Affecting one in every eight Canadian men, prostate cancer is the most common type of cancer among males. As with other forms of cancer, men with prostate cancer are much more likely to develop comorbid depression than the general population without cancer diagnoses. Depression negatively affects these men’s quality of life and increases mortality rates among cancer patients. Therefore, effective therapies to manage depression in this unique subpopulation are needed. This project sets out to assess the efficacy of glutamate-targeting drugs as antidepressants. The major excitatory neurotransmitter in the central nervous system, glutamate is released in excessive quantities by cancer cells. It is thought that this abundance of glutamate leads to excitotoxicity and neurodegeneration, affecting neurons in important regions of the brain relating to mood and mood regulation. A validated mouse model of depression was established using RM1 murine prostate cancer cells. This model was then used to test the properties of three drugs: sulfasalazine (SSZ), (S)-4-carboxyphenylglycine ((S)-4-CPG), and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide (NBQX). Results show that these drugs were able to improve depressive-like behaviours and symptoms to varying degrees, at least partially reversing the negative effects of tumours. This project showed that disrupting glutamate release and/or signaling could be an effective approach for an antidepressant therapy or adjuvant in prostate cancer patients. / Thesis / Master of Science (MSc) / Prostate cancer affects one in every eight Canadian men. Cancer patients are at a much higher risk of developing depression than the rest of the population. Unfortunately, current antidepressants are limited in their ability to improve depressive symptoms in cancer patients. Therefore, this project sets out to identify new options for treating depression in prostate cancer patients. Glutamate is a signalling molecule that is released in abundance by cancer cells and is largely responsible for communication between neurons in the central nervous system. This project showed that limiting the amount of glutamate released by cancer cells and limiting glutamate-based signaling improves depressive-like symptoms in mice with prostate cancer tumours. These results suggest that targeting glutamate could be an effective antidepressant therapy in the cancer population.

Patients with prostate cancer (PC) are at high risk of developing migratory thrombosis compared to healthy individuals. This is due to the haemostatic abnormality as a result of the presence of cancer, and is referred to as Trousseau’s syndrome. Trousseau's syndrome leads to increased mortality among cancer patients, and is considered the second cause of death after cancer itself. We investigated the role of microvesicles (MVs), which are circular membrane compartments shed from cancer as well as from healthy cells, in the development of Trousseau’s syndrome. We compared the pro-coagulant activities between MVs derived from PC cell lines with different oncogenic and metastatic characteristics, using chromogenic assays to determine their thrombin generation. Microvesicles from the more aggressive DU145vIII and more metastatic PC3-MLN4 show increased thrombin generation compared to MVs derived from DU145 and PC3. We also compared thrombin generation in MVs extracted from plasma of PC patients of various cancer stages. MVs from PC patients with a metastasized tumour had increased thrombin generation compared to patients with localized tumours. Finally, we transfected the CHO cell line with the human protease-activated receptor 1 (hPAR1), the principal receptor of thrombin. PC MVs led to the activation of PAR1 in CHO (hPAR1), indicating thrombin generation. Our in vitro studies suggest a potential role of PC MVs in the migratory thrombosis observed in Trousseau’s syndrome, due to their independent ability to generate active thrombin. We also demonstrated that thrombin generation of PC-derived MVs correlated with the oncogenic and metastatic characteristics of prostate cancer. / Thesis / Master of Science (MSc)

Aim: Several studies have tried to quantify overdiagnosis of prostate cancer with Prostate-specific antigen(PSA) screening, but estimates vary widely. This study aims to evaluate the degree of overdiagnosis of prostate cancer with 10 or 14 follow-up years after the stop of screening in Finland. Methods: We selected 80379 men aged 55-69 years who were randomized to a screening or a control arm, distinguishing four birth cohorts: 1941-44,1937-40, 1933-36 and 1929-32. The first PSA screening test occurred during1996-1999. Men without detected as prostate cancer in the previous screening would be invited to the next screening 4 years later. The estimate of overdiagnosis is the ratio of the cumulative excess incidence to the cumulative incidence of prostate cancer in the screened group after the year-specific incidence became stable. Results: The patterns of all incidences in these four cohorts have not become stable yet, and the difference of cumulative incidence in the current longest follow- up years is the best estimate of overdiagnosis so far. Conclusion: Overdiagnosis rates of prostate cancer in people who received screening in Finland was estimated as 2.27%,15.4%, 11.4%, and 10.2% for 1929-32, 1933-36,1937-40, and 1941-44 cohorts, respectively. / Thesis / Master of Science (MSc)

