Τα πενταμερή ιοντικά κανάλια που ενεργοποιούνται από τη δέσμευση ενός προσδέτη (pLGICs-pentameric Ligand Gated Ion Channels) της υπεροικογένειας των υποδοχέων Cys-θηλιάς είναι διαμεμβρανικές γλυκοπρωτεΐνες που εμπλέκονται σε ποικιλία βιολογικών λειτουργιών. Στην παρούσα εργασία παρουσιάζεται η NMR μελέτη της εξωκυττάριας περιοχής ενός προκαρυωτικού pLGIC, της GLIC (Gloeobacter ligand-gated ion channel), που προέρχεται από το κυανοβακτήριο Gloeobacter violaceus και εμφανίζεται ως μονομερές σε διάλυμα. / Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution.

The pentameric ligand gated ion channel super family plays a central role in fast synaptic communication between neurons and at the neuromuscular junction. Extensive studies on the prototypic pLGIC, the Torpedo nicotinic acetylcholine receptor (nAChR) have revealed an exquisite lipid sensitivity, with the nAChR adopting a novel uncoupled conformation in membranes lacking activating anionic and neutral lipids. The lipid-exposed transmembrane alpha-helix, M4, in each homologous subunit likely acts as a lipid sensor. One model proposes that activating lipids promote M4 “binding” to the adjacent alpha-helices, M1 and M3, to enhance interactions between the M4 C-terminus and the Cys-loop of the agonist-binding domain, with such interactions promoting coupling between the agonist site and channel gate. The first part of my thesis indirectly tests this hypothesis by exploring the effects of membrane hydrophobic thickness on nAChR function. Specifically, I tested the hypothesis that thicker membranes, which should promote alignment of M4 parallel to M1/M3 and thus helix-helix interactions, favor a coupled conformation. Although I found that the nAChR is uncoupled in all membranes tested, regardless of hydrophobic thickness, thicker membranes promote transitions from uncoupled to ultimately the desensitized state over the minutes to hours time frame. In contrast to anionic lipids, which influence function primarily via a conformational selection mechanism, membrane hydrophobic thickness influences function via a kinetic mechanism - thick membranes lower the activation energy between uncoupled and coupled conformations to promote conformational transitions. In the second part of my thesis, I used the two prokaryotic homologs, GLIC and ELIC, to explore how amino acid interactions at the interface between M4 and M1/M3 influence channel activity. Alanine scanning mutagenesis of this interface shows that disruption of almost any interaction in GLIC leads to a loss of folding and/or function, while analogous mutations in ELIC typically lead to no change or produce gains in function. Sequence comparisons with other members of the pLGIC superfamily suggest that the transmembrane domains of GLIC and ELIC represent two distinct archetypes. Each archetype may strike a different balance between the need for strong M4 binding to M1/M3 to promote folding and pentamer assembly, and the need for weaker interactions that allow for greater conformational flexibility during function.

Pentameric ligand-gated ion channels (pLGICs) are membrane receptors that play a crucial role in every living organism. The pLGIC protein structure forms a pore through the membrane of a cell that can let specific ions pass through, upon activation by endogenous agonists. pLGICs are allosterically modulated by ligands binding at allosteric sites, that either stabilize a certain conformation or change the binding affinity of the endogenous agonist. However, much remains unknown about the exact way in which these modulators bind to and affect pLGICs. An increased understanding could help in the search for novel and/or more effective target drugs. With this masters thesis, I hope to contribute by investigating the modulatory effect of ethanol on the bacterial Gloeobacter ligand-gated ion channel (GLIC). This has been done by performing oocyte electrophysiology recordings and analysis of molecular dynamics simulations, both with and without ethanol, and of four separate variants of GLIC that are either potentiated or inhibited by ethanol. Two possible allosteric sites were discovered in a transmembraneintrasubunit pocket: a potentiating allosteric site close to the M2 helix and residue V242, as well as an inhibitory membrane- and M4 helix-close intrasubunit site. Finally, evidence was found that could support a previously suggested inhibitory allosteric site in the pore around the 9’ hydrophobic gate. / Pentameriska ligandstyrda jonkanaler (pLGICs) är membranreceptorer som utgör vitala delar av varje levande organism. pLGICs proteinstruktur formar en por genom cellmembranet, som kan släppa igenom specifika joner efter aktivering av endogena agonister. pLGICs är allostermodulerade av ligander som binder vid allostera säten och som därigenom antingen stabiliserar en viss form eller förändrar den endogena agonistens bindningsstyrka. Emellertid saknas fortfarande mycket