研究背景和研究目的 / 前列腺癌是許多西方國家男性人群中最常見的惡性腫瘤。最新癌症統計結果表明，前列腺發病例和致死率在亞洲國家尤其是中國和香港地區呈迅猛上升趨勢(2009年，本港前列腺癌發病率列所有腫瘤發病率中第三位，致死率列第五位)。目前前列腺癌治療策略主要集中在拮抗雄激素信號通路。然而，臨床實踐表明，這種治療方式除了引起由於體內激素水平失調產生的一系列副作用之外，往往導致疾病進展到令人棘手的去勢治療無效階段。因此，從分子水平更為深入的理解前列腺癌疾病進展過程對於最終攻克前列腺癌具有重要的研究價值。雌激素相關受體是孤兒核受體的亞組之一，包括 α, β, γ三個亞型。該組受體在結構上與α亞型雌激素受體具有很高的同源性。已有研究表明，α亞型雌激素相關受體直接调控涉及氧化磷酸化，線粒體生物發生和脂肪酸氧化的相關基因表達，從而在細胞能量代謝調節中發揮至關重要作用。最新研究發現， α亞型雌激素相關受體的高表達在包括乳腺癌和前列腺癌在內的一系列腫瘤中與疾病的進展和不良預後高度相關。這提示該受體可能參與這些腫瘤的惡性進展。腫瘤細胞對低氧環境的耐受是實體腫瘤的標誌性表型之一，同時也有研究表明這一機制可能在癌細胞的惡性克隆選擇中發揮了重要作用。在眾多低氧耐受的機制中，細胞能量代謝方式轉換被研究人員看作重要的調節通路之一。考慮到前列腫瘤的低氧微環境以及α亞型雌激素相關受體在能量代谢過程的重要調節作用，有理由推測在該受體可能在前列腺癌細胞低氧耐受中發揮了積極的作用進而促進前列腺癌的惡性進展。 / 材料和方法 / 為了研究α亞型雌激素相關受體在前列腺癌細胞低氧耐受中的功能，本次研究採取了下列實驗方法：1）用免疫組化方法考察α亞型雌激素相關受體在人前列腺癌組織中的表達情況；2）用合適的前列癌細胞系建立α亞型雌激素相關受體穩定過表達細胞系同時研究這些穩轉細胞系的體外生長表型；）研究雌激素相關受體穩定過表達細胞系在低氧环境下的體外生長表型；）研究雌激素相關受體穩定過表達細胞系在免疫缺陷小鼠中的致瘤能力同時用免疫組化方法考察其腫瘤血管生成情況；）用定量 PCR和免疫印跡(Western blot)方法檢測低氧誘導因子-1α亞基(HIF-1α)及其信號通路中相關基因在α亞型雌激素相關受體穩定過表達細胞系中的表達水平，同時用雙螢光素酶報告基因方法考察α亞型雌激素相關受體對低氧誘導因子‐1(HIF-1)靶基因啟動子的轉錄激活效應；5）用 shRNA介導的基因阻斷的方法進一步考察α亞型雌激素相關受體對前列腺癌細胞低氧耐受的影響；6）通過觀考察用α亞型雌激素相關受體選擇性抑製劑 XCT790處理細胞對其在低氧環境下的體外生長情況的作用，進一步闡明 α亞型雌激素相關受體對前列腺癌細胞低氧耐受的影響；7）用免疫印跡 (Western blot)，免疫共沉澱 (Co-IP)和熒光能量共振轉移(FRET)分析的方法考察α亞型雌激素相關受體對低氧誘導因子‐1α亞基表蛋白表達和穩定性以及對低氧誘導因子 -1信號通路的影響。 / 結果 / 本研究所得得到的結果簡要總結如下：1）α亞型雌激素相關受體在前列癌組織中的免疫反應性呈現隨著惡性程度升高而增加的趨勢；2）α亞型雌激素相關受體在人前列腺癌細胞系 LNCaP中的過表達能提升其在常氧和低氧環境下的體外細胞增殖，細胞集落形成，細胞對胞外基質的粘附以及細胞侵襲能力； 3) α亞型雌激素相關受體在人前列腺癌細胞系 LNCaP中的過表達能促進其體內腫瘤形成及腫瘤血管生成； 4）過表達 α亞型雌激素相關受體能上調低氧誘導因子-1α亞基的蛋白水平並提高其轉錄活性；5）shRNA介導的α亞型雌激素相關受體 mRNA阻斷可以削弱人前列腺癌細胞系 LNCaP細胞在低氧環境下的體外生長能力；6）在体外用α亞型雌激素相關受體選擇性抑製劑 XCT790处理人前列腺癌細胞系 LNCaP細胞可能通過減少低氧誘導因子‐1α亞基蛋白表達水平從而抑制其在低氧環境下的細胞生長能力；7）α亞型雌激素相關受體可以直接與低氧誘導因子-1α亞基相互作用，並且這種相互作用可能有助於抑制低氧誘導因子-1 α亞基的蛋白降解。 / 結論 / 本研究獲得結果提示，α亞型雌激素相關受體可能通過提高低氧誘導因子-1α亞基的蛋白水平及激活低氧誘導因子-1信號通路從而促進前列腺癌細胞在低鹽環境下的細胞生長能力。体外用 shRNA介導的α亞型雌激素相關受體 mRNA阻斷方法和α亞型雌激素相關受體選擇性抑製劑处理都有可能通過阻止低氧誘導因子‐1α亞基以削弱前列腺癌細胞在低鹽環境下的細胞生長能力。同時， α亞型雌激素相關受體能直接與低氧誘導因子-1 α亞基相互作用而這種相互作用有可能有助於抑制其蛋白降解，這些結果提示 α亞型雌激素相關受體可能在前列腺癌進展過程中的低氧耐受中發揮積極作用。 / Background and aims of study / Prostate cancer is the most common cancer in many Western counties among the male populations. Latest cancer statistics also show that its incidence and mortality rates are rapidly increasing in China and Hong Kong (Prostate cancer ranked the 3rd common cancer and 5th cancer causing death in Hong Kong in 2009). Current therapeutic strategies of prostate cancer mainly target to the antagonizing androgen signaling pathway, which usually drives the disease to the impasse of castration resistance albeit the side effects caused by the imbalance of hormone. The substantial clinical significance of prostate cancer is urgent to better understand the progression of this disease. Estrogen-related receptors (α,β,γ) are a subgroup of ligand-independent orphan nuclear receptors, which is constitutively activated without binding any physiological ligands and all share high homology with the estrogen receptor alpha (ER α) structurally. Previous studies indicates that ERR α plays a pivotal role in cellular energy home stasis regulation, target genes of which are involved in the procedures of oxidative phosphorylation, mitochondrial biogenesis and fatty acid oxidation. Recent studies reveals that high expression of ERR α may be useful as a poor prognostic marker in both hormone-dependent and hormone-independent cancers (including breast cancer and prostate cancer), which implicates this nuclear receptor may be involved in the advanced malignant progression of these cancers. Adaptation to hypoxia is one of the hallmark features of solid tumors and it is conceived to play an important role in malignant clonal selection of cancer cells. Among the diverse mechanisms on cellular hypoxia adaptation, energy metabolism reprogramming is characterized and considered as a critical regulatory pathway. Given the hypoxic microenvironment of prostate cancer and the energy regulatory role of ERR α, it is hypothesized that ERR α might play an active role in the cellular hypoxic adaptation of prostate cancer hence advancing the progre sion of this disease. / Materials and methods / To investigate the functional significance of ERR α in cellular hypoxic adaptation of prostate cancer, the following experimental approaches were employed and performed in my thesis study: 1) to survey the expression pattern of ERR α in human prostate cancer tissues by immunohistochemical staining; 2) to generate ERR α-stable expressing cell lines in selected prostate cancer cell lines and functionally characterize their in vitro phenotypes under normoxia condition; 3) to characterize in vitro hypoxic-response phenotypes of ERR α-infectants; 4) to determine the tumorigenicity of ERR α-infectants in immuno-deficient SCID mice and to investigate their tumor angiogenesis by immunohistochemical staining; 5) to determine the HIF-1α signal cohort in ERR α-infectants by both RT-PCR and immuno blot analysis and to investigate the transactivation effect of ERR α on HIF-1 targeting genes promoters by dual luciferase reporter assay; 6) to further characterize the hypoxic adaptation phenotypes induced by ERR α transduction using shRNA-mediated gene knockdown approach; 7) to further elucidate the effect of ERR α on the hypoxic cell growth regulation of prostate cancer by treating ERR α-infectants with an ERR α-selective antagonist XCT790; 8) to further investigate the mechanisms via which