Prostate cancer is the most common cancer occurring in males. The identification of novel microRNAs (miRs) that contribute to tumor progression represents prospective treatment targets. miRs are small non-coding RNAs important in gene regulation with specific tissue expression patterns. Each miR is thought to affect the expression of hundreds of different RNA targets. Two putative oncomiRs, miR-155 and miR-146a, were shown to be differentially expressed in the human derived, prostate cell sublines M12 and F6. Quantification of endogenous miR expression showed high levels in the metastatic M12 cell line versus low in its weakly tumorigenic F6 variant. The restoration of miR expression to M12 levels was evaluated on F6 growth, morphology, and in vitro behavior. F6 plus miR-155 or miR-146a displayed increased growth, motility and invasiveness when compared to M12, with less organized structural morphology when grown embedded in matrigel. Altogether these results suggest that the overexpression of miRs 155 and 146a could contribute to tumor progression in vivo.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Master of Science / Department of Kinesiology / Steven W. Copp / Cancer patients have a reduced exercise capacity compared to age-matched healthy counterparts which contributes to premature fatigue. The reductions in exercise capacity are multifactorial and vary depending on the type of treatments and the specific cancer. Given that cancer treatments have been shown to impair cardiovascular and/or skeletal muscle function, it is difficult to determine if cancer itself reduces exercise capacity. We used a rat prostate tumor model to test the hypothesis that cancer independently reduces endurance exercise capacity. Methods: In male Copenhagen rats (COP/CrCrl), an initial treadmill test to exhaustion was used to determine endurance exercise capacity. Subsequently, the prostates of the rats were injected with either prostate carcinoma cells (R-3327 AT-1) in Matrigel (cancer: n = 9) or Matrigel only (sham: n = 7). Treadmill tests to exhaustion were repeated four and eight weeks post-surgery. Results: Time to exhaustion decreased over the course of the experimental protocol in both the sham and cancer groups. However, the overall reduction in time to exhaustion in the cancer group (-16.7 ± 1.9 min) was significantly greater (p = 0.038) than the sham group (-10.1 ± 2.2 min). Despite no differences in total body mass at the end of the experimental protocol, heart, left ventricle, and gastrocnemius muscle mass were significantly lower in the cancer group compared to the sham group (p < 0.05 for all). Moreover, within the cancer group heart and left ventricle mass, but not gastrocnemius mass, were significantly inversely correlated with prostate tumor mass. Conclusion: Endurance exercise capacity was reduced in rats with untreated prostate cancer to a greater extent than it was reduced in sham operated rats. Although multiple mechanisms likely contributed to the reduced exercise capacity, reductions in heart and gastrocnemius muscle mass likely played an important role.

