This PhD aimed to elucidate the mechanisms by which polymorphisms may alter androgen-induced transactivation of androgen receptor (AR) target genes which may be important in prostate cancer aetiology. The second aspect of this PhD focused on identifying and characterising functional polymorphisms that may have utility as predictive risk indicators for prostate cancer and which may aid in earlier therapeutic intervention and better disease management. Analyses were carried out on the kallikrein-related peptidase 3 (KLK3), also known as the prostate specific antigen (PSA), gene and the kallikrein-related peptidase 4 (KLK4) gene. The PSA and KLK4 genes are part of the serine protease family that have trypsin or chymotrypsin like activity and are thought to play a role in the development of hormone-dependent cancers in tissues such as those in the prostate, breast, endometrium and ovaries. In the prostate, PSA is regulated by androgens and three androgen response elements (AREs) have been described in the promoter and upstream enhancer region. The PSA ARE I harbours a polymorphism at -158 bp from the transcription initiation site (TIS) that results in a G to A transition (G-158A). This PhD investigated the functional significance of the PSA G-158A polymorphism which has been reported to be associated with prostate cancer risk. Electromobility shift assays (EMSAs) investigating the interaction of ARE I variants with the AR DNA binding domain (AR-DBD) demonstrated that the A allele had a two-fold increased binding affinity for the AR-DBD when compared with the G allele. This was confirmed with endogenous AR in limited proteolysis-EMSA experiments. The limited proteolysis-EMSA experiments also demonstrated differential sensitivities of PSA ARE I alleles to trypsin digestion, which suggests that the G-158A polymorphism has an allosteric effect on the AR that alters AR/ARE I complex stability. Furthermore, Chromatin Immunoprecipitation (ChIP) assays suggest that the A allele more readily recruited the AR in vivo when compared with the G allele and is consistent with the in vitro binding data. Luciferase reporter assays carried out in both LNCaP and 22Rv1 prostate cancer cells, and using the natural (dihydrotestosterone; DHT) ligand demonstrated that the A allele was more responsive to androgens in LNCaP cells. Hence, this study has elucidated the potential mechanisms by which the G-158A polymorphism may differentially regulate PSA expression (of which up-regulation of PSA is thought to be important in prostate cancer development and progression). KLK4 has similar tissue-restricted expression as PSA and is up-regulated by steroid hormones in many endocrine cells including those in the prostate. A putative ARE (KLK4-pARE) located at -1,005 to -1019 relative to the more predominantly used transcription initiation site, TIS3, was initially found in supershift assays using AR antibodies to interact with endogenous AR. However, subsequent EMSA analysis using purified AR-DBD suggest that KLK4-pARE may be interacting with the AR indirectly. To investigate this hypothesis, a tandem construct of KLK4-pARE was cloned into the pGL3-Promoter vector for hormone-induced reporter assays. However, reporter assays did not demonstrate any responsiveness of KLK4-pARE to androgens, estradiol or progestins. Consequently, Real-Time PCR was carried out to reassess the hormonal regulation of KLK4 at the mRNA level. Consistent with the literature, data from this study suggests that KLK4 may be up-regulated by androgens, progestins and estradiol in a cyclical manner. Hormone-induced luciferase reporter assays were then carried out on seven promoter constructs that span 2.8 kb of the KLK4 promoter from TIS3. However, none of the seven promoter constructs demonstrated any significant responsiveness to androgens, estradiol or progestins. This study suggests that hormone response elements (HREs) that may drive the hormonal regulation of KLK4 in prostate cancer may be located further upstream from the promoter region investigated in this PhD, or alternatively, may lie 3' of TIS3. The characterisation of KLK4 promoter polymorphisms and their flanking sequences were also carried out in parallel to the functional work with the intent to assess the functional significance of any polymorphisms that may be located within HREs. In total 19 polymorphisms were identified from the public databases and from direct sequencing within 2.8 kb of the