Le cancer de la prostate est le cancer le plus fréquent chez les hommes dans la plupart des pays développés. C'est le cancer le plus fréquent chez les hommes en France (73.609 cas en 2014) et en Colombie (9564 cas en 2014). En outre, c'est la troisième cause de décès par cancer chez les hommes dans les deux pays (9,3 % en France et 7,1 % en Colombie en 2014). L'une des techniques de traitement est la radiothérapie externe, qui consiste à délivrer un rayonnement ionisant à une cible clinique, à savoir la prostate et les vésicules séminales. En raison des variations anatomiques au cours du traitement, qui consiste en environ 40 fractions de rayonnement délivrant une dose totale allant de 70 à 80Gy, des marges de sécurité sont définies autour de la cible tumorale lors de la planification du traitement. Ceci entraîne des portions d'organes sains voisins de la prostate - la vessie et le rectum - à être inclus dans le volume cible, pouvant conduire à des événements indésirables affectant les fonctions urinaires (hématurie et cystite, entre autres) ou rectale (saignement rectal, incontinence fécale, Etc.). La vessie présente les plus grandes variations de forme entre fractions de traitement, provoquées par des changements continus de volume. Ces variations de forme introduisent des incertitudes géométriques qui rendent difficile l'évaluation de la dose réellement délivrée à la vessie pendant le traitement. Ces incertitudes limitent la possibilité de modéliser une relation dose-volume pour la toxicité génito-urinaire tardive (GU). Le projet QUANTEC (Quantitative Analysis of Normal Tissue Effects in the Clinic) a déclaré que la relation dose-réponse pour la toxicité gastro-intestinale tardive (GI) était loin d'être établie. Les variables dosimétriques obtenues à partir de la tomodensitométrie de planification peuvent être faiblement représentative de la dose effectivement administrée. En conséquence, il est crucial de quantifier les incertitudes produites par les variations inter-fraction de la vessie afin de déterminer les facteurs dosimétriques qui affectent les complications GU tardives. Le but de cette thèse était donc de caractériser et de prédire les incertitudes produites par les variations géométriques de la vessie entre les fractions de traitement, en utilisant uniquement la tomodensitométrie de planification comme information d'entrée. En pratique clinique, une seule tomodensitométrie est disponible au moment de la planification du traitement pour un patient typique, alors que des images supplémentaires peuvent être acquises en cours de traitement. Dans cette thèse une approche population a été utilisée pour obtenir suffisamment de données pour apprendre les directions les plus importantes du mouvement et de la déformation de la vessie en utilisant l'analyse en composante principales (ACP). Comme dans les travaux de référence, ces directions ont ensuite été utilisées pour développer des modèles basés population pour prédire et quantifier les incertitudes géométriques de la vessie. Cependant, nous avons utilisé une analyse longitudinale afin de caractériser correctement la variance du patient et les modes spécifiques du patient à partir de la population. Nous avons proposé d'utiliser un modèle à effets mixtes (ME) et une ACP hiérarchique pour séparer la variabilité intra et inter-patients afin de contrôler les effets de cohorte confondus. Finalement, nous avons présenté des modèles sur l'APC comme un outil pour quantifier des incertitudes de la dose produit par le mouvement et déformation de la vessie entre fractions. / Prostate cancer is the most common cancer amongst the male population in most developed countries. It is the most common cancer amongst the male population in France (73.609 cases in 2014) and in Colombia (9564 cases in 2014). It is also the third most common cause of cancer deaths in males in both countries (9.3% and 7.1% in France and in Colombia in 2014, respectively). One of the standard treatment methods is external radiotherapy, which involves delivering ionizing radiation to a clinical target, namely the prostate and seminal vesicles. Due to the uncertain location of organs during treatment, which involves around forty (40) radiation fractions delivering a total dose ranging from 70 to 80Gy, safety margins are defined around the tumor target upon treatment planning. This leads to portions of healthy organs neighboring the prostate or organs at risk — the bladder and rectum — to be included in the target volume, potentially resulting in adverse events affecting patients’ urinary (hematuria and cystitis, among others) or rectal (rectal bleeding, fecal incontinence, etc.) functions. The bladder is notorious for presenting