O reconhecimento e interação intermoleculares são cruciais na patogênese de doenças desmielinizantes autoimunes, como a esclerose múltipla (EM). A EM é uma doença que acomete o sistema nervoso central (SNC) e leva à desmielinização e axonopatia. Os alvos da resposta não são claros, mas proteínas da mielina, como a glicoproteína oligodendrocítica da mielina (MOG) e a proteína básica da mielina (MBP), são potenciais candidatas ao reconhecimento por células e autoanticorpos durante o processo autoimune. Assim, métodos de modelagem e simulações de dinâmica molecular (MD) e steered molecular dynamics (SMD) foram empregados para detalhar o reconhecimento e ligação do domínio externo da MOG e do peptídeo imunogênico MBP85-99 por anticorpos específicos. Para a obtenção das estruturas 3D dos anticorpos, particularmente do anti-MBP, um protocolo computacional envolvendo mutações sequenciais da região determinante de complementaridade (CDR) de estruturas-molde foi proposto. Dados obtidos evidenciaram grande contribuição das ligações de hidrogênio na manutenção dos complexos antígeno-anticorpo. Treze resíduos da MOG foram identificados como âncoras da ligação com o anti-MOG, os quais se relacionaram a peptídeos importantes descritos na literatura, principalmente o MOG92-106. No caso da MBP, os resíduos do MBP85-99 com maior interação com o anti-MBP envolveram a Arginina 99, Lisina 93, Asparagina 94 e Histidina 90, corroborando achados na literatura acerca da resposta celular e análises do anti-MBP em casos postmortem. Dados de SMD envolvendo os sistemas moleculares foram confirmados por dados de microscópio de força atômica, sugerindo grande participação do peptídeo MOG92-106 na manutenção da ligação com o anti-MOG. Com relação à MBP, os estudos computacionais indicaram que o ponto de interação da região da Arginina 99 é muito importante para a ligação com o anti-MBP. A consonância entre dados computacionais e dados experimentais resultantes de décadas de pesquisas da MOG e a MBP, bem como com dos experimentos de AFM, ficou evidente. Desta forma, as aproximações teórico-experimentais aplicadas neste trabalho para a caracterização de moléculas ainda não estudadas é uma via em potencial para otimização de passos iniciais e pré-clínicos de investigações de doenças autoimunes, guiando experimentos, reduzindos custos e o uso de modelos animais. / Intermolecular recognition and interaction are crucial in autoimmune demyelinating diseases pathogenesis as multiple sclerosis (MS). MS causes demyelination and axonopathy in the central nervous system (CNS). The targets of immune cells and autoantibodies are not clear, but myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP), are potential candidates. Thus, methods of molecular modeling, molecular dynamics (MD), and steered molecular dynamics (SMD) simulation were applied to detail the recognition and binding of MOG external domain and the immunogenic MBP85-99 peptide by specific antibodies. A computational protocol based on mutations of complement determinant regions (CDR) in template structures was proposed to obtain antibodies 3D structures, especially the anti-MBP. The obtained data evidenced a significant contribution of hydrogen bonds in the maintenance of antigen-antibody complexes. Thirteen anchor residues were found in the MOG structure. These residues were related to three well-known epitopes recognized by immunologic components, mainly MOG92-106. In the case of MBP, the most interactive residues of the MBP85-99 with the anti-MBP were Arginine 99, Lysine 93, Asparagine 94, and Histidine 90. These data complied with several studies concern cellular recognition of MBP and postmortem cases involving anti-MBP. SMD information of both molecular systems was confirmed by atomic force microscopy and suggested the MOG92-106 acting as an anchor for the complex with the anti-MOG. Regarding MBP, the computational force study evidenced the importance of Arginine 99 interaction region for the antigen-antibody binding. The agreement between the obtained computational data and experimental information resulted of decades of MOG and MBP research was evident. In this context, theoretical and experimental approaches application as described here for characterizing novel molecules in autoimmune disease is a potential pathway to optimize early-stage and pre-clinical steps of investigations, guiding experiments, reducing costs, and animal model usage.

