Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A Bothrops leucurus (jararaca do rabo branco) à uma serpente peÃonhenta que habita a regiÃo Nordeste do Brasil. Os efeitos biolÃgicos devido ao envenenamento por B. leucurus tÃm perfil semelhante Ãqueles apresentados por outras serpentes do gÃnero Bothrops, tais como, importantes efeitos locais e sistÃmicos graves como a InsuficiÃncia Renal Aguda (IRA). O veneno de Bothrops leucurus induziu nefrotoxicidade em sistema de perfusÃo de rim isolado de rato, associado com citotoxicidade em cÃlulas tubulares epiteliais renais. Neste trabalho foi avaliado o efeito nefrotÃxico direto da enzima L-aminoÃcido oxidase isolada do veneno de Bothrops leucurus (LAAO-Bl) sobre cÃlulas epiteliais renais (MDCK e HK2) e o seu potencial nefrotÃxico em rim isolado de rato. O tratamento com LAAO-Bl, 1.56 â 100 Âg/mL induziu significativa morte celular de maneira concentraÃÃo dependente em ambas Ãs linhagens celulares apÃs 12 horas de tratamento. Nas cÃlulas MDCK nÃo foi observada liberaÃÃo de LDH apÃs 12 horas de exposiÃÃo à LAAO-Bl, enquanto nas cÃlulas HK2 LAAO-Bl induziu ruptura de membrana nas maiores concentraÃÃes estudadas quando comparado ao controle nÃo tratado. Nas cÃlulas MDCK o tratamento com LAAO-Bl aumentou significativamente a porcentagem de cÃlulas em apoptose (Anexina-V+, IP-), necrose (Anexina-V-, IP+) e necrose secundÃria (Anexina-V+, IP+). Nas cÃlulas HK2 LAAO-Bl induziu um aumento na porcentagem de cÃlulas em necrose (IP+) e necrose secundÃria (Anexina-V+, IP+) de maneira concentraÃÃo dependente. A induÃÃo de apoptose nas cÃlulas MDCK foi acompanhada de liberaÃÃo de Ca2+ do retÃculo endoplasmÃtico, aumento de espÃcies reativas de oxigÃnio, disfunÃÃo mitocondrial e aumento de expressÃo de Bax. O tratamento com LAAO-Bl induziu ativaÃÃo de caspase 3 e 7 em ambas as linhagens celulares. LAAO-Bl (10 Âg/mL) exerce efeitos significativos no rim isolado de rato aumentando a pressÃo de perfusÃo e o fluxo urinÃrio e diminuindo a taxa de filtraÃÃo glomerular e os transportes tubulares de sÃdio, potÃssio e cloreto. Os nossos resultados sugerem que LAAO-Bl contribui para nefrotoxicidade observada no envenenamento por Bothorps leucurus. AlÃm disso, os efeitos citotÃxicos de LAAO-Bl nas cÃlulas epiteliais renais podem ser responsÃveis, pelo menos em parte, pela nefrotoxicidade observada no rim isolado. / The pit viper Bothrops leucurus (White-tailed-jararaca) is a poisonous snake habituating area in the northeast of Brazil. The biological effects due envenomation have similar profile than those observed with other Bothrops, such as important severe local and systemic effects such as Acute Renal Failure (ARF). Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney of rats associated with cytotoxicity against renal tubular epithelia cells. In this study, it was evaluated the direct nefrotoxicity of L-aminoacid oxidase isolated of B. leucurus venom (LAAO-Bl) on renal epithelial cells (MDCK and HK2) and their potential nephrotoxic in isolated rat kidney. In these cells treated with LAAO-Bl, 1.56 â 100 Âg/mL for 12 h, there was a decrease in their viability in a concentration-dependent manner after 12 hours of treatment. In MDCK cells LDH release was not observed after 12 h of LAAO-Bl exposure while LAAO-Bl induced membrane rupture in HK-2 cells at the highest concentrations studied when compared with untreated cells. In MDCK cells, LAAO-Bl significantly increased the percentage of early apoptotic (Annexin-V+, PI-), necrotic (Annexin-V-, PI+) and secondary necrotic cells (Annexin-V+, PI+) when compared with control untreated cells. In HK-2 cells LAAO-Bl induced an increase in necrotic (PI+ cells) and secondary necrotic cells (Annexin-V+, PI+) in a concentration-dependent manner. MDCK apoptosis induction was accompanied with Ca2+ release from the endoplasmic reticulum, reactive oxygen species (ROS) generation, mitochondria dysfunction with enhanced expression of Bax protein levels. LAAO-Bl induced caspase-3 and caspase-7 activation in both cell lines. LAAO-Bl (10 Âg/mL) exerts significant effects on the isolated kidney perfusion increasing perfusion pressure and urinary flow and decreasing the glomerular filtration rate and sodium, potassium and chloride tubular transport. Taken together our results suggest that LAAO-Bl contributes for the nephrotoxicity observed in the envenomation by Bothrops leucurus. Moreover, the cytotoxic of LAAO-Bl to renal epithelial cells might be responsible, least in part for the nephrotoxicity observed in isolated kidney.