Yes / The aim of this study was to assess the relationship between pre-prostatectomy urinary Engrailed-2 (EN2), a transcription factor secreted by prostate cancer cells, with tumour volume and pathological characteristics in resected prostate specimens. First pass urine samples (10 ml) without prior prostatic massage were collected and stored at –80°C. EN2 levels were measured using an enzyme-linked immunoabsorbent assay. Tumour volume in the prostatectomy specimens was determined histologically. 57 men undergoing RP in one urological cancer network were evaluated. EN2 was detected in 85% of RP patients. EN2 correlated with tumour volume (but not total prostatic volume) in a linear regression analysis, with increasing pathological T stage and margin positivity. Using three “cutoff levels” of tumour volume (0.5 ml, 1.3 ml and 2.5 ml) to define “significant disease”, men with “significant disease” had markedly higher levels of urinary EN2 (p < 0.001 for each cut off level). Levels of urinary EN2 may be useful in predicting tumour volume in men with prostate cancer by potentially identifying men with small volume “insignificant” disease. This study justifies a larger multicentre evaluation of urinary EN2 levels as a biomarker of PC significance using cancer volume, pathological and PSA criteria.

Le cancer de la prostate est le premier cancer en incidence et le troisième au niveau de la mortalité chez les hommes canadiens. Selon les estimations de la société canadienne du cancer pour l'année 2020, près de 23 300 hommes recevront un diagnostic et 4 200 mourront de la maladie. Le cancer de la prostate est l'un des rares cancers à évolution lente. Toutefois, il présente beaucoup de variabilité dans les risques de récidive, de réponse aux traitements et de décès spécifique chez des patients ayant des caractéristiques clinico-pathologiques similaires. Il y a donc un grand besoin pour le développement d'outils prédictifs plus précis pour l'évolution et la classification de la maladie. Dans les dernières années il y a de plus en plus d'indices qui suggèrent qu'une analyse exhaustive des cellules immunitaires infiltrant les tumeurs pourrait aider à prédire l'évolution des cancers. Nous avons donc émis l'hypothèse que l'étude de l'infiltration de cellules immunes dans le microenvironnement du cancer de la prostate chez des patients traités par prostatectomie radicale pourrait prédire l'évolution de la maladie. Nous avons utilisé deux techniques pour analyser l'environnement immunitaire dans le cancer de la prostate. Tout d'abord, nous avons établi le profil d'infiltration immunitaire du microenvironnement prostatique cancéreux par des marquages immunohistochimiques, en nous intéressant non seulement au type, mais aussi à la quantité et à la localisation des cellules immunes dans les différents compartiments du microenvironnement tumoral. Nos expériences ont montré tout d'abord, que l'infiltration dans la zone centrale de la tumeur par des cellules présentatrices d'antigènes ne permet pas de prédire l'évolution du PCa. Toutefois, l'infiltration dans l'épithélium péritumoral normal et dans la marge tumorale par ces cellules est associée avec l'évolution de la maladie. Soit, en augmentant les risques de progresser vers une récidive biochimique, pour des fortes infiltrations de cellules dendritiques immatures CD209+ dans la marge tumorale et un PCa létal pour une forte infiltration de macrophages M2 CD163+ dans l'épithélium péritumoral normal. Ou bien en diminuant les risques de traitements d'hormonothérapie définitive ou de PCa létal, pour une forte infiltration de cellules dendritiques matures CD83+ dans l'épithélium péritumoral normal et la marge tumorale. Par la suite, nos analyses ont montré que la valeur prédictive de l'infiltration des lymphocytes se concentre principalement dans la zone centrale de la tumeur, tel que démontré par les risques accrus de récidive biochimique par des ratios intra tumoraux faibles de CD45RO/CD3 et hauts des FoxP3/CD45RO. Nous avons observé aussi des risques plus faibles de traitements d'hormonothérapie définitive pour des faibles ratios dans la tumeur de FoxP3/CD3 et FoxP3/CD45RO ainsi que, des risques diminués de PCa létal pour un haut ratio de CD45RO/CD3 dans l'épithélium tumoral. En complément, nous avons effectué une analyse du profil d'expression génique en ciblant une série de gènes liés à la fonction immunitaire à l'aide d'expériences de RT-qPCR dans le but de bonifier les expériences d'immunohistochimie. Ces expériences nous ont permis d'identifier un sous-groupe des gènes liés à la fonction des cellules présentatrices d'antigènes pour lequel une surexpression est associée à des taux de survie sans PCa léthal diminués. De plus, nous avons mis en évidence la protéine TIM-3 comme étant un point contrôle immunologique important dans la progression du PCa, puisque des taux élevés d'expression de ce