published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy

Thesis (M.Sc. (Microbioligy))--Mahidol University, 1970.

Th. 3e cycle--Pharm.--Paris 5, 1981. N°: 25.

The objectives of this study are to characterize two carrier proteins in the horse that significantly decrease in humans following anabolic androgenic steroid administration: sex hormone-binding globulin (SHBG) and thyroxine-binding globulin (TBG). For SHBG characterization, qPCR, RNA sequencing, and immunohistochemistry were performed on testes and equine livers. Free and total testosterone immunoassays were utilized to confirm the presence of a carrier protein in equine circulation. SHBG was detected in the testes using qPCR, RNA sequencing, and IHC, indicating the presence of the isoform androgen-binding protein (ABP). SHBG was not detected in any liver samples. Evidence of a carrier protein was shown by free testosterone being significantly lower than the total testosterone that was detected in stallions (p < 0.0001) and pregnant mares (p < 0.0001). TBG characterization was completed using an equine specific TBG ELISA. Equine serum was analyzed across seasons, reproductive statuses, sexes, and ages. TBG concentrations were also measured following anabolic steroid administration (Stanozolol) and increased endogenous androgen production via hCG administration in stallions and aromatase inhibition via Letrozole administration in pregnant mares. TBG did not significantly differ across season, reproductive status, sex, or age Alterations of androgen concentrations did not result in any significant changes to circulating TBG concentrations.

La mucoviscidose (ou cystic fibrosis, CF) est une maladie caractérisée par une inflammation pulmonaire chronique qui contribue à la dégradation progressive de l’épithélium des voies aériennes des patients. Les glucocorticoïdes (GC) représentent un outil essentiel pour le traitement du patient mais leur efficacité et leur rapport bénéfice/risque restent cependant controversés. Les effets secondaires provoqués par l’administration de ces molécules pourraient être diminués par l’utilisation de leur protéine d’adressage, la corticosteroid-binding globulin (CBG). L’objectif de ce travail était d’étudier l’expression de la CBG chez les patients CF, afin d’optimiser leur traitement anti-inflammatoire par GC. Nous avons montré, dans un premier temps, que la synthèse hépatique de CBG était augmentée, tandis que son taux plasmatique était conservé chez les patients CF, comparé aux patients non-CF. Dans un second temps, nous nous sommes intéressés à l’expression de la CBG au niveau pulmonaire. L’inflammation chez les patients CF étant principalement pulmonaire, l’expression locale de CBG à ce niveau pourrait moduler l’efficacité du GC, en le recaptant. Nos données montrent que l’expression de cette CBG pulmonaire est diminuée chez les patients CF. Les études sur des modèles in vitro hépatiques et pulmonaires n’ont pas permis d’expliquer les résultats obtenus et ont souligné la limite des modèles et des outils à disposition. Le maintien de la concentration plasmatique de CBG et la diminution de la CBG pulmonaire chez les patients CF suggèrent ainsi que la CBG pourrait être utilisée comme outil thérapeutique dans le contexte de la mucoviscidose, afin d’optimiser le traitement par GC. / Cystic fibrosis (CF) is characterized by a chronic pulmonary inflammation, responsible of the progressive degradation of the airways epithelium. In CF, glucocorticoids (GC) are widely used but their efficiency and benefit/risk ratio are still discussed. The side effects, caused by the administration of these molecules, might be decreased by the use of their delivery protein, corticosteroid-binding globulin (CBG). The aim of the work was to study the expression of CBG in CF patients, in order to optimise their anti-inflammatory treatment by GC. First, we showed that the hepatic synthesis of CBG was increased while its plasmatic level was preserved in CF patients, compared to non-CF. Second, we were interested by the expression of CBG at the pulmonary level. The inflammation in CF patients being primarely pulmonary, the local expression of CBG in the lung could modulate the efficiency of GC through recapture. Our data showed that this expression of this pulmonary CBG was decreased in CF patients. The studies conducted in vitro on hepatic and pulmonary models did not explained our results and highlighted the limit of the models and tools at our disposal. The maintained plasmatic concentration of CBG and the decrease of pulmonary CBG in CF patients suggest that CBG might be useful as a therapeutic tool in the CF context, in order to optimise the GC treatment.