O objetivo deste trabalho é estudar a afinidade e especificidade de sítios EF-hand, e correlacionar estas propriedades com a estrutura primária do sítio, com as interações entre aminoácidos nas posições de coordenação, e com prováveis características da estrutura terciária da proteína. Os efeitos de três mutações no sítio EF-hand da cadeia leve regulatória de miosina (RLC) foram estudados: D5S, em que o aspartato presente na posição 5 do sítio foi substituído por uma serina; D9E, substituindo o aspartato da posição 9 por um glutamato, e D12E, substituindo o aspartato da posição 12 por um glutamato. Todas as combinações destas três mutações foram produzidas. Os mutantes simples D5S e D9E e o duplo mutante D5S/D9E têm baixa afinidade por cálcio. Todos os mutantes contendo a mutação D12E são específicos para cálcio, com afinidades maiores que RLC tipo selvagem. Todos os mutantes estudados possuem menor afinidade por magnésio que RLC tipo selvagem. As mudanças na energia livre de ligação e as energias de acoplamento sugerem que há interações inespecíficas entre todas as posições, e uma interação específica entre uma serina na posição 5 e um glutamato na posição 9. Esta interação ocorre somente na presença de magnésio, e quando há um aspartato na posição 12. O glutamato na posição 9 pode ser capaz de coordenar a ligação de magnésio diretamente no duplo mutante D5S/D9E. Embora um aminoácido ou um certo arranjo deles possa determinar características específicas do sítio EF-hand, o conjunto de propriedades depende da estrutura terciária, uma vez que sítios homólogos podem possuir afinidades e especificidades bastante diferentes. / The aim of this thesis was to study affinity and specificity in EF-hand sites, and how these properties are related to the site primary structure, interactions between amino acids in coordinating positions, and probable tertiary structure properties. The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9, and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All the mutants studied have lower affinity for Mg2+ than wild type RLC. Coupling energies and changes in binding free energy suggest that all positions interact in a non-specific way, and a specific interaction occurs between a serine in position 5 and a glutamate in position 9. This interaction can be seen only in the presence of magnesium, and with an apartate in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E. Even though an amino acid or a few amino acids in certain positions can determine specific characteristics for an EF-hand site, the site properties depend on the tertiary structure, since homologue sites can have very different affinities and specificities.

O objetivo deste trabalho é estudar a afinidade e especificidade de sítios EF-hand, e correlacionar estas propriedades com a estrutura primária do sítio, com as interações entre aminoácidos nas posições de coordenação, e com prováveis características da estrutura terciária da proteína. Os efeitos de três mutações no sítio EF-hand da cadeia leve regulatória de miosina (RLC) foram estudados: D5S, em que o aspartato presente na posição 5 do sítio foi substituído por uma serina; D9E, substituindo o aspartato da posição 9 por um glutamato, e D12E, substituindo o aspartato da posição 12 por um glutamato. Todas as combinações destas três mutações foram produzidas. Os mutantes simples D5S e D9E e o duplo mutante D5S/D9E têm baixa afinidade por cálcio. Todos os mutantes contendo a mutação D12E são específicos para cálcio, com afinidades maiores que RLC tipo selvagem. Todos os mutantes estudados possuem menor afinidade por magnésio que RLC tipo selvagem. As mudanças na energia livre de ligação e as energias de acoplamento sugerem que há interações inespecíficas entre todas as posições, e uma interação específica entre uma serina na posição 5 e um glutamato na posição 9. Esta interação ocorre somente na presença de magnésio, e quando há um aspartato na posição 12. O glutamato na posição 9 pode ser capaz de coordenar a ligação de magnésio diretamente no duplo mutante D5S/D9E. Embora um aminoácido ou um certo arranjo deles possa determinar características específicas do sítio EF-hand, o conjunto de propriedades depende da estrutura terciária, uma vez que sítios homólogos podem possuir afinidades e especificidades bastante diferentes. / The aim of this thesis was to study affinity and specificity in EF-hand sites, and how these properties are related to the site primary structure, interactions between amino acids in coordinating positions, and probable tertiary structure properties. The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9, and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All the mutants studied have lower affinity for Mg2+ than wild type RLC. Coupling energies and changes in binding free energy suggest that all positions interact in a non-specific way, and a specific interaction occurs between a serine in position 5 and a glutamate in position 9. This interaction can be seen only in the presence of magnesium, and with an apartate in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E. Even though an amino acid or a few amino acids in certain positions can determine specific characteristics for an EF-hand site, the site properties depend on the tertiary structure, since homologue sites can have very different affinities and specificities.

