Purpose: The purpose of this study was to assess whether the factors historically presented in the literature to differentiate vocal cord dysfunction (VCD) from breathing difficulties of other etiologies accurately predict and identify patients who have VCD. The researchers were also interested in whether patients with VCD have a higher risk of misdiagnosis than patients with breathing difficulties of other etiologies. Finally, the present study investigated whether patients with VCD were more likely to have their symptoms attributed to psychological factors than patients with breathing difficulties of other etiologies. Method: A survey comprised of 23 questions regarding the participants’ previous and current diagnoses, triggers that precede breathing difficulty, and whether or not the participants have ever been misdiagnosed was posted to internet support groups, websites, blogs, and forums. The final participant pool included 20 participants with VCD and 25 participants with asthma. Results: None of the factors investigated accurately differentiated participants with asthma from participants with VCD one hundred percent of the time. However, participants with VCD were more likely to report throat tightness during an attack, association of an attack with symptoms of acid reflux, and rapid resolution of symptoms without treatment. Conversely, participants with asthma were more likely to report expiratory stridor and chest tightness, full resolution of symptoms with use of asthma medications, nocturnal symptoms or symptoms just after waking, and symptoms that seemed to be triggered by environmental agents or allergens. Preliminary findings from the present study suggest that patients with VCD are both more likely to receive a misdiagnosis and have their symptoms attributed to psychological factors than participants with asthma. Conclusion: A diagnosis of VCD must be made very carefully, ideally with instrumental evaluation of the vocal mechanism during an acute “attack” of breathing difficulty. The factors identified in the literature to differentially diagnose patients with asthma from patients with VCD do not accurately differentiate these populations. These findings suggest that continued education about the nature of VCD and differential diagnosis should be paramount to medical professionals. / text

Menopause is associated with disorders such as osteoporosis and ovarian cancer. It is unclear whether the postmenopausal ovary retains steroidogenic capacity and how it can impact the development of these disorders. The present studies used the VCD-treated follicle-depleted mouse model of menopause to test the hypothesis that residual ovarian tissue retains steroidogenic capacity following ovarian failure and, thus, affects the development of these disorders. Microarray technology was used to evaluate gene expression in residual ovarian tissue of follicle-depleted mice compared to that in ovaries from cycling animals. Among the genes identified were those encoding proteins for synthesis of androgens. Steroidogenic capacity of residual ovarian tissue was further evaluated by determining the expression of genes and proteins involved in ovarian steroidogenesis, and by measuring levels of circulating and rostenedione and gonadotropins. Follicle-depleted ovaries were enriched in mRNAs for androgenic enzymes, receptors involved in the internalization of cholesterol, and luteinizing hormone receptor. Increased circulating levels of FSH and LH and detectable and rostenedione were measured throughout the study. Protein for 3β-hydroxysteroid dehydrogenase, 17α- hydroxylase/17,20-lyase and luteinizing hormone receptor was detected in follicledepleted ovaries by Western blot analysis and localized by immunofluorescence staining. The contribution of retaining residual ovarian tissue to accelerated bone loss following ovarian failure was evaluated by comparing bone mineral density from young and aged VCD-treated mice to that in age-matched ovariectomized (OVX) animals. Retaining residual ovarian tissue resulted in protection against accelerated bone loss in young but not aged VCD-treated mice. Whether residual ovarian tissue is more susceptible to development of ovarian neoplasms compared to ovaries from cycling animals was addressed by combining the VCD-treated mouse with the DMBA model of ovarian carcinogenesis. VCD-treated follicle-depleted mice that received DMBA developed Sertoli-Leydig cell tumors while no tumors were observed in cycling animals. Residual ovarian tissue following ovarian failure appears to have a protective effect against loss of bone integrity, but a detrimental effect on development of ovarian neoplasms. Findings from these studies: provided evidence of a physiological role for residual ovarian tissue following ovarian failure, and furthered the use of the VCD-treated mouse as a relevant model for menopause and associated disorders.

