression are at high risk of biochemical relapse and are at higher risk of prostate cancer specific mortality. In addition, coupling this high EGFR – low ERBB3 combination to a low ERBB2 expression helps classify patients at high risk of developing metastases. My doctoral research project will have made it possible to identify and validate biomarkers of interest for predicting the progression of prostate cancer and demonstrating the interest of combining these biomarkers in order to achieve better stratification of patients with prostate cancer. In the context of clinical utility, these results need to be validated on an independent and multicenter cohort in order to confirm these findings. This would eventually provide clinicians with a greater number of tools at their disposal to correctly anticipate patient trajectories and possibly identify new therapeutic targets for the control of the disease.

O melanoma cutâneo é a neoplasia de pele de maior mortalidade. A imprevisibilidade de sua evolução é uma de suas características principais, o tratamento do tumor primário é, atualmente, de pouca morbidade e, na doença disseminada, as opções terapêuticas são pouco eficazes. É fundamental a pesquisa de marcadores tumorais que permitam a previsão da evolução, melhor compreensão da patogênese do melanoma e possibilitem a descoberta de alvos moleculares. Nesse contexto, estudos genéticos mostraram a importância da regulação do ciclo celular, especialmente a passagem da fase G1-S. Importantes fatores envolvidos compõem a cascata da proteína Rb (p16, ciclina D1, CDK4 e pRb) e da proteína p53 (p53 e p21). Objetivo: verificar a frequência da expressão de p16, ciclina D1,CDK4, pRb, p53 e p21 em melanomas cutâneos de cabeça, pescoço e tronco e sua relação com prognóstico. Métodos: Estudo retrospectivo envolvendo 46 pacientes (sendo 67,3% homens, idade média 57,7 ± 15,8 anos) com melanoma cutâneo de cabeça, pescoço e tronco que foram tratados pela mesma equipe com seguimento mínimo de dois anos. Foram estudados fatores clínicos (topografia do tumor primário, tempo de seguimento, ocorrência de metástases e óbito relacionado), histopatológicos (tipo histológico, índice de Clark, índice de Breslow) e análise imuno-histoquímica pela técnica de micro-array para as proteínas reguladoras do ciclo celular p16, ciclina D1, CDK4, pRb, p53 e p21. Resultados: Houve proporção igualitária entre as topografias (23 casos em tronco, 23 em cabeça e pescoço). Treze pacientes com Clark I (29,5%), cinco com II (11,3%), 16 com III (36,5%), 10 com IV (22,7%) e nenhum com Clark V. A média das medidas de Breslow foi 0,96 (DP=1,01). O seguimento médio foi de 77 meses (DP=47). Oito dos 46 pacientes (17,3%) tiveram evolução desfavorável, com seis óbitos relacionados. A idade foi mais elevada no grupo com evolução desfavorável (p=0,04). Houve expressão de p16 em 80%, ciclina D1 em 58,9%, CDK4 em 43,5%, pRb em 58,5%, p53 em 53,6% e p21 em 52,3% dos melanomas. Em análise univariada, a expressão do p21 foi relacionada com evolução desfavorável (p=0,04), o que não foi observado com a expressão dos outros marcadores (p>0,05). Conclusão: A expressão da proteína p21 nos melanomas cutâneos de cabeça, pescoço e tronco foi relacionada com evolução desfavorável, o que não ocorreu com outros fatores envolvidos na regulação do ciclo celular / Melanoma is the most lethal skin cancer. The outcome of melanoma is not predictable in most cases. Although the treatment for the primary tumor is well tolerated, there are no effective therapeutic options in disseminated disease. Efforts are being made in the search for tumoral markers that may predict outcome, increase the comprehension of melanoma pathogenesis, and may also help the search for molecular targets. In this issue, genetic studies concerning the regulation of cell cycle, including the G1-S checkpoint, are important. The retinoblastoma protein (pRb) pathway (p16, cyclin D1, CDK4 and pRb) and the p53 pathway (p53 and p21) are part of this regulation. Objectives: to verify the expression of p16, cyclin D1, CDK4, pRb, p53 and p21 in head, neck and trunk melanomas, and its correlation with prognosis. Method: Retrospective study approved by institution ethics committee. Fourtysix head, neck and trunk melanoma patients (67.3% men, mean age 57.7±15.8) treated by a single surgeon with minimum 2-years follow-up were enrolled. Clinical factors (primary tumor location, follow-up period, metastasis or related deaths), pathologic (histological subtype, Clark and Breslow index) and microarray immunohystochemical analysis of the cell cycle proteins p16, cyclin D1, CDK4, pRb, p53 and p21. Results: Location of the primary tumor was equal for head/neck and trunk (50% each). Thirteen patients were classified as Clark I (29.5%), five as Clark II (11.3%), 16 as Clark III (36.5%), 10 as IV (22.7%), none as Clark V. Mean Breslow measure was 0.96±1.01. Mean follow-up was 77±47 months. Eight patients (17.3%) had bad outcome, with six related deaths. Patients with worse outcome had a higher mean age at diagnosis (p=0,04). Expression of p16 was positive in 80%. Cyclin D1 was positive in 58.9%. CDK4 was positive in 43.5%. pRb was positive in 58.5%. p53 was positive in 53.6%. p21 was positive in 52.3%. Univariated analysis showed that p21 expression was related to worse outcome (p=0,04), while the other markers were not (p>0,05). Conclusion: p21 expression in head, neck and trunk melanomas was related to worse outcome. Expression of the other cell cycle regulators proteins was not