The aim of this semestral project is to try to study and describe work with sequences of proteins of different organisms, namely above all alligment sequences and evaluation of similarities of sequences. At the beginning of this work we deal with biochemistry of proteins, their constitution and structure. Further text go on with theory for work with sequential data, global, local and multiple assignment. At the last part we investigate possibilities of programme Matlab for aforesaid assignment.

Relatos sobre a toxicidade da fruta carambola (Averrhoa carambola) em pacientes com disfunção renal, com alguns desses pacientes chegando ao óbito, foram atribuídos a uma toxina denominada caramboxina, um aminoácido não peptídico derivado da fenilalanina, recentemente isolada por pesquisadores da Universidade de São Paulo. Com isso, este estudo foi planejado com o objetivo principal de investigar estratégias de síntese que permitiriam a síntese total da toxina. A partir do desenvolvimento inicial de uma rota sintética que empregou um aril-triflato para uma reação de acoplamento cruzado de Heck e a olefina acrilaldeído, que não se mostrou efetiva, outras rotas sintéticas foram planejadas empregando uma olefina derivada do aminoácido serina ou fazendo uso de produtos sintetizados durante o estudo, através de reações que conduzissem à molécula alvo. Apesar dos resultados negativos obtidos, a variação de estratégias permitiu maior compreensão acerca da síntese, além de contribuir para o estudo de diferentes metodologias. Entretanto, uma rota que fez uso da reação de acoplamento cruzado de Negishi com composto organozinco oriundo da serina como etapa chave, foi capaz de levar à síntese total da caramboxina, permitindo inclusive obter o produto assimétrico, conforme observado em análises de quantificação de enantiômeros. Com bons rendimentos na obtenção dos blocos de construção e na etapa chave de acoplamento, a rota exigiu dez etapas, desde os materiais de partida até a desproteção total, apresentando 33% de rendimento global, com boa reprodutibilidade e escalabilidade. Assim, com a obtenção da caramboxina sintética poderão ser respondidas várias lacunas, tanto em nível molecular (mecanismo de ação), quanto sobre seu possível metabolismo e farmacocinética / Reports on the toxicity of star fruit (Averrhoa Carambola) in patients with renal dysfunction, with some of them coming to death, were attributed to a toxin named caramboxin, a non-peptidic amino acid derived from phenylalanine, recently isolated by researchers from the University of São Paulo. Thus, this study was designed with the main objective of investigate strategies that would allow the total synthesis of the toxin. Starting from the initial development of a synthetic route that used an aryl-triflate for a cross coupling reaction of Heck with the olefin acrylaldehyde, which did not show effectiveness, other routes were planned using an olefin derived of the serine amino acid or making use of synthetized products obtained through the study, employing reactions that would lead to the target molecule. Despite the negative results, the variation of strategies allowed greater comprehension of the synthesis contributing to the study of different methodologies. However, a route that used the cross-coupling reaction of Negishi with an organozinc compound derived of serine as key step, led to the total synthesis of caramboxin including the obtention of the asymmetric product, as observed in enantiomers quantification analysis. With good yields for the building blocks and the cross-coupling key step the route required ten steps, from the starting materials to the total deprotection, exhibiting 33% of overall yield with good reproducibility and scalability. Thus, with the obtainment of synthetic caramboxin, will be possible to answer different gaps, both at the molecular level (mechanism of action) as well as on its possible metabolism and pharmacokinetics

