Xiao, Lijia. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 139-158). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The introduction of prostate-specific antigen (PSA) testing about 3 decades ago led to the possibility of early detection of prostate cancer (PCa). Although PSA testing reduced the mortality rate, it is also associated with high risk of over diagnosis in patients with and without PCa. Despite the current drawbacks, it would be a challenge to replace PSA testing entirely. Instead, there is a need to develop parallel testing of other potential biomarkers that can complement the results from PSA tests. To address alternative biomarker sensing, this thesis highlights on the development of oligonucleotide-based biosensors for the detection of different biomarkers of PCa. Using PSA as a gold standard, the first study of this dissertation investigates the use of DNA aptamers to detect PSA using electrochemical impedance spectroscopy (EIS). The study compares 6-mercapto 1-hexanol chemistry with sulfo-betaine chemistry for the development of PSA aptasensor in terms of performance and selectivity. The second study focuses on glycoprofiling in order to complement PSA quantification as an additional information for reliable PCa diagnosis. This strategy was developed in a microfluidic channel with an optical read out using chemiluminescence. This study addresses one of the major problems of cross-reactivity with lectins in glycoprofiling, which can be solved using DNA aptamers. A third study concentrates on the development of an aptasensor for Alpha-Methylacyl-CoA Racemase (AMACR). AMACR has been reported for its high specificity and sensitivity to PCa. For the fabrication of the biosensor, a new strategy using polyethylene glycol was developed by electrochemical grafting it to a polypyrrole film. Since PCa diagnosis can be improved by looking at different biomarkers, an electrochemical platform for miRNA/DNA detection using a gold nanoparticle amplification strategy was also investigated. The sensor was fabricated using peptide nucleic acids (PNA) probes on gold electrodes. The study presents non-Faradaic EIS and amperometric techniques in order to exploit the inherent charges of nucleic acids. In conclusion, this thesis wants to serve as a potential orientation for overcoming the shortcomings of the current PCa testing and contribute towards the development of oligonucleotide-based biosensors for PCa biomarker detection and hopefully enhance the diagnosis and prognosis of PCa.

Intra-operative imaging is sometimes available to assist in needle biopsy, but typical open- loop insertion does not account for unmodeled needle deflection or target shift. Robotic closed-loop compensation for deviation from an initial straight-line trajectory can reduce the targeting error, using image-guidance for rotational control of an asymmetric bevel tip. By pairing closed-loop trajectory compensation with a hands-on cooperatively controlled needle insertion, a physician's control of the procedure can be maintained while incorporating benefits of robotic accuracy. Additionally, if puncture of a membrane can be detected, an enhanced haptic response can assist the physician in perceived anatomical localization of the needle tip. Functionality was implemented on a needle placement robot suitable for use in the MR environment and capable of holding a typical clinically used biopsy gun. The robot is configured for cooperatively controlled needle insertion with continuous closed-loop image- guided needle rotation. The robot and custom controller were tested for their effect on the Signal-to-Noise ratio (SNR) of MR images, and the results showed an approximate drop of only 12% in signal when the robot was present, and no additional signal drop when the robot was powered on or moving. The hardware and software subsystems were developed for clinical translation, and after each was validated in the lab they were integrated into the clinical environment to mimic the workflow of MRI-guided targeted biopsy. The full system was evaluated in-bore at Brigham and Women’s Hospital in Boston, MA where experiments for real-time puncture detection and MR image-guided targeted needle insertions under cooperative control were performed. Results showed overall targeting accuracy was 3.42mm RMS, improving accuracy by approximately 50% as compared to clinical trials of prostate biopsy using manual needle insertion. A cooperatively controlled robotic biopsy is more likely to gain acceptance by physicians over teleoperation due to maintaining proximity to the surgical site, but regulatory hurdles regarding robotic needle insertion still exist. The current robotic system framework is suitable for clinical use as it was fully validated in-bore, but some modifications could be made to increase the likelihood of regulatory approval. With these modifications the system could be ready for cadaver and pre- clinical animal trials within one year, and ready for in-human clinical trials in the next two to three years.