KLK4 promoter from TIS3. However, the functional and clinical significance of these 19 polymorphisms were not further pursued given the negative findings from the functional work. The PSA AR enhancer region was also assessed for potential polymorphisms that may be associated with prostate cancer risk. A total of 12 polymorphisms were identified in the PSA enhancer of which two (A-4643G and T-5412C) have been reported to alter functionality of the enhancer region and thus, prioritised for further analysis. Association analysis for prostate cancer risk was then carried out on these PSA enhancer polymorphisms as none of the KLK4 promoter polymorphisms were found in functional HREs. No significant association for either the A-4643G or T-5412C polymorphism with prostate cancer risk was found at the P = 0.05 level. However, under an age-adjusted dominant model a 1.22- (95% CI = 1.16-1.26) and 1.23-fold (95% CI = 1.17-1.29) increased risk for prostate cancer was found for the A-4643G or T-5412C polymorphisms, respectively. Both polymorphisms were also assessed for association with tumour grade and stage and PSA levels. Genotypes were significantly different for the A-4643G and T-5412C polymorphisms with tumour stage and PSA levels, respectively. However, these results are likely to be biased by the case population which consist primarily of men who presented with incidental (pT1) and organ-confined (pT2) tumours. To summarise, the A-4643G and T-5412C polymorphisms are unlikely to be associated with prostate cancer risk, PSA levels or stage/grade of disease. However, further analyses in a larger cohort is warranted given that these polymorphisms alter androgen responsiveness of the PSA enhancer and that elevated PSA levels are indicative of men with prostate cancer. To summarise, this PhD has elucidated the functional significance of the PSA G-158A polymorphism in prostate cancer and which may be important in prostate cancer patho-physiology. This PhD has also furthered the understanding of the hormonal regulation of KLK4 in prostate cancer cells. Finally, this PhD has carried out a pilot study on two functional PSA enhancer polymorphisms (A-4643G and T-5412C) with prostate cancer risk.

The inhibitor of DNA binding protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors.1 Recently, Patel et al., demonstrated that inhibitor of differentiation 4 (ID4) acts as a tumor suppressor and its loss, frequently observed in prostate cancer, promotes castration-resistant prostate cancer (CRPC) through constitutive androgen receptor (AR) activation.2 However, the mechanism by which loss of ID4 promotes constitutively active AR signaling in the CRPC conditions is unknown. The rationale of the present study was to unravel the underlying molecular mechanisms through which loss of ID4 potentiates AR signaling in this setting. Initially, chromatin immunoprecipitation (ChIP) assay results demonstrated a significant increase in binding of AR to its respective response elements on PSA, FKBP51, TMPRSS2, and ETV1 promoters in L(-)ID4 cells, further implicating constitutive AR activity. Among the notable findings, proteomic profiling between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated protein levels of Heat shock protein 27 (Hsp27) and the 52-kDa FK506-binding protein (FKBP52), suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies further confirmed a direct interaction between ID4 and FKBP52 in vitro but not with AR. Recent evidences suggest that FKBP52 is a positive regulator of AR signaling in cellular and whole animal models.3-6 Thus, we hypothesized that ID4 acts as a tumor suppressor by selectively regulating AR activity through interaction with FKBP52. To address the underlying mechanism, we blocked the FKBP52-AR signaling using a specific inhibitory compound known as MJC13.4, 6-7 The results demonstrated that MJC13 effectively inhibited AR-dependent expression and activity in a dose-dependent manner. In addition, xenograft studies further confirmed that inhibiting FKBP52-regulated AR activity via MJC13 significantly attenuated the growth of subcutaneous L(-)ID4 xenografts in vivo. Collectively, our results suggested that ID4 selectively regulates AR activity through direct interaction with FKBP52 in vitro, and, its loss promotes CRPC through FKBP52-mediated AR signaling. Increased AR signaling along with a subsequent decrease in ID4 expression levels in prostate cancer strongly supports this model.

