Fundulus is a diverse and widespread genus of small teleost fish of North America. Due to its high tolerance for physiochemical variation (e.g. temperature, oxygen, salinity), Fundulus is a model organism to study physiological and molecular adaptations to environmental stress. The thesis focuses on patterns of protein expression in Fundulus heteroclitus and F. grandis.The patterns of protein expression were investigated using traditional methods of enzyme activity measurements and recent proteomic approaches. The findings of the study can be used to guide future studies on the proteomic responses of vertebrates to environmental stress. Chapter 2 focuses on measurement of the temporal effects of oxygen treatments on the maximal specific activities of nine glycolytic enzymes in liver and skeletal muscle during chronic exposure (28d) of Fundulus heteroclitus. The fish was exposed to four different oxygen treatments: hyperoxia, normoxia, moderate hypoxia, and severe hypoxia. The time course of changes in maximal glycolytic enzyme specific activities was assessed at 0, 8, 14 and 28 d. The results demonstrate that chronic hypoxia alters the capacity for carbohydrate metabolism in F. heteroclitus, with the important observation that the responses are both tissue- and enzyme-specific. Chapter 3 studies the effect of tissue storage on protein profile of tissues of F. grandis. The technique of one dimensional gel electrophoresis (1D-SDS-PAGE) was used to assess the effects of tissue sampling, flash frozen in liquid nitrogen versus immersion of fresh tissue in RNA later, for five tissues, liver, skeletal muscle, brain, gill, and heart, followed by LC-MS/MS to identify protein bands that were differentially stabilized in gill and liver. The study shows that, in F. grandis, the preferred method of preservation was tissue specific. xi Chapter 4 focuses on the use of advanced 2DE-MS/MS to characterize the proteome of multiple tissues in F. grandis. Database searching resulted in the identification of 253 non-redundant proteins in five tissues: liver, muscle, brain, gill, and heart. Identifications include enzymes of energy metabolism, heat shock proteins, and structural proteins. The protein identification rate was approximately 50 % of the protein spots analyzed. This identification rate for a species without a sequenced genome demonstrates the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.

Therapeutic proteins, a.k.a. biopharmaceuticals, are most commonly produced in expression systems derived from Chinese Hamstery Ovary (CHO) cells, thanks to great capacity of post-translational modifications like secretation, folding and glycosylation. The engineering of cells for regulation of protein expression has many options including knock-in and knock-out of genes, epigenetic studies or improvement of the expression casette of the protein of interest by e.g. promotor variants or modifications of the 5’ and 3’ untranslated region (UTR). The 3’-UTR is therefore a good optimization candidate for attempting to achieve increased expression of therapeutic proteins. The final aim of this study was to identify and design 3’-UTRs for improved expression of therapeutic proteins in HyClone™ CHO cells from GE Healthcare Bio-Sciences AB (GEHC). The impact goal is to increase the efficiency and lower the costs for pharmaceutical companies when producing biopharmaceuticals in the HyClone™ CHO cell line, leading to increased accessibility of monoclonal antibodies (mAbs) on the pharmaceutical market. The study was initiated with bioinformatic analysis of the CHO cell transcriptome from a set of RNA-seq data of HyClone™ CHO to find high expressing, context independent genes. The 3’-UTRs from the best candidate genes were used for construction of plasmids for expression of a Fc-eGFP fusion protein. Nine selected 3’-UTRs were designed, synthesized and cloned into a parent plasmid (pGE0520) creating nine plasmid variants (pGE0523-531). The constructed plasmids were used for evaluation with site directed integration (SDI) into the HyClone™ CHO cell line and expression analysis were performed by flow cytometry and antibody titer measurements from cells with successfully integrated plasmid sorted by fluorescence-activated cell sorting (FACS).   Result show a significant effect on protein expression when using different variants of 3’-UTRs. Two variants, pGE0524 and pGE0526, competing with the parent plasmid in expression levels and integration efficiency from SDI, making them candidates for further investigations against the parent plasmid. Results also show good correlation between flow cytometry data from pre- and post-sorting, which can make research for further 3’-UTRs more efficient by evaluations and prediction of expression levels before cell sorting.