the largest inter-fraction shape variations during treatment, caused by continuous changes in volume. These variations in shape introduce geometric uncertainties that render assessment of the actual dose delivered to the bladder during treatment difficult, thereby leading to dose uncertainties that limit the possibility of modeling dose-volume response for late genitourinary (GU) toxicity. The Quantitative Analysis of Normal Tissue Effects in the Clinic (QUANTEC) project has stated that a similar dose-response to that of late gastrointestinal (GI) toxicity is far from being established. The dosimetric variables obtained from the planning CT prove to be very poor surrogates for the real delivered dose. As a result, it appears crucial to quantify uncertainties produced by inter-fraction bladder variations in order to determine dosimetric factors that affect late GU complications. The aim of this thesis was thus to characterize and predict uncertainties produced by geometric variations of the bladder between fractions, using solely the planning CT as input information. In clinical practice, a single CT scan is only available for a typical patient during the treatment planning while on-treatment CTs/CBCTs are seldom available. In this thesis, we thereby used a population approach to obtain enough data to learn the most important directions of bladder motion and deformation using principal components analysis (PCA). As in groundwork, these directions were then used to develop population-based models in order to predict and quantify geometrical uncertainties of the bladder. However, we use a longitudinal analysis in order to properly characterize both patient-specific variance and modes from the population. We proposed to use mixed-effects (ME) models and hierarchical PCA to separate intra and inter-patient variability to control confounding cohort effects. . Subsequently, we presented PCA models as a tool to quantify dose uncertainties produced by bladder motion and deformation between fractions.

Submitted in fulfillment of the requirements of the Degree in Chemistry, Durban University of Technology, 2017. / High prevalence and mortality cases of prostate cancer (PCa) have increased around the world, particularly in developing countries. Several forthcoming factors have been revealed nowadays, one of them is due to the incapability of the diagnostic methods to produce reliable results, which impacts negatively on cancer-treatment. However, a sensitive diagnosis of PCa cells remains a challenge in the field of biosensors. Emerging whole-cell detection as biosensing targets has opened up avenues for successful cancer diagnostics, due to high selectivity among other cells. A switchable and flexible surface-based graphene material is one of the techniques that revolutionized smart biodevice platforms in biosensor technology. In this present study, a covalently linked poly-(N-isopropylacrylamide) (PNIPAM) to graphene oxide surface has been employed as “on/off”-switchable aptamer-based sensor for the detection of PC3 whole-cancer cell. The constructed surface has benefitted from PNIPAM, as the thermal-stimulus agent, which allows the coil-to-globule transitions by triggering temperature changes. When the system is above its lower critical solution temperature (LCST) of 32oC, PNIPAM will exist as hydrophobic -globular state providing an “on” binding region for the whole-cell, reaching the interactions on the biosurface. The “off” binding systems is only possibly when the PNIPAM turns into extended-state by lowering its temperature below LCST. The first principle studies have successfully characterized the electronic behavior with particular emphasis of PNIPAM monomer functions along with the description of the structural energetics of complex through density functional theory (DFT). Docking studies have further been performed to predict a plausible binding aptamer toward the protein-representative PCa cell. To better understand the prospect of an aptamer-based tunable biosensor, molecular dynamics (MD) highlighted the behavior of PNIPAM-grafted GO in exhibiting a globular and extended conformations at above and below LCST, permitting the biomolecules to interact with each other as well as to avoid interactions, respectively. Experimental studies have been included to validate the theoretical predictions by fabricating real-biosensor systems using electrochemical impedance technique, resulting a low-detection limit down to 14 cells/mL. Engagement between theoretical and experimental studies delivered an enhanced tunable-biosensor performance for the detection of whole cell prostate cancer. / M

