Dissertations / Theses on the topic 'Orphan nuclear receptor NURR1'

In this work, we characterized the novel mouse orphan receptor, Estrogen-Related Receptor gamma (ERRgamma). ERRgamma is closely related to the other ERR family members. ERRalpha and ERRbeta. ERRgamma binds to both an ERR response element (ERRE) and an estrogen response element (ERE) and displays constitutive transcriptional activity which is enhanced when ERRgamma is fused to the VP16 activation domain. ERRalpha, ERRbeta, and ERRgamma heterodimerize both on an ERE and in solution in absence of DNA. This may allow for a more versatile mode of gene regulation. Furthermore, the ligand-binding domain (LBD) of ERRgamma interacts with coactivators and corepressors in vitro. Expression of ERRgamma in mouse embryogenesis starts at 7.5 until 10.5 day stage and is observed in the ventricular zones of the brain and the spinal cord. This suggests a role for ERRgamma in the developing nervous system. Although ERRgamma is more closely related to ERRbeta than ERRalpha, the expression pattern of ERRgamma is similar to ERRalpha and distinct to ERRbeta, suggesting a unique role for ERRgamma in development.

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing ptrifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-CpPhC6H5) substituents have been identified as a new class of peroxisome proliferatoractivated receptor γ (PPARγ) agonists that exhibit antitumorigenic activity. In this study, the PPARγ-active compounds decreased HT-29, HCT-15, RKO, HCT116 and SW480 colon cancer cell survival and KU7 and 253JB-V33 bladder cancer cell survival. In HT- 29, HCT-15, SW480 and KU7 cells, the PPARγ agonists induced caveolin-1 expression and this induction was significantly downregulated after cotreatment with the PPARγ antagonist GW9662. Since overexpression of caveolin-1 is known to suppress cancer cell and tumor growth, the growth inhibitory effects of the DIM compounds in these cell lines are associated with PPARγ-dependent induction of caveolins. These PPARγ-active compounds did not induce caveolin-1 in HCT-116 cells. However, these compounds induced NSAID-activated gene-1 (NAG-1) and apoptosis in this cell line. This represents a novel receptor-independent pathway for C-DIM-induced growth inhibition and apoptosis in colon cancer cells. In SW480 colon cancer cells 2.5-7.5 μM C-DIMs induced caveolin-1 whereas high concentrations (10 μM) induced pro-apoptotic NAG-1 expression. In athymic nude mice bearing SW480 cell xenografts DIM-C-pPhC6H5 inhibited tumor growth and immunohistochemical staining of the tumors show induction of apoptosis and NAG-1 expression. Thus, the PPARγ-active compounds induce both receptor-dependent and-independent responses in SW480 cells which are separable over a narrow range of concentrations and this dual mechanism of action enhances their antiproliferative and anticancer activities. Similar results were obtained for another structural class of PPARγ agonists namely 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and the corresponding methyl (CDDO-Me) and imidazole (CDDO-Im) esters. Structure-activity studies show that 1,1-bis(3'-indolyl)-1-(psubstitutedphenyl) methanes containing p-trifluoromethyl (DIM-C-pPhCF3), hydrogen (DIM-C-pPh) and p-methoxy (DIM-C-pPhOCH3) substituents activate Nur77 and induce apoptosis in pancreatic, prostate, and breast cancer cell lines. Nur77 agonists activate the nuclear receptor, and downstream responses include decreased cell survival, induction of cell death pathways including tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and PARP cleavage. Nur77 agonists also inhibit tumor growth in vivo in athymic nude mice bearing Panc-28 cell xenografts.

The neuron-derived orphan receptor 1 (NOR1) belongs to the NR4A nuclear receptor subfamily. As an immediate early response gene, NOR1 is rapidly induced by a broad spectrum of physiological and pathological signals. Functional studies demonstrate NOR1 as a constitutively active ligand-independent nuclear receptor whose transcriptional activity is dependent on both expression level and posttranslational modifications. To date, an increasing number of studies have demonstrated a pivotal role of NOR1 in the transcriptional control of metabolism and the development of cardiovascular diseases. In this dissertation, we demonstrate NOR1 expression in endothelial cells and sub-endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through an NF-κB-dependent signaling pathway. Functional studies reveal that NOR1 increases monocyte adhesion by inducing the expression of adhesion molecules VCAM-1 and ICAM-1 in endothelial cells. Transient transfection and chromatin immunoprecipitation assays identify VCAM-1 as a bona fide NOR1 target gene in endothelial cells. Finally, we demonstrate that NOR1-deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE-/- mice by decreasing the macrophage content of the lesion. In smooth muscle cells (SMC), NOR1 was previously established as a cAMP response element binding protein (CREB) target gene in response to platelet-derived growth factor (PDGF) stimulation. CREB phosphorylation and subsequent binding of phosphorylated CREB to the NOR1 promoter play a critical role in inducing NOR1 expression. In this dissertation, we further demonstrate that histone deacetylase (HDAC) inhibition potentiates and sustains PDGF-induced NOR1 mRNA and protein expression in SMC. This augmented NOR1 expression is associated with increased phosphorylation of CREB, recruitment of phosphorylated CREB to the NOR1 promoter, and trans-activation of the NOR1 promoter. Additionally, HDAC inhibition also increases NOR1 protein half-life in SMC. Collectively, these findings identify a novel pathway in endothelial cells underlying monocyte adhesion and expand our knowledge of the epigenetic mechanisms orchestrating NOR1 expression in SMC. Finally, we establish a previously unrecognized atherogenic role of NOR1 in positively regulating monocyte recruitment to the vascular wall.

The orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a major role in transcription regulation of metabolic genes. Its role in glycolysis regulation is well known. However, we ignore yet if it could be implicated in a signaling pathway connected to glycolysis, the Hexosamine Biosynthetic Pathway (HBP). The HBP is an important energy and nutrient-sensing pathway, which modulates O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification. We demonstrate here that ERRα is involved in transcriptional regulation of HBP genes. ERRα can be localized to promoters of several HBP genes, such as Ogt and Oga. Both encode two enzymes that directly modulate the O-GlcNAc cycling. Moreover, ERRα activates HBP genes transcription in collaboration with its coactivator PGC-1α. In ERRα-null mice, the HBP genes expression is downregulated. However, we can observe more O-GlcNAcylated proteins in absence of ERRα. This was demonstrated more specifically on the mitochondrial chaperone Mortalin. Mortalin is encoded by Hspa9, and we show that this gene is an ERRα target. Moreover, Mortalin is much more O-GlcNAcylated in absence of the ERRα. This suggests that in addition to Mortalin transcriptional regulation, ERRα is involved in Mortalin post-translational modification through regulation of the HBP.<br>Le récepteur nucléaire orphelin relié à l'estrogène (ERRα, NR3Β1) joue un rôle majeur dans la régulation de gènes métaboliques. Son rôle dans la régulation de la glycolyse est très bien connu. Cependant, nous ignorons encore s'il pourrait être impliqué dans la régulation d'une voie de signalisation directement liée à la glycolyse, la voie de signalisation des hexosamines (HBP). La HBP est une importante voie de signalisation servant de détecteur d'énergie et de nutriments et régulant la modification post-traductionelle O-lié N-acétylglucosamine (O-GlcNAc). Nous démontrons qu'ERRα est impliqué dans la régulation des gènes de HBP. ERRα se lie aux promoteurs de plusieurs gènes de HBP, comme Ogt et Oga, codant pour deux enzymes qui régulent directement le cycle des O-GlcNAc. De plus, ERRα active la transcription des gènes de HBP en collaboration avec son coactivateur PGC-1α. Dans les souris déficientes pour le gène ERRα, l'expression des gènes de HBP se trouve réduite. Cependant, nous pouvons observer plus de protéines O-GlcNAcylées en absence d'ERRα. Ceci a été démontré plus spécifiquement sur la chaperone mitochondriale Mortalin. Mortalin est codée par le gène Hspa9 et nous démontrons que ce gène est une cible d'ERRα. De plus, Mortalin est beaucoup plus O-GlcNAcylée en absence d'ERRα. Ceci suggère qu'en plus de réguler la transcription de Mortalin, ERRα est impliqué dans la régulation des modifications post-traductionelles de Mortalin, en régulant la voie de signalisation des hexosamines.

