Made available in DSpace on 2014-10-09T12:42:03Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:44Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-01T16:41:18Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Priscila.pdf: 3187592 bytes, checksum: a982eac78d937dd89e264fd88f2d7be9 (MD5) / Made available in DSpace on 2016-04-01T16:41:18Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Priscila.pdf: 3187592 bytes, checksum: a982eac78d937dd89e264fd88f2d7be9 (MD5) Previous issue date: 2015-02-26 / FACEPE / O câncer de próstata (CaP) é um dos tipos de tumor maligno mais incidente na população mundial, sendo também uma das principais causas de mortalidade por câncer. Geralmente ocorre em homens idosos e não é percebido em seus estágios iniciais. Para detectar o câncer de próstata, é de grande importância a avaliação pela dosagem sérica do antígeno específico da próstata (PSA), considerado o marcador tumoral da próstata. Porém, alterações nos níveis séricos do PSA são observadas também em doenças benignas da próstata, reduzindo a sensibilidade e especificidade do teste para se obter o diagnóstico. Uma peculiaridade do câncer que pode ser relevante no diagnóstico é a alteração no padrão de glicosilação de glicoproteínas secretadas e de superfície celular. A identificação de mudanças na expressão de glicanos permite a detecção precoce de vários tipos de câncer, inclusive o câncer de próstata. As lectinas são ferramentas empregadas para detectar tais alterações, graças à propriedade de reconhecer carboidratos com especificidade. Uma grande novidade no campo de estudos com lectinas é o desenvolvimento de pequenos dispositivos conhecidos como biossensores eletroquímicos baseados em lectinas para análise do perfil de glicoproteínas em processos fisiopatológicos. Esses dipositivos são elaborados utilizando eletrodos geralmente modificados com polímeros e nanomateriais, onde a lectina pode ser imobilizada e sua interação com o carboidrato quantificada com elevada sensibilidade e em tempo real. Neste trabalho foi realizado um estudo bioeletroquímico da lectina de Cratylia mollis (CramoLL) para caracterizar o padrão de glicosilação em soros de indivíduos saudáveis, com hiperplasia benigna da próstata (HBP) e CaP, utilizando biossensor eletroquímico baseado em CramoLL, imobilizada sobre eletrodo de carbono vítreo modificado com o polímero catiônico poli-Llisina (PLL) e nanotubos de carbono (NTC). Análises eletroquímicas e ópticas mostraram a formação de um filme de PLL e NTC na superfície eletródica, onde os NTC proporcionaram um aumento na área sensora de 71,2%, e nos valores de corrente anódica e catódica, e aumentaram a estabilidade da superfície, graça às propriedades físico-químicas dessas nanoestruturas. Foram investigadas as concentrações ótimas de PLL e NTC. CramoLL foi imobilizada com sucesso, numa concentração ótima de 200 μg mL-1. A interação entre CramoLL e metil-α-D-manopiranose (MeαMan)/ fetuína foi confirmada utilizando a técnica de voltametria de onda quadrada, mostrando que a lectina manteve sua atividade biológica após imobilização. Foram obtidas curvas de calibração para diferentes concentrações dos glicanos, com faixa linear de 0,96 e 38,4 μg mL-1 de MeαMan (r= 0,982, p < 0,001) e limite de detecção (LD) de 0,04 μg mL-1e de 0,5 a 25 μg mL-1 de fetuína (r= 0,994, p< 0,001) e LD = 0,05 μg mL-1. O biossensor baseado em CramoLL foi exposto à amostras de soro de indivíduos sadios (controle), com HBP e CaP com escores de Gleason 6, 7 e 9 para analisar a interação entre CramoLL e glicoproteínas dos soros de cada grupo. Os sinais de correntes obtidos por voltametria de onda quadrada mostraram diferenças significativas entre o grupo controle e os grupos HBP, CaP escore 6 e 7; entre HBP e CaP escore 6; entre CaP escore 6 e os grupos CaP escore 7 e 9 e entre CaP escore 7 e CaP 9. O potencial de reconhecimento da lectina permitiu identificar alterações de glicosilação associadas ao câncer de próstata e distinguir das amostras não cancerosas e entre os diferentes escores de CaP. Esses resultados sugerem a possibilidade de aplicação em estudos futuros envolvendo caracterização de glicosilação em câncer. / Prostate cancer (PCa) is a most frequently type of malignancy in global population and is also a major cause of cancer mortality. It usually occurs in older men and is not perceived in early stages. To detect the prostate cancer, this is of great importance the evaluation of serum prostate specific antigen (PSA), considered the prostate tumor marker. But, changes in serum PSA levels are also observed in benign prostate diseases, reducing the sensitivity and specificity of the test to obtain the