Plaquetas apresentam um papel importante no desenvolvimento de metastases tumorais. Os eventos que levam à ativação plaquetária, como agragação e secreção de proteínas, podem significar etapas importantes neste papel. O agonista plaquetário trombospondina está envolvido no processo de agragação plaquetária. Com o intuito de clonar o receptor de trombospondina GpIIIb, produziu-se um soro policlonal contra uma banda eluída de SDS PAGE de extrato proteico de plaquetas, que apresentava peso molecular igual ao de GpIIIb (denominada banda 80kD). Uma biblioteca de cDNA de endotélio de cordão umbilical humano construída em lambda gt11 foi varrida com este soro anti-80kD. Dois clones diferentes foram isolados, seus insertos foram subclonados no vetor pGEM-3Z e sequenciados. Através de consulta ao Genbank observou-se que um dos clones não apresentou homologia significativa com nenhuma proteína até então clonada. O outro clone, por sua vez, apresentou 100% de homologia com a proteína 80K-H, substrato de proteína quinase C. Levando em consideração o fato de que as vias detransdução de sinal que utilizam PKC apresentam extrema importância nos processos de ativação plaquetária decidiu-se prosseguir com a caracterização de 80K-H. Para isto foi produzido um soro policlonal contra a proteína de fusão 80K-H, que foi utilizado em ensaios bioquímicos e imunoquímicos que permitiram caracterizar a proteína 80K-H quanto a alguns aspectos como distribuição em diferentes tipos celulares, localização celular e fosforilação. Além de estar presente em plaquetas, a proteína 80K-H foi encontrada em todas as linhagens celulares testadas, parecendo portanto ser uma proteína ubíqua. Os dados obtidos indicaram que, apesar de apresentar uma sequência N-terminal que é clivada \"in vivo\" muito semelhante a um peptídeo sinal, 80K-H não é secretada nem é de membrana plasmática, mas sim citoplasmática. Em ensaios de fosforilação \"in vivo\" não se detectou fosforilação de 80K-H. Portanto, apesar de 80K-H ser um bom substrato para PKC \"in vitro\", ela não o é \"in vivo\", ao menos nas células analisadas, ou é fosforilada de uma forma extremamente rápida e transiente. / Abstract not available.

The 14-3-3 family of proteins are important signalling proteins involved in a number of cellular processes. These include cell cycle regulation, apoptosis, signal transduction and cell signalling. There is also considerable evidence in the literature that 14-3-3 proteins play a vital role in the pathology of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, Huntington’s and Prion disease. The neurodegenerative disease of focus in this research is Spinocerebellar Ataxia Type 1 (SCA1). SCA1 is a polyglutamine-repeat disease and the interaction of the disease protein ataxin-1 with 14-3-3 proteins leads to the toxic accumulation and subsequent protein aggregation which is characteristic of this disease. This study focused on attempting to elucidate the structure of various domains of the disease protein and also in identifying potential inhibitors of this deleterious interaction. Unfortunately, structural studies were not successful due to a number of caveats encountered in the expression and purification of the ataxin-1 protein domains. By utilising computational methods and small molecule inhibitors, a number of potential lead compounds which possess the ability to at least partly disrupt the interaction of 14- 3-3ζ have been identified. As 14-3-3 proteins play roles in other neurodegenerative diseases, successful identification of potential drug lead treatments can have far reaching benefits in a number of neurodegenerative diseases including SCA1. Lipid rafts are also involved in neurodegenerative disease pathology. Lipid rafts are cholesterol and sphingolipid rich domains which organise the plasma membrane into discrete microdomains and act as signalling platforms and processing centres which attach specific proteins and lipids. A number of disease proteins are processed at these membrane regions, including those involved in Alzheimer’s, Parkinson’s and Prion disease. This processing is a step which is critical in the pathology of disease and abnormal processing leads to the formation of toxic protein aggregates. Previous research in the lab identified the association of low levels of the five main brain isoforms of 14-3-3 proteins with rafts. This study expanded on this to positively identify the presence of the two phospho-forms of 14-3-3, α and δ. The mechanism by which 14-3-3 proteins associate with rafts was also investigated, indicating that 14-3-3 associates with rafts via an unidentified raftbound protein(s). In addition, the phosphorylation status and quaternary structure of 14-3-3 in the presence of sphingolipids has been explored.

