Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • The UPLC-MS/MS analyses and quantification of novel 5α-reduced steroids using response factors. • The kinetic characterisation of human steroid 5α-reductase type 1 (SRD5A1), expressed in HEK-293 cells, towards 11OHA4 and 11OHT and their keto derivatives by progress curve analysis. • The subcloning, transformation and functional expression of SRD5A1 in the yeast expression system, P. pastoris. • The conversion of 11OHA4 and 11OHT and their keto derivatives by SRD5A1 expressed in P. pastoris. • The endogenous enzymatic activity in P. pastoris towards the 5α-reduced metabolites in the 11OHA4- and alternate 5α-dione pathways. • The potential application of P. pastoris as a biocatalyst in the production of 5α- reduced C19 steroids. / AFRIKAANSE OPSOMMING: Hierdie ondersoek beskryf: • Die UPLC-MS/MS analise en kwantifisering van nuut-ondekte 5α-gereduseerde steroïede met behulp van responsfaktore. • Die kinetiese karakterisering van menslike steroïed 5α-reduktase tipe 1 (SRD5A1), uitgedruk in HEK-293 selle, vir 11OHA4 en 11OHT en hul ketoderivate deur middel van progressiekurwe-analise. • Die subklonering, transformasie en funksionele uitdrukking van SRD5A1 in die gis P. pastoris. • Die omsetting van 11OHA4 en 11OHT en hul ketoderivate deur SRD5A1 uitgedruk in P. pastoris. • Die omsetting van 5α-gereduseerde steroïede in die 11OHA4 en alternatiewe 5α-dioon paaie deur endogene ensieme in P. pastoris • ‘n Ondersoek na die toepassing van die gisuitdrukkingstelsel as ‘n moontlike OR potensiële biokatalis vir die produksie van 5α-gereduseerde C19 steroïede.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

Parthenolide (PN), a major active component of the traditional herbal medicine feverfew, has been shown to have anti-inflammatory and anti-tumor properties. More remarkably, the cytotoxicity of PN seems selective to tumor cells but not their normal cell counterparts. In the present study, we investigate whether and how PN selectively enhances tumor sensitivity to radiation therapy by using prostate cancer cells LNCaP, DU145 and PC3, as well as normal prostate epithelial cells PrEC. Our study demonstrates that inhibition of NF-κB pathway and suppression of its downstream target MnSOD are common mechanisms for the radiosensitization effect of PN in prostate cancer cells. The differential susceptibility to PN in two radioresistant cancer cells, DU145 and PC3, is due, in part, to the fact that in addition to NF-κB inhibition, PN activates the PI3K/Akt pro-survival pathway in both cell lines. The presence of PTEN in DU145 cells enhances the radiosensitization effect of PN by suppression of the steady state level of activated p-Akt. We also demonstrate that PN selectively exhibits a radiosensitization effect on prostate cancer PC3 cells but not on normal prostate epithelial PrEC cells. PN causes oxidative stress in PC3 cells but not in PrEC cells, as determined by the oxidation of the ROS-sensitive probe H2DCFDA and intracellular reduced thiol and disulfide levels. In PC3 but not PrEC cells, PN activates NADPH oxidase leading to a decrease in the level of reduced thioredoxin, activation of PI3K/Akt and consequent FOXO3a phosphorylation, which results in the downregulation of FOXO3a targets, antioxidant enzyme MnSOD and catalase. Importantly, when combined with radiation, PN further increases ROS levels in PC3 cells, while it decreases radiation-induced oxidative stress in PrEC cells, possibly by increasing GSH level. Overall, our data support the concept that increasing oxidative stress in cancer cells, which are already under high constitutive oxidative stress, will lead to cell death, while the same stress may allow normal cells to maintain redox homeostasis through adaptive response. Thus, modulating cell redox status may be a novel approach to efficiently and selectively kill cancer cells.

The purpose of this study was to determine the factors related to young black men’s intention to screen for prostate cancer as well as their engagement in prostate cancer risk-reduction behaviors. The study tested the significance of the constructs – age, attitude (direct and indirect), social influence, comfortability, cues to action, health screening experiences and knowledge – in predicting young black men’s intention to screen for prostate cancer; as well as the significance of the constructs – age, cues to action, exercise and knowledge – in predicting engagement in prostate cancer risk-reduction behaviors. Demographic/personal factors were also explored in related to the model predictors. Web-based and paper-pencil surveys were administered to 279 black men aged between 18 – 40 years from the Austin area. Three focus groups were conducted to collect information regarding young black men’s behavioral beliefs toward prostate cancer screening as well as their comfortability with prostate examinations. The number of usable surveys was 267. Using direct and indirect measures, the combination of attitude, social influence, comfortability (indirect model), and knowledge explained 41.0 and 43.0 percent of the variance in intention to screen for prostate cancer, respectively; with social influence being the strongest predictor ([Beta]=0.41; p <0.01 for the direct model and [Beta]=0.47 for the indirect model). For the model with prostate cancer risk-reduction as the outcome variable, the model accounted for 10.0 percent of the variance in behavior with only knowledge ([Beta]=0.19; p=0.03) as significant predictor. Interventions that address young black men’s attitude, social influence, comfortability, and knowledge may be necessary to increase young men’s intention to screen for prostate cancer when it is recommended by a physician. Additionally, factors surrounding exercise and knowledge may be important in increasing young men’s engagement in prostate cancer risk-reduction behaviors. Future studies using intention as a predictor of young men’s behavior are needed to assess the influence of intention on prostate cancer screening. / text