diagnosis. A peculiarity of cancer that may be relevant in the diagnosis is the change in the pattern of glycosylation in glycoproteins secreted and cell surface. The identification of changes in the expression of glycans permits detection early of various types of cancer, including prostate cancer. Lectins are tools used to analyze such changes in diseases, through to the property to recognize carbohydrates with specificity. A great innovation in the field of studies with lectins is the development of smalls devices known as electrochemical biosensors based on lectins for analysis of the glycoproteins profile in physiopathological processes. These devices are typically produced using modified electrodes containing polymers, nanomaterials, where in the lectin may be immobilized and the interaction with carbohydrate quantified with high sensitivity in real time. This work presents a bioelectrochemical study of lectin of Cratylia mollis (CramoLL) to characterize the glycosylation pattern in sera from healthy individuals, with benign prostatic hyperplasia (BPH) and PCa, using electrochemical biosensor based on CramoLL immobilized on glassy carbon electrode modified with the polymer cationic poly-L-lysine (PLL) and carbon nanotubes (CNT). Electrochemical and optical analysis showed the formation of a PLL and CNT film on the electrode surface, where the CNT provided an increase in sensor area of 71.2%, and the anodic and cathodic current values, and increased stability of the surface due to the physical and chemical properties of these nanostructures. The optimal concentrations of PLL and NTC were investigated. CramoLL was successfully immobilized at a great concentration of 200 μg mL-1. The interaction between CramoLL and methyl-α-Dmannopyranose (MeαMan)/ fetuin was confirmed using voltammetry square wave technique, showing that the lectin retained its biological activity after immobilization. Calibration curves were plotted for different concentrations of the glycans with a linear range of 0.96 and 38.4 μg mL-1 for MeαMan (r = 0.982, p <0.001) and limit of detection (LOD) of 0.04 μg mL-1, and from 0.5 to 25 μg mL-1 for fetuin (r = 0.994, p <0.001) and LOD = 0.05 μg mL-1. The CramoLL-based biosensor was exposed to serum samples from healthy individuals (control), and patients with BPH and PCa with Gleason scores of 6, 7 and 9 to analyze the interaction between CramoLL and glycoproteins of sera from each group. The signals of current by square wave voltammetry show significants differents between the control group and the groups of BPH, PCa score 6 and 7; between BPH and PCa score 6; between PCa score 6 and groups PCa score 7 and 9 and between PCa score 7 ad PCa score 9. The potential of specific recognition of the lectin allowed to identify changes in glycosylation associated at prostate cancer and to distinguish of non-cancerous samples and between the differents scores of PCa. The results suggest the possibility of using in future studies involved characterization of glycosylation in cancer.

Made available in DSpace on 2014-10-09T12:42:03Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:44Z (GMT). No. of bitstreams: 0 / A tireoide é uma das principais glândulas do nosso sistema endócrino e responsável pela produção dos hormônios Triodotirosina (T3) e Tetraiodotironina (T4) que são responsáveis pelo controle metabólico basal. A tireoide pode ser acometida por neoplasias benignas e malignas que pode levar a uma produção anormal de hormônios e levando a sérios problemas de saúde. O diagnóstico de algumas destas neoplasias por citologia ainda não possuem altas taxas de sensibilidade e especificidade para os casos com padrão folicular, portanto o desenvolvimento de novos métodos que se baseiam na analise de características bioquímicas dos tecidos tireoidianos se faz necessário. O presente trabalho caracterizou tecidos tireoidianos normais e com bócio nodular por meio da espectroscopia no infravermelho. As imagens espectrais foram adquiridas com alta resolução e aliadas a um grande processamento das mesmas utilizando métodos de estatística multivariada se obteve diferenças bioquímicas dos tecidos. Nos resultados obtidos no presente trabalho, a maior diferença observada está na região da amida I que está relacionada a estrutura secundária da tiroglobulina devido ao processo de incorporação de iodo. Outras observações que foram feitas são diferenças em glicosilaçao do tipo N. A partir dos resultados obtidos nesta tese conclui-se que a técnica de imageamento por microespectroscopia no infravermelho é capaz de observar diferenças bioquímicas importantes entre tecidos tireoidianos sadios e com bócio, apresentando grande potencial para o desenvolvimento no futuro de novas metodologias, baseadas em espectroscopia vibracional. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