Neste trabalho descrevemos o isolamento e a caracterização bioquímica, estrutural e funcional de uma L-aminoácido oxidase isolada do veneno da serpente Bothrops jararacussu, proteína esta denominada BjussuLAAO. O isolamento foi realizado pela combinação de três etapas cromatográficas, utilizando filtração molecular em Sephadex G-75, seguida de cromatografia de afinidade em Benzamidina-Sepharose, seguida pela última etapa cromatográfica, realizada com interação hidrofóbica em Fenil-Sepharose. A BjussuLAAO é uma proteína com massa molecular de 61 kDa e pI 5,8, e apresentou alta similaridade sequencial com as LAAOs já isoladas dos venenos de serpentes. A enzima BjussuLAAO induziu edema apenas na maior dose (100 ?g/animal), não apresentou atividade miotóxica, também não apresentou atividade hemorrágica, mesmo nas doses mais elevadas. A BjussuLAAO, demonstrou ter efeito coagulante e ação fibrinogenolítica e, mesmo utilizando inibidores de metaloproteases e serino proteases, continuou apresentando tal atividade, demonstrando assim não existir contaminantes destas classes de proteínas na amostra. As atividades mais interessantes do ponto de vista farmacológico, onde podemos visualizar a BjussuLAAO como um futuro fármaco, foram as atividades antitumoral, bactericida e antiparasitária. Esta apresentou, em baixas concentrações, citotoxicidade sobre células tumorais, ação leishmanicida e tripanocida, demonstrando assim ser uma substância candidata a fármaco multifuncional. / In this research we described the isolation and biochemical, structural and functional characterization of an L-amino acid oxidase from Bothrops jararacussu venom, named BjussuLAAO. The purification was carried out through three chromatography steps, using a molecular filtration on Sephadex G-75, followed by an affinity chromatography on Benzamidine-Sepharose, and finally a hydrophobic chromatography on Phenyl-Sepharose. BjussuLAAO is a protein with a molecular mass of 61 kDa and pI 5.8. This protein showed high sequencial similarity with other LAAOs from snake venoms. BjussuLAAO induced edema only in the highest dose (100 ?g/animal), did not show any myotoxic or hemorrhagic activity. BjussuLAAO, showed clotting and fibrinogenolitic activity, even in the presence of metalloproteases and serino-proteases inhibitors. The more interesting pharmacological activities, where from we can look a future application, were the anti-tumoral, bactericide, leishmanicidal and tripanocidal actions. The enzyme displayed a bactericide effect too, showing to be a multifunctional drug.

Este trabalho consiste em uma introdução teórica dos princípios de difração de raios-X e em métodos de determinação de estruturas moleculares e cristalinas aplicados a dois complexos de metal-aminoácido: Cobre (II) bis (D,L-alaninato) mono-hidratado e Cobre(II) bis (L-valinato) mono-hidratado. O complexo Cu(D,L-ala)2&#183H2O tem fórmula química Cu(H2NCHCO2CH3)2&#183 H2O e cristaliza no sistema monoclínico, grupo espacial C2/c com a= 12,087(3)&#197, b= 9,583(3)&#197, c= 8,973(3)&#197, &#946= 110,85(2)&#176, dcalc= 1,762 g/cm3, Z= 4, F(000) 532(elétrons). Foram utilizadas 737 reflexões independentes com I &#62 3&#948(I), que resultaram em um fator R final 0,032. Nesta estrutura, cada íon cobre (II), localizado em um centro de inversão, está ligado ao nitrogênio da amina e a um dos oxigênios carboxilatos de duas moléculas de alanina relacionadas por simetria em um arranjo planar cristalograficamente perfeito. Dois átomos de oxigênio de moléculas de água relacionadas por simetria completam uma configuração de octaedro alongado em volta do cobre. O átomo de oxigênio da molécula de água está em um vértice comum a dois octaedros vizinhos e está fortemente ligado por ponte de hidrogênio a oxigênios carboxilatos de duas moléculas vizinhas. Para o complexo Cu(L-val)2&#183H2O de fórmula química Cu(H2NCHCO2CH(CH3)2)2&#183H2O, a cristalização se deu no sistema monoclínico, grupo espacial C2, com a= 21,314(5)&#197, b= 9,586(2)&#197, c= 7,417(2)&#197, &#946= 108,89(2)&#176, dcalc= 1,454g/cm3, Z= 4, F(000) 660(elétrons). Esta estrutura foi resolvida usando 1605 reflexões independentes com I &#62 3&#948(I). A cadeia lateral do aminoácido não pode ser localizada devido à