Como resultado do seu passado evolutivo, a família dos camarões Palaemonidae reúne representantes de ambientes osmóticos dos mais variados. Sejam de ambientes marinhos, estuarinos ou dulcícolas, estáveis ou variáveis, as espécies destes camarões mantêm a concentração osmo-iônica da hemolinfa independente da concentração do meio. Essas espécies hiper-regulam a osmolalidade e íons da hemolinfa em meio diluído e água doce e hipo-regulam em meio concentrado ou água do mar. Um importante local de transporte iônico envolvido na regulação osmo-iônica é o epitélio brânquial, pois nas membranas de seus ionócitos constituintes encontra-se um conjunto de transportadores que efetuam o movimento transepitelial de íons. Dentre estes transportadores, o simportador Na+, K+, 2Cl- (NKCC) é considerado ter um papel na secreção de sal, objetivo primário dessa investigação. Espécies representativas de habitat marinho do gênero Palaemon foram coletadas em regiões de estuário e de poça de maré, e de habitat dulcícola do gênero Macrobrachium foram coletadas em rios que desembocam no mar e também em riachos continentais sem influência do aporte salobro. Avaliou-se os limites letais de salinidade superior (LSS50) das espécies marinhas P. northropi e P. pandaliformis e dulcícolas diádromas M. acanthurus, M. olfersi, M. amazonicum que dependem de água salobra para completo desenvolvimento larval, e hololimnéticas M. potiuna e M. brasiliense com ciclo reprodutivo completo em água doce. Objetivou-se aqui (i) caracterizar os mecanismos de hiperregulação (condição controle de 18 S P. northropi, 17 S P. pandaliformis, água doce <0,5 S nas espécies de Macrobrachium) e hiporegulação [a curto (24 h) e longo prazo (120 h) em salinidade correspondente a 80% da LSS50] da osmolalidade e [Cl-] da hemolinfa, da expressão gênica e proteica e a localização por imunofluorescência do NKCC nos ionócitos branquiais; (ii) da existência de um padrão filogenético nesses parâmetros; e (iii) testar as hipóteses de um efeito da salinidade na evolução da expressão gênica e proteica desse simportador. As espécies de Palaemon apresentaram os maiores limites de tolerância ao aumento da salinidade, assim como exibiram uma maior capacidade hiporegulatória a longo prazo (120 h) comparada aos representantes de Macrobrachium. Dentre as espécies de Macrobrachium, os limites de tolerância foram maiores nas espécies diádromas do que nas hololimnéticas. Os parâmetros LSS50, osmolalidade e [Cl-] da hemolinfa demonstraram-se estruturados na filogenia, sendo as semelhanças compartilhadas justificadas pela estreita proximidade entre as espécies. As análises filogenéticas revelaram que a capacidade hiper-regulatória da [Cl-] da hemolinfa foi correlacionada com a expressão gênica do simportador NKCC nas brânquias, enquanto que a síntese proteica do NKCC parece estar associada à hiper-regulação da osmolalidade da hemolinfa. A avaliação da localização do NKCC por imunofluorescência demonstrou que o simportador está distribuído em ambas as células que compõem o epitélio das brânquias, as células pilares e células do septo intralamelar. A localização na porção inferior das franjas e no corpo da célula pilar e por toda célula do septo não diferiu entre as espécies, e também não difereiu entre as condições controle e a curto e longo prazo em salinidade elevada. Esses resultados em conjunto sugerem a importância do NKCC também na captação de sal pelas brânquias. Houve um aumento da síntese proteica do NKCC nas brânquias dos representantes de Macrobrachium, exceto M. potiuna, quando em salinidade elevada. Observou-se que este aumento é explicado pela proximidade filogenética entre as espécies. Não houve mudança na transcrição de RNAm para o NKCC apesar do aumento na síntese proteica, o que sugere uma possível regulação pós-transcricional. A reconstrução da história evolutiva da osmorregulação, incorporando o conceito de filofisiologia, revelou a existência de mecanismos em nível molecular, celular e sistêmico que evoluíram acompanhando os eventos cladogenéticos