ERR α interferes with the protein expression or stabilization of HIF-1α as well as HIF-1 signal cohort using immuno blot analysis, immunoprecipitation assays and fluorescence resonance energy transfer (FRET) analysis. / Results / My results are briefly summarized as follows: 1) ERR α exhibited an increased immuno expression pattern in high-grade prostate cancer; 2) Ectopic expression of ERR α in LNCaP prostate cancer cell line could promote its in vitro cell proliferation, clonal formation, cell-extracellular matrix attachment and cell invasion capacities under both normoxic and hypoxic conditions; 3) Ectopic expression of ERR α in LNCaP prostate cancer cell line could promote its in vivo tumorigenicity and tumor angiogenesis; 4) Overexpression of ERR α could up-regulate protein level of hypoxia regulatory transcriptional factor-1(HIF-1) α subunit (HIF1-α) and enhance its transcriptional activity; 5) mRNA knock-down of ERR α could attenuate in vitro cell growth capacity of LNCaP prostate cancer cell line under hypoxic condition; 6) Treatment with an ERR α specific antagonist XCT790 could inhibit in vitro hypoxic cell growth of LNCaP cells via its effect on decreasing the protein level of HIF-1α; 7) ERR α could physically interact with HIF-1α and such ERR α-HIF1-α interaction might help to inhibit protein degradation of HIF-1α. / Conclusion / The results obtained in this study indicated that ERR α could promote the hypoxic cell growth of prostate cancer via its enhancing the protein level of HIF-1α and activation of HIF-1 signal cohort. Both treatment with ERR α selective antagonist and down-regulating of ERR α by shRNA-mediated gene knockdown approach could attenuate the hypoxia adaptation of prostate cancer cells, which might be mediated by their suppression of the protein level of HIF1α. ERR α could directly interact with HIF-1α and such interaction might help to suppress the protein degradation of HIF1α, suggesting that ERR α may play an active role in hypoxic adaptation in advancing of prostate cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zou, Chang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 138-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.viii / PUBLICATIONS --- p.ix / CONTENTS --- p.x / ABBREVIATIONS --- p.xiii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Prostate cancer --- p.2 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Risk factors --- p.3 / Chapter 1.1.3 --- Patho-physiology --- p.6 / Chapter 1.1.4 --- Diagnosis and treatment --- p.8 / Chapter 1.2 --- Androgen,androgen receptor and prostate cancer --- p.10 / Chapter 1.2.1 --- Androgen and androgen receptor --- p.10 / Chapter 1.2.2 --- Castration Resistance Prostate Cancer (CRPC) --- p.12 / Chapter 1.2.2.1 --- Overexpression of AR --- p.13 / Chapter 1.2.2.2 --- Increasing sensitivity to and rogen --- p.13 / Chapter 1.2.2.3 --- AR mutation --- p.14 / Chapter 1.2.2.4 --- Deregulation of AR regulator factors --- p.15 / Chapter 1.2.2.5 --- Outlaw pathway --- p.15 / Chapter 1.2.2.6 --- AR-independent pathway --- p.16 / Chapter 1.3 --- Estrogen and prostate cancer --- p.17 / Chapter 1.3.1 --- Overview of estrogen and estrogen receptors --- p.17 / Chapter 1.3.2 --- Estrogen signaling pathway andprostatecancer --- p.18 / Chapter 1.4 --- Nuclear receptors --- p.20 / Chapter 1.4.1 --- Overview of NRs superfamily --- p.20 / Chapter 1.4.2 --- Classification --- p.21 / Chapter 1.4.3 --- NRs as therapeutic targets for cancer treatment --- p.23 / Chapter 1.5 --- Estrogen-related receptors --- p.25 / Chapter 1.5.1 --- NR3B subgroup --- p.25 / Chapter 1.5.2 --- Isoforms --- p.26 / Chapter 1.5.3 --- Structure --- p.27 / Chapter 1.5.4 --- Ligand --- p.28 / Chapter 1.5.5 --- Co-regulators --- p.31 / Chapter 1.5.6 --- Tissue-specific expression pattern and identifiedfunction --- p.32 / Chapter 1.5.6.1 --- Tissue-specific expression pattern --- p.32 / Chapter 1.5.6.2 --- Identified physiological function of ERRs --- p.33 / Chapter 1.5.7 --- ERRs and cancer --- p.35 / Chapter 1.5.7.1 --- ERRβ/γ and cancer --- p.35 / Chapter 1.5.7.2 --- Expression of ERRα in cancer --- p.37 / Chapter 1.5.7.3 --- Identified functional roles of ERRα in cancer --- p.40 / Chapter 1.5.7.4 --- Regulation of ERRα in cancer cells --- p.42 / Chapter 1.6 --- Hypoxiaadaptation andcancer --- p.47 / Chapter 1.6.1 --- HIFs isoforms and structure --- p.47 / Chapter 1.6.2 --- Structure --- p.48 / Chapter 1.6.3 --- Regulation of HIF-1α expression --- p.49 / Chapter 1.6.3.1 --- Regulation of HIF-1α mRNA transcription --- p.49 / Chapter 1.6.2.2 --- Regulation of HIF-1α mRNA transcription --- p.50 / Chapter 1.6.2.3 --- O₂-dependent regulation of stability of HIF-1α protein --- p.51 / Chapter 1.6.2.4 --- O₂-independent regulation of HIF-1α --- p.52 / Chapter 1.6.2.5 --- Genetranscriptional regulation role of HIFs --- p.54 / Chapter 1.6.3 --- HIFs and cancer --- p.55 / Chapter 1.6.3.1 --- Overview --- p.55 / Chapter 1.6.3.2 --- Expression of HIF-1α in cancer progression --- p.55 / Chapter 1.6.3.2 --- Functional roles of HIF-1α in cancer progression --- p.56 / Chapter CHAPTER 2 --- Aims of study --- p.58 / Chapter CHAPTER 3 --- Materials and methods --- p.61 / Chapter 3.1 --- Cell lines and cell culture --- p.62 / Chapter 3.2 --- Human Prostatic Tissues --- p.64 / Chapter 3.3 --- RNA isolation and Reverse transcriptional-PCR --- p.64 / Chapter 3.3.1 --- Total RNA extraction --- p.64 / Chapter 3.3.2 --- Reverse transcription reaction --- p.65 / Chapter 3.3.3 --- Polymerase Chain Reaction for gene expression detection --- p.66 / Chapter 3.4 --- Plasmids construction --- p.69 / Chapter 3.4.1 --- Genomic DNA extraction --- p.69 / Chapter 3.4.2 --- PCR for cloning and sub-cloning --- p.70 / Chapter 3.4.3 --- PCR for mutant generation --- p.70 / Chapter 3.4.4 --- Restriction enzymes cut and ligation --- p.71 / Chapter 3.5 --- Antibody and reagents --- p.73 / Chapter 3.6 --- Immunohistochemistry --- p.74 / Chapter 3.7 --- Western Blot Analysis --- p.75 / Chapter 3.7.1 --- Protein extraction --- p.75 / Chapter 3.7.