Prostate adenocarcinoma (PCa) is one of the most prevalent cancers in the world. Second only to lung cancer, the key to its successful treatment is in its early detection. With the introduction of prostate-specific antigen in the early 1990s, a screening test involving measuring levels of this protein was developed to detect PCa in asymptomatic individuals. This test is also known as the PSA test. PCa-specific mortalities have been in decline since the test's introduction. Despite this decline, recent studies have called the efficacy of the PSA test into question. Two large randomized controlled trials conducted in the US and Europe reveal contradicting results as to PSA's accuracy and usefulness. Concerns of overdiagnosis and overtreatment as the result of using PSA screening has led to many national organizations recommending caution or even recommending against its use. Through a thorough review of a large collection of current PCa literature, this study reviews the flaws of using PSA to screen for PCa and investigates alternative approaches currently being pursued through active research to make PCa early detection more accurate. These approaches include improving the accuracy of the PSA screen using PSA-derived testing methods, using PCa-induced epigenetic modifications as a new target for PCa screening, and using urine biomarkers. All of these methods were compared using area under the curve (AUC) values obtained via receiver operating characteristic analysis. Each method has its own flaws but by comparing each of the different approaches, I was able to conclude that out of the currently available screening methods, screening for Engrailed-2 protein in urine is the most promising screening method with the highest AUC values compared to the other methods. Although this method has been introduced in the UK, it has not been introduced in the US yet. Epigenetic screening methods hold the most promise for accurate PCa screening in the future as it confers the highest accuracy in detecting PCa. However, as it hasn't been shown that epigenetic modifications can be easily obtained in the urine or blood serum for easy and accurate screening, I believe more work has to be done in order for it to be successful in being applied as a screening test. By determining the most promising screening type, we can focus resources and efforts towards finding a way to detect PCa early, allowing for successful treatment.

Prostate cancer (PC) is characterised by dependence upon androgen receptor (AR) as its driving oncogene. When organ-confined, radical treatment can be curative, however there is no cure for advanced, castration-resistant prostate cancer (CRPC). There is therefore a need to better understand the biology of PC, and how influencing AR can modify disease progression. Estrogen is essential for prostate carcinogenesis with evidence from epidemiological, in vitro, human tissue and animal studies. Most suggests that estrogen receptor beta (ERβ) is tumour-suppressive, but trials of ERβ-selective agents have not improved clinical outcomes. ERβ has also been implicated as an oncogene, therefore its role remains unclear. Additional evidence suggests interplay between ERβ and AR, the mechanisms of which are uncertain. The study hypothesis ‘ERβ is an important modulator of prostate carcinogenesis’ was developed to establish whether targeting ERβ could affect PC progression. Much of the confusion around ERβ stems from use of inadequately validated antibodies and cell line models. The first phase of this work was to test ERβ antibodies using an ERβ-inducible cell system. Eight ERβ antibodies were assessed by multiple techniques, showing that commonly used antibodies are either non-specific or only specific in one modality. Two reliable antibodies were identified. Next, cell lines previously used to study ERβ were assessed using validated antibodies and independent approaches. No ERβ expression was detected; an important finding that casts doubt on previously published ERβ biology. Subsequently, a PC cell line with inducible ERβ expression (LNCaP-ERβ) was developed and validated to enable controlled experiments on the effects of ERβ on proliferation, gene expression and ERβ/AR genomic cross-talk. Phase three of this work focused on ERβ biology in PC and its relationship to AR. Interrogation of clinical datasets showed that greater ERβ expression associated with favourable prognosis. Gene expression data from men treated with androgen deprivation therapy revealed that AR represses ERβ. This was confirmed in vitro. The LNCaP-ERβ cell line was treated with androgen and/or ERβ-selective estrogen. Activated ERβ in the presence of androgen-stimulated AR inhibited cell proliferation and down-regulated androgen-dependent genes. Genome-wide mapping of ERβ binding sites reveals that ERβ antagonises AR through competition for shared DNA binding sites. In conclusion, ERβ expression is down-regulated by AR during malignant transformation of prostate epithelium. We reveal an antagonistic relationship between ERβ and AR whereby sustaining or replacing ERβ may inhibit tumour growth through down-regulation of AR-target genes. In future, an ERβ-selective compound may be used to slow or abrogate PC progression.