kunskap på detaljnivå om hur dessa modulatorer binder sig till och påverkar kanalerna. En ökad förståelse skulle hjälpa forskningen efter nya och/eller mer effektiva mediciner. Mitt examensarbetehoppas bidra genom att studera hur etanol modulerar den bakteriella ligandstyrda jonkanalen GLIC från Gloeobacter. Det har gjorts genom elektrofysiologimätningar på oocyter och analys av molekulärdynamiksimuleringar, båda av fyra olika GLIC-varianter, som antingen potentieras eller hämmas av etanol, och med eller utan etanol. Två allostera säten upptäcktes i det transmembrana intrasubenhetområdet: ett säte för potentiering nära M2 helixen och aminosyran V242, och ett hämmande säte nära membranet och helix M4. Slutligen hittades tecken som kan styrka existensen av det tidigare föreslagna hämmande allostera sätet i poren kring den hydrophoba porten.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-27T14:12:30Z No. of bitstreams: 1 DIS_CRISTINA_LORENSKI_FERREIRA_COMPLETO.pdf: 3410093 bytes, checksum: 23798c7a6d0f4dcae08a1e2a111ebd1b (MD5) / Made available in DSpace on 2017-07-27T14:12:31Z (GMT). No. of bitstreams: 1 DIS_CRISTINA_LORENSKI_FERREIRA_COMPLETO.pdf: 3410093 bytes, checksum: 23798c7a6d0f4dcae08a1e2a111ebd1b (MD5) Previous issue date: 2017-03-27 / Peripheral nerve injury continues to be an important research topic in the scientific community as it may cause lifelong disability. Biocompatible polymers are materials potentially capable of aiding the regeneration of peripheral nerves being used for the production of biocompatible tubes. The aim of this work is to prepare and characterize polymeric nanocomposites based on polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) and polypyrrole nanofibers (PPy) capable of acting as a guidewire in the regeneration of peripheral nerves. PPy was synthesized by oxidative chemical polymerization with p-toluenesulphonic acid monohydrate (PTSA) as a doping agent. PCL:PLGA blends films and PCL:PLGA:PPy nanofibers nanocomposites films were prepared by the solvent casting method, in the ratio of PCL:PLGA 100:0, 90:10, 80:20 and 70:30 (m/m); to the films with nanoload were added 10% PPy. In order to characterize the films, the following techniques were used: SEM, DSC, TGA, determination of electric conductivity and contact angle, citotoxicity test and hydrolytic degradation test, in vitro, based on ASTM F1635-11 standards. The PPy nanofibers presented electrical conductivity equal to 2.0.10-1 S.cm-1. The presence of PLGA and PPy did not change, meaningfully, the thermal properties of the films. However, in the degradation process, there was a tendency to a greater loss of mass for the blends with higher percentage of PLGA when submitted to longer incubation periods (150 days); when PPy was added to these blends, mass loss occurred in shorter periods (90 days). The nanocomposites films showed nontoxic and porous morphology surface, with hydrophilic intermediary character, good thermal stability and adequate degradation time for potential use in the treatment of injury in peripheral nerves. / Les?o de nervos perif?ricos continua sendo um importante tema de pesquisas no meio cient?fico, podendo causar defici?ncia no paciente por toda a vida. Pol?meros biocompat?veis s?o materiais potencialmente capazes de auxiliarem a regenera??o de nervos perif?ricos sendo utilizados para a produ??o de tubos biocompat?veis. O objetivo deste trabalho ? preparar e caracterizar nanocomp?sitos polim?ricos baseados em policaprolactona (PCL), poli(?cido l?ctico-co-glic?lico) (PLGA) e nanofibras de polipirrol (PPy) capazes de atuarem como conduto guia na regenera??o de nervos perif?ricos. PPy foi sintetizado via polimeriza??o qu?mica oxidativa com ?cido p-toluenosulf?nico monohidratado (APTS) como agente dopante. Foram preparados filmes de blendas PCL:PLGA e de nanocomp?sitos PCL:PLGA:nanofibras de PPy pelo m?todo de evapora??o de solvente, nas raz?es de PCL:PLGA 100:0, 90:10, 80:20 e 70:30 (m/m); aos filmes com a nanocarga, foram adicionados 10% de PPy. Para caracteriz?-los, foram utilizadas as t?cnicas: MEV, DSC, TGA, determina??o da condutividade el?trica e do ?ngulo de contato, teste de citotoxicidade e de degrada??o hidrol?tica, in vitro, com base na norma ASTM F1635-11. As nanofibras de PPy apresentaram condutividade el?trica igual a 2,0.10-1 S.cm-1. A presen?a de PLGA e de PPy n?o modificou, significativamente, as propriedades t?rmicas dos filmes. Por?m, no processo de degrada??o, houve uma tend?ncia ? maior perda de massa para as blendas com maior percentual de PLGA quando submetidas a maiores tempos de incuba??o (150 dias); ao adicionar PPy ?s blendas, a perda de massa ocorreu em menores tempos (90 dias). Os filmes dos nanocomp?sitos apresentaram superf?cie at?xica e de morfologia porosa, com car?ter hidrof?lico intermedi?rio, boa estabilidade t?rmica e tempo de degrada??o adequado para o potencial uso no tratamento de les?es em nervos perif?ricos.