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.76 / Chapter 3.8 --- Retroviral transduction and generation of ERRα poolandstable clones --- p.77 / Chapter 3.9 --- In vitro Cell Growth Assays --- p.77 / Chapter 3.9.1 --- Cell counting --- p.77 / Chapter 3.9.2 --- 5-Bromodeoxyuridine (BrdU) incorporation assay --- p.78 / Chapter 3.9.3 --- MTT assay --- p.79 / Chapter 3.9.4 --- In vitro clonal formation assay --- p.79 / Chapter 3.10 --- Cell attachment assay --- p.80 / Chapter 3.11 --- Transwell cell invasion assay --- p.81 / Chapter 3.12 --- In vivo tumorigenicity assay --- p.81 / Chapter 3.13 --- RNA interference --- p.82 / Chapter 3.14 --- Transient Transfection and Luciferase Reporter Assay --- p.83 / Chapter 3.15 --- Immuno-precipitation (IP) assay --- p.84 / Chapter 3.16 --- Fluorescence Resonance Energy Transfer (FRET) detection --- p.85 / Chapter 3.17 --- In vitro treatment with XCT790, cycloheximide and MG-132 --- p.86 / Chapter CHAPTER 4 --- Reuslts --- p.88 / Chapter 4.1 --- ERRα exhibits an increased expression pattern in high grade prostate cancer --- p.89 / Chapter 4.2 --- Ectopic expression of ERRα in LNCaP prostate cancer cell line can promote its in vitro cell proliferation, clonal formation, cell attachment and cell invasion capacity under normoxic condition --- p.91 / Chapter 4.3 --- Ectopic expression of ERR α in LNCaP prostate cancer cell line can promote its in vitro cell proliferation, clonal formation, cell attachment and cell invasion capacities under hypoxic condition --- p.94 / Chapter 4.4 --- Ectopic expression of ERR α in LNCaP prostate cancer cells can promote their in vivo tumorigenicity and tumor angiogenesis. --- p.97 / Chapter 4.5 --- Overexpression of ERRα can up‐regulate protein level of HIF-1α and enhance its transcriptional activity --- p.99 / Chapter 4.6 --- mRNA Knock-down of ERRα can attenuate in vitro cell growth of LNCaP prostate cancer celll line under hypoxic condition --- p.107 / Chapter 4.7 --- Treatment with an ERRα specific antagonist XCT790 can inhibit in vitro hypoxic cell growth of LNCaP cells via its effect on decreasing the protein level of HIF-1α --- p.110 / Chapter 4.8 --- ERRα can physically interact with HIF-1α and such ERRα-HIF-1α interaction helps to inhibit protein degradation of HIF-1α --- p.114 / Chapter CHAPTER 5 --- Discussion --- p.119 / Chapter CHAPTER 6 --- Summary --- p.134 / References --- p.138

Prostate cancer is a leading cause of cancer-related death in men, and while many men diagnosed with the disease will have an indolent clinical course, 20-25% of men will experience disease recurrence which is invariably lethal. There is an urgent need for prognostic biomarkers that will predict disease recurrence and risk-stratify patients upon diagnosis, allowing for personalised therapies. This thesis attempts to identify new prognostic biomarkers for prostate cancer and investigates their patterns of protein expression in human primary prostate tumour tissue. Cancer stem cells are cancer cells thought to be uniquely capable of self-renewal and tumorigenicity, and may have a role in tumour recurrence. Using a literature searching approach, potential biomarkers related to stem cells, cancer stem cells or recurrence in prostate cancer were identified, and ALDH7A1, BMI1, SDC1, MUC1-C, Nestin and ZSCAN4 were chosen for investigation. An in silico approach was also used for biomarker identification, with RS1 and SLC31A1 selected as their mRNA was found to be upregulated in recurrent tumours. The expression patterns of all 7 potential biomarkers were examined by immunohistochemistry on prostate tumour tissue and benign tissue from prostate biopsies and prostatectomies. BMI1, ALDH7A1, MUC1-C and Nestin showed no relationship to recurrence or other clinical features. RS1 protein levels increased in patients with recurrence within 5 years, negatively correlated with AR expression, and a meta-analysis showed that the RS1 gene was amplified in up to 32% of castration-resistant prostate tumours. ZSCAN4 was heterogeneously expressed in a subset of 26% of prostate tumours with unclear characteristics and was not expressed in benign tissue, but was not associated with recurrence. Finally, SDC1 expression was lost in tumour epithelium, but a population of unidentified SDC1-expressing cells were found in the stroma of a third of tumours, and an increased burden of these cells was associated with primary Gleason pattern 5 tumours. These cells do not overlap with common epithelial, mesenchymal or stromal lineages, but may be migratory. In summary, the data presented in this thesis identifies 3 potential new biomarkers for prostate cancer, and provides the basis for future characterisation of their wider roles in the disease.