Kidney disease is a major complication of diabetes and accounts for one-third of all diabetes-related deaths. Estrogen is considered protective against cardiovascular and non-diabetic renal disease, however it is unclear if this protection extends to diabetes and diabetic kidney disease.To address these questions, we have used a new model of menopause in which repeated daily injections of 4-vinylcyclohexene (VCD) induces gradual ovarian failure in mice. Unlike with ovariectomy, the VCD model preserves the gradual transition into ovarian failure (OF) (modeling perimenopause). Also, following OF, the residual ovarian tissue is retained and secretes androgens, similar to the androgen production by postmenopausal human ovaries.The VCD model of menopause was combined with the streptozotocin (STZ) model of type 1 diabetes, and the development of diabetes and diabetic kidney damage were studied over the subsequent 6 weeks. We observed that blood glucose levels are higher in post-OF diabetic mice compared to cycling diabetic and peri-OF diabetic mice. Renal cell proliferation, an early marker of kidney damage, is increased in post-OF diabetic mice compared to cycling diabetic mice, as measured by expression of proliferating cell nuclear antigen. We also demonstrate that expression of α-smooth muscle actin is increased in post-OF diabetic mice compared to cycling diabetic mice. Five weeks after STZ injection, post-OF diabetic mice had higher rates of urine albumin excretion than cycling diabetic mice.Using real-time PCR, we identified changes in expression between post-OF diabetic and cycling diabetic mice of genes which have previously been associated with diabetic kidney damage. We also show that some of these changes occur in peri-OF diabetic mice as well. Using microarray, we identified 119 new genes which are regulated by the combination of ovarian failure and diabetes in the mouse kidney.These data support our hypothesis that the changes in hormones which occur during the transition into ovarian failure exacerbate the development and progression of diabetic kidney damage in mice. These data also highlight the utility and importance of the VCD model of menopause in the study of diabetic kidney damage.

Ovarian toxicants 4-vinylcychlohexene (VCH) and 7,12-dimethylbenz[a]anthracene (DMBA) requires bioactivation to induce follicle loss. VCH is bioactivated to monoepoxides (1,2-VCM and 7,8-VCM), and subsequently to an ovotoxic diepoxide (VCD) by hepatic CYP 2A and CYP 2B. DMBA is sequentially bioactivated to the ovotoxicant DMBA-3,4-diol-1,2-epoxide by hepatic CYP 1B1, microsomal epoxide hydrolase (mEH), and CYP 1A1/1B1 enzymes. Even though the liver is the primary organ metabolizing VCH and DMBA to reactive intermediates, several studies suggest that the ovary can also metabolize these two compounds. Studies were designed to investigate the role of ovarian metabolism in the resulting ovotoxicity of these two compounds using a novel mouse ovarian culture system. The hypothesis was that the ovary can participate in bioactivation and detoxification of VCH/VCM and DMBA and thereby influence the resulting ovotoxicity.Postnatal day 4 CYP 2E1 wild-type, null and B6C3F1 mouse ovaries were incubated with 1,2-VCM, VCD or DMBA for various lengths of time. 28 day old female CYP 2E1 wild-type and null mice were dosed (15d, i.p) with VCH, 1,2-VCM, VCD, or sesame oil (control). Following incubations and dosing, ovaries were prepared for histological evaluation of follicle numbers, mEH mRNA level, or mEH protein level. Medium from cultures were analyzed by LC/MS for VCD-GSH adducts.DMBA was found to be a potent ovotoxicant compared to VCH/VCM/VCD. In the ovarian culture system, VCM-induced toxicity required the CYP 2E1 enzyme. However, in vivo dosing studies indicated that in the presence of hepatic metabolism the ovary plays a minimal role in VCH/VCM-induced toxicity. Studies utilizing LC/MS showed that once bioactivated to VCD, this ovotoxic metabolite can be detoxified by glutathione conjugation in the ovary. Follicle loss induced by the ovotoxicant DMBA was found to involve mEH enzyme in culture.Collectively, these studies show that the ovary has the capacity to bioactivate and detoxify ovotoxicants. In the presence of hepatic metabolism ovarian effects might play only a minimal role in the resulting toxicity. The role of ovarian metabolism in the whole animal needs to be further investigated, especially for potent toxicants such as DMBA that can induce ovotoxicity at nanomolar concentrations.

