Resumo: As plaquetas são fragmentos do citoplasma do megacariócito e possuem papel fundamental na manutenção da hemostasia. Ao aderirem umas às outras, ativam-se, mudam de forma e liberam grânulos que contêm fatores de crescimento, proteínas que atuam na divisão celular. Os grânulos α- plaquetários secretam fatores de crescimento que regulam eventos, tais como, síntese de DNA, quimiotaxia e citodiferenciação. A estocagem prolongada das plaquetas a 22ºC é prejudicial à viabilidade e à função, mesmo quando estocadas em condições adequadas nos bancos de sangue. Observa-se diminuição da concentração do glutation intraplaquetário, representando diminuição da proteção contra o stress oxidativo, do qual resulta a perda do grupo sulfidril. As plaquetas desempenham funções com consumo de energia, como exemplo a diapedese, que pode ser recuperada após cinco dias de armazenamento pelo azul de metileno. Durante a divisão celular há necessidade de bloqueio das funções citoplasmáticas com aumento da produção de proteínas de adesão para que ocorra a fixação da célula que vai se dividir. Os resultados mostraram que quando adicionamos azul de metileno, a quantidade de células viáveis diminui com aumento de apoptose e necrose, muito provavelmente devido à liberação de energia / Abstract: Platelets are megakaryocyte cytoplasmic fragments that play a fundamental role in maintaining hemostasis. When they adhere to each other, they become activated, change shape and release granules containing growth factors, proteins involved with cell division. Platelet α-granules secrete growth factors that regulate events such as DNA synthesis, chemotaxis and cytodifferentiation. Long platelet storage at 22ºC is harmful to their viability and function, even when they are stored under proper conditions at blood banks. A reduced concentration of intraplatelet glutathione is observed, representing decreased protection against oxidative stress, from which the loss of the sulfhydryl group results. Platelets perform actions with energy consumption, such as diapedesis, which can be recovered after five storage days by methylene blue. During cell division, cytoplasmic functions must be blocked by increased adhesion protein so that the fixation of the dividing cell can occur. Results showed that when methylene blue is added, the number of viable cells decreases with apoptosis and necrosis increase, which is very likely due to energy release / Orientador: Paulo Eduardo de Abreu Machado / Coorientador: Márjorie de Assis Golim / Banca: Izolete Aparecida Thomazini Santos / Banca: Sueli Gonçalves Saez / Mestre

No description available.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Kathrin Schrick / Class IV homeodomain leucine-zipper transcription factors (HD-Zip TFs) are master regulators of cell-type differentiation in the plant epidermis. These transcription factors contain a putative START (STeroidogenic Acute Regulatory (StAR)-related lipid Transfer) lipid/sterolbinding domain that is hypothesized to link metabolism to gene expression in plant development. This study is focused on two class IV family members that serve as models in many of the experiments: GLABRA2 (GL2) is a key regulator of differentiation in hair cells called trichomes as well as other epidermal cell types in various plant tissues. The second member addressed in this study is PROTODERMAL FACTOR2 (PDF2), which plays a crucial role in epidermal cell specification in shoots. A leading hypothesis is that the START domain, by binding a ligand, controls transcription factor function, analogously to nuclear receptors from mammals. Domain swap experiments indicated that the START domain from both plants and mammals is a conserved ligand-binding motif that is required for transcription factor activity. To further address its function in ligand binding, mutational analysis of the START domain of GL2 was performed. Several of the mutations remove charged residues in the predicted ligand-binding pocket and resulted in loss-of-function phenotypes, suggesting that ligand binding is critical for HD-Zip TF activity. Chromatin immunoprecipitation–based sequencing (ChIP-seq) revealed that the START domain is dispensable for transcription factor binding to DNA. Using a high throughput thermal shift assay to screen a library of pure natural compounds, specific secondary metabolites were identified as putative START domain ligands for PDF2. Experiments in both yeast and N. benthamiana demonstrated that the START domain is required for homodimerization of GL2 through its Zip domain. It was also found that the START domains physically interact with RHAMNOSE SYNTHASE I (RHM1). Further, this work provided evidence for a previously elusive redundancy between GL2 and another class IV HD-Zip TF, and unveils a positive feedback loop in the maintenance of the GL2 activity during trichome differentiation. Taken together, these findings support the premise that START domains are central players in metabolic regulatory networks that can modulate transcription factor activity by binding ligands and mediating protein-protein interactions.