Chapter 1 includes the Introduction and literature review which describes current developments within the field of in vitro/vivo imaging of cancers, with a particular emphasis on the techniques employing fluorescence emission-based spectroscopy and imaging modalities. Examples are cited whereby graphene and its congeners have been used in conjunction with various fluorophores and peptide sequences as a means of achieving highly specific imaging probes. This section discusses aspects of energy transfer and the possibility that molecular probes can be designed to achieve both therapeutic goals and diagnosis (Theranostics). This review concludes with a discussion of the use of organic supramolecularly assembled imaging agents as a means of achieving thermodynamically controlled nano-constructs for the functionalisation of graphenes and their potential future applications as theranostic agents. Chapters 2, 3 and 4 describe the synthesis of chiral and naphthalene diimides (NDIs) which are fluorescent. Spectroscopic investigations in the solution phase are described and the propensity for aggregation in these systems is discussed. The specific nature of the self-assembly processes involved is explored in different solvent systems and in the solid state. Fluorescence lifetime imaging microscopy (FLIM) and laser scanning confocal microscopy (LSCM) are used to investigate the cellular uptake of the NDI molecules and their capacity to image living prostate cancer cells (PC-3). The NDIs are subsequently complexed supramolecularly to poly-aromatic carbon systems such as C60 and coronene (Chapter 3), as well as thermally reduced graphene oxide (Chapter 4). Chapter 3 describes the explorations into the modelling of the donor-acceptor interactions between the NDIs and the C60/coronene in order to establish binding stoichiometry and association constants. Both Chapters 3 and 4 discuss fluorescence titration and time correlated single photon counting (TCSPC) experiments which were performed as a means of establishing the presence of excited state energy transfer mechanisms. The chapters conclude with investigations in living cells in order to establish retention of in vitro fluorescence, with particular attention being paid to confirming the graphene complex stability. Chapter 5 describes the synthesis and functionalisation of a seven amino acid sequence peptide known as the G-receptor protein (GRP) binding unit of the polypeptide bombesin. The sequence binds GRPs that are known to be up-regulated in prostate cancer carcinoma and it has been widely utilised in the literature as a means of enhancing the up-take of various cancer imaging agents that employ a variety of imaging modalities. The peptide was attached to the fluorescent NDIs via carbodiimide activation protocols with the purpose of providing added specificity to the imaging agent with respect to PC-3 cells. Prior to NDI derivatisation with bombesin, electrochemical impedance spectroscopy (EIS) has been performed to establish the extent to which the peptide sequence binds to prostate cancer cells over healthy ones. The chapter concludes with confocal microscopy of the bombesin derivative NDI complexed to thermally reduced graphene oxide as a means of validating the utility of the fluorescent targeted bioconjugate as synthetic scaffolds for future early diagnosis and sensing devices for prostate cancer. Chapter 6 constitutes the summary of this work and highlights several possible areas of future developments of relevance to the results discussed and related future experiments proposed to fully validate the device assembly for prostate cancer sensing. Chapter 7 contains the Experimental section and the relevant data gathered over the course of the investigations. Additional supporting figures or data referred to but not included in the main text of the thesis are reported in the Appendices.

"Prostate cancer (Pca) is the number one cancer in U.S. men and a leading cause of cancer-related deaths. The disease is initially dependent upon active male androgens for survival, such as testosterone or dihydrotestosterone. However, relatively inactive androgens like 5-androstenediol (5Adiol) have been considered to play larger roles in disease progression than previously considered. 5Adiol is synthesized in the prostate, is not affected by current anti-androgen therapies, and activates the androgen receptor in human prostate cancer cell lines more effectively than active male androgens. The causal relationship between androgens and prostate cancer progression has significantly risen due to anabolic steroid abuse in competitive sports and recreational athletes. The evidence surrounding an association between androgen abuse and prostate cancer, as well as various other carcinomas, is of great concern within this demographic because of its preventability. Despite their prohibited use, anabolic products continue to be marketed and available for purchase as nutritional supplements. Surveys on current steroid user trends have documented high dosing regimens, extended periods of use, multiple self-diagnosed side effects, and unsafe injection practices. Because few studies have investigated the effects of advertised 5Adiol products on prostate cancer progression, the goal of this study was to investigate whether Value Nutrition’s 5Adiol product possesses comparable mitogenic stimulant activity to testosterone in the androgen-dependent cell line, LNCaP. It should be noted that while this compound is banned by the U.S. Drug Enforcement Administration and is no longer sold through this company, alternative steroids continue to be marketed to consumers. Additional goals included determining whether this product would affect the proliferation of liver cells using the HepG2 cell line, if HepG2 metabolism of the product would play a role in the proliferation of LNCaP cells, and whether the LNCaP cells are capable of converting the 5Adiol product into testosterone. Multiple assays determined that the LNCaP cells were androgen responsive to the 5Adiol product, HepG2 proliferation was stimulated, and HepG2 metabolism of the 5Adiol product increased LNCaP mitogenicity, although significant differences were unattainable primarily due to the LNCaP cells reduced adhesion properties. Because reverse-phase HPLC was unable to detect compounds within 5Adiol even at high concentrations, the mitogenicity observed in both cell lines is unable to be correlated with a particular component in this product. While it is possible that 5Adiol is present at extremely low levels, contamination with other factors, hazardous or not, cannot be ruled out. In conclusion, these results suggest that the use of nutraceuticals have inherent risks for men at risk of developing prostate cancer. The mitogenic effects observed from small levels of unknown compounds in the tested 5Adiol product raise serious questions as to the legitimacy of allowing companies to market and self-regulate their own products."