dos Palaemonidae durante a irradiação e ocupação de diferentes nichos osmóticos. / Owing to their evolutionary history, the shrimp family Palaemonidae includes species from widely distinct osmotic environments. Whether from marine, estuarine, or fresh waters, inhabiting stable or variable osmotic niches, these shrimps maintain the osmotic-ionic concentration of their hemolymph independently of the concentration of the external medium. These species hyper-regulate hemolymph osmolality and ions in dilute medium and fresh water and hypo-regulate this fluid in concentrated medium or seawater. The gill epithelium constitutes an important interface of ion transport, and its constituent ionocytes express an ensemble of ion transporters that enable active transepithelial ion movements. The Na+, K+, 2Cl- cotransporter (NKCC) is thought to play a significant role in compensatory salt secretion. Species representative of the marine habitat (Palaemon) were collected from estuaries and tidal pools; diadromous species from the fresh water habitat (Macrobrachium) were collected near the mouths of rivers that flow into the sea, while hololimnetic species were collected in continental streams lacking the influence of brackish waters. The critical upper salinity limits (LSS50) of the marine species P. northropi and P. pandaliformis and the diadromous freshwater species M. acanthurus, M. olfersi, M. amazonicum that depend on brackish water for complete larval development, and the hololimnetic M. potiuna and M. brasiliense that complete their reproductive cycle entirely in fresh water were established. Our objectives were to characterize the mechanisms of hyper-regulation (control condition 18 S P. northropi, 17 S P. pandaliformis, fresh water <0.5 S for Macrobrachium) and hypo-regulation [short-term (24 h) and long-term 120 h) at salinities corresponding to 80% of LSS50] of hemolymph osmolality and [Cl-], gene and protein expression, and NKCC localization for immunofluorescence in the gill ionocytes; (ii) the existence of a phylogenetic pattern in these parameters; and (iii) to test hypotheses for a salinity effect on the evolution of the gene and protein expression of this symporter. The species of Palaemon had the highest tolerance limits to increased salinity, and also exhibited a greater hypo-regulatory capacity for long-term acclimation compared to the species of Macrobrachium. Among the Macrobrachium species, the LSS50 were higher in the diadromous species than in the hololimnetic species. The parameters LSS50 and osmolality and [Cl-] of the hemolymph were phylogenetically structured, similarities being shared by closely related species. The hyper-regulatory capacity of hemolymph [Cl-] correlated with NKCC gene expression in the gills, while NKCC protein synthesis appears to be associated with hyper-regulation of hemolymph osmolality. Immunofluorescence analysis showed that the NKCC was located in both cell types that constitute the gill epithelium, the pillar cells and the septal cells. The location of the NKCC in the lower flanges and perikarya of the pillar cells and throughout the septal cells, did not differ among species, and also did not differ among control conditions or short and long-term exposure at high salinity. These results together also suggest the importance of the NKCC in salt uptake by the gills. When in high salinity there was an increase in NKCC protein synthesis in the gills of the Macrobrachium species, except for M. potiuna. This increase can be explained by the phylogenetic proximity among those species, which excludes adaptive inferences. There was no change in NKCC mRNA transcription, which suggests possible post-transcriptional regulation. The reconstruction of the evolutionary history of osmoregulation, incorporating the concept of phylophysiology, revealed the existence of mechanisms at the molecular, cellular and systemic levels that have evolved accompanying the cladogenetic events of the Palaemonidae during their radiation and occupation of different osmotic niches.