desordem ocupacional, o que resultou em um fator R final 0,12. O íon cobre (II) está coordenado a duas moléculas de valina no centro de uma unidade ligante praticamente planar N2O2, que forma a base de uma pirâmide quadrada um pouco distorcida com um átomo de oxigênio da água no ápice. Os íons cobre (II) pentacoordenados estão dispostos em camadas paralelas ao plano bc. Cada molécula Cu (L-val)2&#183H2O está ligada dentro da camada por um par de pontes de hidrogênio relativamente fortes entre o átomo de oxigênio da água e oxigênios carboxilatos de duas moléculas vizinhas. / This work consists of a theoretical introduction to the X-ray diffraction principles and methods of molecular and crystal structure determination applied to two metal-aminoacid complexes: Bis (D,L-alaninato) copper (II) monohydrate and Bis (L-valinato) copper (II) monohydrate. The complex Cu(D,L-ala)2&#183H2O has chemical formula (H2NCHCO2CH3)2&#183 H2O and crystallizes in the monoclinic space group C2/c, with a=12,087(3)&#197, b= 9,583(3)&#197, c= 8,973(3)&#197, &#946= 110,85(2)&#176, dcalc= 1,762 g/cm3, Z= 4, F(000) 532(electrons). 737 independent reflections with I &#62 3&#948(I) resulted in a final R-factor 0,032. The Cu (II) ion, at an inversion center, is bonded to the amine nitrogen and to one of the carboxylate oxygen of two symmetrically related alanine molecules in a crystallographically perfect planar arrangement. Two centro-symmetrically related water-oxygen atoms complete an elongated configuration around copper. The water oxygen is at a common apical corner of neighboring coordination octahedral and it is strongly hydrogen-bonded to carboxylate oxygens of other two neighboring molecules. The complex Cu(L-val)2&#183 H2O, with chemical formula Cu(H2NCHCO2CH(CH3)2)2&#183H2O, crystallizes in the monoclinic space group C2 with a=21,314(5)&#197, b= 9,586(2)&#197, c= 7,417(2)&#197, &#946= 108,89(2)&#176, dcalc= 1,454g/cm3, Z= 4, F(000) 660(electrons). The structure was solved employing 1605 independent reflections with I &#62 3&#948(I). The aminoacid side chain could not be located in the electron density map due to positional disorder, which resulted a final R-factor 0,12. The Cu (II) ion is in a cis coordination with two valine molecules at a center of an approximately planar N2O2 ligand set, which forms the basis of a somewhat distorted square pyramid with a water oxygen at its apex. The five-fold coordinated cooper ions are arranged in two-dimensional sheets parallel to the BC plane. Each Cu (L-val)2&#183 H2O molecule is linked within a sheet by a pairo f relatively strong O(N)-HO hydrogen bond with carboxylate oxygens of two neighboring molecules.

Introduction Heteromeric amino acid transporters (HATs) are composed of a heavy subunit (rBAT or 4F2hc) and a light subunit (b0 + AT, ASC1, LAT1, LAT2, y + LAT1, y + LAT2 and xCT), joined by a disulfide bridge (Chillaron et al. 2001). rBAT and 4F2hc are type II membrane glycoproteins (N-terminal cytoplasmic). Both have a single transmembrane segment, an N-terminal intracellular tail and an extracellular domain (ectodomain). As far as we know, the role of the heavy subunit is facilitating the transit of the light subunit to the plasma membrane. The light subunits are polytopic proteins unglycosylated, with 12 transmembrane segments, and the N-and C-terminal intracellular. The light subunit is the catalytically active subunit which confers specificity to the heterodimer on the transport system (Reig et al. 2002): LAT1 and LAT2 for system L , y+LAT1 and y+LAT2 for system y + L, asc for system ASC1; xCT for system Xc -, and b0+at for system b0, + (Chillaron et al. 2001). Results Overexpression of these human proteins was carried out with the methylotrophic yeast Pichia pastoris (strain KM71H) as expression system (based on Long et al. 2005). The main objective was to generate enough protein in a high level of purity to study the structure and check their function by transport assays. The different subunits, light and heavy, were cloned into the expression vector pPICZ (Invitrogen). To facilitate the purification of the different proteins, a cluster of 10 histidines was introduced by PCR at the N-terminus of the heavy subunits and a StrepTagII (IBA) at the N-terminus of the light subunits. 