by Kwong Joseph. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 142-164). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prostatic Secretory Proteins --- p.1 / Chapter 1.2 --- Rat Prostatic Secretory Proteins --- p.1 / Chapter 1.2.1 --- Prostatic Secretory Proteins in Ventral Prostate --- p.2 / Chapter 1.2.1.1 --- Prostatic Binding Protein (PBP) --- p.2 / Chapter 1.2.1.2 --- Androgen-Suppressed Proteins of Rat Ventral Prostate --- p.6 / Chapter 1.2.1.3 --- The 20-kDa Protein --- p.8 / Chapter 1.2.1.4 --- Spermine-Binding Proteins --- p.9 / Chapter 1.2.1.5 --- Prostatic Acid Phosphatase (PAP) --- p.10 / Chapter 1.2.2 --- Prostatic Secretory Proteins in Dorsal Prostate --- p.12 / Chapter 1.2.2.1 --- Dorsal Proteins I and II (DP I and DPII) --- p.12 / Chapter 1.2.2.2 --- Seminal Vesicle Secretion II (SVSII) --- p.14 / Chapter 1.2.2.3 --- Probasin --- p.16 / Chapter 1.2.3 --- Prostatic Secretory Proteins in Lateral Prostate --- p.18 / Chapter 1.3 --- Human Prostatic Secretion --- p.18 / Chapter 1.4 --- Human Prostatic Secretory Proteins --- p.18 / Chapter 1.4.1 --- Prostatic Acid Phosphatase (PAP) --- p.19 / Chapter 1.4.2 --- Prostate Specific Antigen (PSA) --- p.22 / Chapter 1.4.2.1 --- Molecular Biology of PSA --- p.22 / Chapter 1.4.2.2 --- Synthesis of PSA --- p.23 / Chapter 1.4.2.3 --- Kallikrein Gene Family --- p.23 / Chapter 1.4.2.4 --- Physiological Function of PSA --- p.24 / Chapter 1.4.2.5 --- PSA as an Immunohistochemical Marker --- p.25 / Chapter 1.4.2.6 --- PSA is not a Prostate-Specific Molecule --- p.26 / Chapter 1.4.3 --- Prostatic Secretory Protein of 94 Amino Acids (PSP94) --- p.27 / Chapter 1.4.3.1 --- Nucleotide Sequence of the PSP94 cDNA --- p.28 / Chapter 1.4.3.2 --- Amino Acid sequence of PSP94 --- p.28 / Chapter 1.4.3.3 --- Biological Properties of PSP94 --- p.29 / Chapter 1.4.3.4 --- Physiological Roles of PSP94 --- p.31 / Chapter 1.4.3.5 --- PSP94 and Its mRNA in Other Non-Prostatic Tissue --- p.31 / Chapter 1.4.3.6 --- PSP94 as a Tumor Marker of Prostate Cancer --- p.32 / Chapter 1.4.3.7 --- Homologous Proteins of PSP94 --- p.34 / Chapter 1.5 --- Aim of Study --- p.35 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and Supply of Noble Rat --- p.37 / Chapter 2.2 --- Chemicals --- p.37 / Chapter 2.3 --- Bilateral Ochidectomy of Animals --- p.37 / Chapter 2.4 --- Androgen Replacement --- p.38 / Chapter 2.5 --- Hormonal and Drug Treatments on Castrated Animals --- p.38 / Chapter 2.6 --- Induction of Prostatic Intraepithelial Neoplasia in Noble Rat Prostate Gland by Long-Term Treatment with Steroids --- p.39 / Chapter 2.6.1 --- Preparation of Steroid Hormone-Filled Silastic® Tubings --- p.39 / Chapter 2.6.2 --- Surgical Implantation of Silastic® Tubings --- p.39 / Chapter 2.6.3 --- Protocols of Hormonal Treatments --- p.40 / Chapter 2.7 --- Androgen-Dependent Rat Dunning Prostatic Adenocarcinoma --- p.40 / Chapter 2.8 --- Androgen-Independent Prostatic Carcinoma Line (AIT) of Noble Rat --- p.41 / Chapter 2.9 --- Plasmids --- p.41 / Chapter 2.10 --- Restriction Enzyme Digestions of pLvB10 and cM-403 --- p.42 / Chapter 2.11 --- Amplification of Rat SVSII cDNA Fragment by RT-PCR and Subcloning --- p.42 / Chapter 2.12 --- Purification of DNA Fragment from Agarose Gel --- p.43 / Chapter 2.13 --- Subcloning of DNA into Vector --- p.44 / Chapter 2.14 --- Tissue Preparation for In-situ Hybridization --- p.47 / Chapter 2.15 --- Synthesis of Digoxigenin (DIG)-Labeled RNA Probe --- p.47 / Chapter 2.16 --- In-situ Hybridization --- p.48 / Chapter 2.17 --- Total RNA Extraction --- p.50 / Chapter 2.18 --- Northern Blotting Analysis --- p.51 / Chapter 2.19 --- Primers and Cycling Conditions --- p.53 / Chapter 2.20 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.54 / Chapter 2.21 --- Southern Blotting Analysis --- p.56 / Chapter 2.21.1 --- Southern Blotting --- p.56 / Chapter 2.21.2 --- Preparation of DIG-dUTP Labeled Rat PSP94 cDNA Probe --- p.56 / Chapter 2.21.3 --- Hybridization --- p.57 / Chapter 2.22 --- Restriction Mapping --- p.58 / Chapter 2.23 --- Semi-Quantitative RT-PCR --- p.59 / Chapter 2.24 --- Statistical Analysis --- p.59 / Chapter 2.25 --- "Protein Extraction, SDS-PAGE and Western Blotting Analysis" --- p.60 / Chapter 2.26 --- Immunohistochemistry --- p.63 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Subcloning of DNAs into Vector --- p.65 / Chapter 3.1.1 --- Subcloning of 18s Ribosomal RNA cDNA Fragment --- p.65 / Chapter 3.1.2 --- Subcloning of Probasin cDNA Fragment --- p.66 / Chapter 3.1.3 --- Subcloning of SVSII cDNA Fragment --- p.66 / Chapter 3.1.4 --- Restriction Enzyme Mapping for PCR Product of SVSII --- p.67 / Chapter 3.2 --- Detection of mRNA and Protein Expression of PSP94 in Normal Rat Prostates --- p.68 / Chapter 3.2.1 --- In-situ Hybridization --- p.68 / Chapter 3.2.2 --- Northern Blotting --- p.68 / Chapter 3.2.3 --- RT-PCR Amplification --- p.69 / Chapter 3.2.4 --- Immunohistochemistry --- p.69 / Chapter 3.2.5 --- Western Blotting --- p.70 / Chapter 3.3 --- Detection of mRNA Expression of Probasin and SVSII in Normal Rat Prostates --- p.71 / Chapter 3.3.1 --- In-situ Hybridization --- p.71 / Chapter 3.3.2 --- RT-PCR Amplification --- p.71 / Chapter 3.4 --- "Androgen Regulation of PSP94,Probasin and SVSII mRNA Expression" --- p.72 / Chapter 3.4.1 --- In-situ Hybridization --- p.72 / Chapter 3.4.2 --- "Relative Expression Levels of PSP94, Probasin and SVSII mRNA in Normal, Castrated and Androgen Replaced Rat Lateral Prostates as Measured by a Semiquantitative RT-PCR Method" --- p.73 / Chapter 3.4.2.1 --- Determination of Exponential Range of PCR --- p.73 / Chapter 3.4.2.2 --- Semi-Quantitative RT-PCR --- p.74 / Chapter 3.4.3 --- Western Blot Analysis --- p.75 / Chapter 3.5 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Prostates" --- p.76 / Chapter 3.5.1 --- Semi-Quantitative RT-PCR --- p.76 / Chapter 3.5.2 --- Western Blot Analysis --- p.77 / Chapter 3.6 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.78 / Chapter 3.6.1 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in T+E2-Induced Prostatic Intraepithelial Neoplasia (PIN) of the Lateral Prostate of Noble Rats by In-situ Hybridization" --- p.78 / Chapter 3.6.2 --- "Detection of PSP94,Probasin and SVSII mRNA Expression in Dunning Tumor and AIT Prostatic Tumor" --- p.79 / Chapter 3.6.2.1 --- In-situ Hybridization --- p.79 / Chapter 3.6.2.2 --- RT-PCR Amplification --- p.79 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Specific Expression of PSP94 in the Lateral Lobe of Rat Prostate --- p.114 / Chapter 4.2 --- Androgen Regulation of PSP94 --- p.118 / Chapter 4.2.1 --- Molecular Mechanism of Androgen Action --- p.118 / Chapter 4.2.2 --- Androgen Regulation of PSP94 in Rat Lateral Prostate --- p.121 / Chapter 4.3 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Lateral Prostate" --- p.126 / Chapter 4.4 --- "Detection of PSP94, Probasin, SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.133 / Chapter 4.5 --- Gene Therapy --- p.139 / Chapter Chapter 5 --- Conclusions --- p.141 / References --- p.142 / Appendixes --- p.165