Endotelinas (ETs) e sarafotoxinas (SRTXs) pertencem a uma família de peptídeos vasconstritores que podem regular a migração e/ou produção de pigmentos em células pigmentares de vertebrados (cromatóforos). Em peixes teleósteos, ETs/SRTXs induzem a migração de pigmentos. Em melanócitos humanos, as ETs promovem a melanogênese e mitogênese. ETs também regulam a transcrição de diversos genes. Esses efeitos são mediados por diferentes vias de sinalização intracelular, dentre elas a via da fosfolipase C (PLC), da proteína quinase C (PKC) e da cascata de sinalização por proteína quinases ativadas por mitógeno (MAPKs). A rodopsina é um fotopigmento responsável pela detecção de fótons presente nos bastonetes dos olhos dos vertebrados. A modulação da transcrição do gene para rodopsina em peixes teleósteos e mamíferos parece ocorrer através de elementos conservados. Cromatóforos podem responder diretamente à luz, resultando no deslocamento dos grânulos de pigmentos através dos processos dendríticos das células. Essas respostas evocadas por luz são provavelmente mediadas por moléculas fotorreceptoras expressas por essas células. A linhagem celular GEM-81, proveniente de eritroforoma do peixe teleósteo Carassius auratus, assim como os melanócitos B16 de Mus musculus expressam rodopsina e receptores para ETs dos subtipos ETB e ETA, respectivamente. O objetivo deste trabalho foi determinar se: 1) os níveis do RNAm para rodopsina poderiam ser modulados por SRTX S6c em GEM-81 e por ET-1 em B16 e quais os mecanismos de sinalização intracelular envolvidos nessa modulação; 2) os níveis protéicos de rodopsina também poderiam ser modulados por SRTX S6c em GEM-81 e por ET-1 em B16. Através de PCR em tempo real (quantitativo), demonstrou-se que SRTX S6c e ET-1 modulam os níveis do RNAm para rodopsina em GEM-81 e B16, respectivamente, de forma temporal e dose-dependente. Em GEM-81, essa modulação envolve a ativação de uma PKC e da cascata das MAPKs. Já em B16, há o envolvimento de PLC, cálcio como mensageiro intracelular, calmodulina, quinase dependente de cálcio/calmodulina e PKC. Através de ensaios de Western blotting, foi demostrado que na linhagem GEM-81 os níveis protéicos de rodopsina não são significativamente alterados por 24 horas de tratamento com SRTX 10-9M S6c, sugerindo o envolvimento de mecanismos de controle pós-transcricional na modulação da expressão protéica de rodopsina. Nas células B16 cuja extração de proteína total ocorreu 0 ou 6h após o fim do tratamento de 24h com ET-1 10-10M, os níveis protéicos de rodopsina não são significativamente alterados. Já nas células cuja proteína total foi extraída 3h após o fim do tratamento com ET-1, observou-se uma diminuição significativa dos níveis da proteína rodopsina. Esses resultados sugerem o envolvimento de mecanismos de controle pós-transcricional na modulação da expressão protéica de rodopsina, mecanismos estes exacerbados nas células B16 cuja extração de proteína ocorreu 3h após o fim do tratamento. / Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which can regulate pigment migration and/or production in vertebrate pigment cells (chromatophores). In teleostean fish, ETs/SRTXs induce pigment migration. In human melanocytes, ETs promote melanogenesis and mitogenesis. ETs also regulate the transcription of several genes. These effects are mediated by different intracellular signaling pathways, such as the phospholipase C (PLC), protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) cascade. Rhodopsin is a photopigment responsible for photon detection, found in vertebrate rod cells. Rhodopsin gene transcription regulation in teleostean fish and mammals seems to occur through conserved elements. Chromatophores can respond directly to light, promoting the migration of pigment granules along the cells dedritic processes. These light-evoked responses are probably mediated by photoreceptive molecules expressed by these cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81 and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. The aim of this study was to determine whether 1) rhodopsin mRNA levels could be modulated by SRTX S6c in GEM-81 cells and ET-1 in B16 cells and the intracellular signaling mechanisms involved; 2) rhodopsin protein levels could also be modulated by SRTX S6c in GEM-81 and ET-1 in B16 cells. Using real time (quantitative) PCR, we demonstrated that SRTX S6c and ET-1 modulate rhodopsin mRNA levels in GEM-81 and B16, respectively, in a time and dose-dependent way. In GEM-81, this modulation involves the activation of a PKC and the MAPK cascade. In B16, it involves PLC, calcium as a second messenger, calmodulin, a calcium/calmodulin dependent kinase and PKC. The Western blotting assays demonstrated that in GEM-81 cells rhodopsin protein levels are not significantly altered by a 24-hour treatment with 10-9M SRTX S6c, suggesting the involvement of post-transcriptional mechanisms in the modulation of rhodopsin expression. In B16 cells, whose total protein was extracted 0 or 6 hours after the 24-hour treatment with 10-10M ET-1, rhodopsin protein levels were not significantly altered. When the cells total protein was extracted 3 hours after the 24-hour treatment with ET-1, a significant reduction in rhodopsin protein levels was observed. These results also suggest the involvement of post-transcriptional mechanisms in the modulation of rhodopsin expression in this cell line. These mechanisms could be somehow exacerbated in B16 cells whose protein was extracted 3 hours after the treatment.