研究背景與研究目的 / 細胞衰老是指細胞進入不可逆的永久化的生長停滯狀態。目前，細胞衰老作為重要的抑癌機制受到廣泛認可，对其相關信號通路的研究為腫瘤的靶向治療提供了新的依據和策略。TLX核受體基因属于核受體亞家族2組E成員1，是一種孤兒受體。雞和老鼠TLX基因最初作為果蠅末端/间隙基(tailless) 的同源基因而被發現，而人TLX 基因是在檢索惡性淋巴癌中的抑癌細胞而從人胚胎的腦cDNA文庫中克隆出來的。TLX基因敲除的轉基因老鼠的研究表明TLX基因對維持胚胎腦和成体腦神經幹細胞的分裂增殖起重要作用。最近的研究發現，TLX在臨床神經胶质瘤組織中高表達。並且，在轉基因鼠中，TLX的高表達会引起神經幹細胞的大量增殖而形成腦腫瘤，提示TLX可能參與腦腫瘤的發生和發展。但是，TLX对包括前列腺癌在内的人類惡性腫瘤的發生發展中所起的功能及作用機制尚不清楚。表達譜研究發現，TLX在前列腺細胞中的表達水平高於永生化的正常前列腺上皮細胞的表達，並且，TLX在臨床惡性程度高的前列腺癌中呈高表達趨勢，預示TLX可能參與促進前列腺癌的惡性進展。因此本研究的主要目的是TLX在前列腺癌細胞中的功能研究。 / 研究材料與方法 / 為了研究TLX對前列腺癌細胞生長的影響以及相關機制，本論文主要採用以下方法：1）運用免疫組化的方法檢測TLX在臨床前列腺癌組織中的表達，並應用實時螢光定量PCR方法檢測TLX在永生化的非惡性前列腺上皮細胞以及前列腺癌細胞株中的表達；2）根據不同的p53表達狀態選擇雄激素依賴（LNCaP）和雄激素非依賴（PC-3, DU145）的前列腺癌細胞株，分別采用慢病毒感染和逆轉錄病毒感染的方法建立TLX-敲除和TLX-過表達的細胞株，並研究這些穩轉細胞系離體和在體的生長表型（包括檢測細胞生長，細胞週期，細胞衰老，細胞的遷移和侵染，化療藥物抗性，缺氧耐受性以及體內成瘤能力）；3）採用檢測β-半乳糖苷酶活性的方法檢測TLX穩轉系細胞在衰老因素誘導和非誘導狀態下TLX缺失和高表達對細胞衰老的影響；4）采用免疫印跡（western blot）的方法檢測TLX穩轉系細胞中參與細胞衰老的關鍵蛋白的表達情況；5）利用雙螢光素酶報告基因方法和染色質免疫沉澱技術，研究TLX對靶基因的調控；6）構建TLX缺失變異體（△ZF1 和 △LBD-AF2），在前列腺細胞系和非前列腺細胞系中外源性表達相應的變異體進一步驗證TLX的功能。 / 結果 / 本論文研究結果總結如下：1）TLX在前列腺癌細株中和惡性程度高的前列腺癌組織中高表達；2）在前列腺癌中進行TLX基因敲除能顯著抑制細胞體外和體內的生長並誘導前列腺癌細胞的衰老；3）相反，TLX的過表達能促進前列腺癌細胞體外和體內的惡性生長，包括促進細胞的錨定和非錨定性生長、促進細胞的遷移與侵染、增強細胞缺氧耐受、對化療藥物抗性、以及增強細胞異位移植瘤的成瘤能力；4）TLX 的高表達抑制了前列腺癌細胞衰老，並保護細胞免受多柔比星誘導的細胞衰老以及持續性激活的癌基因H-RAS（H-RAS{U+1D33}¹²{U+2C7D}）誘導的細胞衰老；5）TLX可以結合到p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹基因（其後縮寫為p21）的啟動子序列並抑制p21的啟動子的轉錄活性，並且在TLX-過表達細胞中外源性高表達p21能誘導前列腺癌細胞重新進入衰老狀態；6）TLX也能結合到SIRT1基因的啟動子序列並激活SIRT1的轉錄活性，在TLX-過表達細胞中對SIRT1進行基因沉默能誘導這些細胞的再次衰老；7）TLX介導的衰老抑制效應以及對其靶基因的轉錄調控作用需要完整的DNA-結合域以及配體結合域，對TLX兩個區域的缺失變異影響TLX在前列腺細胞和非前列腺細胞中的生理功能及轉錄調控活性。 / 結論 / 本論文的研究結果提示TLX通過抑制前列腺癌細胞的衰老在前列腺癌發生發展過程中起重要作用，並且這種衰老抑制作用是通過介導p21基因的轉錄抑制以及對SIRT1基因的轉錄激活而實現的。此研究首次證實了TLX在前列腺癌中高表達，並且TLX能夠抑制前列腺癌細胞的衰老從而促進前列腺癌的發生發展，提示TLX有可能成為前列腺癌治療潛在的重要靶點。 / Background and aims of the study / Cellular senescence represents an irreversible form of permanent cell-cycle arrest and it acts a key process of tumor suppression, while targeting to pathways involved in this process can provide potential and promising therapeutic strategies to cancer treatments. TLX belongs to the NR2E1 orphan nuclear receptor subfamily. The chicken and mouse TLX genes were initially isolated as a vertebrate homolog to the Drosophila terminal-gap gene tailless (tll), while the human TLX was cloned from a fetal brain cDNA library in a search for putative tumor suppressor genes in lymphoid malignancies. Functional studies in transgenic mouse model of TLX-knockdown show that TLX plays important regulatory roles in the maintenance and self-renewal control of both embryonic and adult neural stem cells. Recent studies of transgenic mice with TLX overexpression combined with its expression studies in human clinical gliomas revealed that TLX is overexpressed in primary human glioblastomas and its dysregulation may contribute to the initiation and development of some brain tumors. However, the exact functional contributions of TLX and the involved mechanism(s) in human malignancies, including prostate cancer, are still far from clear. In an expression profile study, it was demonstrated that TLX exhibited an up-regulated expression pattern in many prostate cancer cell lines and also the high-grade clinical prostate cancer, suggesting that TLX might play a positive regulatory role in the advanced progression of prostate cancer. The overall aim of this study was to elucidate the functional role of TLX in prostate cancer cell growth. / Materials and methods / In order to elucidate the functional roles of TLX in prostate cancer growth and the involved mechanisms, the following experiments were conducted: 1) To investigate and determine the expression pattern of TLX in clinical prostatic tissues by immunohistochemistry, and to survey the expression profile of TLX in a panel of prostatic immortalized epithelial and prostate cancer cell lines by quantitative real-time PCR analysis; 2) To generate stable TLX-knockdown prostate cancer cells by lentiviral transduction and TLX-stable expressing cells by retroviral transduction in both hormone-sensitive (LNCaP) and -insensitive (DU145 and PC-3) prostate cancer lines with different expression status of p53; and to conduct growth phenotype characterization studies (including cell growth, cell cycle, cellular senescence, cell migration and invasion, resistance to chemotherapy drugs, hypoxic cell growth assays, and tumorigenesis) on these TLX-transfectants in vitro and in vivo; 3) To characterize cellular senescence phenotype of TLX-infectants by senescence-associated β-galactosidase (SA-β-Gal) staining method with or without senescence inducers; 4) To investigate the expression status of markers involved in cellular senescence in TLX-infectants by immunoblotting; 5) To demonstrate the transcriptional regulation targets of TLX by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay; 6) To confirm the cellular function of TLX in prostatic and non-prostatic cells expressing different TLX deletion mutants (△ZF1 and △LBD-AF2). / Results / Results obtained in this study are summarized as follows: 1) TLX displayed an increased expression pattern in many prostate cancer cell lines and also high-grade (Gleason score ≥ 7) prostate cancer tissues; 2) Depletion of TLX mRNA by RNA interference dramatically suppressed in vitro and in vivo tumor cell growth and triggered cellular senescence (SA-β-Gal histochemical marker) in prostate cancer cells; 3) On the contrary, TLX overexpression significantly enhanced multiple advanced malignant growth capacities (including enhanced anchorage-dependent and -independent cell growth, cell migration and invasion, hypoxia adaptation, resistance to chemotherapy drug Doxorubicin as well as in vivo tumorigenicity) in prostate cancer cells; 4) TLX overexpression significantly suppressed cellular senescence and protected cells against doxorubicin-induced or oncogenic H-RAS (H-RAS{U+1D33}¹²{U+2C7D})- induced senescence; 5) TLX could directly bind to p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹ gene (hereafter p21) promoter and repress the transcriptional activity of p21 promoter, while ectopic restoration of p21 expression in TLX-overexpressed cells could rescue cellular senescence with enhanced SA-β-Gal staining; 6) protein deacetylase SIRT1 gene was also activated by TLX through its direct transcriptional regulation, while knockdown of SIRT1 in TLX-overexpressed cells could rescue cellular senescence; 7) TLX-induced suppression of cellular senescence and also its direct gene regulation would require an intact DBD and LBD domain, as truncated deletion of DBD or LBD domain could both abolish the cellular function and transcriptional activity of TLX in prostatic and non-prostatic cells. / Conclusions / The results obtained in this study suggested that TLX could play a positive growth regulatory or tumor-promoting role in prostate cancer development by its suppression of cellular senescence and this senescence suppression was mediated via its direct transcriptional regulation of both p21 (repression) and SIRT1 (transactivation) genes. Moreover, this study also showed for the first time that TLX, which was overexpressed in prostate cancer tissues, might function to suppress premature senescence in prostate cancer progression and also targeting to TLX could be a potential therapeutic approach for prostate cancer treatment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Dinglan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 135-151). / Abstract also in Chinese. / Thesis /Assessment Committee --- p.I / ABSTRACT --- p.II / 摘 要 --- p.VI / ACKNOWLEDGEMENT --- p.IX / PUBLICATIONS