>Magister Scientiae - MSc / Prostate Cancer is the leading cause of cancer-related death in males in the Western world. It is a common biological disease originating from the reproductive system of the male namely, the prostate gland, usually in older patients (over the age of 50) and with a family history of this disease. The disease shows clinical aggressiveness due to genetic alterations of gene expression in prostate epithelial cells. Prostate cancer is currently diagnosed by biopsy and prostate cancer screening via the Prostate-Specific Antigen (PSA) blood test. Early detection is critical and although PSA was discovered to aid in the diagnoses of this cancer at its early stages, it has a disadvantage due to its low specificity thus causing unnecessary biopsies of healthy individuals and overtreatment of patients. Although various studies and efforts have been made to identify the ideal biomarker for prostate cancer and many even being applied to clinical use, it is still challenging and has not replaced the best-known biomarker PSA. PSA test has minimal invasive characteristics, at relatively low cost together with high sensitivity but low specificity. Biomarker discovery is a challenging process and a good biomarker has to be sensitive, specific and its test highly standardized and reproducible as well as identify risk for or diagnose a disease, assess disease severity or progression, predict prognosis or guide treatment. Computational biology plays a significant role in the discovery of new biomarkers, the analyses of disease states and the validation of potential biomarkers. Bioinformatic approaches are effective for the detection of potential micro ribonucleic acid (miRNA) in cancer. Altered miRNA expression may serve as a biomarker for cancer diagnosis and treatment. Small non-protein coding RNA, miRNA are small regulatory RNA molecules that modulate the expression of their target genes. miRNAs influence numerous cancer-relevant processes such as proliferation, cell cycle control, apoptosis, differentiation, migration and metabolism. Discovery and existence of extracellular miRNAs that circulate in the blood of cancer patients has raised the possibility that miRNAs may serve as novel diagnostic markers. Since a single miRNA is said to be able to target several mRNAs, aberrant miRNA expression is capable of disrupting the expression of several mRNAs and proteins. Biomarker discovery for prostate cancer of mRNA and miRNA expression are strongly needed to enable more accurate detection of prostate cancer, improve prediction of tumour aggressiveness and facilitate diagnosis. The aim of this project was to focus on functional analyses of genes and their protein products regulated by previously identified miRNA in prostate cancer using bioinformatics as a tool. Most proteins function in collaboration with other proteins and therefore this study further aims to identify these protein-protein interactions and the biological relevance of these interactions as it relates to Prostate cancer. Various computational databases were used such as STRING, DAVID and GeneHub-GEPIS for functional analyses of these miRNA regulated genes. The main focus was on the 21 genes regulated by several miRNAs identified in a previous study. Results from this study identified six genes; ERP44, GP1BA, IFNG, SEPT2, TNFRSF13C and TNFSF4, as possible diagnostic biomarkers for prostate cancer. These results are promising, since the targeted biomarkers would be easily detectable in bodily fluids with the Gene Ontology (GO) analysis of these gene products showing enrichment for cell surface expression. The six genes identified in silico were associated to transcription factors (TFs) to confirm regulatory control of these TFs in cancer promoting processes and more specifically prostate cancer. The CREB, E2F, Nkx3-1 and p53 TFs were discovered to be linked to the genes IFNG, GP1BA, SEPT2 and TNFRSF13C respectively. The expression of these TFs show strong association with cancer and cancer related pathways specifically prostate cancer and thus demonstrates that these genes can be assessed as possible biomarkers for prostate cancer. The prognostic and predictive values of the candidate genes were evaluated to assess their relationship to prognosis of this disease by means of several in silico prognostic databases. The results revealed expression differences for the majority of the candidate genes were not significantly sufficient to be distinguished as strong prognostic biomarkers in several prostate cancer populations. Although one marker, GP1BA was supported as having prognostic value for prostate cancer based on it's statistical pvalue in one of the prostate cancer patient datasets used. Another candidate gene SEPT2 showed promise as it has some prognostic value in the early stages of the disease. Although the results yielded, based on the in silico analysis, were not the discovery of an ideal diagnostic marker based on the set criteria in this study, further analysis using a molecular approach qRT-PCR can be considered for a detailed followup study on selected candidate genes to evaluate their roles in disease initiation and progression of prostate cancer using cell lines as well as patient samples. / CSIR