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.

A Interleucina 8 (IL-8) é uma quimiocina CXC angiogênica que tem papel importante no desenvolvimento e progressão de vários tumores malignos, incluindo o câncer de próstata (CaP). O polimorfismo de nucleotídeo único (SNP) -251 T/A da região promotora do gene da IL-8, relativo ao local de início da transcrição deste gene, está associado com a produção desta citocina. O efeito da IL-8 é mediado através de dois receptores de alta afinidade, CXCR1 e CXCR2. O presente estudo investigou a influência da variação dos genes IL-8 e CXCR2 na susceptibilidade e nas características clinicopatológicas do CaP em um grupo de brasileiros. Duzentos e um pacientes e 185 controles saudáveis foram selecionados neste estudo casocontrole. Amostras de sangue foram coletadas para extração de DNA; a tipagem da IL-8 -251 T/A e CXCR2 +1208 C/T foi realizada através da reação em cadeia da polimerase com sequência específica de primers (PCR-SSP), seguida pela eletroforese em gel de agarose. O risco associado entre os genótipos, a susceptibilidade do CaP e as características do tumor, foi estimado pelo odds ratio (OR), com intervalo de confiança de 95%, usando análise de regressão logística e ajustando para idade ao diagnóstico. Encontramos uma associação estatisticamente significativa entre o genótipo heterozigoto CT do gene CXCR2 +1208 e CaP. Este genótipo foi significativamente menos frequente em pacientes com estádio clínico T3-T4 comparado com T1-T2 (56.7% versus 80.5%). Nossos achados sugerem que os portadores do genótipo CT CXCR2 +1208 tiveram um efeito protetor para estádio avançado de CaP (CT versus CC: OR ajustado = 0.25; P = 0.02). Não foi encontrada associação significativa entre o polimorfismo -251 T/A da IL-8 e os parâmetros clinicopatológicos do CaP. Estes resultados indicam que o genótipo CT do CXCR2 +1208 é menos frequente em estádios avançado de CaP, sugerindo que este receptor de quimiocina tenha um papel na patogênese desta doença. / Interleukin-8 (IL-8) is an angiogenic CXC chemokine that plays an important role in both the development and progression of several human malignancies including prostate cancer (PC). A single nucleotide polymorphism (SNP) at -251 upstream of the transcriptional start site of the IL-8 gene has been shown to influence its production. The effects of IL-8 are mediated by two highly related chemokine receptors, CXCR1 and CXCR2. The present study investigated the influence of the IL-8 and CXCR2 gene variation on susceptibility and clinicopathological characteristics of PC in a group of Brazilian subjects. Two hundred and one patients and 185 healthy controls were enrolled in a case-control study. Blood was collected for DNA extraction; typing of IL-8 -251 T/A and CXCR2 +1208 C/T genes was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP), followed by agarose gel electrophoresis. Risk association between the genotypes, PC susceptibility and tumor characteristics was estimated by odds ratio (OR) and 95% confidence intervals (95% CI) using logistic regression analysis, after adjusting for age at diagnosis. A significant association was found between the heterozygous CXCR2 +1208 CT genotype and PC. The CXCR2 +1208 CT genotype was significantly less frequent in patients with clinical stage T3-T4 compared to T1-T2 (56.7 versus 80.5%). Our findings suggest that carriers of the CXCR2 +1208 CT genotype had a protective effect for advanced PC (CT versus CC: adjusted OR = 0.25; P = 0.02). No association was observed between the SNP for IL-8 -251 T/A and clinicopathological parameters of PC. These results indicated that the CXCR2 +1208 CT genotype is less frequent in advanced stages of PC, suggesting that this chemokine receptor plays a role in the pathogenesis of this disease.