O cão é a única espécie, além do homem, em que o câncer de próstata (CP), a neoplasia intraepitelial prostática (PIN) e a hiperplasia prostática benigna (HPB) ocorrem espontaneamente, permitindo dessa forma que se realize estudo comparativo de afecções benignas e malignas da próstata. Acredita-se que a existência de stem cells malignas, localizadas na camada de células basais da próstata, seja um dos fatores responsáveis pelo insucesso da terapia por ablação androgênica que ocorre na maioria dos carcinomas prostáticos avançados. O objetivo deste estudo foi caracterizar a próstata canina quanto a aspectos envolvidos na evolução para o carcinoma prostático, tentando identificar a origem celular e as alterações das lesões pré-neoplásicas. Foram obtidas 44 próstatas na necrópsia. Amostras prostáticas foram fixadas em metacarne, embebidas em parafina e seccionadas a 5µm para a coloração com hematoxilina eosina (HE) e avaliadas em relação à presença de hiperplasia, prostatite, PIN e neoplasia. Além disso, cortes corados em HE representando cada afecção foram utilizados na determinação da área nuclear média por morfometria computadorizada. Cortes histológicos obtidos em lâminas silanizadas foram utilizados na imunoistoquímica para células basais (p63 e 34E-12), conexinas 32 e 43, receptor de andrógeno (AR) e antígeno nuclear de proliferação celular (PCNA). Amostras foram coletadas também em nitrogênio líquido e mantidas a 80o C para a realização do PCR quantitativo em tempo real, para a determinação da expressão do RNAm do AR, e para a realização do Western blot, para a determinação da expressão da conexina 43. As afecções mais freqüentes foram a prostatite e a hiperplasia prostática benigna. Foi observada uma maior porcentagem de células basais e um alto índice proliferativo, como demonstrado pela imunoistoquímica para o PCNA, na neoplasia intraepitelial prostática. Além disso, observou-se nessas lesões marcação nuclear heterogênea para o AR, menor em relação à dos ácinos benignos. Ao contrário do observado na próstata humana, não foi observada expressão das conexinas 32 e 43 na próstata canina (normal ou com PIN). A área nuclear média, obtida pela morfometria computadorizada, foi maior em células epiteliais de ácinos apresentando PIN e/ou neoplasia em relação à de células epiteliais de ácinos benignos. Observou-se expressão variável do RNAm para o AR nas PINs e neoplasias, utilizando-se o PCR em tempo real. Estes achados sugerem que células basais malignas desempenham papel na origem da neoplasia intraepitelial prostática e possuem capacidade de proliferar a despeito da expressão heterogênea do receptor de andrógeno. / Dogs are the only animal other than man to develop prostate cancer, prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (HPB) spontaneously, allowing the comparison between benign and malignat affections of prostate. Malignant stem cells among the basal cell layer of the prostate are believed to play an important role in the failure of androgen-ablation therapy that occurs in most advanced prostate cancer. The goal of this study was to characterize the canine prostate in relation to evolution to prostatic carcinoma, trying to identify the cellular origin and the alterations of pre-neoplastic lesions. Forty-four canine prostates were obtained at necropsy. Prostatic samples were fixed in methacarn, embedded in paraffin wax and sectioned into 5µm-thick slices for hematoxylin eosin (HE) staining and evaluated for the presence of hyperplasia, prostatitis, PIN and neoplasia. Moreover, HE stained sections representing each affection were used to determine the mean nuclear area by computerized morphometry. Tissue sections obtained in silanized slides were used in immunohistochemical staining for basal cells (p63 and 34E-12), connexins 32 and 43, androgen receptor (AR) and proliferating-cell nuclear antigen (PCNA). Quantitative real-time PCR to determine the expression level of AR at the mRNA level and Western blot to protein levels of connexin 43 were examined in samples collected using liquid nitrogen and kept at 80o C. The most common lesions were prostatitis and benign prostatic hyperplasia. The prostatic intraepithelial neoplasia exhibited a higher percent of basal cells and was highly proliferative, as demonstrated by PCNA immunohistochemistry. Moreover, these lesions exhibited heterogeneous nuclear AR staining, lower in comparision with benign acini. In contrast to human prostate, the canine prostate (normal or harboring PIN) did not express the connexins 32 and 43. The mean nuclear area measured by computerized morphometry was greater in epithelial cells of PIN and neoplastic acini than that of benign acini. We found variable RNAm AR expression in prostatic intraepithelial neoplasia and neoplasia by real-time PCR. These findings suggest that malignant basal cells may play a role in the origin of PIN and can proliferate despite the heterogeneous AR expression.