Le phénomène métastatique est important à comprendre puisque de manière irrémédiable, une fois engagé, il conduit le plus souvent au décès des patients. Le cancer de la prostate représente un bon modèle car sa progression du stade hormono-dépendant vers le stade d'hormono-échappement s’accompagne par l’apparition de métastases. Les Protease Activated Receptors (PAR1-4) sont des récepteurs qui jouent un rôle important dans l'hémostase et l'inflammation et dont l’implication dans la prolifération et l'invasion des cellules tumorales a été décrite dans plusieurs tissus. L’étude comparative de l'expression in vitro de PAR1 a confirmé son rôle dans la prolifération et l'invasion des lignées prostatiques normales et tumorales hormono-sensibles comme hormono-indépendantes. Son expression in vivo dans des tissus prostatiques à différents stades pathologiques a montré une surexpression de PAR1 chez les patients ayant atteint le stade d'hormono-échappement, associée à un mauvais pronostic. Son absence s’avère par contre de bon pronostic chez les patients hormonodépendants. L'ensemble des résultats obtenus nous permet de proposer PAR1 comme un nouveau marqueur pronostique pour le cancer de la prostate. L’activation des PARs, comme celle de plusieurs récepteurs de chémokines, apparaît comme un élément fondateur de la transition vers l’état métastatique. Le décryptage de cette combinatoire permettra de mieux comprendre les phénomènes impliqués dans cette transition et permettra de développer des thérapies ciblées pour prévenir l’apparition délétère de métastases / Metastasis is nowadays an important field of research as, once engaged, patients will generally die from their metastatic cancer. Prostate cancer represents an interesting model as its progression from hormone-naïve to hormone-independent status lead to metastatic disease. Protease Activated Receptors (PAR1-4) are G-protein-coupled receptors that play crucial roles in blood coagulation and inflammation but that are likely to play fundamental role in tumor cells proliferation and invasion. In vitro analysis of PAR1 expression in prostate cancer cell lines has confirmed the role of PAR1 in prostate cancer proliferation and invasion. Its expression in vivo in prostate cancer tissues have shown a constant surexpression in hormonerefractory ones, associated with a worse prognosis. However, its absence in hormone-naïve tissues is associated with a good prognosis. These results prompted us to recommend PAR1 as a new prognostic marker associated with prostate cancer progression. PAR activation, as well as several chemokine receptors, seems to be a founder feature of cancer transition to metastasis. Deciphering the pattern of receptor activation will allow a better understanding of the events that drive transition to metastasis and thus the development of new specific targeted therapeutics aimed at stopping deleterious metastatic evolution

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The prostate is prone to three pathological processes that include inflammation, benign prostate hyperplasia (BPH) and tumors. According to the center for Disease and Control 1999-2012 report, prostate cancer is the second leading cause of death in the United States. Scientific evidence suggests that up to 30% of men in the general population aged from 50 years and above, irrespective of geographic origin, have foci of prostate neoplastic growth. Unbalanced ROS production and a dysregulated antioxidant defence system have been implicated in prostate cancer development. The transformation of a normal cell into cancer takes a very long period. This observation provides the advantage of using nutraceuticals to prevent, arrest or reverse the cellular and molecular processes of carcinogenesis. Based on scientifically observed positive health roles of green tea (Cameli sinensis) and rooibos (Aspalathus linearis) on major diseases like atherosclerosis, hepatitis and certain types of cancer, this thesis evaluated the effects of these two teas on benign (RPWE 1) and malignant (LNCaP) prostate cells. This was done through the quantification of reactive oxygen species (ROS) using a fluorescence dye 5,6 CM-H2DCFDA, total prostate specific antigen (PSA) levels using a PSA ELISA kit, cell viability using the MTT assay, apoptosis using Tali annexin V stain and cell imaging studies using a Zeiss axiovert 200M inverted fluorescence microscope. Statistical analysis was done using graphpad prism. The findings of this study show that aqueous extracts of green and black tea, fermented and unfermented rooibos and their active compounds epigallocatechin gallate (EGCG) and aspalatin, respectively, are cytotoxic in malignant (LNCaP) prostate cells but exert protective effects in benign (RPWE 1) prostate cells. This thesis implicates the pro-oxidant and anti-oxidant properties of the plant extracts, respectively, for the above mentioned effects. In this regard, tea and rooibos promoted ROS production in malignant (LNCaP) prostate cells, which subsequently promoted cell death of the malignant cells through apoptosis and necrosis. Further to this, tea and rooibos used in this thesis, protected normal prostate cells from the adverse effects of ROS. In this regard, fluorescence microscope photographs showed RPWE 1 cells with low DCF fluorescence compared to the malignant prostate cells. Low magnification light microscope photographs showed RPWE 1 cells with flat polygonal shapes and increased adherence both at low and high concentrations of tea and rooibos. On the contrary, high concentrations of tea and rooibos on malignant (LNCaP) prostate cells induced stress, which made the cells attain irregular shapes and as the stress levels increased, cells became detached and appeared dead. Flow cytometry confirmed the presence of apoptotic and necrotic cell in malignant (LNCaP) prostate cells. In this thesis, EGCG and aspalathin were responsible for the high rates of apoptosis observed whereas green tea and unfermented rooibos induced the highest rate of necrosis. Further to this, tea and rooibos and the main active compounds EGCG and aspalathin, respectively, significantly promoted the reduction of total serum prostate specific antigen (PSA) in malignant prostate cells. In normal prostate cells, these plant extracts maintained the total serum PSA at its basal physiological level. In this thesis, to the best of our knowledge, we report for the first time the cell-specific effects of fermented rooibos, unfermented rooibos and their main active component aspalathin, on prostate cancer cells. We showed that rooibos and aspalathin exert pro-oxidant effects on malignant LNCaP cells and anti-oxidant effects on benign RPWE 1 cells. In conclusion, tea (C. sinensis) and rooibos (A. linearis) and their respective main active compounds, epigallocatechin gallate and aspalathin, are cytotoxic to malignant prostate cells whereas in normal prostate cells, they have protective effects against ROS induced stress. The pro-oxidant and anti-oxidant effects are responsible for the aforementioned effects respectively. The decrease in total serum PSA demonstrate the strong therapeutic effects that tea and rooibos have on malignant (LNCaP) prostate cells. / Malawi Government: Department of Human Resources Development and Management

Les options thérapeutiques d'intention curative ont une faible probabilité de succès chez les patients atteints d'un cancer de la prostate avec métastases ganglionnaires. Ainsi, l'obtention d'une méthode de prédiction de la présence ou l'apparition de métastases ganglionnaires est souhaitable. Ce document présente le développement d'un modèle de prédiction de la présence de métastases ganglionnaires à la prostatectomie radicale. Le modèle utilise les marqueurs radiomiques extraits de l'examen FDG-TEP/CT fait en préopératoire. Ces marqueurs issus de la radiomique - définie comme le processus d'extraction quantitative de données exploitables de haute dimension à partir d'images médicales - permettent de quantifier des caractéristiques invisibles à l'œil tels que les paramètres de texture et d'intensité. Dans un premier volet, afin de limiter les erreurs de recalage entre la TEP et le CT, une évaluation de l'extraction des marqueurs radiomiques a été effectuée en comparant la valeur maximale d'absorption standardisée (SUVₘₐₓ) sur l'imagerie TEP. Une méthode de segmentation semi-automatique de la vessie en TEP a été développée de manière à soustraire la vessie de la segmentation de la prostate, ce qui permet une extraction plus juste des marqueurs radiomiques. Dans un second volet, une méthode de sélection des marqueurs radiomiques a été développée afin de réduire les marqueurs redondants et de sélectionner les plus importants, ce qui a permis de sélectionner 17 marqueurs TEP. Lorsque combinés avec l'intelligence artificielle, les marqueurs radiomiques peuvent être utilisés pour la prédiction de différents