Usando incorporação \"in vivo\" de 35S metionina tradução \"in vitro\" de RNA e eletroforese bidimensional, iniciamos um estudo do controle da síntese de proteínas durante duas fases distintas de diferenciação celular, a esporulação e a germinação, do fungo Blastocladiella emersonii. Durante a esporulação ocorre uma intensa variação no padrão de síntese proteica. Foi analisada a síntese de 108 proteínas, sendo verificado que o aumento na síntese de várias proteínas está associado com estágios definidos da esporulação. Um grande número de proteínas básicas é sintetizado exclusivamente no final da esporulação, que corresponde à fase de diferenciação dos zoósporos. Também foram detectadas drásticas variações na população de mRNAs ao longo de toda a esporulação. A síntese de várias proteínas típicas da esporulação parece ser controlada ao nível da transcrição. Além disso a maioria dos RNAs mensageiros específicos da esporulação não é conservada nos zoósporos maduros; o zoósporos contém mRNAs armazenados que provavelmente são sintetizados nos últimos 30 minutos da esporulação. Durante a transição dos zoósporos a células redondas, que ocorre nos primeiros 25 minutos após a indução da germinação em meio inorgânico, não foram verificadas diferenças qualitativas no padrão de síntese proteica, tanto na ausência como na presença de actinomicina D, indicando que os eventos precoces da germinação são inteiramente pré-programados pelo mRNA que está armazenado nos zoósporos. Contudo, na germinação tardia são verificadas profundas variações no padrão de síntese proteica. A síntese de algumas dessas proteínas (seis polipeptídios), provavelmente corresponde a uma tradução seletiva de mensagens armazenadas nos zoósporos, enquanto que a maioria das novas proteínas expressas (vinte e dois polipeptídios) corresponde a tradução de novos mRNAs. Assim, durante a germinação dos zoósporos, ocorrem múltiplos níveis de regulação da síntese proteica, envolvendo controles ao nível da tradução e transcrição. Durante o início da germinação também foi observado um controle ao nível de pós-traduçãoo, com várias proteínas dos zoósporos sendo especificamente degradadas ou modificadas. Também analisamos o padrão das proteínas sintetizadas durante a germinação em meio nutriente sendo observada a síntese de polipeptídios específicos desta condição de germinação e crescimento. Algumas proteínas cuja síntese é controlada pelo desenvolvimento foram identificadas. Utilizando anticorpos monoclonais comerciais contra actina, α e β-turbulinas foip-tubulinas foi possível identificar estas proteínas no perfil eletroforético de proteínas sintetizadas durante a esporulação. Comparando a cinética da síntese \"oin vitro\" destas proteínas com o acúmulo de seus respectivos mRNAs traduzidos \"in vitro\", o, foi possível demonstrar que o intenso aumento na síntese de actina, α e β-tubulinas que ocorre durante a esporulação apresenta uma correlação temporal com o aumento dos mRNAs correspondentes. Em paralelo ao aumento da síntese destas três proteínas citoesqueLéticas pôde ser detectado um aumento dos seus conteúdos em massa. Durante a germinação e crescimento ocorre uma sensível diminuição no conteúdo destas proteínas. Além disso, verificamos que as proteínas identificadas como α e β-tubulinas estão presentes no flagelo dos zoósporos. Muito interessante foi a observação de que três proteínas sintetizadas durante a esporulação correspondiam aparentemente a três proteínas, Hsp70, Hsp76 e Hsp39a, cuja síntese é induzida pelo choque térmico. Esta verificação decorreu do fato de estarmos investigando se em Blastocladiella a resposta ao choque térmico teria algum controle do desenvolvimento, uma vez que alguns dados da literatura sugeriam o envolvimento de certas proteínas de choque térmico (Hsps) no desenvolvimento normal de alguns organismos. Em Blastocladiella a resposta ao choque térmico é dependente do estágio do desenvolvimento. Células expostas a temperaturas elevadas nos diferentes estágios do desenvolvimento (esporulação, germinação e crescimento) mostram uma síntese diferencial de proteínas de choque térmico. Conjuntos específicos de Hsps (de um total de 22 Hsps) são induzidos em cada fase, demonstrando uma expressão não coordenada dos genes de choque térmico. A proteína de 70 kDa, sintetizada espontaneamente durante um certo intervalo da esporulação, apresenta mobilidade eletroforética em géis bidimensionais idêntica à Hsp70. A confirmação da identidade entre estas proteínas foi obtida através de análise dos seus peptídios resultantes de digestão enzimática parcial bem como pelo reconhecimento de ambas as proteínas por anticorpos contra a proteína DnaK (homóloga à Hsp70> de E. coli e contra a proteína Hsp70 de