4F2hc is a glycoprotein with 4 possible targets for glycosylation. The glycosylations confer heterogeneity to protein, thus glycosylation targets were eliminated by directed mutagenesis. From all these human heavy and light subunits and heterodimers, only 4F2hc for the heavy subunits, LAT2 for the light subunits, and the heterodimer 4F2hc/LAT2 were overexpressed and extracted from the yeast membrane in enough amounts to continue with the purification step. The light subunit LAT2 was successfully purified but when the stability was analysed by size exclusion chromatography showed a clear profile of aggregation, concluding further studies. In contrast, the heavy subunit 4F2hc was stable after the exclusion chromatography for two days. The heterodimer 4F2hc/LAT2 proved to be stable after gel filtration analysis during one day. Thus, the heterodimer was significantly more stable than the light subunit alone, which allowed us to make an important statement. The catalytic subunit LAT2 needed their heavy subunit (i.e. 4F2hc) to increase the stability. This statement contrasted with the results for the heterodimer rBAT/b0+AT, in which was the heavy subunit rBAT the one who needed its light subunit b0+AT to a correct folding during its biogenesis (Bartoccioni et al. 2008; Rius et al. 2011). Functional studies with human heterodimer 4F2hc/LAT2 were set up to check the role of 4F2hc in the transport. Firstly the functionality of the heterodimer 4F2hc/LAT2 and the light subunit LAT2 in the living cell was checked successfully, meaning a correct folding at expression level. The apparent KM in both cases was the same, remaining unanswered the role of the heavy subunit 4F2hc in the transport function. Next, reconstitution in liposomes was carried out successfully for 4F2hc/LAT2 but not for LAT2, due to the high aggregation tendency. 4F2hc/LAT2 showed the typical overshoot for an amino acid transporter. To carry out the structural studies and due to the difficulty to maintain a stable soluble heterodimer, it was decided to carry out the technique of Single particle -negative staining (SP-NS) in the laboratory of Prof. Fotiadis in the University of Bern (Switzerland). The 3D model technique based on transmission electron microscopy (TEM) is relatively new and has been imposed for mammalian membrane proteins, allowing structural analysis with relatively small concentration of protein. The pure heterodimer was stained in a grid with uranyl formate at 0.75% (two drops optimized for 1 second, washing with water twice). This sample was analysed by transmission electron microscopy (TEM). Different images of projections in different orientations for 4F2hc/LAT2 were kept in a library of 11,000 picks. The refinement of the whole library allowed the 3D reconstruction of this protein by Mr. Meury. The model showed two asymmetric particles, one smaller, in which the crystal of the human ectodomain 4F2hc (Fort et al. 2007) fitted pretty well, and other bigger, which showed a black hole. Thus, the smaller particle was recognized as the heavy subunit, located on top of the light subunit. The resolution was 19 amstrongs, which was in the normal range for this method (from 16 amstrongs to 25 amstrongs). Discussion It was observed that the heavy subunit was located on top of the light subunit LAT2, and not in contact with the cell membrane as was firstly though. The size for the heavy subunit coincided with the existing 3D crystals of the human ectodomain which can fit quite accurately, always assuming the presence of the transmembrane segment in the 3D model. By contrast, the light subunit did not fit with the crystal structure of the prokaryotic homolog AdiC in the APA family (APC superfamily) (Gao et al. 2009) (Kowalczyk et al. 2011) due to the large amount of detergent surrounding this highly hydrophobic subunit in SP-NS method. In spite of that, when the size was compared with AdiC and Stet (a