Cette recherche propose d’étudier l’expérience du cancer de la prostate. Elle a pour objectif de documenter le vécu et la prise en charge de cette maladie à partir d’une sociologie qui entre dans la fabrique des sujets. Dans quelle mesure les hommes atteints d’un cancer de la prostate et leur entourage peuvent-ils produire de nouvelles formes de subjectivités compte tenu de la vulnérabilité à laquelle ils sont exposés ? Pour répondre à cette question, l’enquête repose principalement sur l’articulation d’observations ethnographiques réalisées pendant une durée de cinq mois au sein de quatre hôpitaux publics avec un corpus de 70 entretiens semi-directifs conduits auprès de patient·e·s, de proches et de professionnel·le·s de santé. L’adoption d’un regard socio-historique permet tout d’abord de mettre en évidence le caractère situé des subjectivités et ouvre la voie à une analyse des modes de subjectivation à l’ère de la biomédecine. L’expérience du cancer de la prostate est ensuite décrite à travers une analyse de son imbrication avec les pratiques et les savoirs biomédicaux. Enfin, les bouleversements du cancer de la prostate au-delà des mondes du soin sont également abordés. Outre les apports de cette thèse à la sociologie du cancer et aux études sur les masculinités, la conclusion met la focale sur sa contribution à l’étude du sujet vulnérable. / This research focuses on the experience of prostate cancer. Its aims to document the lived experience and the medical care of this illness from a sociological perspective that enters into the making of subjects. To what extent can men with prostate cancer and relatives produce new forms of subjectivity given the vulnerability they are enduring? To answer this question, the study relies mainly on the articulation of ethnographic observations carried out over a period of five months in four public hospitals with a corpus of 70 semi-directive interviews conducted with patients, relatives and health professionals. Firstly, the adoption of a socio-historical approch allows us to highlight the situated expression of subjectivities and opens the way to an analysis of processes of subjectivation in the age of biomedicine. Secondly, the experience of prostate cancer is described as intertwined with knowledges and practices of biomedicine. Thirdly, the lived experience of prostate cancer in everyday life is analysed. Besides the contributions of this reaserch to the sociology of cancer and masculinities studies, the conclusion focuses on the construction of vulnerable subjects.

In summary, the present findings have demonstrated the important roles of CFTR in prostatitis and cancer development, which may provide new insight into the understanding of the prostate in health and disease. The present findings may also have potential application in diagnosis and prognosis of cancer. / In the first part of the study, the possible role and a bacterial killing mechanism involving CFTR-mediated bicarbonate secretion in prostatitis were investigated in a rat prostate model. CFTR was found to be expressed in the epithelium of rat ventral prostate. Experiments using cultured rat primary prostate epithelial cells demonstrated that CFTR was involved in mediating bicarbonate extrusion across the prostate epithelium. The expression of CFTR and carbonic anhydrase II (CAII), a key enzyme involved in cellular HCO 3- production, along with several pro-inflammatory cytokines including IL-6, IL-1beta, TNF-alpha, was significantly up-regulated in the primary culture of rat prostate epithelial cells upon E.coli-LPS challenge. Inhibition of CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO3- content (>50mM), rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO 3- on bacterial killing was confirmed by its ability to suppress bacterial initiation factors in E coli. The relevance of the CFTR-mediated HCO3- secretion in human was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues. The present results have demonstrated that CFTR plays a previously undefined role in prostatitis and could be up-regulated during the inflammation in prostate as a host defense mechanism to increase bicarbonate secretion for bacterial killing. / In the second part of the study, the possible role of CFTR in prostate cancer development and the underlying mechanisms were investigated. Our results showed that the expression of CFTR and CAII in prostate was remarkably decreased in aged rat prostate. We observed that testosterone could up-regulate the expression of CFTR and CAII in vitro and in vivo , indicating that the declined male hormones during aging may be responsible for the observed age-dependent expression of CFTR. In the present study, we found that inhibition of CFTR enhanced cell proliferation/anti-apoptosis in the prostate primary epithelial cells. CFTR was detected in all examined