Drosophila. Utilizando tradução o\"in vitro\"o de RNA e hibridização de RNA com uma sonda do gene hsp70 de Drosophila, demonstramos que o aumento de síntese da Hsp70 que ocorre durante o choque térmico e espontaneamente durante a esporulação, está associado com a acumulação do mRNA desta proteína. Embora a síntese de Hsps seja controlada pelo desenvolvimento em Blastocladiella, a aquisição de termotolerância pode ser induzida em qualquer estágio do seu ciclo de vida. A indução da termotolerância em Blastocladiella é dependente da síntese de proteínas e está correlacionada com o aumento da síntese de algumas Hsps: Hsp82a, Hsp82b, Hsp76, Hsp70, Hsp60,Hsp25 e Hsp17b. As outras Hsps parecem não estar envolvidas especificamente com a termotolerância. As observações anteriores de que o estado de fosforilação da proteína ribossômica 56, em Blastocladiella, varia durante o desenvolvimento e em resposta a alterações do meio ambiente (Bonato et al., 1984, Eur. J. Biochem. 144:597-606) e a verificação de que a resposta ao choque térmico, em Blastocladiella, também está sob o controle do desenvolvimento proporcionou a oportunidade de verificar se os diferentes níveis de fosforilação de 56 poderiam ser correlacionados com a tradução de mensageiros específicos isto é, mRNAs normais ou de choque térmico durante o choque térmico, recuperação do choque térmico e indução de termotolerância nos diferentes estágios do ciclo de vida deste fungo. Assim foi observado que, independente do estado inicial de fosforilação de 56 (máximo ou intermediário>, ocorre uma rápida e completa desfosforilação de 56 durante o choque térmico, sendo que a 56 permanece desfosforilada durante a termotolerância. Durante a recuperação do choque térmico, ocorre a refosforilação de 56 para os níveis característicos de cada estágio do desenvolvimento, coincidentemente com a interrupção da síntese de proteínas de choque térmico. / Using 35S methionine pulse labeling in vitro translation and two-dimensional gel electrophoresis, we investigated the regulation of protein synthesis during two distinct phases of cell differentiation, sporulation and germination, in the aquatic fungus Blastocladiella emersonii. We have found dramatic changes in the spectrum of proteins synthesized during sporulation. Synthesis of 108 polypeptides was analyzed and a large increase in the synthesis of several proteins is associated with particular stages. A large number of basic proteins are synthesized exclusively during late sporulation. Changes in translatable mRNA species were also detected by in vitro translation of RNA prepared at different stages of sporulation. The synthesis of several proteins during sporulation seems to be transcriptionally controlled. Most of the sporulationspecific messages are not present in the mature zoospores; the zoospores contain stored mRNA, which is apparently synthesized in the last 30 min of sporulation.We analyzed the pattern of proteins synthesized during zoospore germination in an inorganic solution, in both the presence and absence of actinomycin D. During the transition from zoospore to round cells (the first 25 min), essentially no qualitative differences were noticeable, indicating that the earliest stages of germination are entirely preprogrammed with stored RNA. Later in germination (after 25 min), however, changes in the pattern of protein synthesis were found. Some of these proteins (a total of 6 polypeptides) correspond possibly to a selective translation of stored messages, whereas the majority of the changed proteins (22 polypeptides) corresponds to newly synthesized mRNA. Thus, multiple levels of protein synthesis regulation seem to occur during zoospore germination, involving both transcriptional and translational controls. We also analyzed the pattern of protein synthesis during germination in a nutrient medium; synthesis of specific polypeptides occurred during late germination. During early germination posttranslational control was also observed, several labeled proteins from zoospores being specifically degraded or charge modified. Some proteins whose expression is developmentally regulated were identified. Actin, α- and β-tubulin have been identified in the two-dimensional pattern of proteins synthesized during sporulation by using well characterized monoclonal antibodies and western blotting. We compared the kinetics of synthesis of these proteins, by pulse-labeling experiments with ‌35S‌methionine, with the accumulation of their corresponding mRNAs, translated in a cell-free system. Large increases occur in the rates of actin and α- and β-tubulin biosynthesis during sporulation and there is an accumulation of the corresponding mRNAs. In parallel to the increased synthesis, these cytoskeletal