abstract: Reproductive hormones are recognized for their diverse functions beyond reproduction itself, including a vital role in brain organization, structure, and function throughout the lifespan. From puberty to reproductive senescence, the female is characterized by inherent responsiveness to hormonal cyclicity. For most women, a natural transition to menopause occurs in midlife, wherein the endogenous hormonal milieu undergoes significant changes and marks the end of the reproductive life stage. Although most women experience natural menopause, many women will undergo gynecological surgery during their lifetime, which can lead to an abrupt surgical menopause. It is of critical importance to better understand how endogenous and exogenous reproductive hormone exposures across the lifespan influence cognitive and brain aging, as women are at a greater risk for developing a variety of diseases after menopause, including dementia. Using rodent models, this dissertation explores how the etiology of reproductive senescence, that is, whether it is transitional or surgical, influences the female phenotype to result in divergent cognitive outcomes dependent upon a variety of factors, with an emphasis on age at the time of intervention playing a key role in brain outcomes. Furthermore, the impact of exogenous hormone therapy on cognition is evaluated in the context of surgical menopause. A novel rat model of hysterectomy is also presented, with results demonstrating for the first time that the nonpregnant uterus, which is typically considered to be a quiescent organ, may play a unique, direct role in modulating cognitive outcomes. Neurobiological mechanisms associated with reproductive hormones and aging are assessed to better recognize neural correlates underlying the observed behavior changes. The overarching goal of this dissertation was to elucidate novel factors contributing to cognitive aging outcomes in females. Collectively, the data presented herein indicate that the age at the onset of reproductive senescence has significant implications for learning and memory outcomes, and that variations in gynecological surgery can have unique, long-lasting effects on the brain and cognition. Translationally, this series of experiments moves the field forward toward the goal of improving the health and quality of life for women throughout the lifespan. / Dissertation/Thesis / Doctoral Dissertation Psychology 2019

A perimenopausa representa a transição da vida reprodutiva para não reprodutiva. É geralmente caracterizada por alterações neuroendócrinas, metabólicas e comportamentais, um possível resultado da depleção folicular ovariana e consequente redução do número de folículos ovarianos. É o período em que as mulheres podem apresentar maior susceptibilidade a manifestar transtornos afetivos e de ansiedade. A exposição de roedores ao resíduo químico 4-diepóxido de vinilciclohexeno (VCD) é um modelo bem estabelecido para estudos sobre perimenopausa, pois o VCD acelera o processo natural de atresia folicular. Embora as concentrações plasmáticas de estradiol estejam normais ou elevadas durante a perimenopausa, a terapia com estradiol pode ser benéfica para mulheres sintomáticas na perimenopausa. Portanto, o objetivo do presente trabalho foi investigar se a depleção folicular gradativa acelerada pelo VCD resulta em alterações na neuroquímica de ratas fêmeas em núcleos cerebrais que controlam o humor, além de avaliar se o estradiol seria capaz de reverter as possíveis alterações. Ratas da linhagem Wistar (28 dias pós-natal) receberam diariamente, durante 15 dias consecutivos, injeções subcutâneas de VCD (160 mg / kg) ou óleo de milho (O). Aproximadamente 55 dias após a primeira injeção, cápsulas de silastic contendo 17?