Nuclear receptors are transcription factors that regulate gene expression in response to small lipophilic molecules, including steroid hormones, thyroid hormone, and vitamin A and D metabolites. RORalpha is an orphan nuclear receptor that was initially cloned based on its similarities with the retinoic acid receptor. The term orphan was coined in reference to a nuclear receptor whose discovery has preceded that of its ligand. Genetic ablation of the Rora gene in mice leads to severe cerebellar ataxia, known as the staggerer phenotype. RORalpha regulates a myriad of genes involved in cellular differentiation, including myogenesis and adipogenesis, as well as various metabolic pathways. Nuclear receptors control the expression of their target genes by binding to short DNA sequences, referred to as hormone response elements, as monomers, homodimers, or heterodimers with the common partner RXR. RORalpha is strictly a monomeric DNA binding protein, lacking key molecular determinants in its DNA binding domain essential for cooperative homodimer formation. This orphan receptor is a potent transcriptional activator, whose activity is dependent on an endogenous ligand and is controlled by the concerted action of coactivator and corepressor proteins. The product of the thyroid hormone-regulated mammalian gene hairless (Hr) is a strong repressor of RORalpha transcriptional activity. In contrast to other corepressor-nuclear receptor interactions, Hr utilizes LxxLL motifs to mediate interaction with RORalpha, a mechanism associated with coactivator interaction. Strikingly, Hr specificity is dictated by the RORalpha AF-2 helix. Moreover, corepressor action is maintained in the presence of ligand, suggesting that Hr is a ligand-oblivious corepressor. The RORalpha AF-2 helix plays a dynamic role in controlling both corepressor and coactivator interactions. The interaction of Hr with RORalpha provides a molecular link converging the thyroid hormone and RO

The orphan nuclear receptor NR5A2 is implicated in a multitude of biological processes including cholesterol homeostasis and development. Its role in cholesterol metabolism and cell proliferation is now well established in vitro and in vivo. Both in vitro and gene expression studies have suggested a role for NR5A2 in ovarian function. In this study, we provide in vivo evidence for its involvement in reproductive function by demonstrating that heterozygosity for a null mutation of NR5A2 leads to a reduction in female fertility. Furthermore, we showed that NR5A2+/- females display a severe reduction in ovarian progesterone production and that progesterone supplementation can rescue the NR5A2+/- subfertility phenotype. We also provide evidence that one of the mechanisms by which NR5A2 regulates ovarian progesterone production is through modulating the expression of SCAR, which controls one of the rate-limiting steps of progesterone synthesis.<br>A targeted disruption of the NR5A2 gene in the mouse leads to early lethality in utero between embryonic days 6.0 and 7.5, showing that NR5A2 plays a crucial role during early embryogenesis. The molecular mechanisms underlying this early lethality, however, are poorly understood. In this study, we used a morphological and marker gene analysis to characterize the NR5A2-/- embryonic phenotype and showed that although initial axis specification occurs in NR5A2-/- embryos, primitive streak and mesoderm fail to form. Using a chimeric approach, we demonstrated a requirement for NR5A2 function in the visceral endoderm (VE), an extra-embryonic tissue, for proper primitive streak morphogenesis and gastrulation. Our results also indicate a reduction in the expression of VE marker genes involved in the nutritive function of this tissue, suggesting that NR5A2 play a dual role in the VE, being implicated in the mediation of both its patterning and nutritive activity.<br>Taking advantage of the LacZ knock-in approach used to inactivate the NR5A2 gene, we also demonstrated that NR5A2 is expressed during craniofacial and nervous system development, suggesting a novel role for NR5A2 in head formation and neural development.

<p> Sterol 12a-hydroxylase (CYP8B1) is involved in cholic acid synthesis and plays a role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone disease and dyslipidemia. In this study, we investigated the underlying mechanism of a fasting-induced and cholesterol activated nuclear receptor and core clock gene RORa in regulation of circadian rhythm and fasting induction of CYP8B1 expression. In free fed mice, CYP8B1 expression was reduced to the lowest level at the onset of the dark cycle when RORa expression was the lowest. However, fasting stimulated, while re-feeding reduced expression of CYP8B1 mRNA and protein expression. Interestingly, fasting and feeding had little effect on the diurnal rhythm of RORa mRNA expression, but fasting increased, whereas feeding decreased RORa protein levels in mouse liver. Adenovirus-mediated transduction of RORa to mice strongly induced CYP8B1 gene expression, increased 12a-hydroxylated bile acids in bile acid pool and serum and liver cholesterol. Reporter assay and mutagenesis analysis of the CYP8B1 promoter identified a functional RORa response element. Mammalian two-hybrid assay showed strong interaction of RORa with cAMP response element binding protein-binding protein (CBP). Chromatin immune-precipitation assay showed that RORa recruited CBP to the CYP8B1 promoter to stimulate histone acetylation. CAMP-activated protein kinase A phosphorylates RORa and increases its half-life. In conclusion, RORa is a key regulator of circadian expression and fasting induction of CYP8B1 to increase 12a-hydroxylated bile acids in the bile acid pool, and serum and liver cholesterol. This study contributes to our understanding of the molecular mechanism by which bile acid synthesis and composition regulates hepatic metabolic homeostasis. Overall this study sheds light on the mechanism of development of hypercholesterolemia in diabetic patients. Therefore, antagonizing RORa activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease.</p>

Le récepteur ERRα (Estrogen Receptor-Related Receptor alpha) appartient à la superfamille des récepteurs nucléaires. Une forte expression de ERRα est corrélée à un mauvais pronostic, suggérant l’implication de ce récepteur dans le processus métastatique. Mon projet est d'analyser le rôle de ERRα dans les mouvements cellulaires. J’ai montré qu’inhiber ERRα perturbe la migration cellulaire. L’étude des mouvements montre que l’absence de ERRα induit une perturbation de l’orientation cellulaire, du nombre des fibres de stress et des protrusions membranaires. Les cellules migrent de façon désorientée. J’ai démontré l’existence d’une cascade de régulation où ERRα stimule transcriptionnellement l'expression de la protéine BACURD2/TNFAIP1, elle-même régulant la stabilité de RhoA. Inhiber ERRα induit également une surexpression de RhoA, sa suractivation et une perturbation de la migration orientée. Cette cascade a été confirmée par des expériences de complémentation. J’ai vérifié ces résultats par des expériences in vivo et ex vivo, chez les souris KO pour ERRα. L’absence de ce récepteur induit une diminution de l’expression de TNFAIP1 inhibant la dégradation de RhoA et entrainant finalement une perturbation des mouvements cellulaires. Ainsi ERRα régule positivement la migration cellulaire conduisant à une forte potentialité métastatique dans les tumeurs surexprimant ce récepteur.L’ensemble de mes résultats pourrait faire de ERRα une nouvelle cible en vue de nouvelles thérapies anticancéreuses et nous pourrions proposer BACURD2/ TNFAIP1 comme un nouveau marqueur de pronostic dans les cancers<br>High expression of the orphan nuclear receptor ERRα is strongly correlated with poor prognosis in various types of tumors, including those of the breast. The fact that high ERRα expression in tumors is also correlated with elevated invasiveness suggests that this nuclear receptor positively regulates cell migration and invasiveness.This possibility was investigated using MDA-MB231 breast cancer cell line as a model. Inactivating ERRα impairs cell migration. Using time-lapse-based cell tracking analysis and Golgi positioning, we show that this impairment is not due to reduced migration speed but rather to cell disorientation. The enhanced number of cell protrusions present in migrating cells and disorganized actin fibers confirm this. In summary cells do migrate but do not sustain persistent linear movement. We observed that upon ERRα inactivation, RhoA, which is instrumental in oriented movement, is overexpressed at the protein level. Further analysis showed that the stability and proteasome-dependent degradation of the protein is affected. To analyze the relationship between ERRα (as a transcription factor) and RhoA protein stability we performed a transcriptomic analysis comparing (by RNA-Seq) wt cells to ERRα-depleted ones. We identified genes regulated by ERRα that are involved in both cell migration (as a biological process) and in protein stability and degradation, more specifically that of RhoA protein (as a molecular process). TNFAIP1/Bacurd2 is stimulated by ERRα and fits these criteria: this protein mediates the Culin3-based, proteasome-dependent of RhoA and its inactivation leads to defects in cell migration.TNFAIP1/RhoA cascade is a major downstream effector of ERRα in cell migration