proteins accumulate during the late stage of sporulation. During germination and early growth there is a strong decrease in the level of these proteins. We also verified that α- and β-tubulin are present in flagellar axonemes of zoopores. Very interesting was the observation that three proteins spontaneously expressed during sporulation correspond possibly to three heat shock-induced proteins CHsp70, Hsp76, Hsp39a). This fact was noticed when we were investigating the heat shock-response during the development of Blastocladiella. The heat-shock response in Blastocladiella is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of life cycle (sporulation, germination or growth) show a differential synthesis of heat-shock proteins (Hsps). Of a total af 22 polypeptides induced, particular subsets of Hsps appear in each phase, demonstrating a non-coordinate heat-shock gene expression. By the criteria of two-dimensional gel electrophoresis and partial proteolysis mapping, the 70-kDa protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat-inducible hsp70. Additional evidence in support of the identity between the 70-kDa protein and Hsp70 was provided by immunological cross-reaction of both proteins with antibodies against DnaK protein from E.coli and Hsp70 from Drosophila. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat-shock treatment and spontaneously during sporulation, is associated with an accumulation of Hsp70 mRNA. Although the Hsps synthesis is developmentally regulated in Blastocladiella, the acquisition of thermotolerance can be induced at any stage of the life cycle. lhe development of thermotolerance is correlated with the enhanced synthesis of some heat-shock proteins: Hsp82a, Hsp82b, Hsp76, Hsp70, Hsp60, Hsp25, Hsp17b. Other Hsps are not specifically involved in thermotolerance. In B. emersonii the state of 56 phosphorylation changes depending on the developmental stage and environmental conditions (Bonato et al., 1984, Eur. J. Biochem. 144, 597-606). On the other hand, we verified that the heat-shock response is developmentally regulated. Then, we examined the changes in 56 phosphorylation during heat shock, thermotolerance, and recovery from heat shock at different stages of life cycle in order to investigate whether the different levels of 56 phosphorylation might be correlated with the translation of specific message subsets. We observed that independently of the initial state of 56 phosphorylation (maximal or intermediate), a rapid and complete dephosphorylation of 56 is induced by heat shock and 56 remains unphosphorylated during the acquired thermotolerance. During recovery from heat shock rephosphorylation of 56 occurs always to the levels characteristic of that particular stage, coincidently with the turn off of heat shock protein synthesis.

Transmembrane signal transduction by ligand-activated G protein-coupled receptors (GPCRs) controls virtually every aspect of mammalian physiology, and this receptor class is the target of 40-50% of currently marketed pharmaceuticals. In addition to the clinical use of direct GPCR agonists and antagonists, it is now believed that GPCR effectors and regulators may also be viable drug targets with improved therapeutic efficacy and specificity. The prototypic role of Regulator of G protein Signaling (RGS) proteins is inhibition of G protein signaling through acceleration of GTP hydrolysis by GΑ, which promotes re-association of GΑ and GΒΓ subunits with the receptor at the cell membrane. In this way, RGS proteins determine the magnitude and duration of the cellular response to GPCR stimulation. Though RGS protein biochemistry has been well elucidated in vitro, the physiological functions of each RGS family member remain largely unexplored. RGS6 belongs to the R7 subfamily of RGS proteins originally identified in brain. Our acquisition of an RGS6-/- mouse allowed us to survey RGS6 expression in all tissues of the body revealing the greatest expression of RGS6 in brain. Despite robust neural RGS6 expression, little is known regarding functional roles of RGS6 in the brain and spinal cord. In addition, we identified several novel, higher molecular weight RGS6 immunoreactive bands specifically present in the nervous system. The plan of this thesis work was multifaceted. We sought to elucidate novel GPCR signaling cascades modulated by RGS6 in brain while simultaneously characterizing the expression patterns and identity of the novel RGS6 species specifically detected in the nervous system. Considering the large diversity of RGS6 isoforms present in brain, the abundance of potential RGS6 binding partners, and the possibility of discovering new mechanisms involved in RGS6 regulation, elucidation of the novel RGS6 