paramètres cliniques. Un modèle de prédiction utilisant un classificateur par forêts aléatoires a permis d'obtenir des résultats démontrant le potentiel de l'algorithme et des marqueurs radiomiques en FDG-TEP/CT. Une fois couplé aux données cliniques, l'algorithme permet une meilleure prédiction. Une AUC de (79 ± 9) % est obtenue lors de la combinaison du modèle de prédiction avec le stade clinique de la tumeur. / Therapeutic options with curative intent have a low probability of success in patients with prostate cancer with lymph node metastases. Therefore, a method for predicting the presence or development of lymph node metastases is desirable. This document presents the development of a model for predicting the presence of lymph node metastases at radical prostatectomy. The model uses radiomic markers extracted from the preoperative FDG-PET/CT examination. These radiomic markers - defined as the process of quantitatively extracting high-dimensional usable data from medical images - allow the quantification of features invisible to the eye such as texture and intensity parameters. In the first part, to limit the misalignment errors between PET and CT, an evaluation of the extraction of radiomic markers was carried out by comparing the standardized maximum absorption value (SUVₘₐₓ) on PET imaging. A semi-automatic PET bladder segmentation method was developed to subtract the bladder from the prostate segmentation, allowing a more accurate extraction of radiomic markers. In the second phase, a method for selecting radiomic markers was developed to reduce redundant markers and to select the most important ones, resulting in the selection of 17 PET markers. When combined with artificial intelligence, radiomic markers can be used for the prediction of various clinical parameters. A prediction model using a random forest classifier provided results demonstrating the potential of the algorithm and radiomic markers in FDG-PET/CT. Once coupled with clinical data, the algorithm allows a better prediction. AUC of (79 ± 9) % is obtained when combining the prediction model with the clinical stage of tumor.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Tumor biologist have long appreciated that both cell to cell and cell to extracellular matrix (ECM) interactions are involved in the invasive and metastatic events that are characteristic of malignancy. Cancer cell attachment to and invasion of an ECM has been associated with metastatic potential of cell lines of the Dunning rat prostate model. It was postulated that differences observed in the metastatic potential of four Dunning cell lines may correlate with cell-matrix interactions. Four cell lines, highly metastatic ML, MLL, AT-3 and non-metastatic AT-1 were studied. The adhesive, invasive and chemoinvasive capability of each cell line was compared. Cell adhesion was examined by plating the cells on plastic dishes coated with various components of the ECM (fibronectin, laminin and collagen) as well as EHS Natrix (a natural ECM) . Invasion was determined by examining cells ability to traverse a matrigel barrier. Correlations were found between the cells' adhesive and invasive abilities in response to the ECM. These observations suggest that ECM components are highly involved in prostate cancer cell activities and loss may contribute to tumor progression and metastasis.

Basal cells in a simple secretory epithelium adhere to the extracellular matrix (ECM), providing contextual cues for ordered repopulation of the luminal cell layer. Early high-grade prostatic intraepithelial neoplasia (HG-PIN) tissue has enlarged nuclei and nucleoli, luminal layer expansion and genomic instability. Additional HG-PIN markers include loss of alpha 6 beta 4 integrin or its ligand laminin-332, and budding of tumor clusters into laminin-511-rich stroma. We modeled the invasive budding phenotype by reducing expression of alpha 6 beta 4 integrin in spheroids formed from two normal human stable isogenic prostate epithelial cell lines (RWPE-1 and PrEC 11220). These normal cells continuously spun in culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells (expressing alpha 6 beta 4 integrin, high-molecular-weight cytokeratin and p63, also known as TP63) and luminal cells that secrete PSA (also known as KLK3). Basal cells were optimally positioned relative to the laminin-332 layer as determined by spindle orientation. beta 4-integrin-defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. This 3D model can be readily used to study mechanisms that disrupt laminin-332 continuity, for example, defects in the essential adhesion receptor (beta 4 integrin), laminin-332 or abnormal luminal expansion during HG-PIN progression.