prostate cell lines, but with relatively higher expression levels in immortalized cell lines (PZ-HPV-7, PNT1A, PNT2C2) than in cancer cell lines (PC-3, DU-145, LNCaP). Immunohistological studies showed that the expression of CFTR was dramatically reduced in prostate cancer specimens as compared to that in normal prostate tissues. Furthermore, our gain and loss of function studies showed that knockdown of CFTR profoundly enhanced cell proliferation, cell adhesion, invasion and migration, while inhibited apoptosis in prostate cancer cell lines, overexpression of CFTR dramatically suppressed tumorigenic phenotype of cancer cells. Soft agar anchorage-independent growth assay showed that knockdown of CFTR in prostate cancer cells increased the number of colonies formed in soft agar. More importantly, we demonstrated that CFTR knockdown promoted the tumor growth in vivo and forced overexpression of CFTR in prostate cancer cells and ultrasound-mediated gene transfer of CFTR inhibited xenograft tumor growth in vivo. Mechanistically, multiple mechanisms were identified to contribute to the CFTR- mediated tumor suppressive effects. Firstly, CFTR chloride channel function was implicated in the regulation of apoptosis in prostate cancer cells. Secondly, CFTR up-regulated the transcription level of miR-34a and miR-193b, both of which have been indicated as tumor suppressors in multiple cancers. Thirdly, 11 cancer-related genes were found to be up- or down-regulated by CFTR using PCR-array. These data demonstrated that CFTR may play an important role in prostate cancer development by acting as a tumor suppressor. / The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel conducting both Cl- and HCO3 -. It is expressed in epithelial cells of a wide variety of tissues. CFTR is also known to be expressed in human prostate; however, the physiological role of CFTR in the prostate and related diseases remains largely unknown. This thesis explored the biological roles of CFTR in prostatitis and cancer development. / Xie, Chen. / Adviser: Chan LiShaw Chang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 175-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In order to further investigate the functions of ERRbeta/gamma in prostate cancer, the following aspects were explored in my study. My results show that: (1) Expressions of ERRbeta/gamma was down-regulated in prostate cancer cell lines and prostate cancer tissues. And only the short-form but not other ERRbeta isoforms was expressed in prostatic cells. (2) Overexpression of ERRbeta/gamma significantly inhibited the cell proliferation in vivo and in vitro. (3) Cell cycle analysis showed that S-phase fraction of ERRbeta/gamma stable clones was significantly decreased, while there was no significantly induced apoptosis by ERRbeta/gamma overexpression. This was confirmed by BrdU incorporation assay. (4) Expressions of two cyclin-CDK inhibitors p21Cip1/Waf1 and p27Kip1 were increased significantly in ERRgamma clones, but only p21 in ERRbeta clones. (5) P21 and p27 gene promoters could be transactived by ERRgamma, but only p21 by ERRbeta. The transactivity of p21 by ERRbeta can be potently enhanced by PGC-1alpha (6) Deletion mutants of ERRgamma showed the transaction of p21 required an intact DNA-binding domain. (7) DY131, the ERRbeta/gamma agonist, further potentiated the growth inhibition in ERRbeta/gamma-stable clones in a dose-dependent manner. (8) There were increase in number of giant potential-active mitochondria and accumulation of lipid droplets in ERRbeta-clones. / Prostate cancer is the most common diagnosed cancers in men in western countries. Despite the substantial clinical significance, the mechanisms underlying the development and progression of prostate cancer are poorly understood. ERRs(alpha, beta, gamma) belong to orphan nuclear. All ERR subtypes share significant homology with estrogen receptors (ERs) in their protein structures. Functionally ERRalpha shares, regulates same target genes with ERalpha and is involved in carcinogenesis, while the ERRbeta and ERRgamma are still unknown. / The results obtained indicate that ERRbeta/gamma inhibit proliferation of prostate cancer cells by arresting of cell cycle progression, suggesting a tumor suppressor function for ERRbeta/gamma in prostate cancer. p21 may be the key mediator of this suppressor function, and the p21 is the target gene of ERRbeta/gamma. The selective ERRbeta/gamma agonist, DY131, potently inhibited the proliferation of ERRbeta/gamma-positive prostate cancer cells, suggesting ERRbeta and gamma could be a potential therapeutic target for prostate cancer therapy. / Yu, Shan. / "July 2007." / Adviser: Franky L. Chan. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0238. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 140-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.