No presente trabalho, desenvolvemos uma estratégia vacinal baseada em vacinas de DNA que codificam proteínas do HPV-16, o tipo viral de maior relevância epidemiológica, fusionadas à glicoproteína D (gD) do vírus herpes simplex tipo 1 (HSV-1). A vacina que contêm o gene da E7 de HPV-16 fusionada à gD de HSV-1 (pgDE7), administrada em regime vacinal de quatro doses, foi capaz de gerar significativa ativação de células T CD8+ E7-específicas e apresentar 40% de efeito protetor anti-tumoral terapêutico em camundongos desafiados com células transformadas que expressam as proteínas E6 e E7 do HPV-16 (células TC-1). A partir das evidências geradas, desenvolvemos um novo vetor vacinal que codifica as proteínas E7, E6 e E5 do HPV-16 (pgD-E7E6E5). Em ensaios em modelo murino, apenas uma dose da vacina foi capaz de gerar ativação de células T CD8+ específicas e 70% dos camundongos previamente desafiados com células TC-1 e inoculados com 3 doses da vacina mantiveram-se livres de tumores. Como tentativa de potencializar o efeito protetor terapêutico encontrado, adotamos duas medidas: a co-administração de plasmídeos que codificam citocinas e a otimização de códons da sequência gênica que codifica a proteína quimérica. A combinação das vacinas pgDE7 ou pgD-E7E6E5 com plasmídeos que carregam os genes das citocinas IL-2, IL-12 ou GM-CSF foi capaz de aumentar a proteção terapêutica para 100% em regime vacinal de dose única. A adequação da sequência antigênica ao sistema de expressão humano, aumentou em cerca de 5 vezes o potencial terapêutico do vetor vacinal pgDE7. Em conjunto, os dados apresentados nesta tese demonstram a evolução do desenvolvimento de uma estratégia vacinal contra tumores induzidos por HPV-16 e encorajam seu potencial para uso em futuros ensaios clínicos. / The development of immunotherapeutic strategies against human papillomavirus (HPV) is a priority for the control of HPV-induced lesions and cervical cancer. In this study, we developed DNA vaccines encoding HPV-16 proteins fused to glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) as an approach to control HPV-16 induced tumors. The vaccine encoding HPV-16 E7 fused to HSV-1 gD (pgDE7), when administered in a four doses vaccine regimen, was able to generate significant activation of E7-specific CD8+ T cells and showed 40% of therapeutic anti-tumor effect in mice previously challenged with tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells). Following these evidences, we developed a new vaccine vector encoding HPV-16 E7, E6, E5 proteins fused to HSV-1 gD (pgD-E7E6E5). Only one vaccine dose generated antigen-specific CD8+ T cell responses and three doses conferred 70% protection to mice previously challenged with TC-1 cells. As an attempt to enhance the observed therapeutic anti-tumor effects, we tested two approaches: co-administration of cytokine-expressing plasmids and codon optimization of the gene encoding the chimeric protein. The combination of the vaccines pgDE7 or pgD-E7E6E5 with plasmids encoding the cytokines IL-2, IL-12 or GM-CSF increased the therapeutic protection to 100% of the vaccinated animals following a single dose. The gene sequence adaptation increased by a factor of 5 the therapeutic potential of the pgDE7 vaccine. In summary, the data presented in this thesis demonstrated the development of a vaccine strategy against HPV-16 induced tumors and reinforces its potential use in future clinical trials.

O Trypanosoma cruzi expressa um grupo de glicoprotcinas de superfície, denominadas Tc-85, que pertencem à superfumília gêmca das gp85/traus-sialidases. Nosso laboratório clonou e caracterizou um membro da fumília Tc85 (Tc85-11), cuja região carboxila tenninal (clone Tc85-1) adere em laminina e em células de mamífero. Usando peptídeos sintéticos, correspondendo em seqüência à Tc85-1, caracterizou-se o motivo mais conservado da superfamilia gênica das gp85/trans-sialidases (VTVxNVFLYNR), o qual não adere em laminina. Esse motivo foi chamado peptídeo J. Por cromatografia de extratos de membrana de cardiomiócitos em coluna de afmidade contendo peptídeo J, foi isolada uma molécula de 30kDa identificada como sendo a subunidade &#946;3 da Na+, K+ ATPase. A porção extracelular da subunidade &#946;3 da Na+, K+ ATPase foi clonada e a interação in vitro desta proteína com peptídeo J foi observada. Deste modo, é sugerido aqui que a subunidade &#946;3 da Na+, K+ ATPase pode ter um papel importante na interação do parasita com a célula hospedeira. / Abstract not available.