Replication-selective oncolytic adenoviruses represent a promising anticancer approach with proven efficacy in cancer cell lines and tumour xenografts in vivo. Anti-tumour efficacy, both in preclinical studies and clinical trials, was significantly improved in combination with chemotherapeutics in numerous cancers, including prostate cancer. It has been established that expression of the viral E1A gene is essential for the enhancement of cell killing in combination with cytotoxic drugs. The overall goal of this project is to identify specific E1A gene regions involved in the sensitization to the cytotoxic drugs mitoxantrone and docetaxel, the current standard of care for late stage prostate cancers, to enable the development of improved anti-cancer therapies. Specific regions in the E1A proteins bind to numerous cellular factors to regulate the host cell function and the viral life cycle, including the p300, p400 and pRb family proteins. This work was aimed at determining the mechanisms involved in the synergistic cell killing in prostate cancer cells in response to the combination of the replication-selective (oncolytic) mutant AdΔΔ with cytotoxic drugs. Previous findings suggested an enhancement of drug-induced apoptosis. I found that the small E1A12S protein, unable to induce viral replication, is sufficient to sensitize the prostate cancer cells, 22Rv-1 (AR+), and PC-3 and DU145 (AR-), to drugs. The non-replicating AdE1A12S-mutant AdE1A1104 (defective in p300-binding) could not sensitize the cells while mutants with intact E1A-p300 binding (AdE1A12S, AdE1A1102, AdE1A1108) and defective in p400- (AdE1A1102) or pRb-binding (AdE1A1108) potently sensitized all tested cell lines. In fact, all mutants except AdE1A1104 potently synergised with mitoxantrone and docetaxel to kill the prostate cancer cells. When comparing the non-replicating E1A12S mutants with the corresponding replicating E1A-deletion mutants (expressing E1A12S and 13S) synergy was demonstrated with all replicating mutants except dl1104, which caused an additive effect with mitoxantrone. We hypothesised that the synergistic cell killing is the result of pathway convergence through E1A-p300 and mitoxantrone-activated DNA-damage/apoptosis events. To address this I employed an extensive miRNA array screen to identify potential pathways. Several miRNAs were found to be differentially regulated in response to the combination of AdE1A12S with mitoxantrone compared to each single agent treatment. The majority of these miRNAs are reported to be part of cell death and survival pathways (e.g. apoptosis and autophagy) and to be differentially regulated in prostate cancer. To further investigate the role of these pathways, I determined changes in expression levels of key proteins that had previously been suggested to be targeted by the identified miRNAs, thereby preventing translation of the respective mRNAs. The greatest changes in protein levels in response to AdE1A12S and mitoxantrone were observed for Bcl-2, p-Akt, LC3BII and p62. Finally, I verified similar mechanisms of action when the oncolytic AdΔΔ was combined with mitoxantrone under synergistic conditions. These findings will direct future investigations aimed at dissecting the mechanisms of action for virus-induced sensitization to cytotoxic drugs and may aid in the development of improved therapies for prostate cancer by design of novel oncolytic mutants and combination strategies and/or identification of targets for small molecules inhibitors.