PCI sont associées à des survies plus courtes sans besoin de traitements d'hormonothérapie définitive. Les résultats de nos travaux ont permis de mettre en évidence l'importance de tenir compte des compartiments de la tumeur dans une étude exhaustive de l'infiltration immune dans le PCa. En effet, nos données suggèrent que la valeur pronostique de l'infiltration immune dépend du type et de la densité des cellules infiltrés mais aussi de leur localisation dans le microenvironnement tumoral. De plus, nous avons pu identifier des acteurs importants dans l'évolution de la maladie, tels que les cellules exprimant FoxP3, CD45RO, CD163, CD209 ainsi que le point de contrôle immunologique TIM-3. La méthodologie de cette étude pourrait être adaptée à la réalité clinique en analysant les biopsies préopératoires. L'utilisation d'un tel outil pronostic en combinaison avec les tests clinico-pathologiques de routine pourrait aider à identifier les patients qui pourraient bénéficier d'immunothérapies intra-prostatiques avant la chirurgie destinée à renverser cet état immunosuppresseur dans un contexte de médecine de précision. / Prostate cancer is the most common cancer and the third leading cause of death from cancer among Canadian men. According to Cancer Canada, in 2020 23 300 men will be diagnosed and 4 200 men will die from prostate cancer. Prostate cancer is one of the rarest cancers to have a slow evolution. However, there is a considerable variation in disease recurrence, treatment response and disease-specific death between individuals showing similar clinico-pathological characteristics. There is therefore a great need for the development of more precise predictive tools for the evolution and the classification of the disease. In recent years, there is a growing evidence suggesting that a comprehensive analysis of tumor-infiltrating immune cells could help to predict cancer progression. We therefore hypothesized that the study of immune cell infiltration in the prostate cancer microenvironment in patients treated by radical prostatectomy can predict the evolution of the disease. We used two techniques to analyze the immune context in prostate cancer. First, we profiled the immune microenvironment of the prostate cancer by immunohistochemistry. By focusing not only on the type, but also the quantity and the localization of immune-infiltrating cells in the various compartments of the tumor microenvironment. Our experiments showed that antigen-presenting cells infiltrating the center of the tumor does not predict the evolution of prostate cancer. On the other hand, infiltration into the normal-like peritumoral epithelium and the tumor margin are associated with the evolutions of the disease either by increasing the risks of progressing to biochemical recurrence for high infiltration by immature CD209+ dendritic cells in the tumor margin and higher risks of lethal PCa for high infiltration by CD163+ M2 macrophages in the tumor margin and normal-like peritumoral epithelium. Or by reducing the risks of definitive androgen deprivation therapy (ADT) or lethal PCa for a high infiltration by mature CD83+ dendritic cells in the normal-like peritumoral epithelium and the tumor margin. Subsequently, our analyzes showed that the predictive value of lymphocyte infiltration is mainly concentrated in the tumor center, as demonstrated by the increased risks of biochemical recurrence by intra-tumor low ratios of CD45RO/CD3 and high ratio of FoxP3/CD45RO. In addition, a lower risk of definitive ADT for low tumor ratios of FoxP3/CD3 and FoxP3/CD45RO and a decreased risk of lethal PCa for a high CD45RO/CD3 ratio in the tumor epithelium was also observed. Secondly, we performed gene expression profile analysis targeting a series of genes related to immune function using RT-qPCR experiments to complement the immunohistochemistry experiments. These experiments allowed us to identify a subgroup of genes related to the function of antigen presenting cells in which, overexpression is associated with decreased rates of lethal prostate cancer-free survival. In addition, we identified TIM-3 as being an important immunecheckpoint in the progression of prostate cancer since high expression levels of TIM-3 are associated with shorter survival to definitive ADT. The findings of our work have highlighted the importance of tumor compartments in a comprehensive study of immune infiltration in prostate cancer. Indeed, our data suggest that the prognostic value of immune infiltration depends on the type and density of the infiltrated cells but also on their location in the tumor microenvironment. In addition, we were able to identify important players in the evolution of the disease, such as cells expressing FoxP3, CD45RO, CD163, CD209 and the immune checkpoint TIM-3. The methodology of this study could be adapted to clinical reality with the analysis of preoperative biopsies. The use of such a prognostic tool in combination with routine clinico-pathologic tests could help identify patients who might benefit from intra prostatic immunotherapies prior to surgery to reverse this immunosuppressive condition in a context of precision medicine.