Rabies is a fatal but preventable neurotropic disease of potentially all mammals. The disease is caused by lyssaviruses. Rabies is recognized as the 10th most common lethal infectious disease in the world, rendering it one of the most feared zoonotic diseases known to man. Nevertheless, rabies can be prevented by application of pre- or post exposure treatments. Rabies vaccines have been available since the time of Pasteur, more that one hundred years ago. Since, vaccine research focused on the development of safer and more effective vaccines. Topics of current interest in the field of rabies vaccinology were addressed in this study. A primary concern regarding the disease is human mortalities, in the range of 60 000, reported every year. Most of these are linked to exposure to rabid dogs. In addition, a great number of post exposure treatments are administered each year at great costs. Despite availability of efficacious biologics, several factors influence the optimal use and accessibility of these agents in the countries of interest, with cost and availability being the major contributing factors. A proven approach is mass oral vaccination of target animals, such as dogs, which indirectly infers protection to susceptible hosts, including man. Currently available vaccines present several disadvantages of use though, including issues of safety or doubtful stability. Safer but effective alternative vaccines that could be used in oral baits would be valuable. Here the use of two candidate host restricted poxvirus vaccine vectors were explored, particularly also in regard to oral innocuity. The construction, convenient isolation and use of a recombinant Lumpy skin disease virus (Neethling strain) expressing rabies virus glycoprotein in a mouse model were investigated. In addition, a recombinant Modified Vaccinia virus Ankara expressing rabies virus glycoprotein was prepared and tested as a vaccine in mice, dogs and raccoons. In both cases it was clear that the severe attenuation of these viruses did affect the efficacy of the recombinant vaccines in the non-permissive hosts. With the recombinant MVA a clear dosage effect could be shown, and equivalent humoral responses could only be attained at much higher titers of vaccine virus as with replication competent counterparts. Secondly, the cross-protection of rabies vaccines across the spectrum of lyssaviruses was addressed. Lyssaviruses can be divided into two groups based on sequence analysis and pathogenesis. Viruses belonging to the so-called phylogroup II, are the Mokola, Lagos and West Caucasian Bat viruses. Classic rabies biologics fail to fully protect against the viruses attributed to a lack of cross-neutralization. Here, cross-protection and cross-reactive immune responses induced by recombinant vaccinia viruses expressing rabies, Mokola or West Caucasian Bat virus glycoproteins, in single or dual combinations, were investigated. As expected, there was a lack of cross-protection of rabies and Mokola glycoprotein vaccines. There was also a clear lack of cross-protection of West Caucasian Bat virus glycoprotein vaccine and rabies and Mokola viruses. The dual antigen expressing vaccines did not appear to offer any additional protective effect in the tested model. The Mokola virus glycoprotein vaccines induced neutralizing antibody responses that significantly cross-neutralized Lagos Bat virus. / Thesis (PhD (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

The genus Candida includes several opportunistically pathogenic species which are common causative agents of the yeast infections in humans. Although current medical research is focused mostly on cancer, AIDS or Alzheimer disease, the problem of systemic candidiases cannot be neglected. These infections represent a real threat to the immunocompromissed patients, they are connected with a high mortality rate and expensive medication with poor prognosis. Pseudomonas aeruginosa could be an inspiration in a way of how to eliminate the pathogenic yeasts. The bacterium can inhibit growth of the most common yeast species of the genus Candida, C. albicans. This effect is based on production of toxic substances by the bacterium and on interaction of the bacterium with the C. albicans cell wall, which leads to the lysis of the yeast cells and which is not fully understood. Nevertheless, coexistence of these microorganisms is also possible and their relationship is affected by various factors. Knowledge of these inter- microbial interactions was obtained from studies of diseases and pathologies, during which C. albicans + P. aeruginosa coinfections occur. In this thesis I studied mechanisms of interaction between pathogenic yeast C. albicans and bacterium P. aeruginosa by a) C. albicans gene expression...