Les protéines de la Famille Ets sont des facteurs de transcription qui reconnaissent par l'intermédiaire d'un domaine de liaison à l'ADN, appelé le domaine ETS, une séquence nucléotidique centrée sur un core consensus 5'-GGAA/T-3'. Le gène fev, un membre de cette famille, a été isolé chez l'humain après avoir été identifié dans une tumeur de Ewing chez l'enfant comme le résultat d'une translocation chromosomique. Une étude préliminaire de 1997 indiquait que chez l'Homme, Fev possède une expression tissulaire restreinte au petit intestin et à la prostate "adulte". Dans la première partie de ce travail, nous avons observé une surexpression de l'ARNm de ce facteur de transcription dans une large série d'adénocarcinomes prostatiques de différenciation variable en comparaison au profil d'expression observé dans un groupe témoin de prostates hyperplasiées. En recherchant des lignées cellulaires qui expriment ce facteur, nous avons mis en évidence la présence de ce messager dans la lignée humaine d'origine prostatique LNCaP. De même, les lignées humaines d'origine hématopoïétiques Dami/HEL92.1.7, K-562, KU-812 F et U-937 expriment ce facteur. Parallèlement, nous avons réalisé l'étude de l'expression de Fev dans le cerveau humain. En effet, chez le rat et chez la souris, l'homologue de Fev (mPet-1/Pet-1) est exprimé spécifiquement dans les neurones sérotoninergiques. Nous avons ainsi montré sur le cerveau humain que l'ARNm de Fev est exclusivement exprimé dans les noyaux du raphé qui contiennent les neurones sérotoninergiques. Il est co-exprimé avec deux marqueurs de ces neurones, la SERT/5-HTT et la TPH2. Parallèlement, nos travaux de caractérisation fonctionnelle de la protéine Fev ont permis de définir ce facteur comme un répresseur transcriptionnel. En effet, ce facteur possède un domaine carboxy-terminal riche en résidus alanines qui lui confère, en partie, sa fonction de répresseur de la transcription (répression active); la délétion de cette région conduisant à une réduction drastique de l'effet répresseur. Parallèlement, nous avons montré que le domaine ETS de Fev est responsable d'une activité répressive passive par l'occupation des sites de liaison aux facteurs Ets. Néanmoins, nous ne connaissons pas, à l'heure actuelle, les gènes-cibles spécifiques qui sont directement réprimés par Fev dans les cellules dans lesquelles le gène est normalement exprimé. Nous avons tenté de développer, par établissement de clones stables, des modèles cellulaires où le gène d'intérêt est surexprimé, mais ces tentatives furent infructueuses. Cet effet drastique de Fev est conféré par la partie carboxy-terminale. Il est ainsi probable que la surexpression de ce répresseur transcriptionnel spécifique de certains gènes cibles de la famille Ets provoque des effets sur la survie des cellules en régulant des gènes indispensables à la croissance cellulaire, et ainsi empêchant la prolifération de clones cellulaires.

Prostate cancer is the most commonly diagnosed cancer and second leading cause of death in American men. Patients with localized prostate cancer may experience unique and multidimensional symptoms that are distressful from treatment and thereafter. This cross-sectional correlational study aimed to investigate the relationships among stress, symptoms, symptom distress, and symptom self-management and identify the effective strategies of symptom self-management in men with localized prostate cancer following prostatectomy or radiation therapy.Eight saliva samples and 3 questionnaires (Perceived Stress Scale, Symptom Indexes, and Strategy and Effectiveness of Symptom Self-Management) were obtained from each participant between 1 and 3 months following their first prostate cancer treatment. The sample consisted of 53 men with localized prostate cancer. Mean salivary cortisol concentrations for the entire sample ranged from 0.3 to 0.08 ug/dL. Cortisol was secreted in a circadian rhythm with heightened activity in the early morning and lowered activity late in the day. The circadian pattern of cortisol secretion was similar in both the prostatectomy and radiation therapy groups, although the values were slightly different. Two areas Under the Curve (AUC) of salivary cortisol were calculated. Three cortisol circadian rhythms were identified, but the majority of the sample had a typical negative consistent circadian rhythm.Patients with localized prostate cancer who underwent radical prostatectomy or radiation therapy had low perceived stress. Perceived stress was positively correlated with AUCg, noon salivary cortisol concentrations, and afternoon salivary cortisol concentrations. Subjects reported a moderate degree of symptoms and symptom distress on urinary, bowel, and sexual dysfunction 1-3 months following treatments. The most effective strategies of urinary symptom management were pad and kegel exercise; the most effective strategy of bowel symptom management was rest or endure; the most effective strategies of sexual dysfunction management included express their feelings or find alternative ways to express their affection. The symptom self-management strategies were significantly and positively correlated with symptom self-management effectiveness.Symptom distress and AUCg were significant and strong predictors of symptom self-management. Findings can help health care providers develop effective strategies for symptom self-management that enhance health related quality of life among men with localized prostate cancer.