Androgen receptor (AR) cell signalling is active in most castration-resistant prostate cancer (PCa) tumours and suppression is hypothesized to impede cell proliferation. Hey1, a corepressor of AR is being investigated as a therapeutic transgene for late-stage PCa. A replication-defective recombinant adenovirus deleted for E1 and E3 and expressing Hey1 under a CMV promoter was constructed (Ad5Hey1). A dual luciferase reporter system demonstrated that Ad5Hey1 repressed AR activity in a dose dependent manner in miboleronestimulated 22Rv1 cells. Ad5Hey1 was cytotoxic in both AR-positive 22Rv1 and LNCaP and AR-negative DU145 cells. The doses required to kill 50% of cells (EC50) were comparable to those of AdE1A12S expressing the cytotoxic E1A12S gene from an identical vector. The mechanisms of Ad5Hey1-induced cell killing were investigated in 22Rv1 and DU145 cells. Using RNA interference towards AR or p53 in 22Rv1 cells we concluded both proteins were required for optimal cell killing by Ad5Hey1. In DU145 cells, with non-functional p53, Ad5Hey1 decreased levels of phospho- STAT3 and total STAT3 suggesting Ad5Hey1 might inhibit STAT3 signalling while the JAK1/2 inhibitor, AZD1480 was ineffective at sensitising DU145 cells to Ad5Hey1. Preliminary data therefore suggests Ad5Hey1 may interfere with JAK/STAT signalling in these cells. Cell-killing efficacy with Ad5Hey1 in combination with cytotoxic drugs currently used in the clinic for the treatment of late-stage PCa, mitoxantrone and docetaxel, resulted in a synergistic enhancement of cell death in 22Rv1 and DU145 cells. LNCaP cells were also sensitised to the drugs. Characterisation of the mode of cell killing demonstrated augmented mitochondrial membrane depolarisation and caspase-3 activation when combined with docetaxel in all cell lines and with mitoxantrone in 22Rv1 and LNCaP cells, typical of apoptotic death. In DU145 cells, the combination of Ad5Hey1 with mitoxantrone decreased the proportion of apoptotic cells suggesting cells are dying by alternative cell death mechanisms. In this thesis I have demonstrated that Ad5Hey1 potently eliminates PCa cells both in the presence and absence of functional AR or p53, and that cell killing is 6 improved in combination with cytotoxic drugs. I demonstrate that the mechanisms by which Ad5Hey1 acts as a cell death enhancer is mainly through cooperation with drugs on apoptotic pathways while other factors such as inhibition of survival are also involved. In conclusion, these data suggest that it is feasible to develop a future replication-selective adenovirus expressing Hey1 as a cytotoxic transgene to improve antitumour efficacy in vitro and in vivo, especially in combination with apoptosis-inducing drugs.

Orientador: Silvia Regina Rogatto / Banca: Spencer L. M. Payão / Banca: Carla Rosemberg / Banca: José Carlos de S. Trindade / Banca: Maria Aparecida M. Rodrigues / Resumo: O câncer de próstata (CaP) é a neoplasia mais comumente diagnosticada entre homens no ocidente. Embora tratamentos efetivos para a doença localizada estejam disponíveis atualmente, não há terapia curativa para tumores metastáticos. Além disso, os marcadores diagnósticos utilizados na clínica não conseguem discriminar totalmente a evolução diferencial da doença. Desta forma, o conhecimento das diferenças biológicas entre tumores primários confinados ao órgão e metástases é essencial para o desenvolvimento de novos marcadores e identificação de alvos terapêuticos. Neste estudo a análise baseada na metodologia de HR-CGH cromossômico foi realizada para identificar alterações de ganhos e perdas genômicas em três grupos de amostras: o grupo I, que compreende amostras pareadas de tumor primário e respectivas metástases (11 casos); o grupo II, constituído de pacientes que apresentaram seguimento clínico favorável por mais de 10 anos (5 casos); e o grupo III, constituído por diferentes biópsias do mesmo paciente (5 pacientes com 2 biópsias cada). As amostras foram microdissecadas (amostras a fresco: a partir de lâminas de referência; em blocos de parafina: a laser) e após a obtenção de DNA foram amplificadas (amostras de arquivo: PCR-SCOMP) ou marcadas por nick-translation para a realização de HR-CGH. Os resultados de HR-CGH foram comparados com os dados obtidos da análise de CGH-array num subgrupo de amostras e revelaram concordâncias significativas. Os resultados obtidos na presente investigação revelaram perdas dos cromossomos 1p, 2, 3q, 4p, 5q, 7, 8, 9q, 10q, 11q, 12q, 14q, 15q, 16q, 17q, 18q, 19, 20q e 22q em 80% dos casos avaliados. Além disso, perdas em 17q11.2-25, por exemplo, foram detectadas exclusivamente nos tumores do grupo I e nas suas metástases, e não nos tumores do grupo II, sugerindo que esta alteração deve ser importante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous malignancy and the second leading cause of cancer mortality in men from Occident. Although effective treatments for the localized disease are available, there is no efficient therapy for metastatic tumors. Additionally, clinical diagnostic markers are not able to completely discriminate the differential evolution of the disease. The knowledge of biological differences between localized primary tumors and metastasis can establish new molecular markers and therapeutic targets. In this study, an analysis based on HR-CGH methodology was performed to identify imbalances genomic in three groups of samples: group I, paired samples of primary tumors and its metastasis (11 cases); group II, patients that exhibited favorable follow-up over 10 years (5 cases); and group III, different biopsies from the same patient (5 patients with 2 biopsies each). The tumor samples were submitted to microdissection procedures (fresh samples: from reference slides; paraffin embedded samples: laser), DNA extracted and amplified (archive sample: PCR-SCOMP) or labeled by nick-translation to HR-CGH. The HRCGH results were compared with data obtained from CGH-array analysis of a subgroup of samples and revealed significant concordances. In the present investigation, there were observed losses on chromosomes 1p, 2, 3q, 4p, 5q, 7, 8, 9q, 10q, 11q, 12q, 14q, 15q, 16q, 17q, 18q, 19, 20q and 22q in 80% of the cases. Losses in 17q11.2-25, for instance, were detected exclusively in tumor from group I and its metastasis, but were not found in tumors from group II, suggesting that this alteration must be important in the progression of the disease. Five genes were selected after the comparison between the HR-CGH and CGH-array data. The tumor suppressor genes ARID1A, MTSS1, NME1 and S100A4 and TOP2A (oncogenes) were evaluated by quantitative real time... (Complete abstract click electronic access below) / Doutor

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Lui, Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-171). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.v / Acknowledgements --- p.vii / Abbreviations --- p.ix / Table of Content --- p.x / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Overview and Endocrinology of hormones and hormone receptors --- p.1 / Chapter 1.2 --- Hormone receptors: membrane bounded receptors --- p.3 / Chapter 1.3 --- Hormone receptors: steroid nuclear receptors --- p.4 / Chapter 1.4 --- "Estrogen, estrogen receptor alpha and beta (ERa, ERβ) and prostate gland" --- p.6 / Chapter 1.5 --- Orphan nuclear receptors --- p.10 / Chapter 1.6 --- The first orphan receptors identified-estrogen receptor related receptors --- p.12 / Chapter 1.6.1 --- Estrogen receptor related receptor alpha (ERRα) --- p.13 / Chapter 1.6.2 --- Estrogen receptor related receptor alpha (ERRβ) --- p.17 / Chapter 1.6.3 --- Estrogen receptor related receptor alpha (ERRγ) --- p.19 / Chapter 1.7 --- Aim of study --- p.21 / Figure 1.1 Mechanism of activation of classical nuclear receptor by