O cão é a única espécie, além do homem, em que o câncer de próstata (CP), a neoplasia intraepitelial prostática (PIN) e a hiperplasia prostática benigna (HPB) ocorrem espontaneamente, permitindo dessa forma que se realize estudo comparativo de afecções benignas e malignas da próstata. Acredita-se que a existência de stem cells malignas, localizadas na camada de células basais da próstata, seja um dos fatores responsáveis pelo insucesso da terapia por ablação androgênica que ocorre na maioria dos carcinomas prostáticos avançados. O objetivo deste estudo foi caracterizar a próstata canina quanto a aspectos envolvidos na evolução para o carcinoma prostático, tentando identificar a origem celular e as alterações das lesões pré-neoplásicas. Foram obtidas 44 próstatas na necrópsia. Amostras prostáticas foram fixadas em metacarne, embebidas em parafina e seccionadas a 5µm para a coloração com hematoxilina eosina (HE) e avaliadas em relação à presença de hiperplasia, prostatite, PIN e neoplasia. Além disso, cortes corados em HE representando cada afecção foram utilizados na determinação da área nuclear média por morfometria computadorizada. Cortes histológicos obtidos em lâminas silanizadas foram utilizados na imunoistoquímica para células basais (p63 e 34E-12), conexinas 32 e 43, receptor de andrógeno (AR) e antígeno nuclear de proliferação celular (PCNA). Amostras foram coletadas também em nitrogênio líquido e mantidas a 80o C para a realização do PCR quantitativo em tempo real, para a determinação da expressão do RNAm do AR, e para a realização do Western blot, para a determinação da expressão da conexina 43. As afecções mais freqüentes foram a prostatite e a hiperplasia prostática benigna. Foi observada uma maior porcentagem de células basais e um alto índice proliferativo, como demonstrado pela imunoistoquímica para o PCNA, na neoplasia intraepitelial prostática. Além disso, observou-se nessas lesões marcação nuclear heterogênea para o AR, menor em relação à dos ácinos benignos. Ao contrário do observado na próstata humana, não foi observada expressão das conexinas 32 e 43 na próstata canina (normal ou com PIN). A área nuclear média, obtida pela morfometria computadorizada, foi maior em células epiteliais de ácinos apresentando PIN e/ou neoplasia em relação à de células epiteliais de ácinos benignos. Observou-se expressão variável do RNAm para o AR nas PINs e neoplasias, utilizando-se o PCR em tempo real. Estes achados sugerem que células basais malignas desempenham papel na origem da neoplasia intraepitelial prostática e possuem capacidade de proliferar a despeito da expressão heterogênea do receptor de andrógeno. / Dogs are the only animal other than man to develop prostate cancer, prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (HPB) spontaneously, allowing the comparison between benign and malignat affections of prostate. Malignant stem cells among the basal cell layer of the prostate are believed to play an important role in the failure of androgen-ablation therapy that occurs in most advanced prostate cancer. The goal of this study was to characterize the canine prostate in relation to evolution to prostatic carcinoma, trying to identify the cellular origin and the alterations of pre-neoplastic lesions. Forty-four canine prostates were obtained at necropsy. Prostatic samples were fixed in methacarn, embedded in paraffin wax and sectioned into 5µm-thick slices for hematoxylin eosin (HE) staining and evaluated for the presence of hyperplasia, prostatitis, PIN and neoplasia. Moreover, HE stained sections representing each affection were used to determine the mean nuclear area by computerized morphometry. Tissue sections obtained in silanized slides were used in immunohistochemical staining for basal cells (p63 and 34E-12), connexins 32 and 43, androgen receptor (AR) and proliferating-cell nuclear antigen (PCNA). Quantitative real-time PCR to determine the expression level of AR at the mRNA level and Western blot to protein levels of connexin 43 were examined in samples collected using liquid nitrogen and kept at 80o C. The most common lesions were prostatitis and benign prostatic hyperplasia. The prostatic intraepithelial neoplasia exhibited a higher percent of basal cells and was highly proliferative, as demonstrated by PCNA immunohistochemistry. Moreover, these lesions exhibited heterogeneous nuclear AR staining, lower in comparision with benign acini. In contrast to human prostate, the canine prostate (normal or harboring PIN) did not express the connexins 32 and 43. The mean nuclear area measured by computerized morphometry was greater in epithelial cells of PIN and neoplastic acini than that of benign acini. We found variable RNAm AR expression in prostatic intraepithelial neoplasia and neoplasia by real-time PCR. These findings suggest that malignant basal cells may play a role in the origin of PIN and can proliferate despite the heterogeneous AR expression.