-estradiol (E) ou O foram inseridas subcutaneamente (Grupos O+O; VCD+O; VCD+E). Cerca de 21 dias após o implante das cápsulas, as ratas dos grupos O+O e VCD+O foram decapitadas na manhã do diestro, enquanto que as do grupo VCD+E foram decapitadas exatamente 21 dias após o implante das cápsulas contendo estradiol, entre 0900 h e 1100 h. O sangue foi colhido para avaliação das concentrações plasmáticas de estradiol e progesterona por radioimunoensaio (RIE). Os cérebros foram removidos para microdissecção do hipocampo, amígdala, Locus coeruleus (LC) e Núcleo Dorsal da Rafe (NDR), para posterior análise dos níveis de RNAm para os receptores de progesterona (PR) e estradiol do tipo beta (ER?) por meio de RT/PCR. Este experimento foi replicado para remoção do hipocampo e amígdala para dosagem dos conteúdos de noradrenalina (NA) e serotonina (5-HT) por meio de cromatografia líquida de alta performance, seguida de detecção eletroquímica (HPLC/ED). Outro conjunto de ratas submetidas às mesmas condições10 experimentais foi perfundido para imunohistoquímica para TPH no NDR e TH no LC. Como esperado, na periestropausa (grupo VCD+O) as concentrações plasmáticas de estradiol não foram diferentes daquelas das ratas controles (O+O). As concentrações plasmáticas de progesterona na periestropausa foram menores que as do grupo controle, o que foi revertido pelo estradiol. No LC, a expressão de PR na periestropausa foi igual à das ratas controles, enquanto a expressão do ER? foi menor; a terapia com estradiol não modificou a expressão de nenhum destes receptores. A densidade de neurônios noradrenérgicos (TH+) no LC não foi alterada nem pela depleção folicular nem pela terapia estrogênica. Na periestropausa, o conteúdo de NA foi menor na amígdala, mas não no hipocampo, e o estradiol não alterou este conteúdo em nenhuma das áreas. No NDR, a expressão de PR e de ER? nas ratas na periestropausa foi menor que nas ratas controles; o estradiol preveniu o declínio da expressão de ER?, mas não de PR. O NDR foi analisado separadamente por toda a extensão rostro-caudal em 3 níveis anatômicos: rostral, médio e caudal, cada um dividido em 3 sub-regiões: lateral, dorsal e ventral. O número de neurônios serotonérgicos (TPH+) no NDR foi menor na periestropausa, e o estradiol foi capaz de reverter esse efeito, atuando principalmente na região caudal. A expressão gênica de PR não foi alterada nem pela depleção folicular nem pela terapia estrogênica tanto na amígdala como no hipocampo. A expressão de ER? também não foi diferente na periestropausa, quando comparada ao grupo controle, mas o estradiol aumentou esta expressão no hipocampo. Tanto na amígdala como no hipocampo houve redução no conteúdo de 5-HT na periestropausa e estradiol foi capaz de reestabelecer os níveis deste neurotransmissor aos valores controles apenas no hipocampo. Estes dados elucidam, pelo menos em parte os mecanismos do efeito positivo da terapia estrogênica nos sintomas de mulheres normoestrogênicas na perimenopausa. Estes efeitos parecem não envolver de forma importante o sistema noradrenérgico central, mas resultar do aumento da biossíntese de progesterona periférica em associação com a regulação positiva de ER? no NDR e hipocampo, que parece potencializar a via serotonérgica NDR/HPC. Portanto, o desenvolvimento de novas terapias que ativem os ER? pode ser uma alternativa para obter os efeitos positivos da ação do estradiol, eliminando os efeitos colaterais das terapias de estradiol que normalmente resultam da ativação do ER?. / Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioural changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, women are more likely to express mood disorders and anxiety. The exposure of animals to diepoxide 4-vinylcyclohexene (VCD) is a well-established experimental model for perimenopause studies, as VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia. Although estrogens levels are normal or even high during perimenopause, estrogen therapy can be beneficial for symptomatic perimenopausal women. The aim of this study was to investigate whether gradual follicular depletion induced by VCD results in changes in the neurochemistry of female rats in brain nuclei that control mood and the role of estradiol on these changes. Female rats (28 days) were daily injected with VCD or corn oil (O) for 15 days. Around 55 days after the first injection, pellets of 17?