Resumo: A formação da musculatura esquelética nos membros de frango é um processo complexo, dividido em várias etapas, desde a formação do somito, progenitor da musculatura, até o músculo propriamente dito. O presente trabalho teve o objetivo de caracterizar o perfil de expressão de genes associados aos fatores miogênicos MyoD, MRF4 e Miogenina (responsáveis pela formação e manutenção da musculatura) e Miostatina. Utilizaram-se 40 animais de duas linhagens (20 de corte e 20 de postura), cujos tecidos das coxas e sobrecoxas foram coletados em duas idades (21 e 42 dias póseclosão). A técnica de PCR quantitativa foi empregada nas análises da expressão gênica e os dados obtidos foram analisados com a utilização do programa REST (Ferramenta para análise de expressão relativa - Relative Expression Software Tool) que se baseia em quantificação relativa. Devido à divergência fenotípica no padrão de crescimento das linhagens estudadas, esperava-se encontrar diferenças na expressão de miostatina, as quais não foram confirmadas. Por outro lado, foram verificadas diferenças para MRF4 nos músculos Gastrocnêmio (coxa) e Biceps (sobrecoxa) em ambas as idades, e 9 para Miogenina, em Gastrocnêmio, aos 42 dias; com maior expressão na linhagem CC. A caracterização dos genes estudados foi parcial, abrindo a possibilidade para que outros genes possam ser selecionados com intuito de compreender o desenvolvimento muscular em frangos. / Abstract: The development of the chicken limb muscles is a complex process, divided into several stages from the initial somite formation to the muscle itself. This study aimed to characterize expression profile of genes associated to myogenic factors MyoD, MRF4 and Miogenin (responsible for muscle formation and maintenance) and Miostatin. It was used 40 animals from two lines (20 broiler and 20 layer), whose thigh and drumstick tissues were collected at two ages (21 and 42 days post-hatch). Gene expression employed techniques of quantitative PCR and data were analyzed by REST program, which is based on a relative quantification. Due to phenotypic divergence in the growth pattern of the strains studied, it was expected to find differences in the expression of miostatin that were not confirmed. On the other hand, differences were found for MRF4, with highest expression in CC line. The characterization of genes studied was partial, opening the possibility that other genes may be selected in order to understand the muscle development in chickens. / Orientador: Patrícia Pasquali Parise-Maltempi / Coorientador: Helena Javiel Alves / Banca: Millor Fernandes do Rosário / Banca: Anderson Luis Alves / Mestre

Introduction: R factor mediated transfer of antibiotic resistance between Enterobacteriaceae has been reported to occur in the mammalian gastrointestinal tract (Farrar et al, 1972; Guinée, 1970; Kasuya, 1964; Reed et al, 1969; Wiedemann et al, 1970). In vivo conjugal transfer of genetic material has also been demonstrated with F¹, F⁺ and Hfr Escherichia coli strains (Jones & Curtiss, 1970). The environment in the lower gastrointestinal tract, where bacteria are abundant, is mainly anaerobic. This is demonstrated by the dominance of obligately anaerobic bacteria such as Bacteroides species (Finegold, 1969; Moore et al, 1969) and direct studies of intestinal gas composition (Askevold, 1956). However, most laboratory investigations of the incidence of R factors and their transfer frequencies have been performed under aerobic conditions using faecal facultative strains. The only investigation of resistance transfer under anaerobic conditions in vitro is that of Mitsuhashi (1965), who reported complete inhibition of transfer of an R factor from a Shigella flexneri donor to an Escherichia coli recipient. In addition, Fisher (1957) reported restriction of chromosomal transfer by an E. coli Hfr strain under anaerobic conditions in various media. On the basis of these results, it could be questioned whether in vivo R factor transfer is in fact possible (Chabbert et al, 1969). The contradictory situation prompted a reexamination of conjugation in facultative strains under anaerobic conditions. Both Fisher (1957) and Mitsuhashi (1965) obtained anaerobic conditions by evacuation. In this investigation, both mating and selection of recombinants were performed under stringent anaerobic conditions using methods developed for the isolation of obligate anaerobes (Hungate, 1969) to obtain a degree of anaerobiosis similar to that found in vivo.