prokaryotic homolog in the LAT family with 30% of homology) studied in the same SP-NS method (Casagrande et al. 2009) the light subunit LAT2 coincided in size with its homologs, demonstrating that the increased volume was due to the detergent effect. Supporting the 4F2hc/LAT2 model, interaction studies with integrins (Feral et al 2005; Feral et al. 2007) and other membrane proteins involved in cell growth (ICAMI; Liu et al. 2003) and / or overexpressed in tumours (CD147/MCT1; Xu et al. 2005) suggest an effect in the transport function through the heavy subunit 4F2hc, which may be explained with an orientation on top of the light subunit and interaction by the external loops. New Evidences: Recently, the 4F2hc/LAT2 heterodimer model in which the heavy subunit is located on top of the light subunit has been corroborated by cross-linking experiments by Miss Helena Alvarez in our laboratory. This fact, allow us to imagine how interactions between both subunits will carry out also when the disulphide bridge is missing. Analyzing the external loops in AdiC atomic structure (the closest paradigm with LATs at present) is found that the external loop 3 and the external loop 4 are the longest (around 25 residues). These loops are even longer in LAT2, which make possible the interaction between both subunits being the separation of 16 amstrongs in the 3D model. Both loops have important roles in the transport cycle based in LeuT fold. The external loop 3 has an important movement in the transition from outward-open conformation to occluded-outward conformation due to the tilt of 40o of the transmembrane 6. The external loop 4, moves down to lid the substrate pathway during the transition from occluded-outward conformation to the occluded-inward conformation. Our new 3D model of a human heteromeric aminoacid transporter offers the opportunity to study new aspects about the role of the heavy subunit in the holotransporter. If the external loops join 4F2hc and LAT2 modulating the transport function in presence of other transmembrane proteins, or if 4F2hc only acts as a bollard of a multiproteic complex, rest to be studied in the future. / Els transportadors heteromèrics d'aminoàcids (HATS) de metazous estan formats per una subunitat pesada (4F2hc o rBAT) (N-glicoproteïna amb 1 segment transmembrana i un gran ectodomini en el seu extrem C-terminal), i una subunitat lleugera (d'entre 10) unides covalentment per un pont disulfur, fent aquests transportadors únics entre els metazous. En humans, 6 subunitats lleugeres es troben formant heterodímers amb 4F2hc (LAT1, LAT2, y+ LAT1, y + LAT2, XCT i asc1) i una (b0, + AT) amb rBAT. Els HATs tenen incidència en la salut, ja que mutacions en qualsevol de les subunitats ocasionen aminoacidúries (cistinúria, lisinúria amb intolerància a proteïnes), són receptors virals o estan sobre expressats en cèl • lules tumorals. El nostre grup va determinar l'estructura de l'ectodomini de 4F2hc a 2.1 Å (Fort J et al. 2007), i recentment ha resolt l'estructura d'un homòleg procariota (AdiC d' E. coli, amb ~17% d´homologia) de les subunitats lleugeres a 3.0 Å de resolució (Kowalczyk et al. 2011). Per contra no hi ha informació estructural sobre els holo-transportadors HAT. El present treball ens mostra el primer model estructural a 19 Å d'un transportador HAT humà, el transportador 4F2hc/LAT2. La importància de 4F2hc, a part de tenir un paper important en immunologia, es troba en la seva sobreexpressió en cèl•lules tumorals, el que la converteix en una important diana per a tractaments i desenvolupament de vacunes contra el càncer. El model ens mostra com en aquest transportador, l´ectodomini de 4F2hc està situat sobre LAT2, suggerint interacció amb els bucles extracel•lulars del transportador i nos sols interacció a través del pont disulfur del segment transmembrana com es pensava anteriorment. Aquesta nova topologia explica la necessitat i la importància de que l'ectodomini de 4F2hc formi part de l´heterodímer 4F2hc/LAT2 i presenta un escenari estructural per al paper "chaperone-like" de 4F2hc sobre les subunitats lleugeres.