Made available in DSpace on 2016-04-26T19:39:29Z (GMT). No. of bitstreams: 1 2007-Rodrigo Grazinolli Garrido.pdf: 601974 bytes, checksum: 814bbefff5f3e886a2c729be5c43e178 (MD5) Previous issue date: 2007-02-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / About half of planet population depend on rice. In Brazil, this cereal crop is widely spread, and influenced by harsh climate conditions such as Humid Tropics one. In this region, certainly nitrogen is the most limiting nutrient. However, low insolation, acid and highly Al3+ satured soils still endangers the culture. In these soils, ammonium can be the single plant s Nitrogen source for plants. Its assimilatory pathway is economic and fast, although it presents strong integration with C metabolism. This work intended to recognize ammonium assimilation integrated with carbon metabolism. Two rice varieties were used: one adapted to Humid Tropics environment - Piau? - and an improved variety represented by IAC-47, selected under high N levels. Assays were carried on with 26 days after emergency plants, cultivated in Hoagland and Arnon (1950) modified solution with N-NH4 + 0.1 mM and 1.0 mM N-NH4 +. Plasmalem (P)-H+ATPases; vacuole (V)-H+ATPases; vacuole (V)-H+PPases activity as well as glutamine sinthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) were analysed. Moreover, it was determined soluble sugars, amino-N, N-NH4 +, total-N, fresh and dry weight, and nutrition solution pH and N-NH4 + variation. Also, seminal root length was measured as well as glycolysis was analyzed through phosphofructokinase (PFK) activity at plants roots tissue. P-H+ATPases activity was higher in IAC-47 roots. On the other hand, at Piaui roots, enhances at microsomal protein were observed, and V-H+ATPases presented higher activity as well as root development was high. This allowed the adapted variety to absorb N like the improved one. However, both varieties accumulated fresh weight in shoots at treatment 1.0 mM N-NH4 + and showed roots/shoots decrease at the same treatment. Moreover, amino N and NH4 + concentration was higher at Piau? shoots under 1.0 mM N-NH4 +. Both Piau? shoots and roots cultivated under 1.0 mM NNH4 + accumulated less soluble sugars. Also, Piaui shoots had higher a+b chorophyl concentration. PFK activity was enhanced at every plant, what accompanied ammonium levels. Piau? plants always presented low sugar oxidation levels. It also seemed to concentrate its sugars. Probably it happened to later availability for grain fullfil. To ensure this, it has high ammonium and amino-N pools. Nitrogen absorbed by 1.0 mM N-NH4 + Piau? seemed to be substrate for GDH-amination, wich activity was superior. Despite this, GDH deamination was high. In this way, Piau? could have GDH activity as an anaplerotic pathway to regulate C skeletons need. Meanwhile, GS increases were observed at IAC-47, toghether with GOGAT increases at 1.0 mM N-NH4 + shoots. IAC-47 assimilated ammonium at GS/GOGAT pathway, wich was fed by glycolysis velocity. These plants would produce constitutive protein, what doesn t mean later grain stock. / Aproximadamente metade da popula??o do planeta depende do arroz. No Brasil, a cultura desse cereal ? largamente difundida, sofrendo a influ?ncia de severas condi??es clim?ticas como ocorre no Tr?pico ?mido. Nessa regi?o, certamente, o fator mais limitante para a cultura do arroz ? a disponibilidade de nitrog?nio. No entanto, ainda pesam sobre a cultura a baixa insola??o e os solos ?cidos com alta satura??o de Al+3. Nesses solos, o am?nio pode ser a ?nica fonte de nitrog?nio mineral para as plantas. Sua assimila??o ? econ?mica e r?pida, por?m, demanda estreita integra??o com o metabolismo de C. Pretendeu-se neste trabalho, reconhecer esta integra??o. Foi utilizada a variedade de arroz Piau?, adaptada ?s condi??es ambientais do Tr?pico ?mido, e a variedade melhorada IAC-47, selecionada sob altos teores de N. Os ensaios foram feitos em pl?ntulas com 26 dias ap?s a germina??o (DAG) cultivadas em solu??o de Hoagland e Arnon (1950) modificada com doses de 0,1 mM e 1,0 mM de NNH4 +. Avaliou-se a atividade de H+ATPases de plasmalema (P); H+ATPases de vac?olo (V); H+PPases de vac?olo (V) e das enzimas glutamina sintetase (GS), glutamato sintase (GOGAT) e glutamato desidrogenase (GDH) respons?veis pela assimila??o de nitrog?nio. Al?m disso, acompanharam-se os teores de a??cares livres, N-amino, N-NH4 +, N-total, massa fresca e massa seca e a varia??o do pH e do N-NH4 + da solu??o nutritiva. Foi ainda medido o comprimento da raiz seminal e o fluxo glicol?tico atrav?s da atividade da enzima fosfofrutocinase (PFK) do tecido radicular das plantas. A atividade das P-H+ATPases mostrouse maior nas plantas IAC-47. No entanto, a variedade Piau? mostrou maior teor de prote?na na fra??o microssomal, maior atividade V-H+ATPases e maior desenvolvimento de ra?zes, o que possibitou uma absor??o de am?nio semelhante ? da planta melhorada. Ambas as variedades apresentaram maior ac?mulo de massa fresca na parte a?rea e decaimento na rela??o raiz/parte a?rea no tratamento com 1,0 mM N-NH4 +. Observou-se ac?mulo de N-amino e NNH4 + na parte a?rea de plantas Piau? no tratamento 1,0 mM de N-NH4 +. Tanto a parte a?rea quanto a raiz da variedade Piau? cultivada a 1,0 mM N-NH4 + apresentaram menor ac?mulo de a??cares sol?veis e maiores teores de clorofila a+b. A atividade da PFK em todas as plantas foi aumentada com os n?veis de am?nio nutricionais. A variedade Piau? apresentou sempre menor oxida??o de a??cares. A variedade adaptada pareceu concentrar seus a??cares possivelmente para disponibilidade em est?dios posteriores de enchimento de gr?o. Para isso, manteve um maior pool de am?nio e N-amino. Houve ind?cios de que o am?nio absorvido por plantas Piau? cultivadas no tratamento 1,0 mM N-NH4 + teria sido drenado pela a??o aminativa da enzima GDH, que se mostrou exacerbada. No entanto, a fun??o desamina??o da GDH tamb?m se mostrou aumentada. Dessa forma, a Piau? poderia ter utilizado-se da GDH como via anapler?tica para regular a necessidade de esqueletos carb?nicos. A atividade de GS foi maior para a variedade IAC-47, acompanhada da atividade de GOGAT de parte a?rea no tratamento 1,0 mM N-NH4 +. As plantas IAC-47 assimilaram o am?nio prontamente a partir da via GS/GOGAT, a qual foi alimentada pela glic?lise acelerada. Essas plantas produziriam prote?na constitutiva n?o aplicando em reservas para o posterior enchimento do gr?o.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-08-29T18:10:46Z No. of bitstreams: 1 2017 - Renata Oliveira Santos.pdf: 1382777 bytes, checksum: 331bbbcbeb5b7d3a9f1d49008191a5b8 (MD5) / Made available in DSpace on 2017-08-29T18:10:46Z (GMT). No. of bitstreams: 1 2017 - Renata Oliveira Santos.pdf: 1382777 bytes, checksum: 331bbbcbeb5b7d3a9f1d49008191a5b8 (MD5) Previous issue date: 2017-02-14 / CAPES / In the past few years the use of prebiotics in the making of dairy products has been gathering many supports, although the search for low cost raw-material to achieve that it still a big challenge, which highlights the potential to the development in the research for alternatives sources or new processes in order to obtain those ingredients. Besides, the need to reduces the additives in the diet has been mandatory to the consumers who wants a health diet, as a growing tendency in the food industry in the development of products with cleaner labels, which are known as clean label. In this context, the search and/or value of natural ingredients that contributes simultaneously for the improvement in the functional properties, sensory and technological of food products that make them essential. Among the sources of these ingredients, stands out the araruta (Maranta arundinacea L.) as unconventional food plants (PANC?s) from Marantaceas Family, which presents a food potential, nutritional, functional that is neglected. Therefore, the aim of this paper was to evaluate the effect of the adding of araruta flours in the physical Chemical characteristics, rheological and in the growing of probiotic bacteria in the vanilla dairy dessert. In the Chapter I was developed and research in the literature in order to evaluate the sensory aspects and technological of prebiotics flours in the dairy products. In the Chapter II was performed a study of the prebiotic potential in the araruta flour, as well as, the effect of different % of these flours in the reological and technological of the mentioned dessert. It is possible to conclude that the particles size of the flours directed affected the texture of the desserts, once the flours with bigger size contributed to the increase in firmness of the dairy desserts. In addition, regarding the functional aspects, the araruta flour presented high level of resistent starch (29,47%) low IG(41,8), beyond the probiotic potential, which contribute to the increase number on the Lactobacillus Casey counting. Also, it was verified that % flour contribute to the reduction in the syneresis, rheological properties(flow behavior in oscillatory tests), texture (firmness and stickiness) and on the microscopical characteristics in the vanilla dairy dessert. / Nos ?ltimos anos, o uso de prebi?ticos na elabora??o de produtos l?cteos tem sofrido grande incentivo, embora a procura por mat?rias-primas de baixo custo para sua obten??o ainda seja um grande desafio, evidenciando um potencial para desenvolvimento de pesquisas de fontes alternativas ou novos processos de obten??o desses ingredientes. Al?m disso, a necessidade de redu??o de aditivos na dieta tem sido mandat?ria para os consumidores que buscam uma alimenta??o mais saud?vel, assim como, uma tend?ncia crescente da ind?stria de alimentos no desenvolvimento de produtos com r?tulos mais limpos, conhecidos como clean label. Neste contexto, a busca e/ou valoriza??o de ingredientes naturais que contribuam simultaneamente para a melhoria das propriedades funcionais, sensoriais e tecnol?gicas dos produtos aliment?cios se torna fundamental. Dentre as fontes desses ingredientes, destaca-se a araruta (Maranta arundinacea L.) uma planta aliment?cia n?o convencional (PANC) da fam?lia das Marantaceas que apresenta potencial aliment?cio, nutricional, funcional e tecnol?gico negligenciado. Assim, o objetivo geral do presente trabalho foi avaliar o efeito da adi??o de farinha de araruta nas caracter?sticas f?sico-qu?micas, reol?gicas e no crescimento de bact?rias probi?ticas de sobremesa l?ctea sabor baunilha. No Cap?tulo I deste trabalho foi realizada uma revis?o de literatura visando avaliar os aspectos sensoriais e tecnol?gicos do uso de farinhas prebi?ticas em produtos l?cteos. No Cap?tulo II foi realizado o estudo das propriedades f?sicas, funcionais (amido resistente e ?ndice glic?mico) e do potencial prebi?tico da farinha de araruta, bem como, o efeito de diferentes % dessa farinha nas propriedades reol?gicas e tecnol?gicas de uma sobremesa l?ctea sabor baunilha. Pode-se concluir que o tamanho de part?cula das farinhas afetou diretamente a textura das sobremesas, uma vez que, as farinhas com maior tamanho m?dio de part?culas contribu?ram para aumento da firmeza das sobremesas l?cteas. Com rela??o aos aspectos funcionais, a farinha de araruta apresentou elevado teor de amido resistente (29,47%), baixo IG (41,8), al?m de potencial prebi?tico contribuindo para o aumento da contagem de Lactobacillus casei. Foi verificado que % farinha contribui para redu??o da sin?rese, al?m de afetar as propriedades reol?gicas (comportamento de fluxo e ensaios oscilat?rio), na textura (firmeza e adesividade) e nas caracter?sticas microsc?picas da sobremesa l?ctea sabor baunilha.

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Made available in DSpace on 2014-12-17T14:16:33Z (GMT). No. of bitstreams: 1 MelinaBL_DISSERT.pdf: 4468439 bytes, checksum: 05a71c9a22430196d211e6d7c8feea61 (MD5) Previous issue date: 2008-09-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Diabetes Mellitus (DM) and osteoposes are chronic diseases with great socioeconomic consequences, mainly due to the late complications and consequent disabilities. The potential effects of DM on bone metabolism remain a very conroversial issue, and disagreement exists with regard to the clinical implications of diabetic osteopenia and the mechanism of its ocurrence. The issue is further complicated by the contribuicion of the especific factors, such as duration of disease an dthe degree of metabolic control. The objective of this study is to identify the osteopathy in children and adolescents with DM 1 assisted in the hospital of pediatrics, UFRN, through biochemical markers of bone and mineral metabolism and the extent of bone mineral density. The study was composed by 74 diabetics type 1 patients (DM1) of both gender and aged 6 to 20 yars. Normoglic?mic group was composed by 97 healthy subjects of both genders, which showed the same age range of DM1, in addition to same socioeconomic class. These individuals qere students from the networks of public education in the city of Natal-RN, randomly invited to paticipate in our study. Both groups DM1 and NG were divided intofour subgroups, according to the classification of tanner , T1, T2, T3, T4 for achieving a benchmark. Diabetic individuals showed up with a poor glycemic control. the group DN1 T4 showed an incresead value for total protein, albumin, urea and microalbumiuria are predictors of grumelura injury in DM1 patients . The total alkaline phosphatase activitywas kept on high levels for both groups because they are in a stature development age. For osteocalcin there were decreased levels for groups Dm1 T1, T2, and T3 when compared to their NG (s), suggesting that this decrease could be associated with reduction in the number and/or differentiation os osteoblasts thereby contributing to reducing bone formation. There were no changes in the activity of TRAP. The serum concentrations of total and ionized calcium, phosphorus and magnesium were included within the RV. It was observed that the BMD (Z- SCORE ) has always been within the RV for both groups, despite to DM1 T4. Taking all together, our results support the hypothesis that children and adolescents with type 1 DM present the risk in the long run to suffer a reduction in the bone mass, associated to poor glicemic control and disease duration. It could limit the bone growth and increase the probality of development of osteopenia, as well as other complications surch as retinopathy and renal failure / Diabetes mellitus (DM) a osteoporose s?o doen?as cr?nicas com grandes consequ?ncias socioecon?micas, sobretudo devido ? complica??es tardias e consequente desabilidades. Os efeitos potenciais do DM no metabolismo ?sseo continua a ser uma quest?o controversa, e ainda n?o existe um consenso no que diz respeito ?s implica??es cl?nicas da osteopenia diab?tica e os mecanismos da sua ocorr?ncia. Entretanto, a contribui??o de fatores espec?ficos, tais como a dura??o da doen?a e o grau de controle metab?lico tem sido muito discutidos. O objetivo do presente estudo foi identificar precocemente a osteopatia diab?tica em crian?as e adolescentes com DM 1 atendidos no Hospital de Pediatria da UFRN atrav?s de marcadores bioqu?micos do metabolismo mineral e ?sseo, marcadores da fun??o renal e da medida da densidade mineral ?ssea (DXA; Z-score L1-L4) . O estudo foi constitu?do por uma amostra de 74 pacientes diab?ticos tipo 1 (DM1) de ambos os sexos, com faixa et?ria entre 6 a 20 anos. O grupo normoglic?mico (NG) foi constitu?do por 97 indiv?duos saud?veis, de ambos os sexos, os quais apresentaram a mesma faixa et?ria do DM1, al?m de compreenderem a mesma classe socioecon?mica. Estes indiv?duos eram alunos de escolas da rede p?blica de ensino da cidade de Natal-RN, convidados aleatoriamente a participar do nosso estudo. Tanto o grupo DM1 quanto o NG foram divididos em quatro subgrupos, de acordo com a Classifica??o de Tanner, T1, T2, T3, T4, para viabilizar uma avalia??o comparativa. Os indiv?duos diab?ticos apresentaram um controle glic?mico insatisfat?rio, com valores de glicemia e HbA1C significativamente superiores aos obtidos para os NG. O grupo DM1 T4 apresentou valores aumentados de prote?nas totais, albumina, ur?ia e microalbumin?ria, sugerindo assim um in?cio de comprometimento renal, visto que os valores elevados de microalbumin?ria s?o preditores de les?o glomerular em pacientes DM1. A atividade da fosfatase alcalina total mostrou-se acima dos VR nos grupos DM1 e NG por estes estarem numa faixa et?ria de desenvolvimento estatural. Observa-se uma diminui??o da concentra??o de osteocalcina para os grupos DM1 T1, T2 e T3 quando comparados aos respectivos NG (s), sugerindo que esta diminui??o estaria associada a diminui??o do n?mero e/ou da diferencia??o dos osteoblastos no seu est?gio final de matura??o, contribuindo consequentemente para a redu??o da forma??o ?ssea. N?o foram observadas altera??es na atividade da TRAP. As concentra??es s?ricas de c?lcio total e ionizado, f?sforo e magn?sio estavam compreendidos dentro dos VR, mas os grupos diab?ticos apresentaram hipozincemia e hiperzinc?ria. A DMO (Z-score L1-L4; DXA) esteve sempre dentro dos VR para os grupos estudados, entretanto os grupos DM1 apresentaram sempre valores abaixo do seu respectivo NG, alca?ando uma diferen?a significativa para DM1 T4. O conjunto de resultados obtidos indicam que o baixo controle glic?mico e o tempo de doen?a representaram fatores de risco importantes para o desenvolvimento precoce da osteopenia diab?tica, bem como para o comprometimento renal e sinais de retinopatia.

GLIC är en pH-känslig pentamerisk ligandstyrd jonkanal (pLGIC) som finns i cellmembranet hos prokaryoten Gloeobacter violaceus. GLIC är en bakteriell homolog till flera receptorer som är viktiga i nervsystemet hos de flesta eukaryotiska organismer. Dessa receptorer fungerar som mallar för utvecklingen av målstyrda bedövnings- och stimulerande läkemedel som påverkar nervsystemet. Förståelsen av ett proteins mekanismer har därför hög prioritet inför läkemedelsutvecklingen. Eukaryota pLGICs är dock mycket komplexa eftersom några av de är heteromera, har flera domäner, och de pågår eftertranslationella ändringar. GLIC, å andra sidan, har en enklare struktur och det räcker att analysera strukturen av en subenhet - eftersom alla subenheter är helt lika. Flertalet möjliga grindmekanismer föreslogs av vetenskapen men riktiga öppningsmekanismen av GLIC är fortfarande oklar. Projektets mål är att genomföra maskininlärning (ML) för att upptäcka nya grindmekanismer med hjälp av datormetoder. Urspungsdatan togs från tidigare forskning där andra ML-redskap såsom molekyldynamik (MD), elastisk nätverksstyrd Brownsk dynamik (eBDIMS) och Markovstillståndsmodeller (MSM) användes. Utifrån dessa redskap simulerades proteinet som vildtyp samt med funktionsförstärkt mutation vid två olika pH värden. Fem makrotillstånd byggdes: två öppna, två stängda och ett mellanliggande. I projektet användes ett annat ML redskap: KL-divergens. Detta redskap användes för att hitta skillnader i avståndfördelning mellan öppet och stängt makrotillstånd. Utifrån ursprungsdatan byggdes en tensor som lagrade alla parvisa aminosyrornas avstånd. Varje aminosyrapar hade sin egen metadata som i sin tur användes för att frambringa alla fem avståndsfördelningar fråm MSMs som byggdes i förväg. Sedan bräknades medel-KL-divergens mellan två avståndfördelningar av intresse för att filtrera bort aminosyropar med överlappande avståndsfördelningar. För att se till att aminosyror inom aminosyrapar som låg kvar kan påverka varandra, filtrerades bort alla par vars minsta och medelavstånd var stora. De kvarvarande aminosyroparen utvärderades i förhållande till alla fem makrotillstånd Viktiga nya grindmekanismer som hittades genom både KL-divergens och makrotillståndsfördelningar innefattade loopen mellan M2-M3 helixarna av en subenhet och både loopen mellan sträckor β8 och β9 (Loop F)/N-terminal β9-sträckan och pre-M1/N-terminal M1 av närliggande subenheten. Loopen mellan sträckor β8 och β9 (Loop F) visade höga KL-värden också med loopen mellan sträckor β1 och β2 loop samt med loopen mellan sträckor β6 och β7 (Pro-loop) och avståndet mellan aminosyror minskade vid kanalens grind. Övriga intressanta grindmekanismer innefattade parning av aminosyror från loopen β4-β5 (Loop A) med aminosyror från sträckor β1 och β6 samt böjning av kanalen porangränsande helix. KL-divergens påvisades vara ett viktigt redskap för att filtrera tillgänglig data och de nya grindmekanismer kan bli användbara både för akademin, som vill reda ut GLIC:s fullständiga grindmekanismer, och läkemedelsföretag, som letar efter bindningsställen inom molekylen för att utveckla nya läkemedel. / GLIC is a transmembrane proton-gated pentameric ligand-gated ion channel (pLGIC) that is found in the prokaryote Gloeobacter violaceus. GLIC is the prokaryotic homolog to several receptors that are found in the nervous system of many eukaryotic organisms. These receptors are targets for the development of pharmaceutical drugs that interfere with the gating of these channels - such drugs involve anesthetics and stimulants. Understanding the mechanism of a drug’s target is a high priority for the development of a novel medicine. However, eukaryotic pLGICs are complex to analyse, because some of them are heteromeric, have more domains, and because of their post-translational modifications (PTMs). GLIC, on the other hand, has a simpler structure and it is enough to study the structure of only one subunit - since all subunits are identical. Several possible gating mechanisms have been proposed by the scientific community, but the complete gating of GLIC remains unclear. The goal of this project is to implement machine learning (ML) to discover novel gating mechanisms by computational approaches. The starting data was extracted from a previous research where computational tools like unbiased molecular dynamics (MD), elastic network-driven Brownian Dynamics (eBDIMS), and Markov state models (MSMs) were used. From those tools, the protein was simulated in wild-type and in a gain-of-function mutation at two different pH values. Five macrostates were constructed: two open, two closed, and an intermediate. In this project another ML tool was used: KL divergence. This tool was used to score the difference between the distance distributions of one open and one closed macrostate. The starting data was used to create a tensor that stored all residue-residue distances. Each residue pair had its own metadata, which in turn was used to yield the distance distributions of all five pre-build MSMs. Then the average KL scores between two states of interest were calculated and were used to filter out the residue pairs with overlapping distance distributions. To make sure that the residues within a pair can interact with each other, all residue pairs with very high minimum and average distance were filtered out as well. The residue pairs that remained were later evaluated across all five macrostates for further studies. Important novel mechanisms discovered in this project through both the KL divergence and the macrostate distributions involved the M2-M3 loop of one subunit and both the β8-β9 loop/N-terminal β9 strand and the preM1/N-terminal M1 region of the neighboring subunit. The β8-β9 loop (Loop F) showed high KL scores with the β1-β2 and β6-β7 (Pro-loop) loops as well with decreasing distances upon the channel’s opening. Other notable gating mechanisms involved are the pairing of residues from the β1-β2 loop (Loop A) with residues from the strands β1 and β6, as well as the kink of the pore-lining helix. KL divergence proved a valuable tool to filter available data and the novel mechanisms can prove useful both to the academic community that seeks to unravel the complete gating mechanism of GLIC and to the pharmaceutical companies that search for new binding sites within the molecule for new drugs.

La motivation première de mes travaux de recherche est de combiner des approches expérimentales et théoriques dans le domaine de la chimie physique pour atteindre une meilleure compréhension des phénomènes à l'échelle atomique. Mes travaux en cours traitent de systèmes d'intérêt biologique concernant les processus membranaires et des phénomènes accessibles par des méthodes de nanomanipulation. Les problèmes de la biophysique et biochimie sont au c?ur de mes recherches. J'ai effectué des simulations complexes de protéines membranaires dans une bicouche lipidique qui se sont montrées tout à fait complémentaires et révélatrices par rapport aux études expérimentales de biologie structurale. Une récente collaboration exploitant cette complémentarité a donné lieu à une publication dans la revue Nature en début 2009. [[i]] Les travaux récents visent à développer des approches combinant la réalité virtuelle avec les simulations moléculaires. [[ii]] Les systèmes biologiques étudiés présentent à la fois un intérêt physico-chimique, biologique et médical et peuvent atteindre un grand nombre d'atomes. En parallèle, je mène un travail de fond sur les méthodes de simulation et des approches novatrices. ----------------------- [[i]] N. Bocquet, H. Nury, M. Baaden, C. Le Poupon, J.P. Changeux, M. Delarue et P.J. Corringer: "X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation", 2009, Nature, 457, 111-114. [[ii]] O. Delalande, N. Férey, G. Grasseau et M. Baaden : "Complex Molecular Assemblies at hand via Interactive Simulations", 2009, J. Comput. Chem., 30, 2009, 2375-2387.