-estradiol (E) or O were inserted s.c (Groups O+O; VCD+O; VCD+E). Around 21 days after, rats O+O and VCD+O were decapitated between 0900 h and 1100 of diestrus while rats VCD+E were decapitated exactly 21 days after the onset of E therapy. Another set of rats followed the same experimental design and were perfused for TH and TPH immunohistochemistry in Locus coeruleus (LC) and Dorsal Raphe Nuclei (DRN), respectively. Blood was collected for estradiol and progesterone measurement by radioimmunoassay (RIA). The brains were removed from decapitated rats to punch out LC, DRN, hippocampus and amygdala to analyse the expression of mRNA for ER? and PR by RT/PCR. This experiment was replicated to punch out the hippocampus and amygdala for the determination of noradrenaline (NE) and serotonin (5-HT) contents by High Performance Liquid Chromatography, followed by Electrochemical Detection (HPLC/ED). As expected, plasma concentrations of estradiol were not different from those of control rats (O + O). Plasma concentrations of progesterone in the periestropause were lower than those in the control group, which was reversed by estradiol. In the LC, the PR expression in the periestropause was similar to that of the control rats, whereas the ER? expression was lower; estradiol therapy did not modify the expression of any of these receptors. The12 density of noradrenergic (TH +) neurons in LC was not altered by either follicular depletion or estrogen therapy. In periestropause, NA content was lower in the amygdala, but not in the hippocampus, and estradiol did not alter this content in any of the areas. In NDR, the expression of PR and ER? in periestropausal rats was lower than in controls; estradiol prevented the decrease of ER? expression, but not PR. The NDR was analyzed separately for the entire rostrocaudal axis in three anatomical levels: rostral, middle and caudal, each divided into three sub-regions: lateral, dorsal and ventral. The number of serotonergic neurons (TPH +) in NDR was lower in the periestropause, and estradiol was able to reverse this effect, acting mainly in the caudal region. PR gene expression was not altered by either follicular depletion or estrogen therapy in either the amygdala or the hippocampus. ER? expression was also no different in periestropause compared to the control group, but estradiol increased this expression in the hippocampus. Both in the amygdala and in the hippocampus there was a reduction in 5-HT content in the periestropause, and estradiol was able to reestablish the levels of this neurotransmitter at the control values only in the hippocampus. These data elucidate, at least in part, the mechanisms of the positive effect of estrogen therapy on the symptoms of normoestrogenic women in perimenopause. These effects do not appear to significantly involve the central noradrenergic system but result from increased peripheral progesterone biosynthesis in association with positive regulation of ER? in the NDR and hippocampus, which appears to potentiate the serotonergic NDR/HPC pathway. Therefore, the development of new therapies that activate ER? may be an alternative to obtain the positive effects of the estradiol action, eliminating the side effects of the estradiol therapies that normally result from the activation of ER?.