Le retard de croissance intra-utérin (RCIU) reste une complication fréquente de la grossesse et expose non seulement à une mortalité néonatale plus élevée, mais également au risque d'un cortège de pathologies chroniques (cardio-métaboliques) à l'âge adulte. Étant un fardeau important pour la santé publique dans le monde entier, il n'existe pas encore à ce jour de traitement curatif autre qu'une extraction plus. précoce avec éventuellement une prématurité. Une étude récente de notre équipe a montré que l'administration de citrulline pendant la gestation améliore la croissance et la synthèse protéique fœtales dans un modèle de RCIU induit chez la rate par la restriction sévère en protéines. L'objectif de ce travail de thèse était donc d'explorer les mécanismes d'action de la supplémentation en citrulline pendant la période périnatale (gestation et allaitement) dans le même modèle. Notre première partie de ce travail confirme que la citrulline stimule la croissance en nous montrant que la supplémentation anténatale en citrulline agit dès le milieu de la gestation sur des gènes codant pour des facteurs de croissance, d’angiogenèse et de transporteurs d'acides aminés pour aboutir à une efficacité placentaire renforcée jusqu'à la fin de la gestation fœtale. Dans la deuxième partie, nous n'avons pas mis en évidence d'effet bénéfique de cette supplémentation postnatale chez des ratons nés dénutris, puis soumis à un régime déséquilibré riche en fructose, ni en termes de croissance, ni sur le métabolisme glucido-lipidique à l'âge adulte jeune. La vigilance impose davantage d'explorations mécanistiques avant d'envisager une étude translationnelle clinique à ce stade sensible de développement qu'est la période néonatale. En revanche, dans le cas de la période gestationnelle, nos résultats incitent à réfléchir à la mise en place d'essais cliniques pertinents pour cette pathologie de la croissance fœtale. / Intra-uterine growth restriction (IUGR) remains a common pregnancy-related complication resulting not only in a significant neonatal mortality, but in an increased risk of chronic cardio-metabolic diseases in adulthood as well. This condition represents a serious burden to public health across the world due to lack of a curative treatment except early cessation of gestation with induced prematurity. Developing alternative strategies aimed towards targeted therapy would thus be highly desirable. In recent studies, we showed that citrulline supplementation during gestation in rats under severe dietary protein restriction enhanced fetal growth and protein synthesis. The objective of this work was to further investigate the mechanisms mediating the effect of citrulline during the perinatal period, i.e. gestation and nursing in the same model. In the first part, we confirmed that citrulline improved fetal growth, and further demonstrated that citrulline activated placental genes coding for growth factors, angiogenesis and amino acid transporters early from mid-gestation, resulting in improved fetal weight. However, in the second part of the current work, we failed to observe any beneficial effect of neonatal citrulline supplementation neither on growth, nor on the prevention of alterations of glucose and lipid metabolism in IUGR rats that were later exposed to an unbalanced, fructose-enriched diet. Therefore, further explorations are needed for a better mechanistic understanding before postnatal citrulline supplementation can be considered in translational trials. Otherwise, the results obtained in the gestational period in this work suggest clinical trails should be envisioned for prenatal citrulline supplementation in targeted populations of patients.

No description available.

The homeodomain transcription factor family of Cdx genes, Cdx1, Cdx2 and Cdx4, are known to play essential roles in many developmental processes including neural tube closure, axial elongation, hematopoiesis and gastrointestinal patterning. In the adult, Cdx1 and Cdx2 are both expressed strictly in the adult intestinal epithelium, but their functions and mechanisms of action at this stage are poorly understood. Cdx transcription factors have also been reported to be lost in intestinal cancers. To circumvent early lethality, a conditional loss of function strategy was used to inactivate Cdx2 in the adult intestinal epithelium. These conditional mutants were crossed to Cdx1-/- mice to examine potential functional compensation between these family members as well as into APC(min/+) mice to study their role in tumorigenesis. Using these models, I have found that Cdx2 regulates adult intestinal homeostasis and differentiation in the small intestinal epithelium, while both Cdx1 and Cdx2 contribute to colon homeostasis. Furthermore, Cdx transcription factors are tumor suppressors in the development of Wnt-induced colorectal cancer, and impact several pathways including TGF-β and Eph-ephrin signaling. Finally, Cdx2 regulates Eph-ephrin signaling through direct activation of the Notch pathway. Altogether, this study underscores critical roles and mechanisms of action for Cdx members in the adult intestine and in intestinal tumorigenesis.

Prospective memory refers to remembering to carry out an intended or planned action, such as keeping a doctor's appointment or telling a friend about an upcoming party. Despite its importance in everyday life, prospective memory has received little empirical or theoretical attention. Rather, much of the literature has focused on retrospective memory, that is, memory for information learnt in the past. The current literature on prospective memory addresses five aspects that are necessary for carrying out an intended action. These are (a) formulating the plan; (b) having the knowledge necessary to carry out the plan; (c) remembering the plan at the appropriate time; (d) carrying out the plan; and (e) remembering that the plan has been performed. The literature also raises three fundamental questions. Namely, whether prospective and retrospective memory involve different processes, whether self-report and behavioral measures of prospective memory are correlated, and whether prospective memory performance varies as a function of age. These questions were the focus of the exploratory studies presented in this dissertation. The first three studies involved the development of a memory diary, a memory questionnaire, and a metamemory questionnaire, respectively. These instruments were used in the fourth and fifth studies. Studies 4 and 5 also included behavioral measures of prospective memory and objective measures of retrospective memory. The fourth study examined how community-dwelling adults feel about, and use, their memory on a daily basis. In accordance with previous research, no age differences were observed on the behavioral measures of prospective memory. Moreover, performance on the self-report measures did not differ as a function of age. As hypothesized, age was correlated with performance on a retrospective memory task. The fifth study involved an extension and replication of Study 4, with the major addition being the use of several standard laboratory tests to assess retrospective memory. This study revealed several interesting findings. First, in contrast to previous studies, performance on some retrospective memory tasks was related to performance on some prospective memory tasks. Second, by and large, self-report and behavioral measures of prospective memory were not correlated. Third, performance on the prospective memory tasks (both self-report and behavioral) did not vary as a function of age, although performance ' on the retrospective memory tasks was clearly age related. The implications of this research are discussed. / Arts, Faculty of / Psychology, Department of / Graduate

No description available.