There is a need to understand how free-ranging animals respond and adapt to stress. However, little is currently known regarding the physiologic adaptations to stress in bears, and there are few tools available to wildlife managers to assess the health and stress status of free-ranging animals, including ursids. The hypothalamus-pituitary-adrenal (HPA) axis plays major roles in the physiological adaptation to stress, leading to the increased secretion of glucocorticoids (e.g. cortisol in most mammals) that mediate adaptive changes in physiology and behaviour. The vast majority of glucocorticoids are bound to its primary carrier protein, corticosteroid-binding globulin (CBG), in most animals, and only the unbound fraction is bioavailable. Thus, CBG plays a major role in modulating glucocorticoid dynamics, and this protein must be characterized to build a more complete understanding of the adaptive role that the HPA axis plays in mitigating stress in bears. The overall objective of this thesis was to characterize the HPA axis activity and CBG levels in bears, and develop tools targeted towards the monitoring of the health and stress status of American black bear (Ursus americanus), grizzly bear (U. arctos), and polar bear (U. maritimus). The binding characteristics of cortisol to CBG in bears were studied via saturation binding experiments, and this information was used to estimate free cortisol concentrations based on CBG concentrations. To quantify CBG concentrations in bears, an enzyme-linked immunosorbent assay (ELISA) was developed. Grizzly bear CBG cDNA was cloned and sequenced, and an antibody was developed against a peptide sequence of the deduced amino acid sequence. The antibody showed good cross-reactivity against black, grizzly, and polar bear CBG, and the ELISA based on this antibody found differences in the mean CBG levels between species. Using this data, free cortisol levels were estimated, and mean levels were elevated in polar bears relative to black and grizzly bears. Having developed these tools, the roles that corticosteroid-binding globulin (CBG) and bioavailable cortisol played in the physiological adaptation to major life history traits and environmental challenges faced by ursids were investigated. Importantly, CBG was not modulated by the acute stress of capture and handling, despite the large differences in the magnitude of acute cortisol responses that are induced by these methods, suggesting that CBG levels may reflect the chronic health and stress status of bears. Altogether, there were few changes in CBG levels throughout much of the annual life cycle of bears, implying that CBG does not play a major adaptive role in the life history traits of bears and, instead, metabolic and environmental factors may be the key modulators of cortisol dynamics. However, CBG was not significantly associated with our measures of dietary patterns and nutrition, including body condition, seasonal dietary patterns, and fasting. The majority of the observed variation in the levels of this protein in bears remains unexplained. However, stress-induced free cortisol levels were negatively associated with urea to creatinine ratio (an indicator of dietary protein content and fasting status in grizzly and polar bears, respectively) and positively associated with lactation in hibernating black bears, suggesting that the variation in adrenal function may be playing an important role in the adaptation to adverse environmental conditions and/or metabolic stress in bears. In addition to serum cortisol dynamics, other proteins were also hypothesized to play adaptive roles in maintaining the hibernating phenotype in bears. Changes in the serum proteome during hibernation in black bears were assessed as a means to discover novel proteins that may be indicative of metabolic stress in bears. The serum proteomes of active and hibernating black bears were compared and analyzed for significant changes by two-dimensional electrophoresis and tandem mass spectrometry. Proteins involved with immune-related function were significantly altered during hibernation, leading to the proposal that the serum protein changes are essential for maintaining immune competence, wound healing, and bone structure. Altogether, this thesis developed a method to quantify CBG and estimated free cortisol concentrations in bears, and characterized their roles in the physiological adaptations associated with the major life history traits and environmental challenges faced by ursids. Also, novel serum proteins were identified as potential markers of immune function and health status in bears. These tools may be tremendously useful for wildlife managers and conservationists in determining how chronic stressors, including anthropogenic activities and climate change, may impact the stress and health performances of individual and populations of free-ranging bears.

There is a need to understand how free-ranging animals respond and adapt to stress. However, little is currently known regarding the physiologic adaptations to stress in bears, and there are few tools available to wildlife managers to assess the health and stress status of free-ranging animals, including ursids. The hypothalamus-pituitary-adrenal (HPA) axis plays major roles in the physiological adaptation to stress, leading to the increased secretion of glucocorticoids (e.g. cortisol in most mammals) that mediate adaptive changes in physiology and behaviour. The vast majority of glucocorticoids are bound to its primary carrier protein, corticosteroid-binding globulin (CBG), in most animals, and only the unbound fraction is bioavailable. Thus, CBG plays a major role in modulating glucocorticoid dynamics, and this protein must be characterized to build a more complete understanding of the adaptive role that the HPA axis plays in mitigating stress in bears. The overall objective of this thesis was to characterize the HPA axis activity and CBG levels in bears, and develop tools targeted towards the monitoring of the health and stress status of American black bear (Ursus americanus), grizzly bear (U. arctos), and polar bear (U. maritimus). The binding characteristics of cortisol to CBG in bears were studied via saturation binding experiments, and this information was used to estimate free cortisol concentrations based on CBG concentrations. To quantify CBG concentrations in bears, an enzyme-linked immunosorbent assay (ELISA) was developed. Grizzly bear CBG cDNA was cloned and sequenced, and an antibody was developed against a peptide sequence of the deduced amino acid sequence. The antibody showed good cross-reactivity against black, grizzly, and polar bear CBG, and the ELISA based on this antibody found differences in the mean CBG levels between species. Using this data, free cortisol levels were estimated, and mean levels were elevated in polar bears relative to black and grizzly bears. Having developed these tools, the roles that corticosteroid-binding globulin (CBG) and bioavailable cortisol played in the physiological adaptation to major life history traits and environmental challenges faced by ursids were investigated. Importantly, CBG was not modulated by the acute stress of capture and handling, despite the large differences in the magnitude of acute cortisol responses that are induced by these methods, suggesting that CBG levels may reflect the chronic health and stress status of bears. Altogether, there were few changes in CBG levels throughout much of the annual life cycle of bears, implying that CBG does not play a major adaptive role in the life history traits of bears and, instead, metabolic and environmental factors may be the key modulators of cortisol dynamics. However, CBG was not significantly associated with our measures of dietary patterns and nutrition, including body condition, seasonal dietary patterns, and fasting. The majority of the observed variation in the levels of this protein in bears remains unexplained. However, stress-induced free cortisol levels were negatively associated with urea to creatinine ratio (an indicator of dietary protein content and fasting status in grizzly and polar bears, respectively) and positively associated with lactation in hibernating black bears, suggesting that the variation in adrenal function may be playing an important role in the adaptation to adverse environmental conditions and/or metabolic stress in bears. In addition to serum cortisol dynamics, other proteins were also hypothesized to play adaptive roles in maintaining the hibernating phenotype in bears. Changes in the serum proteome during hibernation in black bears were assessed as a means to discover novel proteins that may be indicative of metabolic stress in bears. The serum proteomes of active and hibernating black bears were compared and analyzed for significant changes by two-dimensional electrophoresis and tandem mass spectrometry. Proteins involved with immune-related function were significantly altered during hibernation, leading to the proposal that the serum protein changes are essential for maintaining immune competence, wound healing, and bone structure. Altogether, this thesis developed a method to quantify CBG and estimated free cortisol concentrations in bears, and characterized their roles in the physiological adaptations associated with the major life history traits and environmental challenges faced by ursids. Also, novel serum proteins were identified as potential markers of immune function and health status in bears. These tools may be tremendously useful for wildlife managers and conservationists in determining how chronic stressors, including anthropogenic activities and climate change, may impact the stress and health performances of individual and populations of free-ranging bears.

Cancer is a leading cause of mortality in the United States. Despite much research on specific carcinogens, the cause of many cancers remains unclear. The identification of novel causative agents offers the potential for cancer prevention. Diseases such as obesity and diabetes mellitus, characterized by hyperinsulinemia, are associated with increased risk of endometrial, colorectal, and breast carcinomas. There is increasing evidence that insulin is a growth factor for tumor formation. The mechanisms underlying insulin-mediated neoplasia may include enhanced DNA synthesis with resultant tumor cell growth, inhibition of apoptosis, and altered sex hormone milieu. The reduced insulin levels seen with physical activity, weight loss, and a high fiber diet may account for decreased cancer risk. The role of newer drugs that restore sensitivity to insulin, thereby reducing hyperinsulinemia, is an exciting potential area of cancer prevention. In this review, we discuss the potential role of insulin as a tumor growth factor.

A transferência de imunidade passiva colostral é muito importante tanto para a saúde do neonato quanto para o seu desempenho. Com o objetivo de estudar a relação entre a eficiência ou não no estabelecimento da imunidade passiva colostral em neonatos e a suas interrelações com aspectos de desempenho e produção destes animais foram colhidas amostras de sangue de 354 bezerras com 30 horas de vida (24 a 36 h) em 10 propriedades localizadas no centro leste do Estado do Paraná, Brasil. Determinações bioquímicas do soro das amostras foram realizadas para avaliar a qualidade de transferência de imunidade passiva. A proteína sérica total foi determinada pelo método do Biureto e a albumina pelo método do verde de bromocresol para o cálculo das taxas de globulinas obtidas pela diferença entre os teores séricos de proteína e albumina. A fração gamaglobulina foi determinada por eletroforese. Uma análise multivariada, incluindo os teores séricos de proteína total, globulinas e gamaglobulinas, foi utilizada para determinar três grupos conforme à qualidade da transferência de imunidade passiva colostral apresentada (Proc cluster, SAS), a saber: baixa, moderada e alta. Durante o estudo, os produtores anotaram as informações referentes a práticas de manejo adotado na atenção a vaca (parturiente) e ao bezerro, incluindo os dados que poderiam ter influência sobre a transferência de imunidade passiva colostral. Os dados das bezerras, enquanto neonatas e depois quando adultas foram colhidos para a determinação da influência a curto e a longo prazo da falha da transferência de imunidade passiva (FTIP) colostral. Observou-se que os dados de escore de condição corporal da mãe da bezerra, da quantidade de colostro ingerida na primeira mamada e o momento em que ela foi realizada, da morbidade e da mortalidade das bezerras estavam relacionados com a FTIP (P<0,05). Os pesos ao nascer e ao primeiro mês de vida, assim como a freqüência de bezerras analisadas que pariram na propriedade não estavam relacionados diretamente com a qualidade da transferência de imunidade passiva. Fatores como a distocia, idade ao primeiro serviço e a produção média diária de leite não diferiram estatisticamente entre os grupos de baixa, moderada e alta transferência de imunidade passiva (P>0,05). Mas quando se correlacionou por regressão os dados de produção de leite das vacas que quando bezerras apresentaram teores de gamaglobulinas menores que 1,6 g/dL obteve-se valores de r2=0,47 (P=0,0005). Com os resultados desta pesquisa pode-se afirmar que práticas muito simples de manejo como fornecer pelo menos 2 L de colostro até 2 horas após o nascimento e a vaca parir numa boa condição corporal (ECC = 3 ou 3,5) podem prevenir a falha na transferência de imunidade passiva. A FTIP tem como conseqüências: maiores taxas de morbidade e mortalidade, primeiro parto mais tardio e diminuição do número de novilhas de reposição, podendo ainda estar correlacionada a menores produções leiteiras. / Adequate passive transfer of maternal immunoglobulin is important for optimal health and performance in newborn calves. Blood samples were collected from 354 dairy calves, ranging from 24 to 36 hours of age, between July 2005 and May 2006 on 10 farms in the middle-eastern region of the state of Parana, Brazil. The objective was to study the relationship and effectiveness of the transfer of colostral passive immunity and its contributing factors as related to the development and production of animals. For each sample collected, total serum protein was determined by the biuret method and albumin by bromocresol green method, and the difference was used to evaluate the globulins. Electrophoresis was used to determine the -globulin fraction of the sample. A multivariable analysis, including total serum protein, globulins and gamma globulin, was used to create 3 groups to classify the quality of the transfer of colostral passive immunity (cluster procedure, SAS): failure or inadequate group, marginal group and adequate group. During the study, breeders were asked to provide information on calf and pre-partum cow management practices, including details on colostrum feeding. Data from the calves while newborn and as heifers was gathered to determine the long and short term effect of the failure of passive immunity transfer (FPIT). Body condition score of the mother at calving, quantity of colostrum ingested, timing of ingestion, morbidity and mortality of calves and age at calving time were related to FPIT (P<0,05). The weight of the calves after birth and at one month of age and the frequency of calves that became cows in the farm were not directly related to failure. Dystocia, age at first service in days, and milk production did not differ statistically (P>0,05). However, when a regression was performed based on data of milk production from calves that had serum gamma globulins levels below 1,6g/dL, a correlation was identified (r2=0.47; P=0.0005). Basic management practices can prevent failure of passive immunity transfer by feeding calves 2 L of colostrum within 2 hours of life and ensuring that the cow calves with a good body condition score (BCS = 3 or 3.5). FPIT is responsible for higher morbidity and mortality rates, a delay in first parturition, a decrease in the number of replacement heifers and it can also be responsible for less milk production.