Androgens are critical for the development and maintenance of adult male characteristics such as muscle mass and sexual function. Consequently, the established decline with age of serum testosterone (T) in males has major health implications. While the androgen receptor (AR) is the major mediator of genomic androgen action and is required for the development of the male phenotype, reproductive organs and the maintenance of male secondary sexual characteristics, it is the entrance of androgens into the cell that mediates the activation of the AR and the subsequent modulation of expression of androgen regulated genes. Testosterone, biologically the most important androgen in male serum, circulates either free, loosely bound to albumin or tightly bound to sex hormone binding globulin (SHBG). Each of these forms of serum T have different abilities to enter cells, and which proportion of serum T is capable of entering cells and initiating the androgen signalling cascade, thereby leading to the activation of the AR has not been precisely defined. The AR amino terminal domain (NTD) is responsible for the majority of the ability of the AR to activate genes but the relative roles of the two activation functions in the AR NTD (activation functions 1 and 5; AF1 and 5) have not been precisely defined while the role of the AF2 surface which forms in the ligand binding domain upon agonist binding is responsible for interactions with key coregulators and also with the NTD in the amino-carboxyl (N/C) interaction. Our laboratory has recently identified a region within AF5 between amino acids 500-535 to which somatic mutations in castrate resistant prostate tumour samples collocate. Due to the lack of functional information on the AF5 region and the NTD in general, the function of this region and the functional consequences of the mutations remain to be defined. The objectives of this thesis were to develop a specific mammalian cell based bioassay capable of reliable measuring T in serum and to determine the ability of this bioassay to measure a physiologically relevant fraction of T in serum. Additionally, this thesis aimed to determine the relative contributions and roles of the activation functions of the AR to overall AR transcriptional activity along with the functional consequences for AR signalling of prostate cancer mutations which have previously been identified in the AF5 region of the AR NTD. The mammalian-cell based bioassay developed in this thesis is capable of sensitively and reliably measuring serum T. However, evaluation of this bioassay utilising approximately 1000 serum samples from the Florey Adelaide Male Aging Study reveals that this bioassay measures a fraction of T in serum that most closely relates to serum T. Furthermore, this measure does not correlate more strongly with grip strength, sexual function or waist circumference than the existing immunoassay-based measures of serum T, highlighting the limitations of utilising a static mammalian cell-based androgen bioassay to measure physiological levels of serum T in males. The investigation of the roles of the activation functions in the AR in this thesis have revealed that while the AF1 domain is responsible for the majority of the transactivation activity of the AR, AF5 and AF2 govern the sensitivity and cellular response of the AR to androgens by providing protein and interdomain interaction interfaces. Furthermore, the evidence in this thesis demonstrates that the AR requires interdomain communication for sensitive AR signalling. Finally, the findings in this thesis demonstrate that the AF5 surface is required for the N/C interaction and coregulator interactions while advanced prostate cancer mutations identified within this region confer increased transactivation activity of the AR in the presence of high cellular levels of coregulators. Collectively, the findings in this thesis provide several novel insights into the mechanism of action of serum androgens and challenges several long held assumptions of androgenic action in males. These findings also delineate a mechanism of treatment failure in advanced prostate cancer, provide a novel model for the events leading to sensitive AR transactivation and contribute to the understanding of physiologically relevant levels of serum T. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Recently reported data demonstrate that Vav3, a Rho Guanine Nucleotide Exchange Factor (Rho GEF) is overexpressed in breast tumors, coexpressed with ER, necessary for proliferation in breast cancer cells, and predictive of response to neoadjuvant endocrine therapies in patients with ER+ tumors. Such data beg the question as to what roles Vav3 plays in modulation of steroid receptor activity in breast cancer and in resistance to current hormonal therapies. Using reporter assays, I provide novel evidence that Vav3 potentiates Estrogen Receptor activity and represses Androgen Receptor activity in breast cancer cells. Vav3 potentiates ligand-dependent estrogen receptor activity in the MCF-7. A truncated, constitutively active form of Vav3, caVav3 potentiates ligand dependent ER activity in both MCF-7 and T47D. Vav3 activates Rho GTPases through its GEF domain. ER potentiation by caVav3 is dependent upon GEF activity. A caVav3 mutant with defective GEF function represses basal and ligand-mediated ER activity in T47D. Although other studies have shown that Vav3 could activate various Rho GTPases, only constitutively active Rac1 mutants potentiated ER activity in both cell lines. Contrastingly, reporter assays were used to show that caVav3 inhibits ligand-mediated AR activity in the AR+ T47D cell line by both R1881 and DHT stimulation. caVav3-mediated repression of AR activity is GEF-dependent, as caVav3 GEF mutants potentiate AR activity. Constitutively active forms of Rho GTPases were found to repress AR activity to different extents, but R1881-mediated AR activity was only significantly repressed by caCdc42. My studies of the effect of androgens on AR protein by western blot show that androgens downregulate AR protein in the highly Vav3 positive T47D cell line. Previous studies have demonstrated that androgens stabilize AR protein in MCF-7, and I now provide evidence that overexpression of Vav3 or caVav3 reverses hormone-mediated AR protein stabilization in MCF-7. These data are especially relevant given recently published data that decreased AR protein levels contributed to failure of response to MPA in patients with metastatic breast cancer. Further breast cancer studies may prove Vav3 to be a potential drug target in hormone dependent, hormone independent, and metastatic disease.

Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

Η συμμετοχή της γενετικής, έναντι της περιβαλλοντικής επίδρασης στη συμπεριφορά αποτελεί θεμελιώδες ερώτημα για τις νευροεπιστήμες και αποτελεί πεδίο έντονου ερευνητικού ενδιαφέροντος. Η σεξουαλικότητα είναι μια σύνθετη αλληλεπίδραση πολλαπλών παραγόντων, συμπεριλαμβανομένων ανατομικών, φυσιολογικών, ψυχολογικών, αναπτυξιακών, πολιτιστικών και σχεσιακών παραγόντων. Παρά την υψηλή συχνότητα εμφάνισης της γυναικείας σεξουαλικής δυσλειτουργίας, λιγότερη έμφαση έχει δοθεί στη μελέτη της από την επιστημονική κοινότητα. Το βιολογικό και ψυχολογικό υπόβαθρό της παραμένει ένα υποσχόμενο πεδίο έρευνας καθώς οι διαθέσιμες θεραπείες είναι πολύ λιγότερες συγκριτικά με την ανδρική σεξουαλική δυσλειτουργία. Η σεξουαλική λειτουργία των γυναικών έχει μελετηθεί κατά καιρούς στο σύνδρομο των πολυκυστικών ωοθηκών, λαμβάνοντας υπόψη ερωτηματολόγια σεξουαλικής δραστηριότητας και επίπεδα φυλετικών ορμονών αλλά όχι τους γενετικούς πολυμορφισμούς που μπορεί να εμπλέκονται και να δημιουργούν συγκεκριμένο βιολογικό υπόβαθρο. Η αλληλεπίδραση ορμονών, νευροδιαβιβαστών και περιβαλλοντικών παραγόντων είναι ευρέως αποδεκτή στη διαμόρφωση του υποστρώματος της γυναικείας σεξουαλικότητας αλλά οι τρόποι παραμένουν ακόμα ασαφείς. Για το λόγο αυτό, ο σκοπός της παρούσας μελέτης ήταν η συσχέτιση των πολυμορφισμών του ανδρογονικού υποδοχέα και του υποδοχέα της βαζοπρεσίνης με τη γυναικεία σεξουαλικότητα στις γυναίκες με σύνδρομο πολυκυστικών ωοθηκών. Η επίδραση των ανδρογόνων στη γυναικεία σεξουαλικότητα αποτελεί πεδίο έντονου ερευνητικού ενδιαφέροντος καθώς