molecular species is of paramount importance. Utilizing RGS6-/- mice we identified RGS6 as a critical modulator of two GPCRs in brain. First, by inhibiting the serotonin receptor 1A (5-HT1AR)-adenylyl cyclase (AC) axis, RGS6 functions to promote anxiety- and depression-related behaviors in mice. As a result, RGS6-/- mice exhibit a robust anxiolytic and antidepressant phenotype remarkably similar to that of animals treated chronically with therapeutic doses of selective serotonin reuptake inhibitors (SSRIs). RGS6 also inhibits GABAB receptor (GABABR)-G protein- activated inwardly rectifying potassium (GIRK) channel current in cerebellar granule cells, and loss of RGS6 results in cerebellar ataxia and gait abnormalities reversible by GABABR blockade. Furthermore, evaluation of voluntary alcohol drinking behaviors in WT versus RGS6-/- mice revealed a striking reduction in alcohol intake resulting from RGS6 loss in both acute and chronic alcohol consumption paradigms due, at least in part, to potentiation of GABABR signaling. Thus, RGS6 inhibitors have potential clinical utility in the treatment of mood disorders and alcoholism. We have shown that one novel RGS6 immunoreactive band expressed in the brain and spinal cord is a phospho-protein sensitive to Λ phosphatase-mediated dephosphorylation. Further, new information acquired from PCR amplification of RGS6 mRNA species from human brain cDNA libraries has necessitated substantial revisions to the RGS6 splicing scheme devised by the Fisher laboratory in 2003. To the 36 isoforms generated from two alternate transcription start sites (RGS6L vs. RGS6), the inclusion or exclusion of exons 14 and 17, and variable splicing to one of 7 different 3' terminal exons, we have added the possible insertion of three novel internal exons (A1, A2, A3), a retained intron, and two new 3' terminal exons. As a result, the number of RGS6 mRNAs present in brain could be as many as 248 unique species, an astonishing diversity unprecedented in the RGS protein family.

<p>In this thesis the oxidative metabolism by blood eosinophils from birch pollen allergic subjects was studied and compared to that by eosinophils from healthy controls, during and out of the pollen season. The effects and mechanisms of <i>in vitro</i> IL-5 priming on blood eosinophils were investigated and compared to the effects of <i>in vivo</i> priming during pollen exposure.</p><p>The main findings of this work were that the oxidative metabolism by blood eosinophils taken from pollen allergic subjects is reduced during the pollen season. The eosinophils taken from asymptomatic allergics have a reduced capacity to produce oxygen free radicals as compared to non-allergic controls. The oxidative metabolism by blood eosinophils from allergic subjects is primed <i>in vivo</i> during the pollen season, as compared to the healthy controls and as compared to out of season. IL-5 primed the oxidative metabolism by eosinophils from allergic subjects in a similar way as eosinophils from healthy controls, both during and out of pollen exposure. The total and tyrosine phosphorylation patterns obtained were identical in eosinophils from allergic subjects and non-allergic controls during the pollen season. Spontaneous phosphorylation was the same in both groups and different from that after IL-5 priming. The oxidative metabolism of blood eosinophils is composed of different stages. The initial stage, measured as the t_½rises of the CL curves, is an indication of the state of priming of the cell, while the end stage, measured as the peaks of the CL curves, is an estimate of the total radical production by the cells. IL-5 priming affected these two stages differently and the two stages are regulated by different signal transduction pathways and IL-5 priming causes a by-passing of MEK.</p><p>In conclusion, in this thesis it is shown that blood eosinophils from allergic subjects are primed <i>in vivo</i> during exposure to their allergen. This <i>in vivo</i> priming leads on one hand to a reduced oxidative metabolism during the pollen season, but also to a faster onset of radical production as a response to certain stimuli. Our data do not provide any evidence of IL-5 involvement in the <i>in vivo</i> priming of blood eosinophils from allergic patients during pollen exposure.</p>