Chez les hommes, le cancer de la prostate est, au niveau mondial, le 2ème cancer le plus diagnostiqué et le 5ème cancer le plus létal. Lorsqu'il est localisé à la prostate, il est principalement traité par prostatectomie ou radiothérapie. Cependant, entre 27 et 53% des patients traités à ce stade développeront éventuellement une récidive. L'hormonothérapie qui vise à réduire l'effet des androgènes sur le cancer, permet d'augmenter significativement la survie de ces patients. Par contre, ces cancers deviendront irrémédiablement résistants à la castration puis évolueront vers la forme métastatique pour laquelle il n'existe aucun traitement curatif. Ainsi de nouvelles thérapies sont nécessaires pour augmenter la survie de ces patients. Un nombre important d'études visant le développement d'immunothérapies du cancer de la prostate ont vu le jour au cours des dernières années. Ces études ont mené entres autres au développement du Sipuleucel-T, la première immunothérapie approuvée pour le traitement du cancer de la prostate, ainsi qu'à l'étude de l'effet antitumoral des inhibiteurs de points de contrôle immunologiques. Ces traitements, principalement testés sur des cancers avancés, ont montré des effets très limités et dans certains cas, des effets indésirables importants. Plusieurs études soulignent l'importance d'administrer les traitements plus tôt dans le cours de la maladie et suggèrent que l'administration locale de combinaisons de molécules immunomodulatrices pourrait être efficace tout en limitant les effets indésirables. L'objectif principal de mon projet était de développer une nouvelle approche immunothérapeutique basée sur l'utilisation du vecteur adénoviral PCA3-TSTA permettant l'expression ciblée à la tumeur de molécules immunostimulatrices. L'expression ciblée à la tumeur est réalisée grâce à la spécificité du promoteur de l'ARN long non-codant PCA3 humain, qui est à ce jour le biomarqueur le plus spécifique du cancer de la prostate, ainsi que l'amplification de l'activité transcriptionnelle par le système TSTA. Ces approches immunothérapeutiques ont été testées dans le modèle murin immunocompétent de cancer de la prostate TRAMP-C2. Dans un premier temps, nous avons montré chez des souris portant des tumeurs TRAMP-C2, la spécificité tumorale et l'activité transcriptionnelle in vivo pendant au moins 21 jours de l'adénovirus PCA3-TSTA codant pour la luciférase. Nous avons ensuite construit et testé des adénovirus PCA3-TSTA codant pour les formes murines de mGM-CSF, mIL-17A ou mIL-12. Seul le traitement précoce de tumeurs TRAMP-C2 par l'adénovirus PCA3-TSTA-mIL-12 a résulté en une régression tumorale complète chez 100% des souris traitées. A un stade plus avancé, ce traitement a induit un ralentissement significatif de la croissance tumorale et une régression complète chez 20% des souris traitées. Par la suite, l'analyse des tumeurs TRAMP-C2 traitées in vivo avec le PCA3-TSTA-mIL-12 a montré l'induction de l'expression de mIL-12 mais aussi d'une réponse cytokinique impliquant l'IFN-γ. L'infection de biopsies de cancer de prostate humain par le PCA3-TSTA-mIL-12 a également induit une sécrétion d'IFN-γ dans certains échantillons, suggérant la stimulation, par l'IL-12 murine, de cellules T CD8+ ou NK infiltrant ces biopsies. Nos analyses en cytométrie de flux ont montré que les cellules immunes infiltrant les tumeurs TRAMP-C2 sont caractérisées par l'expression de PD-1 et plus faiblement de CTLA-4 et que cette infiltration est caractérisée par une importante proportion de MDSC. Nous avons donc analysé l'effet antitumoral du PCA3-TSTA-mIL-12 en combinaison avec des anticorps anti-PD-1 et anti-CTLA-4. La combinaison du PCA3-TSTA-mIL-12 avec un anticorps bloquant le CTLA-4 a montré une régression tumorale complète chez 33% des souris traitées contre 16% lorsque que le virus était combiné avec un anticorps inhibant le PD-1. Finalement, nous avons testé la combinaison d'un inhibiteur de PD-1 avec le tasquinimod, un composé inhibiteur de la fonction des MDSC. Nous n'avons cependant pas pu mettre en évidence d'effet significatif de cette combinaison sur la croissance des tumeurs TRAMP-C2. Globalement, nos résultats ont démontré que le vecteur adénoviral PCA3-TSTA permet d'induire l'expression localisée et durable de molécules immunomodulatrices par des cellules tumorales prostatiques. L'injection du PCA3-TSTA-mIL-12 dans des tumeurs TRAMP-C2 in vivo et des biopsies de cancer de prostate humain in vitro nous ont permis de mettre en évidence la production de cytokines par les cellules immunes infiltrant ces tumeurs en réponse à la production de mIL-12. Ces protocoles permettraient dans des travaux futurs de tester différents vecteurs PCA3-TSTA afin de sélectionner des combinaisons optimales de signaux immuns capables d'activer une réponse antitumorale pouvant mener à des effets immunothérapeutiques cliniques. / In men, prostate cancer is, worldwide, the 2nd most diagnosed cancer and the 5th most lethal cancer. When localized to the prostate, it is mainly treated with prostatectomy or radiation therapy. However, between 27 and 53% of patients treated at this stage will eventually develop a recurrence. Hormone therapy, which aims to reduce the effect of androgens on cancer, significantly increases the survival of these patients. On the other hand, these cancers will become irremediably resistant to castration and then evolve into the metastatic form for which there is no cure. Thus, new therapies are needed to increase the survival of these patients. A significant number of studies aimed at the development of immunotherapies for prostate cancer have emerged in recent years. These studies led, among other things, to the development of Sipuleucel-T, the first immunotherapy approved for the treatment of prostate cancer, as well as the study of the antitumor effect of immunological checkpoint inhibitors. These treatments, mainly tested on advanced cancers, have shown very limited effects and in some cases, significant adverse effects. Several studies highlight the importance of administering treatments earlier in the course of the disease and suggest that local administration of combinations of immunomodulatory molecules may be effective while limiting adverse effects. The main objective of my project was to develop a new immunotherapeutic approach based on the use of the adenoviral vector PCA3-TSTA allowing the targeted expression to the tumor of immunostimulatory molecules. Targeted expression to the tumor is achieved by the specificity of the promoter of the human PCA3 long non-coding RNA, which is to date the most specific biomarker of prostate cancer, as well as the amplification of transcriptional activity by the TSTA system. These immunotherapeutic approaches were tested in the immunocompetent mouse model of prostate cancer TRAMP-C2. As a first step, we showed in mice carrying TRAMP-C2 tumors, tumor specificity and transcriptional activity in vivo for at least 21 days of the PCA3-TSTA adenovirus encoding luciferase. We then built and tested PCA3-TSTA adenoviruses encoding the murine forms of mGM-CSF, mIL-17A or mIL-12. Only early treatment of TRAMP-C2 tumors with PCA3-TSTA-mIL-12 adenovirus resulted in complete tumor regression in 100% of the treated mice. At a more advanced stage, this treatment induced a significant slowdown in tumor growth and complete regression in 20% of the treated mice. Subsequently, the analysis of TRAMP-C2 tumors treated in vivo with PCA3-TSTA-mIL-12 showed the induction of mIL-12 expression but also of a cytokine response involving IFN-γ. Infection of human prostate cancer biopsies with PCA3-TSTA-mIL-12 also induced secretion of IFN-γ in some samples, suggesting stimulation by murine IL-12 of CD8+ or NK T cells infiltrating these biopsies. Our flow cytometry analyses have shown that immune cells infiltrating TRAMP-C2 tumors are characterized by the expression of PD-1 and weaker expression of CTLA-4 and that this infiltration is characterized by a large proportion of MDSC. We therefore analyzed the antitumor effect of PCA3-TSTA-mIL-12 in combination with anti-PD-1 and anti-CTLA-4 antibodies. The combination of PCA3-TSTA-mIL-12 with a CTLA-4-blocking antibody showed complete tumor regression in 33% of treated mice compared to 16% when the virus was combined with a PD-1-inhibiting antibody. Finally, we tested the combination of a PD-1 inhibitor with tasquinimod, a compound that inhibits MDSC function. However, we could not show a significant effect of this combination on the growth of TRAMP-C2 tumors. Overall, our results demonstrated that the adenoviral vector PCA3-TSTA induces the localized and sustained expression of immunomodulatory molecules by prostate tumor cells. Injection of PCA3-TSTA-mIL-12 into TRAMP-C2 tumors in vivo and human prostate cancer biopsies in vitro allowed us to demonstrate the production of cytokines by immune cells infiltrating these tumors in response to the production of mIL-12. These protocols would allow in future work to test different PCA3-TSTA vectors in order to select optimal combinations of immune signals capable of activating an antitumor response that can lead to clinical immunotherapeutic effects.