A perimenopausa representa a transição da vida reprodutiva para não reprodutiva. É geralmente caracterizada por alterações neuroendócrinas, metabólicas e comportamentais, um possível resultado da depleção folicular ovariana e consequente redução do número de folículos ovarianos. É o período em que as mulheres podem apresentar maior susceptibilidade a manifestar transtornos afetivos e de ansiedade. A exposição de roedores ao resíduo químico 4-diepóxido de vinilciclohexeno (VCD) é um modelo bem estabelecido para estudos sobre perimenopausa, pois o VCD acelera o processo natural de atresia folicular. Embora as concentrações plasmáticas de estradiol estejam normais ou elevadas durante a perimenopausa, a terapia com estradiol pode ser benéfica para mulheres sintomáticas na perimenopausa. Portanto, o objetivo do presente trabalho foi investigar se a depleção folicular gradativa acelerada pelo VCD resulta em alterações na neuroquímica de ratas fêmeas em núcleos cerebrais que controlam o humor, além de avaliar se o estradiol seria capaz de reverter as possíveis alterações. Ratas da linhagem Wistar (28 dias pós-natal) receberam diariamente, durante 15 dias consecutivos, injeções subcutâneas de VCD (160 mg / kg) ou óleo de milho (O). Aproximadamente 55 dias após a primeira injeção, cápsulas de silastic contendo 17?-estradiol (E) ou O foram inseridas subcutaneamente (Grupos O+O; VCD+O; VCD+E). Cerca de 21 dias após o implante das cápsulas, as ratas dos grupos O+O e VCD+O foram decapitadas na manhã do diestro, enquanto que as do grupo VCD+E foram decapitadas exatamente 21 dias após o implante das cápsulas contendo estradiol, entre 0900 h e 1100 h. O sangue foi colhido para avaliação das concentrações plasmáticas de estradiol e progesterona por radioimunoensaio (RIE). Os cérebros foram removidos para microdissecção do hipocampo, amígdala, Locus coeruleus (LC) e Núcleo Dorsal da Rafe (NDR), para posterior análise dos níveis de RNAm para os receptores de progesterona (PR) e estradiol do tipo beta (ER?) por meio de RT/PCR. Este experimento foi replicado para remoção do hipocampo e amígdala para dosagem dos conteúdos de noradrenalina (NA) e serotonina (5-HT) por meio de cromatografia líquida de alta performance, seguida de detecção eletroquímica (HPLC/ED). Outro conjunto de ratas submetidas às mesmas condições10 experimentais foi perfundido para imunohistoquímica para TPH no NDR e TH no LC. Como esperado, na periestropausa (grupo VCD+O) as concentrações plasmáticas de estradiol não foram diferentes daquelas das ratas controles (O+O). As concentrações plasmáticas de progesterona na periestropausa foram menores que as do grupo controle, o que foi revertido pelo estradiol. No LC, a expressão de PR na periestropausa foi igual à das ratas controles, enquanto a expressão do ER? foi menor; a terapia com estradiol não modificou a expressão de nenhum destes receptores. A densidade de neurônios noradrenérgicos (TH+) no LC não foi alterada nem pela depleção folicular nem pela terapia estrogênica. Na periestropausa, o conteúdo de NA foi menor na amígdala, mas não no hipocampo, e o estradiol não alterou este conteúdo em nenhuma das áreas. No NDR, a expressão de PR e de ER? nas ratas na periestropausa foi menor que nas ratas controles; o estradiol preveniu o declínio da expressão de ER?, mas não de PR. O NDR foi analisado separadamente por toda a extensão rostro-caudal em 3 níveis anatômicos: rostral, médio e caudal, cada um dividido em 3 sub-regiões: lateral, dorsal e ventral. O número de neurônios serotonérgicos (TPH+) no NDR foi menor na periestropausa, e o estradiol foi capaz de reverter esse efeito, atuando principalmente na região caudal. A expressão gênica de PR não foi alterada nem pela depleção folicular nem pela terapia estrogênica tanto na amígdala como no hipocampo. A expressão de ER? também não foi diferente na periestropausa, quando comparada ao grupo controle, mas o estradiol aumentou esta expressão no hipocampo. Tanto na amígdala como no hipocampo houve redução no conteúdo de 5-HT na periestropausa e estradiol foi capaz de reestabelecer os níveis deste neurotransmissor aos valores controles apenas no hipocampo. Estes dados elucidam, pelo menos em parte os mecanismos do efeito positivo da terapia estrogênica nos sintomas de mulheres normoestrogênicas na perimenopausa. Estes efeitos parecem não envolver de forma importante o sistema noradrenérgico central, mas resultar do aumento da biossíntese de progesterona periférica em associação com a regulação