RELATED TO THIS THESIS --- p.XI / CONTENTS --- p.XII / ABBREVIATION --- p.XV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Prostate cancer --- p.2 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Nature history --- p.4 / Chapter 1.1.3 --- Androgen Axis prostate cancer --- p.7 / Chapter 1.1.3.1 --- Androgen receptor --- p.7 / Chapter 1.1.3.2 --- Function of the androgen receptor in prostate cancer --- p.7 / Chapter 1.1.3.3 --- Mechanisms of CRPC progression --- p.8 / Chapter 1.1.3.4 --- Androgen receptor pathway-directed therapies --- p.10 / Chapter 1.1.4 --- Treatment of prostate cancer --- p.11 / Chapter 1.2 --- Cellular senescence --- p.13 / Chapter 1.2.1 --- What is senescence --- p.13 / Chapter 1.2.1.1 --- Replicative cellular senescence --- p.13 / Chapter 1.2.1.2 --- Oncogene induced senescence (OIS) --- p.15 / Chapter 1.2.1.3 --- Tumor suppressor loss-induced senescence --- p.17 / Chapter 1.2.2 --- Establishment of cellular senescence --- p.19 / Chapter 1.2.3 --- The p16/pRb and ARF/p53/p21 pathway of senescence induction --- p.21 / Chapter 1.2.3.1 --- p16/pRb senescence pathway --- p.22 / Chapter 1.2.3.2 --- ARF/p53/p21 senescence pathway --- p.23 / Chapter 1.2.4 --- Markers of senescence --- p.24 / Chapter 1.2.4.1 --- Cell cycle arrest and morphology --- p.24 / Chapter 1.2.4.2 --- Senescence-associated β-galactosidase --- p.25 / Chapter 1.2.4.3 --- p16/pRb and p53/p21 pathways --- p.26 / Chapter 1.2.4.4 --- γ-H2AX staining as a marker for DNA damage --- p.27 / Chapter 1.2.4.5 --- Senescence-associated heterochromatin foci (SAHF) --- p.27 / Chapter 1.2.5 --- Pro-senescence therapy for cancer treatment --- p.29 / Chapter 1.2.5.1 --- Why pro-senescence therapy --- p.29 / Chapter 1.2.5.2 --- Critical factors of pro-senescence therapy --- p.31 / Chapter 1.2.5.3 --- Strategies of senescence induction --- p.32 / Chapter 1.2.5.4 --- Targeting to senescence-associated secretory phenotype (SASP) --- p.38 / Chapter 1.2.6 --- Future direction --- p.40 / Chapter 1.3 --- TLX --- p.41 / Chapter 1.3.1 --- Nuclear receptor --- p.41 / Chapter 1.3.2 --- Identification of tailless/TLX --- p.42 / Chapter 1.3.3 --- Tailless in drosophila --- p.43 / Chapter 1.3.4 --- Functional role of tll/TLX --- p.45 / Chapter 1.3.4.1 --- Role of tll/TLX in brain development --- p.45 / Chapter 1.3.4.2 --- Role of tll/TLX in visual system developments --- p.46 / Chapter 1.3.4.3 --- Role of TLX in neural stem cell self-renewal --- p.47 / Chapter 1.3.5 --- Target genes of TLX --- p.49 / Chapter 1.3.6 --- Transcriptional regulation of tll/TLX --- p.51 / Chapter 1.3.7 --- TLX in cancer --- p.52 / Chapter CHAPTER 2 --- STUDY AIMS --- p.54 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.57 / Chapter 3.1 --- Human prostatic tissues and Immunohistochemistry --- p.58 / Chapter 3.2 --- Cell lines and cell cultures --- p.59 / Chapter 3.3 --- Antibody and reagents --- p.63 / Chapter 3.3.1 --- Generation of rabbit anti-TLX polyclonal antibody --- p.63 / Chapter 3.3.2 --- Commercial antibody --- p.64 / Chapter 3.4 --- RNA isolation and Reverse transcriptional-PCR --- p.65 / Chapter 3.4.1 --- RNA isolation --- p.65 / Chapter 3.4.2 --- Reverse transcription reaction (RT) --- p.66 / Chapter 3.4.3 --- Polymerase Chain Reaction (PCR) --- p.66 / Chapter 3.5 --- Western blotting --- p.68 / Chapter 3.5.1 --- Protein extraction --- p.68 / Chapter 3.5.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.69 / Chapter 3.6 --- Plasmids construction --- p.70 / Chapter 3.6.1 --- PCR for sub-cloning --- p.70 / Chapter 3.6.2 --- PCR for mutant generation --- p.71 / Chapter 3.6.3 --- Restriction enzymes digestion and ligation --- p.72 / Chapter 3.7 --- Retroviral, lentiviral transduction and generation of TLX-stable cells --- p.73 / Chapter 3.8 --- RNA interference --- p.75 / Chapter 3.9 --- In vitro cell growth assay --- p.76 / Chapter 3.9.1 --- Cell counting --- p.76 / Chapter 3.9.2 --- MTT assay --- p.76 / Chapter 3.9.3 --- Soft agar assay for anchorage independent growth --- p.77 / Chapter 3.10 --- Cell cycle assay --- p.77 / Chapter 3.11 --- Cell invasion assay --- p.78 / Chapter 3.12 --- In vivo tumor growth assay --- p.78 / Chapter 3.13 --- In vitro and in vivo SA-β-Gal staining --- p.79 / Chapter 3.14 --- In vitro treatment with doxorubicin --- p.80 / Chapter 3.15 --- Transient Transfection and Luciferase Reporter Assay --- p.81 / Chapter 3.16 --- Chromatin immunoprecipitation (ChIP) assay --- p.82 / Chapter 3.16.1 --- Cross-linking and harvesting cells --- p.82 / Chapter 3.16.2 --- Cell lysis --- p.83 / Chapter 3.16.3 --- Sonication --- p.83 / Chapter 3.16.4 --- Immunoprecipitation --- p.83 / Chapter 3.16.5 --- Washing --- p.84 / Chapter 3.16.6 --- Elution --- p.85 / Chapter 3.16.7 --- Reverse cross-linking and DNA purification --- p.85 / Chapter 3.16.8 --- PCR --- p.86 / Chapter 3.17 --- Statistical analysis --- p.86 / Chapter CHAPTER 4 --- RESULTS --- p.87 / Chapter 4.1 --- TLX is up-regulated in prostate carcinoma and prostate cancer cell lines --- p.88 / Chapter 4.2 --- Knockdown of TLX suppresses in vitro cell growth and triggers cellular senescence in prostate cancer cells --- p.93 / Chapter 4.3 --- Knockdown of TLX inhibits in vivo tumor growth and induces cellular senescence of prostate cancer cells --- p.97 / Chapter 4.4 --- Ectopic expression of TLX enhances in vitro cell growth and multiple advanced malignant phenotypes in prostate cancer cells --- p.100 / Chapter 4.5 --- Ectopic expression of TLX suppresses cellular senescence in prostate cancer cells --- p.105 / Chapter 4.6 --- TLX suppresses cellular senescence via its direct transcriptional repression of p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹ gene --- p.110 / Chapter 4.7 --- TLX also suppresses cellular senescence via its transcriptional regulation of SIRT1 gene --- p.116 / Chapter CHAPTER 5 --- DISCUSSION --- p.121 / Chapter CHAPTER 6 --- SUMMARY --- p.131 / REFERENCES --- p.135

A partir de um melhor entendimento sobre a radiossensibilidade do câncer de próstata, com a redefinição de parâmetros radiobiológicos, atualmente os esquemas hipofracionados de próstata tornaram-se um dos principais desafios da radioterapia moderna. Associado a este conhecimento, o emprego de técnicas precisas de radioterapia possibilitaram a entrega de maiores doses por fração, com manutenção da toxicidade e melhor controle de qualidade dos planos de tratamento. Pretendemos avaliar a implementação da radioterapia tridimensional computadorizada (3D-CRT) ou intensidade modulada do feixe (IMRT) utilizando o regime de hipofracionamento moderado (70 Gy em 28 frações) em pacientes com neoplasia localizada da próstata na rotina do departamento de radioterapia do Hospital das Clínicas da FMRP-USP. Avaliação da viabilidade técnica e do impacto financeiro na utilização da radioterapia hipofracionada na instituição. / Evaluate the implementation of computerized three-dimensional radiation therapy (3D-CRT) or intensity modulated beam (IMRT) using moderate hypofractionation schedule (70 Gy in 28 fractions) in patients with localized prostate cancer (no metastasis lymph node or distant metastasis) in routine of department of radiotherapy of the Hospital of FMRP-USP. Assessment of technical feasibility and financial impact on the use of hypofractionated radiotherapy in the institution.