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Prostate cancer (CaP) has the highest incidence of any malignancy affecting South African males. The aetiology of prostate carcinoma indicate that ethnicity is one of the most important risk factors. The causes of these ethnic differences are unknown but presumably involve both environmental and genetic factors. Carcinoma of the prostate is androgen dependent, and it has been suggested that variations in androgen metabolism and synthesis may affect an individuals' risk. Therefore, genes involved in these pathways are candidates for determining CaP susceptibility. In this study two candidate genes in the androgen biosynthetic and metabolic pathway were analysed, viz., the androgen receptor gene (AR), involved in androgen transport and transcriptional activation, and the cytochrome p450c17a gene (CYP17), important for testosterone biosynthesis. Comprehensive mutation detection assays were designed (appropriate for analysis of archival paraffin-embedded material) for almost the entire coding region (excluding polymorphic repeat sequences), and including all splice site junctions of the AR gene, as well as the entire coding region of CYP17. The aim of this study was thus to determine the type and frequencies of genetic variants of these androgen metabolism genes within the diverse South African population, and to determine if the observed ethnic variation in the incidence and progression of CaP can be explained by ethnic-based genetic differences. For high sensitivity mutation detection, the most powerful of the pre-screening methods was used, namely denaturing gradient gel electrophoresis (DGGE). 20 CaP and 25 control benign prostatic hyperplasia (BPH) tissue samples were screened in order to identify possible mutations. Blood samples from the same patients were analysed in order to determine whether mutations are germline and therefore present in all cells of the body. Additional blood samples from the Western Province Blood Transfusion Service (WPBTS) (Refer to section 2.1.2, Table) were also analysed in order to determine the frequency of identified polymorphisms within the general population. Certain polymorphisms were further analysed in paraffin-embedded wax material (exclusively from Blacks) to determine the distribution of these polymorphisms in the Black population. Direct sequencing of mutant-containing DNA fragments was performed to determine the exact location and nature of mutation. Using the AR- DGGE assay 4 novel mutations were identified as well as a previously reported codon 211 (E211) polymorphism. With the CYP17- DGGE assay, 3 novel single nucleotide polymorphisms (SNPs) were detected. Three base variants occured, in codons 36 (L36), 46 (H46) and 65 (S65), as well as intronic substitutions in intron 4 (IVS+58G4C) and intron 6 (IVS-25C7A). Frequencies of SNPs were measured in the CaP and BPH samples. In conclusion, the identified polymorphisms could be used as markers in determining CaP susceptibility and may thus facilitate the identification of individuals with a high- or low-risk of developing carcinoma of the prostate. / AFRIKAANSE OPSOMMING: Prostaatkanker vertoon die hoogste voorkoms van enige kwaardaardigheid wat Suid-Afrikaanse mans aantas. Die etiologie van prostaatkarsinoom dui aan dat etnisiteit een van die mees belangrike risikofaktore is. Oorsake van hierdie etniese verskille is onbekend, maar vermoedelik is omgewing en genetiese faktore albei betrokke. Karsinoom van die prostaat is androgeenafhanklik en daar is voorgestel dat variasies in androgeenmetabolisme en androgeensintese 'n persoon se risiko mag affekteer. Gevolglik, is gene betrokke in hierdie paaie kandidate vir die bepaling van prostaatkanker vatbaarheid. In hierdie studie het ons twee kandidaat gene in die androgeen biosintetiese en metaboliese pad geanaliseer, naamlik, die androgeen reseptor geen (AR), betrokke in androgeen vervoer en aktivering van transkripsie, en die sitokroom p450c17a geen (CYP17), belangrik vir testosteroon biosintese. Ons het omvattende mutasie-bespeurings-essai-sisteme ontwikkel (ook uitvoerbaar op argivale paraffien-bewaarde materiaal), wat amper vir die