ligand --- p.23 / Figure 1.2 Distribution of ERa and ERβ in human body --- p.24 / Chapter Chapter 2. --- Methods and Materials / Chapter 2.1 --- Origin and supply of Noble rats --- p.25 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Cell lines and culture media --- p.26 / Chapter 2.2.2 --- Cell culture onto cover slips for immunohistochemistry --- p.27 / Chapter 2.3 --- RNA preparation / Chapter 2.3.1 --- Total RNA extraction --- p.27 / Chapter 2.3.2 --- mRNA extraction by Oligote´xёØ procedure --- p.29 / Chapter 2.3.3 --- mRNA extraction by Fast Track 2.0 procedure --- p.30 / Chapter 2.4 --- Molecular cloning by Rapid Amplification of cDNA Ends (RACE) / Chapter 2.4.1 --- Molecular cloning of rERRα --- p.31 / Chapter 2.4.2 --- Molecular cloning of rERRβ --- p.36 / Chapter 2.4.3 --- Molecular cloning of rERRγ --- p.42 / Chapter 2.5 --- Molecular cloning into pCRII TOPO cloning vector --- p.47 / Chapter 2.6 --- Sequencing analysis of DNA sequence by dRodamine® or BigDye® --- p.47 / Chapter 2.7 --- DNA sequence analysis --- p.49 / Chapter 2.8 --- Reverse transcription and RT-PCR --- p.49 / Chapter 2.9 --- Southern blotting analysis / Chapter 2.9.1 --- Preparation of DNA blot membrane --- p.51 / Chapter 2.9.2 --- Purification of DNA fragment from agarose gel for DIG-DNA labeling --- p.52 / Chapter 2.9.3 --- Preparation of the DIG-labeled DNA probe --- p.53 / Chapter 2.9.4 --- Membrane hybridization and colorimetric detection --- p.53 / Chapter 2.10 --- In-situ hybridization histochemistry / Chapter 2.10.1 --- Linearization of DNA plasmid --- p.55 / Chapter 2.10.2 --- Synthesis of riboprobe --- p.56 / Chapter 2.10.3 --- Hybridization and detection --- p.56 / Chapter 2.11 --- Western blotting analysis / Chapter 2.11.1 --- Protein extraction --- p.59 / Chapter 2.11.2 --- Casting of SDS-PAGE electrophoresis --- p.59 / Chapter 2.11.3 --- Polyacrylamide gel electrophoresis --- p.61 / Chapter 2.11.4 --- Protein blotting analysis --- p.61 / Chapter 2.12.1 --- Immunohistochemistry / Chapter 2.12.1 --- Histological preparation --- p.63 / Chapter 2.12.2 --- Immunohistochemistry --- p.64 / Table 1. List of culture media --- p.66 / Table 2. Primer sequences for RACE-PCR --- p.67 / Table 3. PCR conditions for RT-PCR --- p.68 / Table 4. Primer sequences for RT-PCR --- p.68 / Table 5. Reagent mixtures for linearization of the plasmid DNA --- p.69 / Table 6. Riboprobe synthesis by in-vitro transcription --- p.70 / Chapter Chapter 3. --- Results / Chapter 3.1 --- Cloning of full-length cDNA of rERRs by RACE-PCR --- p.71 / Chapter 3.2 --- Cloning of full-length cDNA of rERRα from rat ovary cDNA library --- p.72 / Chapter 3.3 --- Cloning of full-length cDNA of rERRβ from rat ventral prostate --- p.76 / Chapter 3.4 --- Cloning of full-length cDNA of rERRγ from rat prostate --- p.80 / Chapter 3.5 --- Expression distribution of ERRs detected by RT-PCR --- p.83 / Chapter 3.6 --- mRNA expression of ERRs detected by in-situ hybridization --- p.86 / Chapter 3.7 --- Protein expression of ERRa and ERRγ detected by western blotting --- p.87 / Chapter 3.8 --- Expression of ERRa and ERRγ detected by immunohistochemistry --- p.88 / Figure 3.1 Full-length DNA sequence of rERRα --- p.92 / Figure 3.2 Predicted amino acid sequence of rERRα --- p.93 / "Figure 3.3 DNA sequence alignment of rat, mouse and human ERRα" --- p.94 / "Figure 3.4 Amino acid sequence alignment analysis of rat, mouse and human ERRα" --- p.95 / Figure 3.5 Full-length DNA sequence of rERRβ --- p.96 / Figure 3.6 Predicted amino acid sequence of rERRβ --- p.97 / "Figure 3.7 DNA sequence alignment of rat, mouse and human ERRβ" --- p.98 / "Figure 3.8 Amino acid sequence alignment analysis of rat, mouse and human ERRβ" --- p.99 / Figure 3.9 Full-length DNA sequence of rERRγ --- p.100 / Figure 3.10 Predicted amino acid sequence of rERRγ --- p.101 / "Figure 3.11 DNA sequence alignment of rat, mouse and human ERRγ" --- p.102 / "Figure 3.12 Amino acid sequence alignment analysis of rat, mouse and human ERRγ" --- p.103 / Figure 3.13 Restriction enzyme cutting of full-length plasmids --- p.104 / Figure 3.14 Expression pattern of rERRα in male sex accessory sex glands by RT-PCR --- p.105 / Figure 3.15 Expression pattern of rERRα in urinary system and female sex organs by RT-PCR --- p.106 / Figure 3.16 Tissue expression of rERRα by RT-PCR --- p.107 / Figure 3.17 In-situ hybridization of ERRα in ovary --- p.108 / Figure 3.18 Western blotting of ERRα --- p.109 / Figure 3.19 Immunohistochemistry of ERRα in ovary --- p.110 / Figure 3.20 Expression pattern of rERRβ in male sex accessory sex glands by RT-PCR --- p.111 / Figure 3.21 Expression pattern of rERRβ in urinary system and female sex organs by RT-PCR --- p.112 / Figure 3.22 Tissue expression of rERRβ by RT-PCR --- p.113 / Figure 3.23 In-situ hybridization of ERRβ in rat prostate --- p.114 / Figure 3.24 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.115 / Figure 3.25 Expression pattern of rERRγ in male sex accessory sex glands by RT-PCR --- p.116 / Figure 3.26 Expression pattern of rERRy in urinary system and female sex organs by RT-PCR --- p.117 / Figure 3.27 Tissue expression of rERRγ by RT-PCR --- p.118 / Figure 3.28 Expression pattern of rERRγ in different prostatic cancer cell lines and xenografts by RT-PCR --- p.119 / Figure 3.29 In-situ hybridization of ERRγ in rat prostate --- p.120 / Figure 3.30 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.121 / Figure 3.31 Western blotting of ERRγ --- p.122 / Figure 3.32 Immunohistochemistry of ERRγ in ERRy-transfected MCF-7 cells --- p.123 / Figure 3.33 Immunohistochemistry of ERRγ in ventral prostate of rat --- p.124 / Figure 3.34 Immunohistochemistry of ERRγ in lateral prostate of rat --- p.125 / Figure 3.35 Immunohistochemistry of ERRγ in dorsal prostate of rat --- p.126 / Figure 3.36 Immunohistochemistry of ERRγ in testis of rat --- p.127 / Figure 3.37 Immunohistochemistry of ERRγ in epididymis of rat --- p.128 / Figure 3.38 Immunohistochemistry of ERRγ in brown adipose tissues of rat --- p.129 / Figure 3.39 Immunohistochemistry of ERRγ in brain of rat --- p.130 / Figure 3.40 Immunohistochemistry of ERRγ in brain of rat --- p.131 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- Sequence analysis of the full-length cDNA sequences of the rat estrogen receptor-related receptors (ERRs) --- p.132 / Chapter 4.2 --- Ligand independence and constitutive self-activation of estrogen receptor-related receptors --- p.133 / Chapter 4.3 --- Board expression pattern of estrogen receptor-related receptors --- p.138 / Chapter 4.3.1 --- Board expression pattern of estrogen receptor-related receptor alpha --- p.138 / Chapter 4.3.2 --- Board expression pattern of estrogen receptor-related receptor beta --- p.140 / Chapter 4.3.3 --- Board expression pattern of estrogen receptor-related receptor gamma --- p.141 / Chapter 4.4 --- Expression of ERRs in the prostate gland --- p.143 / Chapter 4.5 --- Expression of ERRs in the prostatic cell lines and cancer xenografts --- p.147 / Chapter 4.6 --- Expression of ERRs in the ERRγ-transfected MCF-7 cells --- p.149 / Chapter 4.7 --- Expression of ERRs in the testis and epididymis --- p.149 / Chapter 4.8 --- Expression of ERRs in the adipose tissue --- p.150 / Chapter 4.9 --- Expression of ERRs in the ovary --- p.151 / Chapter 4.10 --- Expression of ERRs in the brain --- p.153 / Figure 5.1 Map of full-length clone of rERRα --- p.155 / Figure 5.2 Map of full-length clone of rERRβ --- p.156 / Figure 5.3 Map of full-length clone of rERRα --- p.157 / Figure 5.4 Comparison of the homology of amino acid sequences amongst ERs and ERRs --- p.158 / Figure 5.5 Phylogeny tree of nuclear receptors --- p.159 / Figure 5.6 Relationship of different prostatic cell lines and xenografts --- p.160 / Chapter Chapter 5. --- Summary --- p.161 / References --- p.163-171