positiva de ER? no NDR e hipocampo, que parece potencializar a via serotonérgica NDR/HPC. Portanto, o desenvolvimento de novas terapias que ativem os ER? pode ser uma alternativa para obter os efeitos positivos da ação do estradiol, eliminando os efeitos colaterais das terapias de estradiol que normalmente resultam da ativação do ER?. / Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioural changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, women are more likely to express mood disorders and anxiety. The exposure of animals to diepoxide 4-vinylcyclohexene (VCD) is a well-established experimental model for perimenopause studies, as VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia. Although estrogens levels are normal or even high during perimenopause, estrogen therapy can be beneficial for symptomatic perimenopausal women. The aim of this study was to investigate whether gradual follicular depletion induced by VCD results in changes in the neurochemistry of female rats in brain nuclei that control mood and the role of estradiol on these changes. Female rats (28 days) were daily injected with VCD or corn oil (O) for 15 days. Around 55 days after the first injection, pellets of 17?-estradiol (E) or O were inserted s.c (Groups O+O; VCD+O; VCD+E). Around 21 days after, rats O+O and VCD+O were decapitated between 0900 h and 1100 of diestrus while rats VCD+E were decapitated exactly 21 days after the onset of E therapy. Another set of rats followed the same experimental design and were perfused for TH and TPH immunohistochemistry in Locus coeruleus (LC) and Dorsal Raphe Nuclei (DRN), respectively. Blood was collected for estradiol and progesterone measurement by radioimmunoassay (RIA). The brains were removed from decapitated rats to punch out LC, DRN, hippocampus and amygdala to analyse the expression of mRNA for ER? and PR by RT/PCR. This experiment was replicated to punch out the hippocampus and amygdala for the determination of noradrenaline (NE) and serotonin (5-HT) contents by High Performance Liquid Chromatography, followed by Electrochemical Detection (HPLC/ED). As expected, plasma concentrations of estradiol were not different from those of control rats (O + O). Plasma concentrations of progesterone in the periestropause were lower than those in the control group, which was reversed by estradiol. In the LC, the PR expression in the periestropause was similar to that of the control rats, whereas the ER? expression was lower; estradiol therapy did not modify the expression of any of these receptors. The12 density of noradrenergic (TH +) neurons in LC was not altered by either follicular depletion or estrogen therapy. In periestropause, NA content was lower in the amygdala, but not in the hippocampus, and estradiol did not alter this content in any of the areas. In NDR, the expression of PR and ER? in periestropausal rats was lower than in controls; estradiol prevented the decrease of ER? expression, but not PR. The NDR was analyzed separately for the entire rostrocaudal axis in three anatomical levels: rostral, middle and caudal, each divided into three sub-regions: lateral, dorsal and ventral. The number of serotonergic neurons (TPH +) in NDR was lower in the periestropause, and estradiol was able to reverse this effect, acting mainly in the caudal region. PR gene expression was not altered by either follicular depletion or estrogen therapy in either the amygdala or the hippocampus. ER? expression was also no different in periestropause compared to the control group, but estradiol increased this expression in the hippocampus. Both in the amygdala and in the hippocampus there was a reduction in 5-HT content in the periestropause, and estradiol was able to reestablish the levels of this neurotransmitter at the control values only in the hippocampus. These data elucidate, at least in part, the mechanisms of the positive effect of estrogen therapy on the symptoms of normoestrogenic women in perimenopause. These effects do not appear to significantly involve the central noradrenergic system but result from increased peripheral progesterone biosynthesis in association with positive regulation of ER? in the NDR and hippocampus, which appears to potentiate the serotonergic NDR/HPC pathway. Therefore, the development of new therapies that activate ER? may be an alternative to obtain the positive effects of the estradiol action, eliminating the side effects of the estradiol therapies that normally result from the activation of ER?.