hele koderende streek van die AR geen gebruik kan word (uitsluitend herhalende polimorfiese reekse) en wat alle splytpunt-aansluitings van die AR geen insluit, asook vir die hele koderende streek van CYP17. Die doel van hierdie studie was dus om die tipe en frekwensies van genetiese variante van androgeen metabolisme gene in ons diverse Suid-Afrikaanse bevolking te bepaal, en om vas te stel of die waarneembare etniese wisseling in die insidensie en vordering van prostaatkanker verstaan kan word deur etnies gebaseerde genetiese verskille. Die mees sensitiewe tegniek wat tans beskikbaar is vir vooraf-sifting vir onbekende mutasies is gekies, naamlik denaturerende gradiënt gel elektroforese (DGGE). Om moontlike mutasies op te spoor, het ons 20 prostaatkanker en 25 benijne prostaathiperplasie (BPH) monsters geanaliseer. Analise was gedoen op bloedmonsters van dieselfde pasiënte om vas te stel of kiemlyn mutasies (in alle liggaamselle) teenwoordig is. Bykomstige bloedmonsters (van die Westelike Provinsie Bloedoortappingsdiens) is ook geanaliseer om die frekwensie van bespeurde polimorfismes in die algemene bevolking te bepaal. Argivale paraffien-bewaarde materiaal (eksklusief van Swartes) is ook geanaliseer om die verspreiding van sekere polimorfismes in die Swart bevolking te bepaal. Direkte DNA volgorde bepaling van mutante DNA fragmente is uitgevoer om die ligging en tipe van mutasies te bepaal. Met die toepassing van ons AR-DGGE mutasiesisteem het ons 4 nuwe mutasies ontdek asook 'n kodon 211 (E211) polimorfisme wat voorheen gevind is. Vyf enkel nukleotied polimorfismes is met die CYP17-DGGE mutasiesisteem opgespoor. Die polimorfismes sluit in: drie basis veranderinge wat voorkom in kodons 36 (L36), 46 (H46) en 65 (S65), asook introniese substitutisies in intron 4 (IVS+58G4C) en intron 6 (IVS-25C7 A). Frekwensies van die polimorfismes was bereken in die prostaatkanker en BPH monsters. Die resultate aangebied in hierdie tesis dui aan dat die gevonde polimorfismes as merkers gebruik kan word om prostaatkanker vatbaarheid te bepaal en daardeur individue te identifiseer met 'n hoë of lae risiko vir prostaatkarsinoom ontwikkeling.

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer death among men in the United States. In African American men, the disease is typically detected at a more advanced stage and mortality is twice the rate of Caucasian men. However, African American men are less likely to participate in prostate cancer screening. The purpose of this descriptive study was to assess the relationship between health beliefs, knowledge, and selected demographic variables (age, income and education) and a man’s decision to participate in prostate cancer screening among African American men dwelling in rural communities. The conceptual framework for the study was the Health Belief Model. Participants for the study were recruited through contacts within rural communities within west central Alabama. A convenience sample of 90 African American men between the ages of 40-82 years of age was recruited. Analysis of the research data indicated that there was a statistically significant difference in motivation (health belief), knowledge, and age of men who participated in prostate cancer screening compared to those who did not participate in prostate cancer screening. Forward logistic regression was used to determine which independent variables [health beliefs (benefits, barriers, motivation); knowledge; age; income; and education] were predictors of prostate cancer screening. Results indicated the overall model of one predictor, motivation, was statistically reliable in predicting prostate cancer screening participation among the rural dwelling men surveyed. The model accounted for 15 to 20% of the variance. The sensitivity of the model in predicting those who would participate in prostate cancer screening was 85%. The odds of those who would participate in prostate cancer screening were 1.3 times greater for each one unit increase in motivation. Results indicate a need for more educational and motivational interventions to promote informed decision making by African American men in regards to prostate screening activities. These interventions need to be culturally sensitive and geared toward African American men, specifically those living in rural areas.

published_or_final_version / abstract / toc / Physiology / Master / Master of Philosophy

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