L’addition nucléophile des réactifs de Grignard sur les nitriles conduit généralement aux cétones après hydrolyse acide. La double addition, menant à des carbinamines tertiaires après traitement, est beaucoup plus difficile et ne s’effectue habituellement qu’avec les organomagnésiens allyliques. Dans ce contexte, nous avons découvert que les organomagnésiens peuvent effectuer une double addition sur la fonction nitrile des acylcyanhydrines, pour fournir des hydroxyamides. Cette réaction est originale par le fait qu’une large gamme d’organomagnésiens peut être utilisée, dans des conditions particulièrement douces. Cette réaction a été appliquée à la synthèse de différents acides α-aminés α,α-disubstitués, par oxydation de la fonction alcool et hydrolyse du motif amide. La divinylglycine a notamment pu être préparée avec un bon rendement. L’addition successive de deux organomagnésiens différents a aussi pu être réalisée, après optimisation des conditions de réaction, pour accéder à des hydroxyamides disymétriques, précurseurs d’acides aminés quaternaires chiraux. Enfin, l’addition des réactifs de Grignard sur les 3-cyano iminocoumarines N-éthoxycarbonylées a été étudiée. Malgré la présence de nombreux sites électrophiles, la réaction est très chimiosélective, et des chromènes originaux substitués en position 4 ont été obtenus. Les propriétés antifongiques et antibactériennes de ces derniers ont été évaluées. / The nucleophilic addition of Grignard reagents on nitriles generally leads to ketones after acidic hydrolysis. The double addition, providing tertiary carbinamines after work-up, is more difficult and usually occurs only with allylic Grignard reagents. In this context, we discovered that Grignard reagents can perform a double addition on the nitrile function of acyl cyanohydrins, to provide hydroxyamides. This reaction is original by the fact that a wide range of Grignard reagents can be used, in particularly mild conditions. This reaction has been applied to the synthesis of different α,α-disubstituted α-aminoacids, by oxidation of the alcohol functionality and hydrolysis of the amide moiety. Especially, divinylglycine has been prepared in good yield. The successive addition of two different Grignard reagents was also carried out, after optimization of reaction conditions, to access unsymmetrical hydroxyamides, which are precursors of chiral quaternary aminoacids. Finally, the addition of the Grignard reagents on N-ethoxycarbonyl 3-cyano-iminocoumarines was studied. Despite the presence of several electrophilic centers, the reaction is highly chemoselective, and novel chromenes displaying substituent on position 4 were obtained. The antifungal and antibacterial properties of these compounds have been evaluated.

Made available in DSpace on 2014-06-11T19:33:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-27Bitstream added on 2014-06-13T21:06:45Z : No. of bitstreams: 1 marin_a_dr_jabo.pdf: 470209 bytes, checksum: 97f3e1c5ad3ee60d8f77b0d316384609 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo do presente trabalho foi verificar o efeito de interação do estresse hídrico e da toxidez do alumínio no crescimento inicial e teores de prolina livre em duas cultivares de guandu, cv. IAPAR 43-Aratã e IAC Fava Larga, cultivadas em hidroponia. As plantas jovens foram submetidas aos estresses em solução nutritiva (pH 3,8), nos potenciais osmóticos de 0,000; -0,004; -0,006; -0,008 e -0,010 MPa com 0,00; 0,25; 0,50; 0,75 e 1,00 mmol Al3+ dm-3. O experimento foi conduzido em sala de crescimento sob luminária com irradiância média de 190 μmol m-2 s-1, fotoperíodo de 12 horas e temperatura de 25°C + 1ºC. O delineamento experimental utilizado foi inteiramente casualizado, em arranjo fatorial 2x5x5, com quatro repetições. Os dados foram submetidos às análises de regressão polinomial, agrupamento e componentes principais. Pelos resultados obtidos verifica-se que, a deficiência hídrica causa redução do crescimento da parte aérea enquanto a toxidez do alumínio provoca diminuição do crescimento radicular. Houve aumento nos teores de prolina livre nas cultivares sob deficiência hídrica e apenas na cv. IAC Fava Larga sob toxidez do alumínio. A análise multivariada mostrou alta correlação no crescimento e no acúmulo de prolina na cv. IAC Fava Larga, evidenciando provável tolerância aos estresses associados. / The objective of the present work was to study the interaction effect of water stress and aluminum toxicity on the initial growth and free proline contents in two cultivars of pigeonpea cv. IAPAR 43-Aratã and IAC Fava Larga cultivated in hydroponics. The seedlings were subjected to stresses in nutritive solution (pH 3.8), osmotic potentials 0.000; -0.004; -0.006; -0.008 and -0.010 MPa with 0.00; 0.25; 0.50; 0.75 and 1.00 mmol Al3+ dm-3. The experiment was carried out in a plant growth room under a luminary unit of average irradiance 190 μmol m-2 s-1, 12-hour photoperiod and 25ºC + 1ºC temperature. A completely randomized experimental design in factorial array 2x5x5 with four replications was used. Data were submitted to polynomial regression, cluster and main components analysis. According to the results it was verified that water stress causes growth reduction of aerial part whereas aluminum toxicity provokes radicular growth reduction. There was increase of the free proline contents in cultivars under water stress and only in cv. IAC Fava Larga under aluminum toxicity. Multivariate analysis showed high correlation in growth and accumulation of proline for the cv. IAC Fava Larga evidencing probable tolerance to associated stresses.