Sex differences exist in the progression of heart disease, as premenopausal women are protected from developing severe hypertension, aortic stenosis, myocardial infarction and hypertrophic cardiomyopathies. The susceptibility and progression of cardiovascular disease increases in post-menopausal women. This is at least partially underlined by a pronounced decrease in circulating estrogen levels. Estradiol (E2), the most abundant estrogen in premenopausal women, is known to be cardioprotective. Recently, AMP-activated protein kinase (AMPK) has emerged as a prominent player in the development of cardiac hypertrophy and heart failure. AMPK is central to the energetic metabolism of the cell and is activated in response to energy deprivation. E2 has been shown to activate AMPK, by yet an unknown mechanism. The first part of this dissertation focuses on describing the molecular mechanism behind this AMPK activation. We found that E2 activates AMPK through a non- genomic pathway and involves direct interaction of classical estrogen receptors (ERα and ERβ) with the α-catalytic subunit of AMPK. These receptors also associate with the upstream kinase LKB1, which is required for E2-dependent activation of AMPK. Furthermore, the two estrogen receptors play opposite roles, where ERα increases AMPK activation, and ERβ acts as a repressor, inhibiting AMPK phosphorylation. To translate our findings to heart disease, the next step was to determine the effect of ovarian failure, underlined by E2 loss, on AMPK signaling during the progression of cardiac hypertrophy. We hypothesized that ovarian failure decreases cardiac AMPK signaling, translating in worsening of hypertrophy. We found that the status of cardiac AMPK signaling depends on the nature of the hypertrophic stimulus and the timing of ovarian failure in relation to the onset of hypertrophy. Furthermore, we did not detect any differences in the development of cardiac hypertrophy between wild type mice and mice in ovarian failure, which most likely occur down the line. In summary we described a novel mechanism of AMPK activation by the hormone E2. We also explored the effect of estrogen loss on cardiac AMPK activity, and found that it is dependent on factors such as the pathological state of the heart and timing of the intervention. These findings add to our understanding of the molecular mechanisms behind sex differences in energy handling and in the future could be translated into better therapeutics for the treatment of cardiac pathologies.

L-3,4-dihyroxyphenylalanine (L-DOPA, levodopa) is a gold standard treatment of Parkinson's disease. Lately, it has been found that some of its deuterated analogues exhibit higher potency in the treatment; thus, they could replace L-DOPA. The subject of this thesis was a study of L-DOPA and its deuterated derivatives by the means of vibrational spectroscopy (Raman, ROA, IR, and VCD) and a comparison of the experimental results to a quantum mechanical simulations of the spectra. ROA and VCD are chiroptical methods, thus they are suitable for measurement of chiral molecules amongst which L-DOPA indeed belongs. Thanks to the quantum chemistry calculations, which yielded spectra with a very good agreement with the experiment, we were able to assign experimental spectral features to individual vibrational modes of the L-DOPA. The use of chiroptical techniques (mainly ROA) enabled an assignment of an absolute configuration of double deuterated derivative of L-DOPA, α,β-D2-L-DOPA. It reviled that it occurs in a (S-α,S-β)-enantiomeric form.

Dans cette thèse les résultats dans le domaine de la synthèse des dérivée des TADDOL et leur capacité d’induction chirale sont présentés. Une librairie des TADDOLs a été synthétisée et une analyse conformationnelle par VCD a été faite. Ces composés enantiopurs ont été testés dans la réaction de cyanosilylation enantioselective en donnant des résultats moyens. En partant de TADDOL nous avons synthétisé des dérivée phosphorés, des amines et des (thio)urées. Les dérivés de P(III) ont été utilisés comme ligands pour le palladium dans l’alkylation allylique asymétrique et les amines dans le réarrangement des époxydes meso. Les (thio)urées ont été testées pour leur capacité de complexation des anions carboxylates. / In this thesis are presented the results in the field of synthesis of TADDOL derivatives and their chiral induction capacity. A family of TADDOLs was synthesized and a conformational analysis was performed by VCD. These enantipure compounds were tested in enantioselective cyanosilylation reactions obtaining moderate results. Starting from TADDOL we obtained phosphorus derivatives, amines and (thio)ureas. The P(III) derivatives were tested as ligands for palladium in asymmetric allylic alkylation and the amines in the rearrangement of meso-epoxides. The (thio)ureas were screened for complexation capacity for carboxylate anions.