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Cheung Chun Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 192-227). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract (English) --- p.II / Abstract (Chinese) --- p.VI / Contents --- p.VIII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Nuclear hormone receptor: a general review --- p.1 / Chapter 1.1.1 --- Classification of nuclear hormone receptors --- p.1 / Chapter 1.1.2 --- Mechanism of action --- p.2 / Chapter 1.1.3 --- Domains structure and functions --- p.3 / Chapter 1.1.4 --- Orphan nuclear receptors --- p.4 / Chapter 1.2 --- Prostate gland - a male accessory reproductive organ --- p.6 / Chapter 1.2.1 --- "Anatomy, histology and physiology of the prostate gland" --- p.6 / Chapter 1.2.2 --- Endocrinology of the prostate gland --- p.8 / Chapter 1.2.3 --- Pathogenesis of the prostate gland --- p.8 / Chapter 1.3 --- The role of estrogen receptors in the prostate gland and prostate cancer --- p.9 / Chapter 1.3.1 --- Estrogens in male --- p.10 / Chapter 1.3.2 --- Effects of estrogens in the prostate gland --- p.11 / Chapter 1.3.3 --- Estrogen receptors - two isoforms --- p.13 / Chapter 1.3.4 --- Expression of ERs in the prostate gland --- p.14 / Chapter 1.3.5 --- Estrogen-modulated transgenic mice 226}0ؤ functional studies of ERs --- p.16 / Chapter 1.4 --- Estrogen receptor-related receptors: orphan receptors --- p.18 / Chapter 1.4.1 --- Estrogen receptor-related receptors: Three isoforms --- p.18 / Chapter 1.4.2 --- Expression of ERRs in different tissues --- p.20 / Chapter 1.4.3 --- Promoter binding and genes regulated by of ERRs --- p.22 / Chapter 1.4.4 --- Coregulators of ERRs --- p.24 / Chapter 1.4.5 --- Ligand of ERRs --- p.25 / Chapter 1.4.6 --- Functional roles of ERRs --- p.29 / Chapter 1.4.7 --- Cross talk between ERRs and ERs --- p.31 / Table 11 --- p.33 / Figure 1.1 - 15 --- p.35 / Chapter Chapter 2 --- Aims of the Study --- p.40 / Chapter Chapter 3 --- Methods and Materials / Chapter 3.1 --- "Expression patterns of ERRs and steroid hormone receptors in the human prostate cell lines, tumor xenografts and prostatic tissues" --- p.41 / Chapter 3.1.1 --- Human prostatic tissues --- p.41 / Chapter 3.1.2 --- Cell cultures --- p.41 / Chapter 3.1.3 --- Human prostate cancer xenografts --- p.42 / Chapter 3.1.4 --- Full length clones of ERR isoforms --- p.42 / Chapter 3.1.5 --- Reverse transcription-polymerase chain reactions (RT-PCR) --- p.43 / Chapter 3.1.6 --- Semi-quantitative RT-PCR analysis --- p.45 / Chapter 3.1.7 --- Southern blot analysis --- p.46 / Chapter 3.1.8 --- Generation and characterization of polyclonal antibodies --- p.49 / Chapter 3.1.9 --- Western blot analysis --- p.55 / Chapter 3.1.10 --- Immunohistochemistry --- p.56 / Chapter 3.2 --- Relationship of ERR and ER expressions in the prostatic cells --- p.57 / Chapter 3.2.1 --- "Expression vectors of ERRa, ERRγ and ERa" --- p.57 / Chapter 3.2.2 --- "Transient transfection of ERRa, ERRγ and ERa expression vectors in PC-3 cells" --- p.58 / Chapter 3.2.3 --- Semi-quantitative RT-PCR analysis --- p.59 / Chapter 3.3 --- Intracellular trafficking and transcriptional activity of GFP-tagged ERRs in the prostatic cells --- p.59 / Chapter 3.3.1 --- Construction of GFP-tagged ERR fusion plasmids --- p.59 / Chapter 3.3.2 --- Examination of transcriptional activity of GFP-tagged ERRs by luciferase assay --- p.61 / Chapter 3.3.3 --- Subcellular localization of GFP-tagged ERRs in the living prostatic cells --- p.63 / Chapter 3.3.4 --- Immunofluorescent staining GFP-tagged ERRs --- p.63 / Chapter 3.4 --- The Role of ERRs in the growth of the prostatic cells --- p.64 / Chapter 3.4.1 --- Evaluation the transfection efficiencies of PC-3 and PNT2 cells --- p.64 / Chapter 3.4.2 --- Cells proliferation assays in ERRs transient transfected prostatic cells --- p.66 / Chapter 3.4.3 --- Flow cytometry of ERRs transient transfected PC-3 cells --- p.66 / Chapter 3.4.4 --- RT-PCR of cell cycle-related genes in ERRs transient transfected PC-3 cells --- p.67 / Chapter 3.4.5 --- Generation of PNT2 and DU145 cells stably transfected with ERRy --- p.68 / Chapter 3.4.6 --- Cell proliferation assay of ERRy stable-transfected PNT2 and DU 145cells --- p.72 / Chapter 3. 4.7 --- Anchorage independent growth assay of ERRy stable-transfected PNT2 and DU 145 cells --- p.72 / Chapter 3.4.8 --- Flow cytometry of ERRγ stable-transfected PNT2 and DU145 cells --- p.74 / Chapter 3.4.9 --- RT-PCR of cell cycle-related genes in ERRγ stable-transfected PNT2 and DU 145 cells --- p.74 / Chapter 3.4.10 --- Western blot analysis of p21 in ERRγ stable-transfected PNT2 cells --- p.75 / Chapter 3.5 --- Statistical analysis --- p.75 / Table 3.1 - 3.2，Figure 3.1 - 35 --- p.76 / Chapter Chapter 4 --- Results / Chapter 4.1 --- "Expression patterns of ERRs and steroid hormone receptors in the human prostate cell lines, tumor xenografts and prostatic tissues" --- p.93 / Chapter 4.1.1 --- "mRNA expression patterns of ERR isoforms in the prostatic cell lines, prostate cancer xenografts and human prostatic tissues" --- p.93 / Chapter 4.1.2 --- mRNA expression patterns of steroid hormone receptors and prostatic differentiation markers in the prostatic cell lines and xenografts --- p.95 / Chapter 4.1.3 --- Characterization of antisera against human ERRs by ERR recombinant proteins --- p.97 / Chapter 4.1.4 --- Protein expression of ERR isoforms in the human prostatic cell lines --- p.98 / Chapter 4.1.5 --- "Immunolocalization of ERR isoforms in the normal, dysplastic and neoplastic prostates" --- p.98 / Chapter 4.2 --- Interrelationship of ERR and ER expression in PC-3 prostate cancer cells --- p.100 / Chapter 4.2.1 --- "Expressions of ERRγ, ERa and ERβ in the ERRa transient transfected PC-3 cells" --- p.100 / Chapter 4.2.2 --- Expression of ERRa in the ERRγ and ERa transient transfected PC-3 cells --- p.101 / Chapter 4.3 --- Intracellular trafficking and transcriptional activities of ERRs in the prostatic cells with fused green fluorescence protein 一 ERRs --- p.102 / Chapter 4.3.1 --- Trans activation of ERE response element 226}0ؤ driven reporter by ERR isoforms in the PC-3 cells in the presence or absence of serum --- p.102 / Chapter 4.3.2 --- Trans activation of SF-1 response element driven reporter by ERR isoforms in the PC-3 cells in the presence or absence of serum --- p.104 / Chapter 4.3.3 --- Subcellular localizations of three ERR isoforms in the PC-3 cells in the