O estudo dos protozoários é importante por diversos motivos, entre eles sua diversidade, sua importância evolucionária e impacto na saúde pública. Muitos aspectos tornam o estudo desses seres vivo ainda mais fascinantes, como por exemplo, fenômenos como o trans-splincing e a presença de um vírus em algumas espécies de leishmanias. A proteína U5-15k é considerada de grande importância fazendo parte do spliciossoma. Neste trabalho tivemos o objetivo de iniciar a caracterização desta proteína empregando ferramentas de bioinformática para analisar sua sequencia de aminoácidos, sua estrutura secundária e modelar sua estrutura por homologia, também foi possível otimizar sua expressão, purificação, caracterizar sua auto clivagem e fazer estudos biofísicos de Espalhamento Dinâmico de Luz e Espectroscopia de Dicroísmo Circular. O Leishmania RNA vírus 1-4 (LRV1-4) é um vírus da família Totiviridae de capsídeo icosaédrico que codifica duas proteínas (proteína capsidial e RNA polimerase). Sendo pouco estudado tivemos o objetivo de iniciar a caracterização molecular deste vírus com objetivos futuros de empreender estudos estruturais e funcionais. Não foi possível obter expressão recombinante em nenhuma das duas proteínas virais, por isso se optou por se estabelecer um novo protocolo de lise celular e purificação com o qual se obteve imagens inéditas de Microscopia Eletrônica de Varredura, na qual o vírus é purificado ainda envolto em material genético. / The study of protozoa is important for several reasons, including their diversity, their evolutionary importance and public health impact. Many aspects make the protozoas study even more exciting, for example, phenomena such as trans-splincing and the presence of virus in some species of Leishmania. The U5-15k protein is considered of great importance as part of the spliciossome machinery. In this work we intended to characterize this protein , this using bioinformatics tools to analyze its amino acid sequence, its secondary structure and its structure by homology modeling, it was possible to optimize expression, purification, characterization and make biophysical studies of their self cleavage of Dynamic Light Scattering and Spectroscopy of Circular Dichroism. The Leishmania RNA virus 1-4 (LRV1-4) is a virus belonging to the Totiviridae family with an icosahedral capsid structure that encodes two proteins (a capsid major protein and an RNA polymerase). It was not possible to obtain the recombinant expression of any of the two viral proteins, so it was decided to establish a new protocol for cell lysis and purification with which it was obtained unprecedented images of Transmission electron microscopy, in which the virus is further purified wrap on genetic material.

As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-&#946; (Transforming Growth Factor &#946; ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto \"in vitro\" como \"in vivo,\" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia para reparo ósseo. A expressão diferencial de genes durante a osteodiferenciação de células C2C12 induzida por rhBMP2 e rhBMP7, foi analisada através de microarranjos de DNA, selecionando 31 genes, dos quais 24 foram validados por qPCR, 13 dos quais são relacionados à transcrição, quatro associados a algumas vias de sinalização celular e sete associados à matriz extracelular. Análise funcional destes genes permitirá conhecer, com maiores detalhes, os eventos moleculares que ocorrem durante a diferenciação osteoblástica de células C2C12 induzida por rhBMPs. Em paralelo, foi perseguida a super-expressão de rhBMP2 e rhBMP7 em células HEK293T, demonstrando-se a atividade de rhBMP7, induzindo osteodiferenciação \"in vitro\" e formação de osso \"in vivo\", demonstrando a viabilidade do objetivo de se produzir estas proteínas para futura aplicação como biofármacos no Brasil. / The BMPs (Bone Morphogenetic Proteins) are members of the TGF-&#946; (Transforming Growth Factor &#946;) superfamily of proteins, regulate growth and differentiation of various cell types in various tissues, and some play a critical role in differentiation of mesenchymal cells into osteoblasts. Particularly, rhBMP2 and rhBMP7, promote osteoinduction \"in vitro\" and \"in vivo\" and both proteins are used therapeutically in Orthopedics and Dentistry. The differential expression of genes during osteodifferentiation induced by rhBMP2 and rhBMP7 in C2C12 cells was analyzed through DNA microarrays, allowing the selection of 31 genes, of which 24 were validated by qPCR, 13 of which are related to transcription, four associated with cell signaling pathways and seven are associated with the extracellular matrix. Subsequent functional analysis of these genes should reveal more details on the molecular events which take place during C2C12 cells osteoblastic differentiation induced by rhBMPs In paralel, rhBMPs 2 and 7 were overexpressed in HEK293T cells and BMP7 activity to induce osteodifferentiation \"in vitro\" and bone formation \"in vivo\" was demonstrated, reinforcing the viability of our objective to produce these proteins for future application as biopharmaceuticals in Brazil.

A Esquistossomose é uma das mais importantes doenças endêmicas do mundo, com mais de 200 milhões de pessoas infectadas em 76 países. É estimado que mais de 600 milhões de pessoas estejam em áreas endêmicas. Em 2003 dados do transcriptoma do Schistosoma mansoni foram disponibilizados. As informações de proteínas codificadas permitiram a análise de suas funções, e auxiliaram na procura de novos candidatos vacinais. A análise do transcriptoma permitiu a identificação de três famílias de proteínas homólogas à dineína de cadeia leve (DLC) de mamíferos. Uma delas é a família L8, com ao menos 18 membros, com proteínas em torno de 10 kDa. Essas proteínas são expressas em diferentes estágios do ciclo de vida do Schistosoma mansoni. Duas DLCs foram reconhecidas no tegumento de S. japonicum, sugerindo que elas são expostas ao sistema imune do hospedeiro. Salmonelas atenuadas como vacinas vivas têm sido descritas como bons veículos para apresentação de antígenos heterólogos. No nosso laboratório uma importante ferramenta tem sido desenvolvida para auxiliar o uso de salmonelas como vacinas vivas recombinantes, foi desenvolvido um vetor plasmididal baseado no regulon soxRS para controlar a expressão de genes heterólogos in vivo. Este sistema de expressão promove a expressão de proteínas recombinantes sob condições de estresse oxidativo, como aquele imposto ao microorganismo dentro do macrófago. Para investigar o potencial vacinal das DLCs, três genes foram selecionados para serem clonados e expressos em E. coli e em salmonelas atenuadas. A antigenicidade e imunogenicidade desses parálogos foram testadas em camundongos depois de serem imunizados com proteínas purificadas ou com salmonelas recombinantes. As DLCs mostraram ser bastante imunogênicas, aumentando os título de IgG. A proteína DLC1 também aumentou os níveis de IgE no soro dos animais, fato que poderia estar relacionado com reações alérgicas observadas em populações infetadas. Altos níveis de IgE também podem ser relacionados com uma marca de resistência existente em pessoas que vivem em áreas endêmicas. Depois de imunizados os animais foram desafiados com cercárias para investigar possível proteção. Foi observado que a imunização com proteínas