In this thesis the oxidative metabolism by blood eosinophils from birch pollen allergic subjects was studied and compared to that by eosinophils from healthy controls, during and out of the pollen season. The effects and mechanisms of in vitro IL-5 priming on blood eosinophils were investigated and compared to the effects of in vivo priming during pollen exposure. The main findings of this work were that the oxidative metabolism by blood eosinophils taken from pollen allergic subjects is reduced during the pollen season. The eosinophils taken from asymptomatic allergics have a reduced capacity to produce oxygen free radicals as compared to non-allergic controls. The oxidative metabolism by blood eosinophils from allergic subjects is primed in vivo during the pollen season, as compared to the healthy controls and as compared to out of season. IL-5 primed the oxidative metabolism by eosinophils from allergic subjects in a similar way as eosinophils from healthy controls, both during and out of pollen exposure. The total and tyrosine phosphorylation patterns obtained were identical in eosinophils from allergic subjects and non-allergic controls during the pollen season. Spontaneous phosphorylation was the same in both groups and different from that after IL-5 priming. The oxidative metabolism of blood eosinophils is composed of different stages. The initial stage, measured as the t½rises of the CL curves, is an indication of the state of priming of the cell, while the end stage, measured as the peaks of the CL curves, is an estimate of the total radical production by the cells. IL-5 priming affected these two stages differently and the two stages are regulated by different signal transduction pathways and IL-5 priming causes a by-passing of MEK. In conclusion, in this thesis it is shown that blood eosinophils from allergic subjects are primed in vivo during exposure to their allergen. This in vivo priming leads on one hand to a reduced oxidative metabolism during the pollen season, but also to a faster onset of radical production as a response to certain stimuli. Our data do not provide any evidence of IL-5 involvement in the in vivo priming of blood eosinophils from allergic patients during pollen exposure.

Fertilization is a process that generates the first cell of a new organism. In mammals, fertilization occurs in the female reproductive tract. The male gametes (spermatozoa) are rendered fertilization-competent only after they undergo capacitation and acrosome reaction (AR). The set of physiological changes, characterised by the acquisition of hyperactivated motility, that render the spermatozoa fertilization competent is known as capacitation. Using in vitro models, the complex intracellular signaling events mediating this process are still being understood. This thesis explores the role of protein tyrosine phosphorylation during capacitation using the golden hamster (Mesocricetus auratus) spermatozoa. The knowledge about the molecular components involved in capacitation, apart from enriching our understanding about a basic cellular process could also provide leads in the management of male (in)fertility. A comprehensive review on the perspectives of male reproduction, spermatogenesis, the structural features of a spermatozoon and sperm maturation, relevant to the content of the thesis is provided in Chapter-1 (General Introduction). Molecular mediators that initiate capacitation include cAMP, Ca2+and HCO3- ions. These signalling molecules regulate activities of protein kinases and phosphatases, which control the level of protein phosphorylation in spermatozoa. Capacitation-associated increase in protein phosphorylation, specifically protein tyrosine phosphorylation (PYP) has been demonstrated in a few species such as mouse, rat and human. The unique nature of PYP signaling during sperm capacitation has been exemplified by discoveries of several male germ cell-specific signalling molecules like soluble adenylate cyclase. However,molecular identities of tyrosine-phosphorylated proteins and their functional role during sperm capacitation are yet to be investigated in detail. In this context, the effect of modulating intracellular levels of signaling molecules upstream of protein phosphorylation was sought using pentoxifylline (PF), a cAMP phosphodiesterase inhibitor. Interestingly, PF-induced capacitation was associated with an early induction of tyrosine phosphorylation of proteins (45-80 kDa) localized to the mid piece of the sperm tail. Interestingly, the ultrastructural localization of tyrosine-phosphorylated proteins in the sperm tail by immunoelectron microscopy (IEM) revealed most intense immunolabelling in the fibrous sheath, followed by outer dense fibers (ODFs)and the axoneme. Data pertaining to the effect of PF on sperm capacitation and the associated protein-phosphorylation is presented in Chapter-2. Since PYP was determined to be extremely critical for hyperactivation in spermatozoa, the involvement of protein tyrosine kinases (PTKs) in this process was assessed using a specific PTK inhibitor, tyrphostin A47 (TP-47: EGFR-TK specific). The third chapter deals with the effect of