La période de réceptivité endométriale chez l’humain coïncide avec la différentiation des cellules stromales de l’endomètre en cellules hautement spécifiques, les cellules déciduales, durant le processus dit de décidualisation. Or, on sait qu’une transformation anormale des cellules endométriales peut être à l’origine de pertes récurrentes de grossesses. LRH-1 est un récepteur nucléaire orphelin et un facteur de transcription régulant de nombreux évènements relatif à la reproduction et comme tout récepteur, son activation promouvoit l’activité transcriptionnelle de ses gènes cibles. Nous avons déjà montré que LRH-1 et son activité sont essentiels pour la décidualisation au niveau de l’utérus chez la souris et nous savons qu’il est présent dans l’utérus chez l’humain au moment de la phase de prolifération mais aussi de sécrétion du cycle menstruel, et que son expression augmente dans des conditions de décidualisation in vitro. Notre hypothèse est alors la suivante : LRH-1 est indispensable à la décidualisation du stroma endométrial, agissant par le biais de la régulation transcriptionnelle de gènes requis pour la transformation de cellules stromales en cellules déciduales. Afin d’explorer le mécanisme moléculaire impliqué dans la régulation transcriptionnelle effectuée par l’intermédiaire de ce récepteur, nous avons mis en place un modèle de décidualisation in vitro utilisant une lignée de cellules stromales de l’endomètre, cellules humaines et immortelles (hESC). Notre modèle de surexpression développé en transfectant les dites cellules avec un plasmide exprimant LRH-1, résulte en l’augmentation, d’un facteur 5, de l’abondance du transcriptome de gènes marqueurs de la décidualisation que sont la prolactine (PRL) et l’insulin-like growth factor binding protein-1 (IGFBP-1). En outre, la sous-régulation de ce récepteur par l’intermédiaire de petits ARN interférents (shRNA) abolit la réaction déciduale, d’un point de vue morphologique mais aussi en terme d’expression des deux gènes marqueurs cités ci-dessus. Une analyse par Chromatin ImmunoPrécipitation (ou ChIP) a démontré que LRH-1 se lie à des régions génomiques se trouvant en aval de certains gènes importants pour la décidualisation comme PRL, WNT 4, WNT 5, CDKN1A ou encore IL-24, et dans chacun de ces cas cités, cette capacité de liaison augmente dans le cadre de la décidualisation in vitro. Par ailleurs, des études structurelles ont identifié les phospholipides comme des ligands potentiels pour LRH-1. Nous avons donc choisi d’orienter notre travail de façon à explorer les effets sur les ligands liés à LRH-1 de traitements impliquant des agonistes et antagonistes à notre récepteur nucléaire. Les analyses par q-PCR et Western blot ont montré que la modulation de l’activité de LRH-1 par ses ligands influait aussi sur la réaction déciduale. Enfin, des études récentes de Salker et al (Salker, Teklenburg et al. 2010) ont mis en évidence que les cellules stromales humaines décidualisées sont de véritables biocapteurs de la qualité embryonnaire et qu’elles ont la capacité de migrer en direction de l’embryon. La série d’expériences que nous avons réalisée à l’aide de cellules hESC placées en co-culture avec des embryons de souris confirme que la migration cellulaire est bien dirigée vers les embryons. Cette propriété quant à l’orientation de la migration cellulaire est notoirement diminuée dans le cas où l’expression de LRH-1 est déplétée par shRNA dans les hESC. Nos données prouvent donc que LRH-1 régule non seulement la transcription d’un ensemble de gènes impliqués dans le processus de décidualisation mais agit aussi sur la motilité directionnelle de ces cellules hESC décidualisées in vitro.<br>The period of endometrial receptivity in humans coincides with the differentiation of endometrial stromal cells into highly specialized decidual cells through a process known as decidualization. This transformation of endometrial cells is abnormal in recurrent pregnancy loss patients. Liver homolog receptor 1 (LHR-1, NR5A2) is an orphan nuclear receptor and a transcription factor that regulates many reproductive events. The activation of this receptor leads to transcriptional activation of its target genes. We have previously shown that it is essential for decidualization in the mouse uterus. LRH-1 is expressed in the human uterus in both proliferative and secretory phases of the menstrual cycle and its expression increases during in vitro decidualization. We hypothesize that LRH-1 is indispensable for proper decidualization of the endometrial stroma, acting through the transcriptional regulation of genes required for transformation of stromal cells into decidual cells. To explore the molecular mechanism of transcriptional regulation mediated by this receptor, we established an in vitro model of decidualization, using an immortal human endometrial stromal cell line (hESC). An overexpression model developed by transfecting the cells with a plasmid constitutively expressing Lrh-1, resulted in 5 fold increases in abundance of transcripts for the decidualization marker genes prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Furthermore, the downregulation of the receptor using short hairpin RNA (shRNA) abrogates the decidual reaction, from both a morphological point of view and in terms of expression of the two marker genes. Chromatin immunoprecipitation (ChIP) analysis showed that LRH-1 binds to genomic regions upstream of genes important for decidualization such as PRL, wingless-type MMTV integration site family, member 4 (WNT4), wingless-type MMTV integration site family, member 5 (WNT5), cyclin-dependent kinase inhibitor 1A (p21, CDKN1A) and interleukin-24(IL-24). For each of these genes, the binding increased during in vitro decidualization. Structural studies have identified phospholipids as potential LRH-1 ligands. We therefore explored the effect of ligand treatment on LRH-1 with an agonist and an inverse agonist for the nuclear receptor. Analysis by quantitative polymerase chain reaction (qPCR) and Western blot demonstrated that the modulation of LRH-1 activity by its ligands also affects the decidual reaction. Recent studies have shown that decidualized human stromal cells are biosensors of embryo quality and that they have the capacity to migrate towards the embryo. Our time-lapse evaluation of hESC cells co-cultured with mouse embryos indicates directed migration of the cells toward the embryo. This effect is markedly diminished when LRH-1 is depleted by shRNA in hESC. Our data provide evidence that LRH-1 regulates not only the transcription of a set of genes involved in decidualization but also the directional motility of these cells in vitro.

研究背景與研究目的<br>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。<br>研究材料與方法<br>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。<br>結果<br>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。<br>結論<br>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。<br>Background and aims of the study<br>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.<br>Materials and methods<br>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).<br>Results<br>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.<br>Conclusions<br>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.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Wu, Dinglan.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 135-151).<br>Abstract also in Chinese.<br>Thesis /Assessment Committee --- p.I<br>ABSTRACT --- p.II<br>摘 要 --- p.VI<br>ACKNOWLEDGEMENT --- p.IX<br>PUBLICATIONS RELATED TO THIS THESIS --- p.XI<br>CONTENTS --- p.XII<br>ABBREVIATION --- p.XV<br>Chapter CHAPTER 1 --- INTRODUCTION --- p.1<br>Chapter 1.1 --- Prostate cancer --- p.2<br>Chapter 1.1.1 --- Epidemiology --- p.2<br>Chapter 1.1.2 --- Nature history --- p.4<br>Chapter 1.1.3 --- Androgen Axis prostate cancer --- p.7<br>Chapter 1.1.3.1 --- Androgen receptor --- p.7<br>Chapter 1.1.3.2 --- Function of the androgen receptor in prostate cancer --- p.7<br>Chapter 1.1.3.3 --- Mechanisms of CRPC progression --- p.8<br>Chapter 1.1.3.4 --- Androgen receptor pathway-directed therapies --- p.10<br>Chapter 1.1.4 --- Treatment of prostate cancer --- p.11<br>Chapter 1.2 --- Cellular senescence --- p.13<br>Chapter 1.2.1 --- What is senescence --- p.13<br>Chapter 1.2.1.1 --- Replicative cellular senescence --- p.13<br>Chapter 1.2.1.2 --- Oncogene induced senescence (OIS) --- p.15<br>Chapter 1.2.1.3 --- Tumor suppressor loss-induced senescence --- p.17<br>Chapter 1.2.2 --- Establishment of cellular senescence --- p.19<br>Chapter 1.2.3 --- The p16/pRb and ARF/p53/p21 pathway of senescence induction --- p.21<br>Chapter 1.2.3.1 --- p16/pRb senescence pathway --- p.22<br>Chapter 1.2.3.2 --- ARF/p53/p21 senescence pathway --- p.23<br>Chapter 1.2.4 --- Markers of senescence --- p.24<br>Chapter 1.2.4.1 --- Cell cycle arrest and morphology --- p.24<br>Chapter 1.2.4.2 --- Senescence-associated β-galactosidase --- p.25<br>Chapter 1.2.4.3 --- p16/pRb and p53/p21 pathways --- p.26<br>Chapter 1.2.4.4 --- γ-H2AX staining as a marker for DNA damage --- p.27<br>Chapter 1.2.4.5 --- Senescence-associated heterochromatin foci (SAHF) --- p.27<br>Chapter 1.2.5 --- Pro-senescence therapy for cancer treatment --- p.29<br>Chapter 1.2.5.1 --- Why pro-senescence therapy --- p.29<br>Chapter 1.2.5.2 --- Critical factors of pro-senescence therapy --- p.31<br>Chapter 1.2.5.3 --- Strategies of senescence induction --- p.32<br>Chapter 1.2.5.4 --- Targeting to senescence-associated secretory phenotype (SASP) --- p.38<br>Chapter 1.2.6 --- Future direction --- p.40<br>Chapter 1.3 --- TLX --- p.41<br>Chapter 1.3.1 --- Nuclear receptor --- p.41<br>Chapter 1.3.2 --- Identification of tailless/TLX --- p.42<br>Chapter 1.3.3 --- Tailless in drosophila --- p.43<br>Chapter 1.3.4 --- Functional role of tll/TLX --- p.45<br>Chapter 1.3.4.1 --- Role of tll/TLX in brain development --- p.45<br>Chapter 1.3.4.2 --- Role of tll/TLX in visual system developments --- p.46<br>Chapter 1.3.4.3 --- Role of TLX in neural stem cell self-renewal --- p.47<br>Chapter 1.3.5 --- Target genes of TLX --- p.49<br>Chapter 1.3.6 --- Transcriptional regulation of tll/TLX --- p.51<br>Chapter 1.3.7 --- TLX in cancer --- p.52<br>Chapter CHAPTER 2 --- STUDY AIMS --- p.54<br>Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.57<br>Chapter 3.1 --- Human prostatic tissues and Immunohistochemistry --- p.58<br>Chapter 3.2 --- Cell lines and cell cultures --- p.59<br>Chapter 3.3 --- Antibody and reagents --- p.63<br>Chapter 3.3.1 --- Generation of rabbit anti-TLX polyclonal antibody --- p.63<br>Chapter 3.3.2 --- Commercial antibody --- p.64<br>Chapter 3.4 --- RNA isolation and Reverse transcriptional-PCR --- p.65<br>Chapter 3.4.1 --- RNA isolation --- p.65<br>Chapter 3.4.2 --- Reverse transcription reaction (RT) --- p.66<br>Chapter 3.4.3 --- Polymerase Chain Reaction (PCR) --- p.66<br>Chapter 3.5 --- Western blotting --- p.68<br>Chapter 3.5.1 --- Protein extraction --- p.68<br>Chapter 3.5.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.69<br>Chapter 3.6 --- Plasmids construction --- p.70<br>Chapter 3.6.1 --- PCR for sub-cloning --- p.70<br>Chapter 3.6.2 --- PCR for mutant generation --- p.71<br>Chapter 3.6.3 --- Restriction enzymes digestion and ligation --- p.72<br>Chapter 3.7 --- Retroviral, lentiviral transduction and generation of TLX-stable cells --- p.73<br>Chapter 3.8 --- RNA interference --- p.75<br>Chapter 3.9 --- In vitro cell growth assay --- p.76<br>Chapter 3.9.1 --- Cell counting --- p.76<br>Chapter 3.9.2 --- MTT assay --- p.76<br>Chapter 3.9.3 --- Soft agar assay for anchorage independent growth --- p.77<br>Chapter 3.10 --- Cell cycle assay --- p.77<br>Chapter 3.11 --- Cell invasion assay --- p.78<br>Chapter 3.12 --- In vivo tumor growth assay --- p.78<br>Chapter 3.13 --- In vitro and in vivo SA-β-Gal staining --- p.79<br>Chapter 3.14 --- In vitro treatment with doxorubicin --- p.80<br>Chapter 3.15 --- Transient Transfection and Luciferase Reporter Assay --- p.81<br>Chapter 3.16 --- Chromatin immunoprecipitation (ChIP) assay --- p.82<br>Chapter 3.16.1 --- Cross-linking and harvesting cells --- p.82<br>Chapter 3.16.2 --- Cell lysis --- p.83<br>Chapter 3.16.3 --- Sonication --- p.83<br>Chapter 3.16.4 --- Immunoprecipitation --- p.83<br>Chapter 3.16.5 --- Washing --- p.84<br>Chapter 3.16.6 --- Elution --- p.85<br>Chapter 3.16.7 --- Reverse cross-linking and DNA purification --- p.85<br>Chapter 3.16.8 --- PCR --- p.86<br>Chapter 3.17 --- Statistical analysis --- p.86<br>Chapter CHAPTER 4 --- RESULTS --- p.87<br>Chapter 4.1 --- TLX is up-regulated in prostate carcinoma and prostate cancer cell lines --- p.88<br>Chapter 4.2 --- Knockdown of TLX suppresses in vitro cell growth and triggers cellular senescence in prostate cancer cells --- p.93<br>Chapter 4.3 --- Knockdown of TLX inhibits in vivo tumor growth and induces cellular senescence of prostate cancer cells --- p.97<br>Chapter 4.4 --- Ectopic expression of TLX enhances in vitro cell growth and multiple advanced malignant phenotypes in prostate cancer cells --- p.100<br>Chapter 4.5 --- Ectopic expression of TLX suppresses cellular senescence in prostate cancer cells --- p.105<br>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<br>Chapter 4.7 --- TLX also suppresses cellular senescence via its transcriptional regulation of SIRT1 gene --- p.116<br>Chapter CHAPTER 5 --- DISCUSSION --- p.121<br>Chapter CHAPTER 6 --- SUMMARY --- p.131<br>REFERENCES --- p.135

前列腺癌是男性常見惡性腫瘤之一，在歐美等國家前列腺癌的發病率及死亡率均位居惡性腫瘤前列。前列腺癌根治術，放療和雄激素治療是前列腺癌治療的主要方法。但是，多數患者都不可避免地在2-3年內進展為去勢抵抗性前列腺癌。對於這些患者，二線內分泌治療和化療成為最後的選擇，但治療效果十分有限。雖然進展為去勢抵抗性前列腺癌的機制尚不完全明確，但是越來越多的證據顯示：腫瘤幹細胞在前列腺癌啟動，進展，轉移和治療抵抗等方面均起作用。因此，闡明腫瘤幹細胞的特性有助於我們理解去勢抵抗性前列腺癌的發生機制。<br>肝受體同源物1 (LRH-1) 屬于核受體超家族中的NR5A亞家族，主要功能包括促進早期發育、膽固醇平衡、類固醇合成以及腫瘤生長調控。近期研究結果表明：LRH-1可以與一些關鍵的幹細胞轉錄因子（例如 OCT4、NANOG和SOX2）相互作用進而調控胚胎幹細胞和間質幹細胞的自我更新和多能性。鑒於LRH-1在幹細胞生物學和腫瘤領域均發揮重要作用，使其成為腫瘤幹細胞特異性治療的潛在靶點。<br>在本研究中，免疫組織化學分析顯示：LRH-1在前列腺癌組織的表達水平明顯高於正常或良性前列腺增生組織，並且與Gleason評分正相關。 前列腺癌細胞系中LRH-1的mRNA水平也明顯高於前列腺非腫瘤上皮細胞系。此外，LRH-1在具有腫瘤幹細胞特性的前列腺腫瘤球中的表達水平明顯高於親本細胞。功能獲得與功能失活研究進一步表明：LRH-1過表達可以促進前列腺細胞的幹細胞特性 （幹性），而下調LRH-1會抑制前列腺癌細胞的幹性，主要體現在自我更新能力，腫瘤幹細胞標記物表達水平，腫瘤幹細胞比例，化療和雄激素治療敏感性以及體內成瘤能力等方面。而LRH-1高表達所增強的幹性能被LRH-1特異性反向激動劑ML-180所抑制。此外，本研究還發現，LRH-1可以在前列腺癌細胞中直接調控腫瘤幹細胞非細胞表面標記物OCT4基因，在LRH-1下調的細胞中高表達OCT4可以部分挽救細胞幹性。最後，在去勢抵抗性前列腺癌移植瘤模型 (VCaP-CRPC)中，LRH-1和一系列腫瘤幹細胞標記物的表達水平以及腫瘤幹細胞比例在去勢後腫瘤進展的早期和晚期階段均有升高。<br>總之，本研究表明：LRH-1能夠維持前列腺癌細胞的幹性，進而促進前列腺癌進展，因此，LRH-1可以作為進展期前列腺癌的治療靶點，尤其是LRH-1特異性反向激動劑ML-180，有可能成為前列腺治療的特異性靶點藥物。<br>Prostate cancer is the most common male-specific cancer and also the second leading cause of cancer-related deaths in men in Europe and United States. In Hong Kong, prostate cancer ranked the 3rd common male-specific cancer and 5th cancer-causing death in 2013, and the prevalence has almost doubled in the past decade. Radical prostatectomy, radiotherapy, and primary hormone therapy are the mainstay therapeutic options. However, many patients will inevitably relapse from the primary therapy and progress to a hormone-independent stage (described clinically as castration-resistant prostate cancer/CRPC) within 2-3 years, to which the current treatment strategies are limited and almost incurable. Although the exact mechanisms underlying the progression to hormone-independent stage are still incompletely understood, emerging evidence has shown that cancer stem cells (CSCs) endowed with capacity of self-renewal and differentiation may be involved in tumour initiation, progression, metastasis and therapy resistance. Therefore, clarifying the unique characteristics of CSCs will enhance our understanding of the mechanisms responsible for the emergency of CRPC and importantly help to identify potential therapeutic targets for this cancer.<br>Nuclear receptor (NR) liver receptor homolog-1 (LRH-1, NR5A2) belongs to NR5A subfamily of NR superfamily, and is characterized to play roles in early development, cholesterol homeostasis, steroidogenesis and growth regulation of many human cancers. Recent work suggests that LRH-1 can modulate the self-renewal and pluripotency of embryonic and mesenchymal stem cells via its interaction with some key stem cell regulatory transcription factors (e.g., OCT4, NANOG, SOX2). Given that LRH-1 plays a role in stem cell biology and human cancers, it may represent a potential target for CSC-specific treatment.<br>In this study, immunohistochemistry analysis showed that the expression of LRH-1 was significantly elevated in human prostate cancer lesions as compared to normal or benign prostatic hyperplasia (BPH) tissues, and its level was positively associated with Gleason Score (GS). LRH-1 mRNA exhibited an increased expression in most prostate cancer cell lines, as compared to immortalized non-tumorigenic prostatic epithelial cell lines. In addition, increased LRH-1 expression was concomitant with an up-regulation of a number of cancer stem cell-associated markers in a well-established CRPC xenograft model (VCaP-CRPC) harboring increased cancer stem cell population during their progression to castration-resistance. Furthermore, LRH-1 was differentially and highly expressed in prostatospheres that were enriched in prostate cancer stem cells. The gain-of-function and loss-of-function studies further demonstrated that ectopic expression of LRH-1 promoted whereas knockdown of LRH-1 suppressed the stem-like properties or phenotypes of prostate cancer cells, in terms of self-renewal capacity, expression levels of cancer stem cell-associated markers, proportion of cancer stem cell subpopulation, sensitivity to chemotherapy and hormone therapy, as well as tumourigenicity. Moreover, the enhanced CSC capabilities induced by LRH-1 over-expression could be reversed by an LRH-1 inverse agonist (ML-180). Lastly, OCT4, a crucial non-cell surface marker for CSCs was identified as a direct target of LRH-1 in prostate cancer cells and restoration of OCT4 expression in LRH-1 silenced prostate cancer cells could partially rescue the impaired stemness.<br>Taken together, results obtained in this study indicate that LRH-1 plays a tumour-promoting role in prostate cancer development, particularly its progression to CRPC, via maintenance of prostate cancer stem cell population, and targeting LRH-1 is of promising therapeutic significance in advanced prostate cancer.<br>Wang, Yuliang.<br>Thesis Ph.D. Chinese University of Hong Kong 2016.<br>Includes bibliographical references (leaves ).<br>Abstracts also in Chinese.<br>Title from PDF title page (viewed on …).<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.

研究背景和研究目的<br>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。<br>材料和方法<br>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。<br>結果<br>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。<br>結論<br>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。<br>Background and aims of study<br>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.<br>Materials and methods<br>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.<br>Results<br>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.<br>Conclusion<br>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.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Zou, Chang.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 138-160).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.<br>ABSTRACT --- p.i<br>ACKNOWLEDGEMENTS --- p.viii<br>PUBLICATIONS --- p.ix<br>CONTENTS --- p.x<br>ABBREVIATIONS --- p.xiii<br>Chapter CHAPTER 1 --- Introduction --- p.1<br>Chapter 1.1 --- Prostate cancer --- p.2<br>Chapter 1.1.1 --- Epidemiology --- p.2<br>Chapter 1.1.2 --- Risk factors --- p.3<br>Chapter 1.1.3 --- Patho-physiology --- p.6<br>Chapter 1.1.4 --- Diagnosis and treatment --- p.8<br>Chapter 1.2 --- Androgen,androgen receptor and prostate cancer --- p.10<br>Chapter 1.2.1 --- Androgen and androgen receptor --- p.10<br>Chapter 1.2.2 --- Castration Resistance Prostate Cancer (CRPC) --- p.12<br>Chapter 1.2.2.1 --- Overexpression of AR --- p.13<br>Chapter 1.2.2.2 --- Increasing sensitivity to and rogen --- p.13<br>Chapter 1.2.2.3 --- AR mutation --- p.14<br>Chapter 1.2.2.4 --- Deregulation of AR regulator factors --- p.15<br>Chapter 1.2.2.5 --- Outlaw pathway --- p.15<br>Chapter 1.2.2.6 --- AR-independent pathway --- p.16<br>Chapter 1.3 --- Estrogen and prostate cancer --- p.17<br>Chapter 1.3.1 --- Overview of estrogen and estrogen receptors --- p.17<br>Chapter 1.3.2 --- Estrogen signaling pathway andprostatecancer --- p.18<br>Chapter 1.4 --- Nuclear receptors --- p.20<br>Chapter 1.4.1 --- Overview of NRs superfamily --- p.20<br>Chapter 1.4.2 --- Classification --- p.21<br>Chapter 1.4.3 --- NRs as therapeutic targets for cancer treatment --- p.23<br>Chapter 1.5 --- Estrogen-related receptors --- p.25<br>Chapter 1.5.1 --- NR3B subgroup --- p.25<br>Chapter 1.5.2 --- Isoforms --- p.26<br>Chapter 1.5.3 --- Structure --- p.27<br>Chapter 1.5.4 --- Ligand --- p.28<br>Chapter 1.5.5 --- Co-regulators --- p.31<br>Chapter 1.5.6 --- Tissue-specific expression pattern and identifiedfunction --- p.32<br>Chapter 1.5.6.1 --- Tissue-specific expression pattern --- p.32<br>Chapter 1.5.6.2 --- Identified physiological function of ERRs --- p.33<br>Chapter 1.5.7 --- ERRs and cancer --- p.35<br>Chapter 1.5.7.1 --- ERRβ/γ and cancer --- p.35<br>Chapter 1.5.7.2 --- Expression of ERRα in cancer --- p.37<br>Chapter 1.5.7.3 --- Identified functional roles of ERRα in cancer --- p.40<br>Chapter 1.5.7.4 --- Regulation of ERRα in cancer cells --- p.42<br>Chapter 1.6 --- Hypoxiaadaptation andcancer --- p.47<br>Chapter 1.6.1 --- HIFs isoforms and structure --- p.47<br>Chapter 1.6.2 --- Structure --- p.48<br>Chapter 1.6.3 --- Regulation of HIF-1α expression --- p.49<br>Chapter 1.6.3.1 --- Regulation of HIF-1α mRNA transcription --- p.49<br>Chapter 1.6.2.2 --- Regulation of HIF-1α mRNA transcription --- p.50<br>Chapter 1.6.2.3 --- O₂-dependent regulation of stability of HIF-1α protein --- p.51<br>Chapter 1.6.2.4 --- O₂-independent regulation of HIF-1α --- p.52<br>Chapter 1.6.2.5 --- Genetranscriptional regulation role of HIFs --- p.54<br>Chapter 1.6.3 --- HIFs and cancer --- p.55<br>Chapter 1.6.3.1 --- Overview --- p.55<br>Chapter 1.6.3.2 --- Expression of HIF-1α in cancer progression --- p.55<br>Chapter 1.6.3.2 --- Functional roles of HIF-1α in cancer progression --- p.56<br>Chapter CHAPTER 2 --- Aims of study --- p.58<br>Chapter CHAPTER 3 --- Materials and methods --- p.61<br>Chapter 3.1 --- Cell lines and cell culture --- p.62<br>Chapter 3.2 --- Human Prostatic Tissues --- p.64<br>Chapter 3.3 --- RNA isolation and Reverse transcriptional-PCR --- p.64<br>Chapter 3.3.1 --- Total RNA extraction --- p.64<br>Chapter 3.3.2 --- Reverse transcription reaction --- p.65<br>Chapter 3.3.3 --- Polymerase Chain Reaction for gene expression detection --- p.66<br>Chapter 3.4 --- Plasmids construction --- p.69<br>Chapter 3.4.1 --- Genomic DNA extraction --- p.69<br>Chapter 3.4.2 --- PCR for cloning and sub-cloning --- p.70<br>Chapter 3.4.3 --- PCR for mutant generation --- p.70<br>Chapter 3.4.4 --- Restriction enzymes cut and ligation --- p.71<br>Chapter 3.5 --- Antibody and reagents --- p.73<br>Chapter 3.6 --- Immunohistochemistry --- p.74<br>Chapter 3.7 --- Western Blot Analysis --- p.75<br>Chapter 3.7.1 --- Protein extraction --- p.75<br>Chapter 3.7.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.76<br>Chapter 3.8 --- Retroviral transduction and generation of ERRα poolandstable clones --- p.77<br>Chapter 3.9 --- In vitro Cell Growth Assays --- p.77<br>Chapter 3.9.1 --- Cell counting --- p.77<br>Chapter 3.9.2 --- 5-Bromodeoxyuridine (BrdU) incorporation assay --- p.78<br>Chapter 3.9.3 --- MTT assay --- p.79<br>Chapter 3.9.4 --- In vitro clonal formation assay --- p.79<br>Chapter 3.10 --- Cell attachment assay --- p.80<br>Chapter 3.11 --- Transwell cell invasion assay --- p.81<br>Chapter 3.12 --- In vivo tumorigenicity assay --- p.81<br>Chapter 3.13 --- RNA interference --- p.82<br>Chapter 3.14 --- Transient Transfection and Luciferase Reporter Assay --- p.83<br>Chapter 3.15 --- Immuno-precipitation (IP) assay --- p.84<br>Chapter 3.16 --- Fluorescence Resonance Energy Transfer (FRET) detection --- p.85<br>Chapter 3.17 --- In vitro treatment with XCT790, cycloheximide and MG-132 --- p.86<br>Chapter CHAPTER 4 --- Reuslts --- p.88<br>Chapter 4.1 --- ERRα exhibits an increased expression pattern in high grade prostate cancer --- p.89<br>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<br>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<br>Chapter 4.4 --- Ectopic expression of ERR α in LNCaP prostate cancer cells can promote their in vivo tumorigenicity and tumor angiogenesis. --- p.97<br>Chapter 4.5 --- Overexpression of ERRα can up‐regulate protein level of HIF-1α and enhance its transcriptional activity --- p.99<br>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<br>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<br>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<br>Chapter CHAPTER 5 --- Discussion --- p.119<br>Chapter CHAPTER 6 --- Summary --- p.134<br>References --- p.138

Parkinson’s disease (PD) is a degressive, neurodegenerative disease that affects approximately four million people worldwide. The disease is characterized by the progressive loss of midbrain dopaminergic (DAergic) neurons, which are highly related with the motor control. As the disease progresses, movement disorders appear such as tremor, rigidity, and bradykinesia, but also disorders in speech and neuropsychiatric disturbances occur.Current therapies for PD focus on symptomatic treatment, while pharmacological methods to prevent or delay the degeneration of neurons have not been discovered yet. The Nurr1 nuclear receptor, which is expressed predominantly in the substantia nigra of the midbrain, has emerged as a target for the treatment of Parkinson’s disease due to its neuroprotective action and contribution in DAergic neuron development. It has been shown that partial loss of Nurr1 function in people due to mutations leads to neuronal death. Thus, the reinforcement of Nurr1 operation via the discovery of novel potent agonists is imperative. Unfortunately, the accomplishment of this task is complicated as Nurr1 ligand binding domain (LBD) lacks a cavity for ligand binding due to the tight packing chains from several hydrophobic amino-acids in the region normally occupied by ligands in other nuclear receptors. However, the activation of Nurr1 can be feasible through heterodimer formation with Retinoid X Receptors (RXR) and especially with RXRα, which are all capable of binding ligands and therefore, mediate Nurr1 expression in midbrain. Therefore, we seek here to identify potent binders of RXRα as a means to increase Nurr1 levels. Based on the fact that multiple RXRα receptor conformations exist depending on binding of RXRα to different heterodimerization partners, we aim to increase the specificity of identified binders for the heterodimer Nurr1/RXRα. For this purpose, we describe here a new computational protocol for the selection of RXRα receptor structures that is used to perform Structure-Based Virtual Screening (SBVS) calculations for the discovery of NURR1 activators. In our study, we developed a computational protocol, where the choice of RXRα conformations for performing the SBVS is based on four criteria: (a) Pairwise comparison of the receptor conformations according to RMSD calculations, (b) analysis and clustering of RXRα structures comparing the binding-site shape and volume using SiteMap, (c) docking of a small-database of known actives for a specific heterodimer partner to the resulting shape-diverse subset of binding sites from (a) and (b) using Glide 5.8 SP and XP, and (d) retrieving representative protein conformations for the structure of interest from MD simulations using GROMACS. Virtual Screening was performed on three different subsets of RXRα receptor conformations, based on their binding to different heterodimerization partners. The final RXRα receptors to be used in SBVS were selected as mentioned above aiming to enhance the success rate and the selectivity of the hits. The Maybridge Hitfinder and Zinc databases were used in this SBVS exercise by first applying the SP filter on the full database and then the XP filter on the top 10,000 compounds of the Maybridge database and the top 40,000 compounds of the ZINC database. Compounds were selected as follows: Molecules that scored high when docked in the RXRα protein ensemble that bind to the heterodimer partner of interest and at the same time scored low for RXRα structures that bind to heterodimer partners of no interest, were selected in order to achieve selectivity. The efficiect selection was also based on their different orientation at the binding site of the various RXRα structures and different interactions with specific surrounding residues in order to maximize their selectivity potential. Finally, a post-processing step was imposed to the top-scoring compounds by using Chembioserver and FAF-Drugs2 filtering tools as well as pharmacological property prediction with the QikProp software. In vitro agonism of these compounds is still pending experimental testing. The workflow of this protocol is shown in Fig. 1. Figure 1: SBVS protocol developed for the discovery of novel selective Nurr1/RXRα agonists.<br>--

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

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

Worldwide, ten percent of adults suffer from a mental illness or behavioural disorder at any given time (Lopez & Murray 1998). Violent behaviour is central to many forms of mental illness (Gothelf et al 1997, APA 2000) and is itself a primary cause of mortality amongst individuals 15-45 years of age (Krug et al 2002). Although 'fierce' mice deleted for nuclear receptor 2E1 (Nr2el) show pathological aggression, it is unknown whether human NR2E1 is similarly involved in the modulation of development and behaviour. To better understand the role of human NR2E1 in brain development and behaviour, I undertook a series of studies employing computational analyses and transgenic mouse technology. A comparative sequence analysis of the NR2E1 locus in human, mouse, and the puffer fish Fugu Rubripes highlighted an unusually high degree of structural conservation across evolution, identified putative regulatory elements, and facilitated the generation of novel transgenic mouse strains. Characterization of a panel of random insertion transgenic mice carrying an Nr2e 1 -spanning clone indicated that standard molecular techniques failed to identify problematic insertions that would have interfered with downstream applications if not identified by molecular cytogenetic analyses. These results have implications for characterization of new transgenic mouse strains and helped shaped my subsequent studies. Finally, to test the ability of human NR2E1 to modulate mouse behaviour, I generated mice carrying human NR2E1 under its endogenous regulatory elements and bred them to fierce mutant mice. I determined that human NR2E1 is sufficient to correct fierce brain-behaviour abnormalities, supporting conserved mechanisms for modulation of behaviour between the two species. These data represent the first example of a human gene correcting mouse behaviour. They also provide a paradigm to enable functional evaluation of candidate psychiatric disease genes, and suggest that variation at NR2E1 may contribute to human behavioural disorders.<br>Medicine, Faculty of<br>Graduate

Le récepteur nucléaire Nr5a2, également connu sous le nom de liver receptor homolog-1 (Lrh-1), est exprimé au niveau de l’ovaire chez la souris, exclusivement dans les cellules lutéales et de la granulosa. La perturbation de Nr5a2, spécifique aux cellules de la granulosa chez la souris à partir des follicules primaires dans la trajectoire du développement folliculaire a démontré que Nr5a2 est un régulateur clé de l’ovulation et de la fertilité chez la femelle. Notre hypothèse veut que Nr5a2 régule les évènements péri- et post-ovulatoires dans une séquence temporelle lors de la folliculogénèse. Afin d'étudier l’implication de Nr5a2 lors de l’ovulation et de la lutéinisation à différents stades du développement folliculaire, nous avons généré deux modèles de souris knockout spécifiques aux cellules de la granulosa pour Nr5a2: 1) Nr5a2Amhr2-/-, avec une réduction de Nr5a2 à partir des follicules primaires et subséquents; 2) Nr5a2Cyp19-/-, avec une réduction de Nr5a2 débutant au stade antral de développement en progressant. L’absence de Nr5a2 à partir des follicules antraux a résulté en une infertilité chez les femelles Nr5a2Cyp19-/-, de même qu’en des structures non-fonctionnelles similaires aux structures lutéales au niveau des ovaires, en une réduction des niveaux de progestérone synthétisée ainsi qu’en un échec dans le support d’une pseudo-gestation. La synthèse de progestérone a été entravée suite à l’absence de Nr5a2 par l’entremise d’une régulation à la baisse des gènes reliés au transport du cholestérol, Scarb1, StAR et Ldlr, démontré par qPCR. Les complexes cumulus-oocytes des femelles Nr5a2Cyp19-/- immatures super-stimulées ont subi une expansion in vivo, mais l’ovulation a été perturbée, possiblement par une régulation à la baisse du gène du récepteur de la progestérone (Pgr). Un essai d’expansion du cumulus in vitro a démontré une expansion défectueuse du cumulus chez les Nr5a2Amhr2-/-, associée à un dérèglement de la protéine des jonctions communicantes (Gja1; Cx43). Cependant, l’expansion du cumulus chez les Nr5a2Cyp19-/- n’a pas été autant affectée. Des résultats obtenus par qPCR ont démontré une régulation à la baisse dans l’expression des gènes Areg, Ereg, Btc et Tnfaip6 chez les deux modèles de cellules ovariennes knockout à 2h et 4h post hCG. Nous avons observé que 85% des oocytes, chez les deux génotypes mutants, peuvent subir une rupture de la vésicule germinative, confirmant leur capacité de maturation in vivo. La technique d’injection intra-cytoplasmique de spermatozoïdes a prouvé que les oocytes des deux génotypes mutants sont fertilisables et que 70% des embryons résultants ont poursuivi leur développement vers le stade de blastocyste, et ce, indépendamment du génotype. En conclusion, Nr5a2 régule la fertilité chez les femelles tout au long du processus du développement folliculaire. Il a été démontré que Nr5a2 est essentiel à la lutéinisation et que sa perturbation dans les cellules somatiques ovariennes ne compromet pas la capacité des oocytes à être fertilisés. En vue d’ensemble, nous avons fourni une investigation inédite et complète, utilisant de multiples modèles et techniques afin de déterminer les mécanismes par lesquels Nr5a2 régule les importants processus que sont l’expansion du cumulus, l’ovulation ainsi que la formation du corps jaune.<br>The nuclear receptor Nr5a2, also known as liver receptor homolog-1 (Lrh-1), is expressed in the mouse ovary, exclusively in granulosa and luteal cells. Granulosa-specific disruption of Nr5a2 in mice from primary follicles onward in the follicle development trajectory has shown that Nr5a2 is a key regulator of ovulation and female fertility. We hypothesized that Nr5a2 modulates peri- and post-ovulatory events in a temporal sequence during folliculogenesis. To examine the role of Nr5a2 in ovulation and luteinization at different stages of the follicular development, we generated two Nr5a2 granulosa-specific knockout mice models: 1) Nr5a2Amhr2-/-, with Nr5a2 depletion from primary follicles forward; and 2) Nr5a2Cyp19-/-, with Nr5a2 depletion from the antral stage of development forward. The lack of Nr5a2 beginning in antral follicles resulted in infertility in Nr5a2Cyp19-/- females, with ovaries displaying non-functional luteal-like structures, synthesizing reduced progesterone levels and failing in supporting pseudopregnancy. Progesterone synthesis was affected by the lack of Nr5a2 through the downregulation of the cholesterol transport-related genes, Scarb1, StAR and Ldlr, as shown by qPCR. The cumulus-oocyte complexes of superstimulated Nr5a2Cyp19-/- immature females underwent expansion in vivo, but ovulation was disrupted, likely due to the downregulation of the progesterone receptor (Pgr) gene. An in vitro cumulus expansion assay showed defective cumulus expansion in Nr5a2Amhr2-/- associated with a dysregulation in the gap junction alpha-1 (Gja1; Cx43). In vitro cumulus expansion in Nr5a2Cyp19-/- was less affected than in Nr5a2Amhr2-/- cumulus-oocyte complexes. Data from qPCR showed a downregulation in the gene expression of Areg, Ereg, Btc and Tnfaip6 in both knockout ovarian cells at 2 h and 4 h post hCG. We found that 85% of the oocytes in both mutant genotypes can undergo germinal vesicle breakdown, confirming their capability to mature in vivo. Intracytoplasmic sperm injection (ICSI) showed the oocytes in both mutant models to be fertilizable and 70% of the resulting embryos proceeded to a blastocyst stage, independent of the genotype. In conclusion, Nr5a2 regulates female fertility along the entire process of the follicular development. Nr5a2 is shown to be essential for luteinization and its disruption in ovarian somatic cells does not compromise oocyte fertilizability. In overview, we provided a novel and comprehensive investigation, using multiple models and techniques to determine the mechanisms by which Nr5a2 regulates the important processes of cumulus expansion, ovulation and formation of the corpus luteum.

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

The nuclear receptor superfamily is responsible for regulating the expression of genes involved in development, reproduction and metabolism. These transcription factors control the expression of their target genes through the binding of small molecule regulators to their ligand binding domains. Classical nuclear receptors include the steroid receptors, which bind endocrine hormones and have been important targets of pharmaceutical intervention. However, approximately one half of the human nuclear receptors remain orphans and are without known cognate ligands. Focusing on the Drosophila orthologues of these orphan receptors, this project used mass spectrometry to identify the chemical diversity associated with the receptors following expression in recombinant systems. In a genome-wide screen of Drosophila nuclear receptors, this approach identified co-purifying molecules with a number of receptors. The physiological relevance of these putative ligand/receptor pairs was determined through biochemical analysis, in vivo characterization and structure determination. Ligand(s) or the ligand state was identified for the Drosophila receptors: DHR3, DHR96, E75, Ftz-f1 and USP. Of these, three were validated through the efforts of this project, and independent groups confirmed the remaining two. The most significant findings were the discoveries that the fly nuclear receptor E75 is regulated by heme, gas and redox, and that there is a similar regulatory scheme in the human orthologues, Reverbα and β. Furthermore, crystallization of the heme-bound Rev-erbβ ligand binding domain was also achieved, and this provided key insights into the mechanism of ligand regulation for the Rev-erbs. This project highlighted the role of nuclear receptors in metabolic surveillance. The ligands/signals identified in association with these receptors include: cholesterol, dehydrocholesterol, heme, NO, CO, redox and phospholipids. Unlike the classical steroid hormones, these are not dedicated signaling molecules, but instead are key substrates or products of metabolism. In the context of nuclear receptor signaling, I hypothesize that these metabolites serve as metabolic indicators/signals in the regulation of development and metabolism. Furthermore, four of these Drosophila receptors comprise the ecdysone-response pathway in the developing fly. Taken together, this suggests that both the metabolic state of the organism and steroid hormones drive nuclear receptor regulation of development.

The orphan nuclear receptor EAR-2 (NR2F6) is a gene that I previously found to be expressed at a higher level in clonogenic leukemia single cells than in leukemia cells that can not divide. For this thesis I undertook to perform the first investigations of the roles EAR-2 may play in normal haematopoiesis and in the pathogenesis of acute myelogenous leukaemia. Here, I show that EAR-2 is overexpressed in the bone marrow of patients with MDS, AML and CMML compared to healthy controls and that EAR-2 is a gatekeeper to hematopoietic differentiation. Over-expression of EAR-2 prevents the differentiation of cell lines, while knock down induces their spontaneous differentiation. In vitro, primary bone marrow cells that over-express EAR-2 do not differentiate into granulocytes in suspension culture, but have greatly extended replating capacity in colony assays. In vivo, overexpression of EAR-2 in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome characterised by hypercellular bone marrow, an increase in blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice. Furthermore, animals that are transplanted with grafts of sorted bone marrow develop a rapidly fatal leukemia that is characterized by pancytopenia, enlargement of the spleen, infiltration of blasts into the spleen, liver and peripheral blood. Interestingly, development of leukemia is preceded by expansion of the stem cell compartment. Overexpression of EAR-2 increases the maintenance of KSL primitive bone marrow cells in ex vivo suspension culture, while knockdown of EAR-2 induces rapid differentiation of KSL cells into granulocytes. These data establish that EAR-2 is a novel oncogene that regulates hematopoietic cell differentiation. Furthermore, I show that EAR-2 is also a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of EAR-2 is important for the survival, proliferation and differentiation of T-cell progenitors. Overall, this work establishes that expression of EAR-2 is an important determinant of cell fate decisions in the hematopoietic system.

Sterols and isoprenoids are vital cellular constituents. In studies of their formation, metabolism, and biological effects, the separation and identification of individual isomers present formidable challenges. Approximately 30 unsaturated C27 sterols were prepared and purified as authentic standards to evaluate and develop chromatographic and spectral methods. Novel chromatographic conditions were devised for silver ion HPLC (Ag+-HPLC), a technique that provided unprecedented separations of these closely related sterols. Ag+-HPLC proved very powerful in addressing several important problems in medicine and biology relating to the biochemical effects of sterols and isoprenoids; described herein. Smith-Lemli-Opitz syndrome (SLOS) is a genetic disorder of development associated with the accumulation of unsaturated C27, sterols. My analysis of SLOS and normal blood samples unequivocally demonstrated that only the Delta5,7, Delta5,8, and Delta5,7,9(11) sterols accumulate significantly in SLOS patients. The 19-nor-Delta5,7,9 sterol, which has been reported to accumulate in SLOS blood by several research groups, was not detected in my analyses and was demonstrated to be a GC artifact arising from the thermal decomposition of cholesta-5,8-dien-3beta-ol. Alternative pathways in the late stages of cholesterol biosynthesis relating to the biological origin and metabolism of the beta5,8, beta6,8, and beta6,8(14) sterols were elucidated by incubation of tritium-labeled substrates in rat liver preparations. Also, a simple and rapid colorimetric method was developed for the clinical screening of SLOS. Ag +-HPLC played a critical role in studying a possible biochemical defect in another genetic disorder, Langer-Giedion syndrome, and in isolating isomers of (20R,22R)-dihydroxycholesterol), all-trans geranylgeranoic acid, other isoprenoids, and 4,4-dimethyl-sterols, compounds obtained from novel and efficient chemical syntheses. (20R,22R)-Dihydroxycholesterol proved to be a moderately potent activator of the nuclear orphan receptor LXRalpha, whereas all trans geranylgeranoic acid was found to be an inhibitor. The synthetic 4,4-dimethyl sterols caused a resumption of meiosis in mouse oocytes. Famesoic acid was demonstrated to be an extraordinarily potent substrate inducer of cytochrome P450BM-3.