purificadas resultou em aproximadamente 40% de diminuição da carga parasitária. A análise dos granulomas hepáticos com 45 dias depois da infecção indicou uma significativa redução, maior que 70% das áreas dos granulomas, sgerindo que a imunização com as DLCs promove uma importante interferência na formação dos granulomas hepáticos. Por outro lado, nossos estudos com salmonelas atenuadas recombinantes, carregando as DLCs, mostraram que foram ineficientes na apresentação dos antígenos ao sistema imune. Relacionando os resultados de diminuição de carga parasitária e das áreas dos granulomas depois da imunização com as DLCs purificadas sugere-se que essas proteínas podem ser consideradas como interessantes candidatos vacinais, uma vez que elas afetam as mais importantes causas da patologia da esquistossomose. / Schistosomiasis is one of the most important endemic diseases in the world, with more than 200 million people infected in 76 countries. It is estimated that more than 600 million people live in endemic areas. In 2003 extensive data from the transcriptome of Schsitosoma mansoni was made available. The information on the encoded proteins allowed the analysis of protein function and improved the search for vaccine candidates. The analysis of the transcriptome allowed the identification of three families of proteins homologs to the mammalian dynein light chain (DLC). One of these was the L8 family, with at least 18 members, all proteins with around 10 kDa. These proteins were found to be expressed in the different stages of the S. mansoni life cycle. Two DLCs were recognized in the tegument of S. japonicum, suggesting that they are exposed to the host immune system. Attenuated salmonellas, as live vaccines, have been described as good vehicles for presentation of heterologous antigens. At our laboratory an important tool has been developed to improve the use of salmonellas as live recombinant vaccines, a plasmidial vector based on the soxRS regulon to control the expression of heterologous genes in vivo. This expression system promotes the expression of recombinant proteins under conditions of oxidative stress, such as that imposed to the microorganism in the macrophage environment. To investigate the vaccine potential of DLCs, three genes were selected to be cloned and expressed in E. coli and in attenuated salmonellas. Antigenicity and immunogenicity of these paralogous were tested in mice after immunization with purified proteins or with the recombinant salmonellas. The DLCs were proven to be very immunogenic, increasing the IgG titers. DLC1 also increased the IgE levels in the sera of animals, what could be related to allergenic reactions observed in infected population. High level of IgE can also be related to the resistance mark of people living in endemic areas. After immunization, the animals were challenged with cercarias to investigate the protective profile. It was observed that immunization with purified proteins resulted in approximately 40% decreasing in the worm burden. The analysis of the hepatic granulomas 45 days after infection indicated a significant, up to 70 %, reduction of the granuloma areas, suggesting that immunization with DLCs promotes important interference in the hepatic granuloma formation. On the other hand, our studies with the attenuated recombinant salmonellas, carrying DLCs, showed no effective antigen presentation to the mice immune system. Taking together, the results of decreasing the worm burden and the granuloma size after immunization with purified DLCs suggest that these proteins could be considered as interesting vaccine candidates, affecting the main causes of the pathology of schistosomiasis.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas

Tip60 is a 60 kDa nuclear protein which exists in three isoforms, belongs to the MYST/HAT family of proteins and was discovered after its interaction with the Human HIV-1 Tat. As a nuclear protein, Tip60 can act as a coactivator or repressor. To understand the HAT action of Tip60, two possible catalytic models exist; the ping-pong and the ternary complex formation models. In correlation with the exploration of HAT catalytic action, mutations of a Cys to Ala and a Glu to Gln on Esa1 (yeast homolog of Tip60 and MYST/HAT prototype), was reported to show wild type-like and decreased acetylating properties, respectively. In this work, Tip60 HAT action was explored. In Tip60, the Cys in the active site is important for acetylation of the H4(1-20) substrate and the Glu showed semi loss in acetylating the H4(1-20) peptide substrate. These data highlight a unique mechanism of Tip60 catalysis.