οι μηχανισμοί αλληλεπίδρασης είναι ιδιαίτερα πολύπλοκοι. Τα ανδρογόνα ασκούν τη δράση τους μέσω πρόσδεσης και ενεργοποίησης των ανδρογονικών υποδοχέων. Το γονίδιο του υποδοχέα των ανδρογόνων αποτελείται από δύο μοτίβα πολυμορφικών επαναλήψεων CAG & GGN που κωδικοποιούν ποικίλου μήκους πολυγλουταμινικών και πολυγλυκινικών περιοχών αντίστοιχα. Έχει επίσης φανεί ότι το αυξημένο μήκος της CAG επαναληπτικής αλληλουχίας πιθανόν να σχετίζεται με μειωμένη δραστικότητα του AR και ως εκ τούτου και με διαταραχές που σχετίζονται με μειωμένη δράση ανδρογόνων Διάφορα νευροπεπτίδια όπως η βαζοπρεσίνη, η αδενοκορτικοτροπίνη, η ωκυτοκίνη κ.α. επιδρούν στην ενήλικο σεξουαλική συμπεριφορά διαφόρων οργανισμών. Η βαζοπρεσίνη και ο υποδοχέας της αποτέλεσαν αντικείμενο μελέτης για την ερμηνεία της ανθρώπινης κοινωνικής και σεξουαλικής συμπεριφοράς. Οι πολυμορφισμοι του AVPR έχουν επίσης σχετιστεί με αλτρουισμό, με μονογαμία και ανάπτυξη σχέσεων δεσμού, γνώση μουσικής και χορού που αντανακλούν αρχέγονες κοινωνικές αλληλεπιδράσεις όπως ιεροτελεστικές κινήσεις και επικοινωνία μέσω ήχων. Το γονίδιο του υποδοχέα της βαζοπρεσίνης διαθέτει τέσσερα μικροδορυφορικά μοτίβα. Ακολουθώντας τις μελέτες στον αρουραίο του αγρού (vole), η προσοχή έχει κυρίως επικεντρωθεί στις μικροδορυφoρικές επαναλήψεις στην περιοχή του υποκινητή. Πρόκειται για τις RS1 {(GATA)14} και RS3 {(CT)4-TT-(CT)8-(GT)24}, που είναι εξαιρετικά πολυμορφικές. Σκοπός της παρούσας εργασίας είναι η διερεύνηση της συσχέτισης της πολυμορφικής CAG περιοχής του ανδρογονικού υποδοχέα και του RS1 πολυμορφισμού του υποδοχέα της βαζοπρεσίνης με την γυναικεία σεξουαλική συμπεριφορά στο σύνδρομο των πολυκυστικών ωοθηκών. Για το λόγο αυτό η παρούσα μελέτη συμπεριέλαβε 40 γυναίκες με σύνδρομο πολυκυστικών ωοθηκών και 94 υγιείς γυναίκες, στις οποίες διενεργήθηκαν ορμονικοί προσδιορισμοί, ψυχομετρικά τεστ για αξιολόγηση της σεξουαλικής λειτουργίας τους και διερεύνηση της συσχέτισης με τα γονοτυπικά τους χαρακτηριστικά (αριθμός επαναλήψεων των πολυμορφικών μοτίβων στα αλληλόμορφά τους). Τα αποτελέσματα της παρούσας εργασίας έδειξαν ότι στην κατηγορία των γυναικών με PCOS η ενεργότητα του υποδοχέα συσχετίστηκε με μειωμένα επίπεδα oιστρογόνων και με αυξημένη ικανοποίηση, γεγονός που υποδηλώνει ότι σε καθεστώς περίσσειας ανδρογονικού ερεθίσματος η γυναικεία σεξουαλικότητα επάγεται. Επίσης στην ίδια ομάδα γυναικών φάνηκε συσχέτιση μεταξύ των υψηλών επιπέδων FSH και των υψηλών αριθμών επαναλήψεων του RS1 πολυμορφισμού, υποδεικνύοντας έναν κεντρικό ρόλο του AVPR στη ρύθμιση της ωοθυλακιορρηξίας των γυναικών με σύνδρομο πολυκυστικών ωοθηκών. / The contribution of genetic versus environmental influence in behavioral analysis is a fundamental question for neuroscience and it is also an area of strong research interest, Sexuality is distinguished by a complex interaction between anatomic, physiologic, psychological, developmental, relational and cultural factors. Despite the high frequency of sexuality disorders in women, scientists have not placed emphasis on this. The biological and psychological background of women’s sexuality disorder still remains a promising field of research, since the available therapies are fewer than those that are used in male sexual dysfunction. Female sexuality has been studied frequently in women with PCOS and has been based on questionnaires of female’s sexual functionality and serum levels of sex steroid hormones. These studies didn’t take account of the genetic polymorphisms which can be involved in a specific biological background. The interaction of hormones, neurotransmitters and environmental factors is widely accepted in the composition of female’s sexual function but the ways that this interaction happens are still unclear. Thus, the aim of our study was to consider the possible association between the genetic polymorphism of androgen receptor gene and vasopressin receptor gene and female sexuality in women with PCOS. The influence of androgens in female sexuality is a field of intense interest in the scientific community, but the ways this interaction occurs are very complicated. Androgens bind and activate androgen receptors.The