presence or absence of serum --- p.105 / Chapter 4.4 --- The role of ERRs in the growth of prostatic cells --- p.106 / Chapter 4.4.1 --- "The growth of ERRs transient transfected PC-3, PNT2 prostatic cells" --- p.107 / Chapter 4.4.2 --- Cell cycle analysis of ERRs transient transfected PC-3 cells --- p.108 / Chapter 4.4.3 --- Expression of cyclin-dependent kinase (CDK) inhibitors and p53 in the ERRs transient transfected PC-3 cells --- p.108 / Chapter 4.4.4 --- Establishment of ERRγ stable-transfected PNT2 and DU145 cells --- p.109 / Chapter 4.4.5 --- Transcriptional activation of ERE response element in ERRγ stable-transfected PNT2 cells --- p.111 / Chapter 4.4.6 --- Effect of over-expression of ERRγ on the growth of PNT2 and DU145 stable-transfected cells --- p.112 / Chapter 4.4.7 --- Efficiencies of colony formation of ERRγ stable-transfected PNT2 and DU145 cells --- p.113 / Chapter 4.4.8 --- Cell cycle analysis of ERRγ stable-transfected PNT2and DU 145 cells --- p.114 / Chapter 4.4.9 --- Expression of cell cycle-related genes in the ERRy stable-transfected PNT2 and DU 145 cells --- p.116 / Figure 4.1 - 4.38，Table 4.1 - 43 --- p.119 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- "Expression study in human prostatic cells, tumor xenografts" --- p.159 / Chapter 5.1.1 --- "Differential expression patterns of ERRs in prostatic cells, cancer xenografts and tissues" --- p.160 / Chapter 5.1.2 --- Co-localization of ERRs and ERβ in the human prostate --- p.166 / Chapter 5.1.3 --- Differential expression patterns of steroid hormone receptors and prostatic specific markers in prostatic cells and xenografts --- p.168 / Chapter 5.2 --- ERRα acts as a expression repressor of ERRγ and ERα in PC-3 cells --- p.173 / Chapter 5.3 --- ERRs are nuclear localized and constitutively active in PC-3 cells --- p.176 / Chapter 5.4 --- ERRs acts as the negative growth regulators in the prostatic cells --- p.179 / Chapter 5.4.1 --- Cell cycle control of mammalian cells --- p.180 / Chapter 5.4.2 --- The roles of AR and ERs in the cell cycle regulation --- p.181 / Chapter 5.4.3 --- Inhibition of cell proliferation in ERRs transient transfected PC-3 cells and ERRγ stable-transfected PNT2 and DU145 cells --- p.184 / Chapter 5.4.4 --- Inhibition of anchorage independent growth in ERRγ stable-transfected PNT2 and DU 145 cells --- p.188 / Chapter Chapter 6 --- Conclusion --- p.191 / Chapter Chapter 7 --- References --- p.192 / Chapter Chapter 8 --- Publications --- p.227

With ever increasing advancements in imaging, there is an increasing abundance of images being acquired in the clinical environment. However, this increase in information can be a burden as well as a blessing as it may require significant amounts of time to interpret the information contained in these images. Computer assisted evaluation is one way in which better use could be made of these images. This thesis presents the combination of texture analysis of images acquired during the treatment of cancer with machine learning in order to improve radiotherapy. The first application is to the prediction of radiation induced pneumonitis. In 13- 37% of cases, lung cancer patients treated with radiotherapy develop radiation induced lung disease, such as radiation induced pneumonitis. Three dimensional texture analysis, combined with patient-specific clinical parameters, were used to compute unique features. On radiotherapy planning CT data of 57 patients, (14 symptomatic, 43 asymptomatic), a Support Vector Machine (SVM) obtained an area under the receiver operator curve (AUROC) of 0.873 with sensitivity, specificity and accuracy of 92%, 72% and 87% respectively. Furthermore, it was demonstrated that a Decision Tree classifier was capable of a similar level of performance using sub-regions of the lung volume. The second application is related to prostate cancer identification. T2 MRI scans are used in the diagnosis of prostate cancer and in the identification of the primary cancer within the prostate gland. The manual identification of the cancer relies on the assessment of multiple scans and the integration of clinical information by a clinician. This requires considerable experience and time. As MRI becomes more integrated within the radiotherapy work flow and as adaptive radiotherapy (where the treatment plan is modified based on multi-modality image information acquired during or between RT fractions) develops it is timely to develop automatic segmentation techniques for reliably identifying cancerous regions. In this work a number of texture features were coupled with a supervised learning model for the automatic segmentation of the main cancerous focus in the prostate - the focal lesion. A mean AUROC of 0.713 was demonstrated with 10-fold stratified cross validation strategy on an aggregate data set. On a leave one case out basis a mean AUROC of 0.60 was achieved which resulted in a mean DICE coefficient of 0.710. These results showed that is was possible to delineate the focal lesion in the majority (11) of the 14 cases used in the study.

A partir de um melhor entendimento sobre a radiossensibilidade do câncer de próstata, com a redefinição de parâmetros radiobiológicos, atualmente os esquemas hipofracionados de próstata tornaram-se um dos principais desafios da radioterapia moderna. Associado a este conhecimento, o emprego de técnicas precisas de radioterapia possibilitaram a entrega de maiores doses por fração, com manutenção da toxicidade e melhor controle de qualidade dos planos de tratamento. Pretendemos avaliar a implementação da radioterapia tridimensional computadorizada (3D-CRT) ou intensidade modulada do feixe (IMRT) utilizando o regime de hipofracionamento moderado (70 Gy em 28 frações) em pacientes com neoplasia localizada da próstata na rotina do departamento de radioterapia do Hospital das Clínicas da FMRP-USP. Avaliação da viabilidade técnica e do impacto financeiro na utilização da radioterapia hipofracionada na instituição. / Evaluate the implementation of computerized three-dimensional radiation therapy (3D-CRT) or intensity modulated beam (IMRT) using moderate hypofractionation schedule (70 Gy in 28 fractions) in patients with localized prostate cancer (no metastasis lymph node or distant metastasis) in routine of department of radiotherapy of the Hospital of FMRP-USP. Assessment of technical feasibility and financial impact on the use of hypofractionated radiotherapy in the institution.