tyrphostins on sperm capacitation and PYP. A dose-dependent inhibition by TP-47 of capacitation and principal piece associated-PYP of ~45-60 kDa proteins was observed. Interestingly, TP-47 treated-spermatozoa exhibited a circular motility pattern; when assessed for kinematic parameters, by computer aided sperm analysis, sperm showed lower values for key kinematic parameters as compared to the controls. While sperm viability in TP-47- treated samples was not affected, the ATP content reduced towards latter (4-5 h) part of culture as compared to the controls. When spermatozoa were treated with two other PTK inhibitors, tyrphostin AG1478 (EGFR-TK specific) and tyrphostin AG1296 (PDGFR-TK specific), they did not show any changes in kinematic parameters or PYP, indicating that the TP-47-effect was compound-specific. The fourth chapter of this thesis involves the molecular analysis of proteins hypo-tyrosine phosphorylated in the presence of TP-47, which started with the enrichment of sperm flagellar proteins that are tyrosine phosphorylated during capacitation, using various detergents. Detergent extractions established that most tyrosine-phosphorylated proteins were non-membranous in nature, which complemented the IEM data. Therefore, phosphoproteome analysis of the untreated and TP-47-treated sperm samples was performed. For this, protein extracts were subjected to 2D-PAGE-phosphotyrosine immunoblots. A 51 kDa spot and two 45 kDa spots, corresponding to the hypo-tyrosine phosphorylated spots, were analyzed by MS/MS. While peptides from the 51 kDa protein matched with tektin-2 (a microtubular protein), those of the 45 kDa spots matched with ODF-2 protein of the sperm flagellum. Validation of the presence of tektin-2 and ODF-2 protein and their tyrosine-phosphorylated forms on sperm capacitation in the hamster spermatozoa has also been performed. In addition to detailing the role of PYP in hamster sperm capacitation, this study revealed the identities of a few of these proteins, whose tyrosine phosphorylated status could be critical for optimal sperm flagellar bending, required for sperm hyperactivation. By understanding causes that lead to altered sperm function, for example, as observed with hamster spermatozoa, new insights could be achieved into molecular regulatory mechanisms that govern sperm function in clinical cases of non-obstructive male infertility in the human.

No description available.

Plaquetas apresentam um papel importante no desenvolvimento de metastases tumorais. Os eventos que levam à ativação plaquetária, como agragação e secreção de proteínas, podem significar etapas importantes neste papel. O agonista plaquetário trombospondina está envolvido no processo de agragação plaquetária. Com o intuito de clonar o receptor de trombospondina GpIIIb, produziu-se um soro policlonal contra uma banda eluída de SDS PAGE de extrato proteico de plaquetas, que apresentava peso molecular igual ao de GpIIIb (denominada banda 80kD). Uma biblioteca de cDNA de endotélio de cordão umbilical humano construída em lambda gt11 foi varrida com este soro anti-80kD. Dois clones diferentes foram isolados, seus insertos foram subclonados no vetor pGEM-3Z e sequenciados. Através de consulta ao Genbank observou-se que um dos clones não apresentou homologia significativa com nenhuma proteína até então clonada. O outro clone, por sua vez, apresentou 100% de homologia com a proteína 80K-H, substrato de proteína quinase C. Levando em consideração o fato de que as vias detransdução de sinal que utilizam PKC apresentam extrema importância nos processos de ativação plaquetária decidiu-se prosseguir com a caracterização de 80K-H. Para isto foi produzido um soro policlonal contra a proteína de fusão 80K-H, que foi utilizado em ensaios bioquímicos e imunoquímicos que permitiram caracterizar a proteína 80K-H quanto a alguns aspectos como distribuição em diferentes tipos celulares, localização celular e fosforilação. Além de estar presente em plaquetas, a proteína 80K-H foi encontrada em todas as linhagens celulares testadas, parecendo portanto ser uma proteína ubíqua. Os dados obtidos indicaram que, apesar de apresentar uma sequência N-terminal que é clivada \"in vivo\" muito semelhante a um peptídeo sinal, 80K-H não é secretada nem é de membrana plasmática, mas sim citoplasmática. Em ensaios de fosforilação \"in vivo\" não se detectou fosforilação de 80K-H. Portanto, apesar de 80K-H ser um bom substrato para PKC \"in vitro\", ela não o é \"in vivo\", ao menos nas células analisadas, ou é fosforilada de uma forma extremamente rápida e transiente. / Abstract not available.

Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation. KEYWORDS Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation