Trypanosoma cruzi, agente causador da Doença de Chagas, é um protozoário intracelular obrigatório. Vários estudos foram realizados visando a caracterização de moléculas de 85-90 kDa, presentes na superfície do parasita bem como de seus possíveis receptores nas células do hospedeiro vertebrado. Um membro da superfamília das gp85/trans-sialidases (Tc85-11), expresso somente na superfície das formas intectivas tripomastigotas e que adere em laminina e em células, foi clonado e caracterizado em nosso laboratório. Peptídeo J, fragmento de Tc85-11 não implicado em adesão a laminina, que contém o motivo conservado na superfamília, com seqüência VTVXNVFLYNR, aqui denominado \"domínio FLY\", foi identificado como sendo responsável pela adesão da porção carboxi-terminal da proteína em células epiteliais e, adicionado ao meio de cultura, promoveu aumento do número de células infectadas por T.cruzi. Seu receptor foi descrito como CK18 (Magdesian et al., 2001). Dando continuidade a esse estudo, em nosso laboratório caracterizamos a porção amino-terminal de CK18 como a região da interação com \"domínio FLY\" e identificamos o provável sítio de ligação, localizado entre os 15 aminoácidos iniciais da proteína. Adicionalmente, com o intuito de determinar a função do \"domínio FLY\", caracterizamos o peptídeo J como molécula extremamente adesiva, interagindo com a superfície de células epiteliais, matriz extracelular e promovendo interação entre tripomastigotas e ECM, possivelmente por meio de interações hidrofóbicas não dependentes de sua estrutura tridimensional adotada na proteína nativa. \"Domínio FLY\" foi caracterizado, ainda, como possível modulador da infecção por tripomastigotas já que, da mesma forma que estimula a invasão quando adicionado ao meio de cultura (Magdesian et al., 2001), promove secreção de proteínas imunorelacionadas com CK18 e ligantes de proteína A para o sobrenadante celular. Esse sobrenadante é capaz de in vitro inibir o processo infectivo do parasita em cerca de 40%. / Trypanosoma cruzi the causative agent of Chagas´ disease is an obligatory intracellular parasite in the mammalian host. Altrough the mechanism of trypomastigotes invasion of host cells has been intensively studied, a final and integrate picture of the process remains elusive. Members of the gp85/trans-sialidase superfamily have been implicated in the parasite-host interaction, with Tc85 family (85 kDa glycoproteins) implicated in the adhesion step. Our laboratory showed that Tc85-11, one member of Tc85 family, is a multi-adhesive molecule, with binding sites located at the amino- and carboxi-portions of the protein. The conserved \"FLY domain\" (peptide J) is present in all members of the family, binds to cytokeratin 18 (CK18) and enhances T. cruzi invasion (Magdesian et al., 2001). Herein, we localized the binding site of \"FLY domain\" on the amino-portion of CK18 and demonstrated an increase of trypomastigotes adhesion to extracellular matrix upon FLY treatment. The \"FLY domain\" can modulate T. cruzi infection in vitro and apparently is responsible for inducing the secretion of molecules by the host that inhibit trypomastigote invasion.

Made available in DSpace on 2014-10-09T12:42:23Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:02:00Z (GMT). No. of bitstreams: 0 / A influência do butirato de sódio (NaBu), do cloreto de manganês (MnCl2), combinados ou isolados, e da cicloheximida (CHX) na síntese de tireotrofina humana recombinante (r-hTSH) derivada de células CHO foi investigada pela primeira vez. Com exceção da CHX, que gerou uma redução de 1,5 vezes na produtividade volumétrica, todos os reagentes geraram um aumento (1,4 vezes para o MnCl2 e 3 vezes para o NaBu e NaBu+MnCl2) na produtividade volumétrica. Também foi observada uma diminuição no número de células viáveis com a utilização de todos os reagentes. A adição destes reagentes ao meio de cultura de células CHO produtoras de hTSH gerou também alterações na glicosilação do r-hTSH. As alterações geradas pela presença de CHX foram todas negativas, resultando em uma diminuição de 5,5% no conteúdo de ácido siálico, 10% na ocupação dos sítios de glicosilação, além de uma diminuição no conteúdo de todos os monossacarídeos neutros. Os demais reagentes, por outro lado, resultaram em alterações positivas. A presença de NaBu no meio de cultura acarretou um aumento de 12% no conteúdo de ácido siálico e de 3% na ocupação dos sítios de glicosilação. Com relação às estruturas de carboidratos presentes no hTSH, foi observado um aumento de 14% nas estruturas bi-antenárias, aumento no conteúdo de manose e fucose e uma diminuição no conteúdo de galactose e N-acetilglucosamina. A presença de MnCl2 no meio de cultura resultou em um aumento de 1,7% no conteúdo de ácido siálico e de 1,3% na ocupação dos sítios de glicosilação. Houve ainda um aumento no conteúdo de manose e N-acetilglucosamina e uma diminuição no conteúdo de fucose e galactose. Um aumento de 7% na freqüência de estruturas bi-antenárias foi também observado quando MnCl2 foi utilizado. O uso simultâneo de NaBu e MnCl2 proporcionou um aumento de 14% no conteúdo de ácido siálico e de 3% na ocupação dos sítios de glicosilação, bem como um aumento no conteúdo de todos os monossacarídeos neutros. Não houve, entretanto, alterações na freqüência das estruturas de carboidratos. Apesar de todas as alterações causadas pelos diferentes reagentes, não foram observadas diferenças na atividade biológica ou no comportamento farmacocinético das diferentes preparações de hTSH estudadas. Este estudo é extremamente relevante, tanto do ponto de vista do desenvolvimento de novos biofármacos, quanto do ponto de vista do controle de qualidade das glicoproteínas hormonais já utilizadas na clínica médica. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-07-25T17:57:36Z No. of bitstreams: 1 2016 - Luiz Gilberto Ambr?sio de Souza.pdf: 3023562 bytes, checksum: b3607c9323156b54bb63bceacd5de74e (MD5) / Made available in DSpace on 2017-07-25T17:57:36Z (GMT). No. of bitstreams: 1 2016 - Luiz Gilberto Ambr?sio de Souza.pdf: 3023562 bytes, checksum: b3607c9323156b54bb63bceacd5de74e (MD5) Previous issue date: 2016-02-29 / CNPq / Glomalin is regarded as a hydrophobic, heat-stable and recalcitrant glycoprotein produced by arbuscular mycorrhizal fungi (AMF). Such characteristics possibly result in high amounts of this protein in the soils, as well as a reduced rate of decomposition, and it is considered an important fraction of soil organic matter. Humic Substances (HS) have been recognized for a long time as the organic component most widely distributed on the planet, present both in terrestrial and aquatic environments. They are formed from chemical and biological degradation of residues from plants, animal and microbial activity. Although with some progress on structural aspects of glomalin and humic substances, there are few studies that describe their structural differences using a spectroscopic and chemometric approach. To search for answers for these questions, having as a reference the existing environmental and ecological relationships, this study aimed to confirm the structural relationship (differences and similarities) between the glomalin and humic substances obtained from different sources, through the isolation and purification of soil protein fractions related to glomalin (Glo), and the humic substances (HSs) fractions from different environments and composted materials; characterizing glomalin and humic substances obtained by chemical, physical and spectroscopic techniques (elemental composition, UV-vis, FTIR, 13 C CP-MAS RMN, EMS), and by using chemometric techniques (Unscrambler? X 10.3). / A glomalina ? considerada como uma glicoprote?na hidrof?bica, termoest?vel e recalcitrante produzida pelos Fungos Micorr?zicos Arbusculares (FMA). Tais caracter?sticas possivelmente implicam em altas quantidades desta prote?na nos solos bem como uma reduzida taxa de decomposi??o, sendo considerada como uma importante fra??o da mat?ria org?nica do solo. As Subst?ncias H?micas (SH) t?m sido reconhecidas durante muito tempo como o componente org?nico mais amplamente distribu?do no planeta, presentes tanto em ambientes terrestres quanto aqu?ticos. Elas s?o formadas a partir da degrada??o qu?mica e biol?gica de res?duos de plantas, animais e da atividade microbiana. Embora existam alguns avan?os sobre aspectos estruturais da glomalina e as subst?ncias h?micas, ainda s?o escassos os estudos que descrevem as suas diferen?as estruturais utilizando uma abordagem espectrosc?pica e quimiom?trica. Buscando encontrar respostas a estes questionamentos, assumindo como base de estudo a rela??o ambiental e ecol?gica existente, este estudo tem como objetivo confirmar a rela??o estrutural (diferen?as e semelhan?as) existente entre a glomalina e as substancias h?micas de diferentes origens, atrav?s do isolamento e purifica??o das fra??es da prote?na do solo relacionada ? glomalina (Glo) e as fra??es de subst?ncias h?micas (SHs) de diferentes ambientes e materiais compostados; caracterizando a glomalina e as subst?ncias h?micas obtidas atrav?s de t?cnicas qu?micas, f?sicas e espectrosc?picas (composi??o elementar, UV-vis, FTIR, CP-MAS 13C RMN, MEV) e atrav?s da utiliza??o de t?cnicas quimiom?tricas (Unscrambler? X 10.3).

Paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica causada pelo fungo dimórfico Paracoccidioides brasiliensis. Aproximadamente 10 milhões de pessoas que vivem em áreas endêmicas estão infectadas com P. brasiliensis e até 2% destas podem desenvolver a PCM. Períodos extensos de quimioterapia são necessários e problemas de recidiva da doença são frequentes. Vacinas anti-PCM ainda não estão disponíveis para uso em humanos, porém, nos últimos anos, testes em modelo animal mostram que estratégias vacinais são viáveis. Formulações vacinais baseadas na proteína gp43 e no peptídeo P10 específico para células T CD4+ mostram-se promissores como antígenos vacinais. No presente trabalho, testamos as propriedades de uma formulação vacinal terapêutica baseada no peptídeo P10 combinado com flagelina de S. enterica, associada ou não a quimioterapia, como tratamento da PCM experimental. Os resultados indicaram que animais tratados apenas com a vacina, combinada ou não com os quimioterápicos, não demonstraram uma redução significativa da carga fúngica no pulmão. No entanto granulomas mais organizados foram observados no pulmão dos animais que receberam a formulação vacinal. / Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Approximately 10 million people living in endemic areas may be infected with this fungus and up to 2% of them may develop the disease. Extended periods of chemotherapy are often necessary to treat PCM and relapses occur frequently. PCM vaccines are not yet available for use in humans, but in recent years tests in animal models showed that vaccine strategies are viable. Vaccine formulations based in protein gp43 and a derived peptide P10, representing a CD4+ T cell-specific epitope, have showed promising results as vaccine antigens. In the present work, we tested the properties of a vaccine formulation based on the P10 peptide admixed with the S. enterica flagellin, in combination or not with chemotherapy, as a PCM therapeutic treatment. Treated mice, combined or not with chemotherapy, showed no significant reduction in lung fungal burden. Nonetheless, more organized lung granulomas were observed in animals that received the vaccine formulation.

O desenvolvimento de vacinas contra o vírus do papiloma humano (HPV) representa uma importante alternativa para o controle da infecção sexualmente transmissível e do câncer cervical. Neste trabalho exploramos uma estratégia vacinal inédita contra tumores induzidos pelo HPV-16 empregando a proteína E7 obtida após fusão genética com a glicoproteína D do vírus herpes tipo 1, uma proteína com propriedades adjuvantes para linfócitos T. A proteína recombinante gDE7 foi expressa em bactérias e em células de inseto e purificada por cromatografia de afinidade. A proteína gerada em bactérias foi administrada nas formas insolúvel e solúvel como vacina em camundongos. A gDE7 insolúvel conferiu 80% de proteção profilática para o crescimento tumoral, a gDE7 que manteve a forma solúvel após refolding protegeu 100% dos animais nas mesmas condições. Já a proteção terapêutica alcançou 30% dos animais. Além disso, verificamos o potencial neutralizante dos soros gerados frente ao HSV-1. As condições de obtenção das proteínas expressas em células de inseto também foram estabelecidas. / The development of vaccines against human papillomavirus (HPV) represents an important alternative to control the sexually transmitted infection and cervical cancer. In this paper we explored a novel vaccine strategy against tumors induced by HPV-16 using the E7 protein obtained after genetic fusion to the glycoprotein D of herpes virus type 1, a protein with adjuvant properties for T lymphocytes. The recombinant protein gDE7 was expressed in bacteria and in insect cells and purified by affinity chromatography. The protein generated in bacteria was administered in soluble and insoluble forms as a vaccine in mice. The insoluble gDE7 gave 80% prophylactic protection for tumor growth, the gDE7 that kept the soluble form after refolding protected 100% of the animals under the same conditions. The therapeutic protection reached 30% of the animals. We also verified the neutralizing potential of sera generated against the HSV-1. The conditions for obtaining the proteins expressed in insect cells were also established.

Ph.D. (Biochemistry) / Genetically modified (GM) crops were first introduced in the 1980s for the production of medicinal products. Since then, areas designated to GM crops have expanded drastically, with the GM crops grown to enhance agricultural productivity, improve agricultural practices, and as a tool to address potential pressures that will be faced by the agricultural sector and to address the issue of food security. Currently, cultivated GM crops include cotton, maize, rapeseed and soybean, carrying agronomic traits such as herbicide tolerance and insect resistance. Following the genetic modification of crops, three possible outcomes can be anticipated: these outcomes include the GM crop produced being equivalent to its untransformed counterpart, the GM crop differing from its untransformed counterpart with several well-defined characteristics, and the GM crop differing from its untransformed counterpart with a multitude of complex characteristics. In cases where the GM crop is equivalent to the untransformed counterpart, no further testing is needed. In instances where several well-defined and characterised differences are found between the GM crop and the untransformed counterpart, safety assessments are performed targeting these differences. The assessments will determine the impact of these unintended and unexpected alterations of the intended enhancement of the GM crops. However, methods currently used to assess GM crops have been found to be lacking, since they only focus on environmental and product-specific risks. Further evidence is essential, as part of GM crop safety assessment, on the molecular characterisation of these crops. This evidence is based on the potential impact of the transformation event, integration of the transgene into the host plant, as well as unintended alterations such as altered gene expression that may occur to the host plant. These events may assist in the further detection of potential dangers of the GM crop. As a result of these highlighted gaps, a project was formulated to study the unintended genomic alterations that may occur during and following the production of a transgenic plant...

Les récepteurs aux hormones glycoprotéiques (rGpHs) rTSH, rFSH et rLH/CG appartiennent<p>à la classe A des GPCRs. Les récepteurs da la classe A des GPCRs sont caractérisés par la<p>similitude de séquence de leur domaine transmembranaire avec la rhodopsine. Outre ce<p>domaine dit « serpentin », qu’ils partagent avec tous les GPCRs, les rGpHs offrent la<p>particularité de présenter un grand domaine extracellulaire (ECD) responsable de la liaison et<p>de l’affinité de leurs ligands respectifs.<p>Les récepteurs couplés aux protéines G (GPCRs) sont les cibles de 50% des médicaments<p>actuellement sur le marché pharmaceutique. La compréhension de leur mode de<p>fonctionnement est donc essentielle au développement de nouvelles molécules capables de<p>cibler spécifiquement ces récepteurs. Ces dernières années, il est apparu clairement que les<p>GPCRs étaient présent à la surface cellulaire sous forme d’oligomères. Le but de ce travail<p>était d’explorer de manière approfondie, le mécanisme d’activation et de dimérisation du<p>rTSH en identifiant les résidus du domaine transmembranaire des récepteurs aux hormones<p>glycoprotéiques impliqués dans le processus d’activation et de dimérisation.<p>Au cours de ce travail de thèse, nous avons dans premier temps généré une banque de mutants<p>aléatoires du rTSH. Ces mutants ont ensuite été criblés par une approche HTRF pour mettre<p>en évidence des mutants dont l’activité constitutive est augmentée ou ayant perdu la capacité<p>de dimériser.<p>Nous avons ainsi déterminé de nouveaux résidus importants pour le mécanisme d’activation<p>du rTSH. Les résultats que nous avons obtenus permettent d’apporter des éléments de réponse<p>et une base de travail sur le mécanisme d’activation. Cependant une description détaillée de<p>l’activation reste toutefois indéterminée. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Contexte : Les désordres congénitaux de la glycosylation (CDGs) sont des maladies rares dues à des mutations dans des gènes codant pour des protéines de la biosynthèse des glycoconjugués. Les CDGs présentent avec des glycoprotéines sériques hypoglycosylées avec un spectre clinique large. Le diagnostic moléculaire des CDG est important dans le cadre du diagnostic prénatal et du développement de stratégies thérapeutiques. Objectif : Déterminer les mutations causales dans une cohorte de cas suspects de CDG. Deux cas ont fait l’objet d’explorations biochimiques afin de comprendre les conséquences des mutations et d’envisager des stratégies thérapeutiques. Sujets/méthodes : Des explorations biochimiques sur des fibroblastes cutanés d’une cohorte de patients présentant des signes cliniques suggérant un CDG et hypglycosylation des protéines sérique. Résultats et conclusions: Le premier patient présentait une maladie multisystémique sévère. Des mutations affectant le gène codant pour la dehydrodolichol diphosphate synthase (DHDDS) ont été trouvées. Une activité diminuée la DHDDS était accompagnée de la diminution du dolichol phosphate. Ce patient est le premier cas de DHDDS-CDG présentant une atteinte multi-viscérale. Dans une deuxième étude deux siblings présentaient une thrombopénie associée à des atteintes neurologiques. Une mutation bi-allélique dans le gène codant pour le transporteur golgien du CMP-acide sialique (SLC35A1) associé avec une hypoglycosylation des protéines sériques a été détectée. Des profils anormaux des glycosphingolipides ont été mis en évidence et supplémentation des cellules de patient par de l’acide sialique a augmenté la biosynthèse des gangliosides. / Background: Congenital disorders of glycosylation (CDGs) are rare inherited diseases caused by mutations in genes required for glycoconjugate biosynthesis. CDG clinical presentations range from monosystemic to multiorgan failure. Often these diseases are diagnosed biochemically by the presence of hypoglycosylated serum proteins. Molecular diagnosis of CDG is crucial for both antenatal diagnostics and development of treatment strategies. Aims: To determine the molecular origins of disease in suspected CDG patients. Two cases were chosen for more extended biochemical explorations in order to investigate the consequences of the mutations and possible treatment strategies. Subjects/Methods: Biochemical explorations of skin biopsy fibroblasts from a cohort of patients presenting with signs suggestive of CDG, and serum protein hypoglycosylation. Results and conclusions: In the first study, a patient presented with multisystemic disease suggesting CDG. Fibroblasts revealed both truncated dolichol-linked oligosaccharides and polymannose-type N-glycans. Mutations in the dehydrodolichol diphosphate synthase (DHDDS) gene were found as well as low DHDDS activity and dolichol phosphate levels. As previous cases of DHDDS-CDG present with retinitis pigmentosa only, we describe the first case of a CDG syndrome associated with mutations in DHDDS. In the second study, two siblings presented with thrombocytopenia and CNS signs. A biallelic mutation in the CMP-sialic acid transporter gene (SLC35A1) was associated with hyposialylated serum glycoproteins. Altered glycosphingolipid profiles were seen and sialic acid supplementation of patient cells increased the appearance of gangliosides

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

Cryopreservation of human red blood cells (RBCs) extends the storage time from 42 days (hypothermic storage limit) to a maximum of 10 years. While this reduces the possibility of RBC shortages in emergency situations, this preservation method is currently limited to individuals with rare blood phenotypes, patients who require autologous blood transfusions, and military applications. Furthermore, cryopreservation is associated with a high degree of cellular damage, which can subsequently reduce the viability of cells post-thaw. The cellular damage incurred upon cryopreservation is primarily attributed to the process of ice recrystallization. To reduce the degree of cellular damage, cryoprotective agents (CPAs) are used. Currently, 10 % dimethyl sulphoxide (DMSO) and 40 % glycerol are used for the cryopreservation of hematopoietic stem cells (HSCs) and human RBCs respectively. Unfortunately, these CPAs do not provide protection against ice recrystallization. The biological antifreezes (BAs) consisting of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) were identified as the first inhibitors of ice recrystallization. Consequently, the Ben laboratory is interested in synthesizing small molecule carbohydrate-based inhibitors of ice recrystallization that can be used as an alternative to glycerol or DMSO for the cryopreservation of various cell types. Therefore, this thesis focuses on elucidating important structural features of carbohydrate-based derivatives that are responsible for IRI activity. The first part of this study examines the importance of the anomeric oxygen atom of aryl glycosides for IRI activity. Our laboratory previously demonstrated that the O-linked aryl glycosides are effective inhibitors of ice recrystallization. However, the influence of stereoelectronic effects at the C1 position of aryl glycosides on IRI activity has not been investigated. As a result, N- and S-linked aryl glycosides were synthesized in this study and their IRI activities were compared to that of the O-linked aryl glycosides. These results suggest that a stronger exo-anomeric effect exhibited by the C1 nitrogen derivatives reduces the IRI activity of aryl glycosides. The second part of this study focuses on the synthesis of AFGP disaccharide analogs. While the β-(1,3) glycosidic linkage found in native AFGP-8 was previously assessed for its influence on IRI activity, an extensive structure-function analysis of AFGP disaccharide analogs has not yet been performed. As a result, an AFGP disaccharide analog was designed whereby a para-methoxyphenyl (PMP) substituent was incorporated. This was done to assess whether the PMP substituent could enhance the lack of IRI activity exhibited previously with AFGP disaccharide analogs. Although the synthesis of this disaccharide target was not completed, a number of advantageous developments have been made regarding the glycosylation of N-acetyl-D-glycosamine derivatives. In addition, the PMP-GlcNAc intermediate encountered in disaccharide synthesis was assessed for its IRI activity, confirming that the acetamido (NHAc) function is not required for IRI activity.