Background and aims of the study. Prostate cancer (PCa) is one of the most common hormone-dependent cancers in men in Western and also Asian countries. The standard treatment options for localized PCa include surgery and androgen-deprivation therapy (ADT). However, most patients upon ADT therapy invariably relapse and progress to a more aggressive and metastatic stage termed as castration-resistant PCa (CRPC). Accumulating studies indicate that androgen receptor (AR) transcriptional activity is dysregulated during the advanced progression of CRPC. One important mechanism responsible for the growth of CRPC includes increased intra-tumoral androgen synthesis in PCa. Recently, a novel androgen-responsive fusion gene TMPRSS2:ERG formed by fusion between the transmembrane protein TMPRSS2 and transcription factor ERG, has been identified in approximately 50% PCa samples, which results in the aberrant expression of ERG function as oncogenic factor in PCa. Currently, TMPRSS2:ERG is regarded as a significant potential diagnostic and prognostic biomarker for PCa. Estrogen-related receptor alpha-ERRα, the first identified ligand-independent orphan nuclear receptor, is characterized to be up-regulated in advanced cancers, suggesting that ERRα might play important regulatory roles in the malignant progression of PCa. Previous studies showed that ERRα can functionally cross-talk with AR signaling via co-targeting to AR targets and regulate the expression of some steroidogenic enzymes in breast cancer. Based on this background, it is hypothesized that ERRα could functionally regulate the TMPRSS2:ERG fusion gene and play a regulatory role in the development and progression of CRPC through activation of the intracellular androgen synthesis pathway.<br>Results. 1) The results obtained in this study showed that suppression of ERRα by its specific inverse agonist XCT790 or shRNA-knockdown could induce down-regulation of TMPRSS2:ERG and also its target genes in AR-positive VCaP PCa cells. 2) Ectopic expression of ERRα and/or its coactivator PGC-1α could increase the expression of TMPRSS2:ERG in AR-negative NCI-H660 PCa cells. 3) Two ERRα-DNA binding elements were identified by ChIP assay and sequence analysis in the promoter of TMPRSS2:ERG and both of these two elements could be transactivated by ERRα and PGC-1α. 4) Ectopic expression of TMPRSS2:ERG under the regulation of ERRα enhanced the prostatic cell invasion capacity as shown in the TMPRSS2:ERG infectants of BPH-1 and PC-3 prostatic cells. 5) ERG expressed by the TMPRSS2:ERG fusion could directly transactivate the ERRα gene in prostatic cells. 6) A positive correlation on the expressions between TMPRSS2:ERG and ERRα was demonstrated in a xenograft model of CRPC (VCaP-CRPC). 7) The expression of TMPRSS2:ERG and ERRα showed significant up-regulation and the transactivation activity of ERRα was also enhanced in castration-resistant VCaP-CRPC cells. 8) Ectopic expression of ERRα could promote resistant growth capacity to androgen-deprivation condition in LNCaP PCa cells, whereas shRNA-mediated silence of ERRα could weaken this resistant capacity. Furthermore, ectopic expression of ERRα in LNCaP-ERRα infectants could promote their in vivo growth resistance to castration in SCID mice. 9) Expression of several androgenic enzyme genes, including CYP11A1, CYP17A1 and ARK1C3, were detected to be up-regulated in castration-resistant VCaP-CRPC cells. Moreover, ectopic expression of ERRα could induce the increased expression of these enzyme genes in LNCaP-ERRα infectants, whereas knockdown of ERRα by shRNA could decrease their expression. 10) ERRα could directly transactivate the gene promoters of CYP11A1, CYP17A1 and ARK1C3 which contain ERRE elements prediction by sequence analysis. These results suggested that ERRα could play a role in de novo or intra prostatic androgen synthesis in the PCa cells.<br>Conclusions. The results obtained in this study suggested that ERRα and TMPRSS2:ERG could form a positive reciprocal loop in PCa cells, and ERRα could also promote the resistant growth capacity of PCa cells resistant to the androgen-deprivation condition in vitro and also castration-resistant growth in vivo via a mechanism of up-regulation of androgenic enzyme genes. The results also suggested that ERRα might play a significant regulatory role in the development and progression of PCa, particularly the advanced CRPC, and also ERRα could be a potential therapeutic target for the treatment of PCa, particularly the advanced PCa-CRPC.<br>研究背景與研究目的：前列腺癌作為激素依賴的一種癌症，經常出現在西方和亞洲國家的男性人群中。對於局限性前列腺癌多採用外科手術和去勢的治療。但是大多數病人經過去勢治療后會再次復發並且形成更加惡心幾轉移的前列腺癌，稱之為去勢難治性前列腺癌（CRPC）。越來越多的研究表明在去勢難治性前列腺癌發病過程中，雄激素受體轉錄活性異性增強。其中一個重要機理解釋為前列腺癌細胞自身合成的雄激素增多。進來，在大約50%的前列腺癌病人中新檢測到一個受雄激素受（AR）體調控的融合基因TMPRSS2:ERG，它是由稱為TMPRSS2的一個跨膜蛋白和一個稱為ERG的轉錄因子融合而成，它的出現導致了在前列腺癌中異常的稱為致癌因子的ERG蛋白的高表達。目前，TMPRSS2:ERG已經被作為一個重要的潛在的診斷和預測的標誌物應用在前列腺癌中。作為第一個鑒定的配體不依賴的孤兒受體-ERRα，被證明在晚期的癌症中有很高的表達，預示著ERRα可能在惡性的癌症中起到一個非常重要的調控作用。之前的研究表明通過共同調控AR的下游基因，ERRα同AR信號通路之間有功能性的交叉調控；除此之外，在乳腺癌中，ERRα還可以調控一些類固醇類化合物的合成相關的一些酶的合成。依據上述，我們推定ERRα可能功能性地調控TMPRSS2:ERG融合基因的表達並且通過調控細胞內的雄激素的合成進而在去勢難治性前列腺癌的發生和發展中起到一個非常重要的作用。<br>結果：本論文研究結果總結如下：1）在有AR表達的前列腺癌細胞-VCaP細胞中，通過ERRα特異性的抑制劑XCT790處理或者shRNA介入的干擾ERRα的mRNA的方法來抑制ERRα，下調了TMPRSS2:ERG和它的一些下游調控基因的表達。2）在沒有AR表達的前列腺癌細胞-NCI-H660細胞中，上調ERRα或者它的特異性的共激活因子PGC-1α表達可以提升TMPRSS2:ERG的表達。3）通過ChIP實驗，在TMPRSS2:ERG的啟動子上面，兩個ERRα的DNA結合位點被鑒定出來。並且這兩個位點可以被ERRα和PGC-1α轉錄激活。4）在兩個前列腺細胞BPH-1和PC-3細胞中，在ERRα的調控下高表達TMPRSS2:ERG融合基因可以增強細胞的侵襲能力。5）融合基因TMPRSS2:ERG導致的ERG蛋白的表達可以直接轉錄激活ERRα的表達。6）我們通過VCaP細胞的異種移植建立VCaP-CRPC的體內模型來模擬CRPC過程，在整個過程中，我們發現TMPRSS2:ERG和ERRα有一致性的表達相關性。除此之外，我們根據上述動物模型通建立了VCaP-CRPC細胞系，並且發現在VCaP-CRPC細胞細胞中，TMPRSS2:ERG和ERRα都有被上調並且ERRα的轉錄活性同樣也提升。7）在LNCaP細胞中高表達ERRα可以提升細胞在去除雄激素的環境中生長的能力。但是當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減弱這種生長的能力。用LNCaP-ERRα穩轉ERRα的細胞異種移植建立SCID老鼠體內腫瘤模型，我們發現和LNCaP-pBABE對照組相比，LNCaP-ERRα細胞生長的更快更大。並且在對老鼠進行睪丸切除術后，LNCaP-ERRα組細胞更快適應這種環境并繼續生長，相比之下，LNCaP-pBABE對照組則持續萎縮減小。8）在上述的VCaP-CRPC細胞中，我們發現一些和雄激素合成相關的關鍵的酶包括CYP11A1，CYP17A1和ARK1C3的表達量有顯著地提升。而且在LNCaP-ERRα細胞中同樣檢測到這些酶的表達量的提升。然而當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減降低上述酶的表達。9）我們在CYP11A1，CYP17A1和ARK1C3基因的啟動子區域發現有ERRα結合位點，並且發現這些位點可以被ERRα轉錄激活。<br>結論：本論文的研究結果提示在前列腺癌細胞中，ERRα和TMPRSS2:ERG可以形成一個相互正向調控的循環。除此之外，上調ERRα可以促進細胞在去除雄激素的環境中生長的能力，並且在動物體內可以提升細胞在睪丸去除的環境中的適應和生長能力。這種體內和體外的能力的提升是通過一種潛在的上調前列腺癌細胞的雄激素合成相關的關鍵的酶的表達，進而提升雄激素的含量而得以實現的。上述的結果預示著ERRα可能在前列腺癌發生機發展的過程中起到非常重要的調控作用，尤其在晚期的CRPC中。同時，ERRα也可能作為一個潛在的重要的前列腺癌尤其是晚期的CRPC的治療靶點，尤其是一些潛在ERRα的特異性抑制劑，比如XCT790，可能作為將來用以作為治療前列腺癌的特異性靶點藥物。<br>Xu, Zhenyu.<br>Thesis Ph.D. Chinese University of Hong Kong 2014.<br>Includes bibliographical references (leaves 126-143).<br>Abstracts also in Chinese.<br>Title from PDF title page (viewed on 05, October, 2016).<br>Xu, Zhenyu.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.