The hemagglutinin-neuraminidase (HN) molecule of Newcastle disease virus (NDV) is an integral membrane glycoprotein that is oriented with its N-terminus in the cytoplasm and its C-terminus external to the infected cell. Single spanning membrane proteins with this type of topology (N-terminus in, C-terminus out) have been classified as Type II glycoproteins, in contrast to the more common Type I glycoproteins, which are oriented in the opposite direction. (C-terminus in, N-terminus out). The membrane integration of HN protein was investigated using a wheat germ translation system to synthesize and integrate HN protein into microsomal membranes in vitro. The insertion and translocation of HN protein into microsomal vesicles was found to occur cotranslationally without signal sequence cleavage. The membrane targeting required both signal recognition particle (SRP) and SRP receptor. Membrane binding assays utilizing HN nascent chain/ribosome/SRP complexes demonstrated that the membrane insertion of HN polypeptide required the presence of GTP, in a way similar to that described for secretory, multispanning and Type I proteins. To investigate further the membrane translocation process of HN protein, the amino terminal region of HN was mutated to determine the role of this region in the membrane integration of HN. The cDNA sequence encoding the bulk of the cytoplasmic tail of the HN glycoprotein was deleted. When transcripts produced from the mutated cDNA were translated in wheat germ extract in the presence of membranes, several abnormalities were identified in the interaction of the mutant protein with membranes. Although translocation and glycosylation of the mutant protein was detected, the efficiency of membrane translocation and the stability of the mutant protein's membrane interaction were reduced. Even though a large proportion of the mutant products remained nontranslocated and unglycosylated, many of these products were inserted into membrane vesicles in a reverse orientation from the wild type HN protein. The aberrant insertion of the mutant protein required both SRP and SRP receptor. Ribosome-bound mutant nascent chains were able to insert into membranes without the addition of GTP or SRP, but this GTP-independent insertion was in reverse. Therefore, the cytoplasmic tail of the HN glycoprotein appears to playa critical role in the maintanence of faithful directionality of the protein's membrane insertion.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / External apical root resorption (EARR) is a common sequela of orthodontic treatment, although it may also occur without orthodontic treatment. Despite rigorous investigation, no single factor or group of factors that directly causes root resorption has been identified. Experiment 1. A sample of 83 pairs of full siblings who had undergone orthodontic treatment was studied. Measurements were made of the longest maxillary central incisor, mandibular central incisor and mesial and distal roots of the mandibular first molars. Heritability estimates were generated by generalized liner models. Our results showed that the heritability estimate of the EARR was 64% on average. It was concluded that there was sufficient heritability for EARR to pursue genetic analysis. Experiment 2. Five polymorphic markers flanking or lying within the IL-IA , IL-JB, TNSALP, TNFA, and TNFRSFJ JA genes were used in a candidate gene approach to assess linkage and association with EARR in 38 pedigrees. Suggestive evidence for linkage between EARR and the polymorphic marker D18S64 was obtained with the analysis program MAPMAKER/SIBS (LOD score 2.51). The Q-TDT program showed highly significant (p = 0.0003) evidence of linkage disequilibrium of IL-1 B polymorphisms with EARR. Our analysis indicates that the JL -1 B polymorphism accounts for 15% of the total EARR variation. Experiment 3. Nine-week-old male mice were randomly selected as controls or for placement under anesthesia of an open coil spring ligated to the left maxillary first molar producing a force of approximately 25 g. The control (C) or treated (T) per strain were A/J (C=3,T=9), C57BL/6J (C=7,T=8), C3H/HeJ (C = 4,T=6), BALB/cJ (C=4,T=6), 129P3 /J (C=6,T=8), DBA/2J (C=8,T=9), SJL/J (C=8,T= 10), and AKR/J (C=9,T =8). Animals were sacrificed after nine days of treatment or control; maxillae were immediately removed, prepared, sectioned, mounted, stained with H&E, and observed microscopically at 1 OOX to determine root resorption. Mice were grouped into root resorption resistant (A/J, C57BL/6J and SJL/J); intermediate (C3H/HeJ and AKR/J); and susceptible (BALB/cJ, DBA/2J, and 129P3/J) strains. It was concluded that there were differential susceptibility or resistance to root resorption among inbred mouse strains, indicating that genotype is an influencing factor.