androgen receptor gene consists of eight exons and encodes a protein with 919 amino acid residues. Exon 1 of the gene consists of two polymorphic repeat (CAG and GGN) motifs, encoding variable lengths of polyglutamine and polyglycine stretches, respectively. Also, it has been proposed that that the increased length of the CAG repeat should associate with decreased AR activity and hence the disorders related to the reduced androgen actions. Many neuropeptides such as vasopressin, oxytocin, adrenocorticotropic hormone (ACTH) etc, affect sexual behavior in many species. Vasopressin and it’s receptor has been well studied in order to interpret human social and sexual behavior. The genetic polymorphisms of the vasopressin receptor gene has been also associated with altruism, monogamy, pair bonding, musical and dancing ability. The latter reflects primitive social interactions such as ritual movements and vocalization. Vasopressin receptor gene is distinguished by three microsatellites in the 5’ flanking region and a fourth in the single intron. Following the vole studies, attention has been primarily focused on two microsatellites in the promoter region, RS1 {{GATA)14} and RS3 {(CT)4-TT-(CT)8-(GT)24}, which are highly polymorphic. The aim of our study is to investigate the association between the polymorphic CAG region of androgen receptor gene and RS1 polymorphism of vasopressin receptor gene with sexual behavior in women with PCOS. Thus, our study included 40 women with PCOS and 94 healthy women. We performed hormonal analysis, psychometric tests to evaluate their sexual functionality and looked into the association with their genetic characteristics (the number of repeats of polymorphic motifs in their alleles). Our results showed that androgen receptor’s activity is associated with low estrogen levels and high sexual satisfaction in women with PCOS. This indicates that in a state of androgen excess, female sexuality is induced. In the same group of women, we noted an association between high levels of FSH and a high number of repeats of RS1 polymorphism. This suggests a central role of vasopressin receptor in the regulation of ovulation in women with PCOS.

This thesis aims to study the effects of endogenous and exogenous hormones and mammographic breast density (BD) on cellular markers in non-cancerous female breast tissue. Women on the waiting list for breast reduction plastic surgery were recruited (n = 79), and randomized to 2 months of hormone therapy or no therapy before surgery. The women had a mammogram and a needle biopsy 2 months before surgery and tissue samples were obtained at the operation. In premenopausal women, estrogen receptor (ER)α levels were associated with age (p = 0.0002), were similar in the follicular and luteal phases of the menstrual cycle and were higher in parous than in nulliparous women (p = 0.009). Current smokers had lower PR levels than non-smokers (p = 0.019). Women on oral contraception had lower ERα (p = 0.048) and PR (p = 0.007) levels than women in the follicular phase. The ERα levels did not differ significantly between postmenopausal estrogen and estrogen-progestogen users, but PR levels were lower among estrogen-progestogen users (p = 0.03). We found lower expression of the genes for decorin and syndecans 1 and 4 in the luteal phase than in the follicular phase, among parous women. Protein levels of the androgen receptor, syndecan-4 and decorin was lower in premenopausal women who were using oral contraceptives (OC) than in those in the follicular phase (p = 0.002 - 0.02), whereas no significant differences between OC use and the luteal phase were found. In premenopausal women, BD was negatively associated with age and body mass index but was similar for the menstrual phases. Breast density was associated with genetic expression of the androgen receptor and remained significant after adjustment for age (rs = 0.56; p = 0.04). After adjustement for age, breast density was also marginally associated with expression of the caspase 3 gene (0.55; 0.053). However, protein levels of caspase 3 was negatively associated (-0.61; 0.03).