O presente projeto teve como proposta estudar a utilização do colostro bovino liofilizado como manejo alternativo para caprinos recém-nascidos. Estudos relacionados com a aquisição de proteção passiva e o desenvolvimento do epitélio intestinal, como aspectos citológicos e histológicos, atividade celular e expressão do receptor específico para o IGF-I (IGF-IR), foram realizados. Às 0, 7 e 14 horas de vida, 15 machos recém-nascidos receberam 5% do peso inicial de colostro bovino liofilizado (CBL) e 14 colostro caprino (CC), ambas as refeições com 55 mg/mL de IgG. Amostras de sangue foram coletadas às 0, 7, 14, 18, 24, 36, 48, 72 e 96 horas de vida para a quantificação da imunoglobulina G (IgG), proteína total (PT) e do fator de crescimento semelhante à insulina tipo I (IGF-I). Amostras do duodeno, jejuno e íleo foram coletadas às 18, 36 e 96 horas de vida para análises utilizando microscopia óptica e microscopia eletrônica de varredura e transmissão. A atividade de enzimas extracelulares, quantificação da proteína, DNA e RNA total e expressão do IGF-IR também foram determinadas. Amostras de um grupo adicional (0 h), que não ingeriu colostro, foram coletadas. No grupo CBL, as concentrações séricas de IgG às 14, 18, 24 e 48 horas foram maiores que às 0 e 7 horas, enquanto no grupo CC, apenas às 18 horas a concentração sérica foi maior que às 0 e 7 horas (P<0,05). A capacidade de absorção de IgG diminuiu entre 7 e 14 horas em CC (P<0,05), enquanto em CBL, manteve-se sem alteração nestes horários (P>0,05). As variáveis PT e IGF-I não foram influenciadas pela ingestão de colostro bovino liofilizado (P>0.05). Os indicativos de atividade celular, concentração de proteína, DNA e RNA total, proteína/DNA, proteína/RNA e RNA/DNA, e a atividade das enzimas aminopeptidase N, dipeptidil peptidase IV, aminopeptidase A, lactase, maltase e sacarase diferiram nos horários experimentais e em relação ao grupo adicional (P<0,05). Apenas as razões proteína/DNA, proteína/RNA no jejuno e a atividade da aminopeptidase A no íleo foram maiores no grupo CBL (P<0,05). A atividade da fosfatase ácida foi observada nas primeiras horas de vida, especialmente no duodeno. Com relação à morfologia do epitélio intestinal e densidade das vilosidades, o fornecimento de colostro bovino liofilizado não determinou alterações. As variações na ultraestrutura dos enterócitos revelaram a presença, no segmento jejuno, de material absorvido e do complexo endocítico apical às 0, 18 e 36 horas. No duodeno, a expressão gênica do IGF-IR ao nascimento foi maior que às 18 e 36 horas nos grupos CC e CBL (P<0,05). Enquanto, a expressão do receptor deste peptídeo bioativo no jejuno do grupo CBL foi maior às 18 e 36 horas em relação a 0 h (P<0,05). Os resultados obtidos do presente trabalho contribuem para que a fonte alternativa de imunoglobulinas, colostro bovino liofilizado, seja utilizada como um substituto para o colostro caprino sem alterações significativas na aquisição de proteção passiva e nas características histofisiológicas do tecido epitelial do intestino delgado de cabritos. / This project was proposed to study the use of lyophilized colostrum as an alternative management for newborn goat kids. Studies related to the acquisition of passive protection and development of the intestinal epithelium, as cytological and histological aspects, cellular activity and expression of the receptor specific for IGF-I (IGF-IR), were performed. At 0, 7 and 14 hours of life, 15 newborn males received 5% of initial weight of lyophilized bovine colostrum (LBC) and 14 goat colostrum (GC), both meals with 55 mg/mL of IgG. Blood samples were collected at 0, 7, 14, 18, 24, 36, 48, 72 and 96 hours of life to quantification of immunoglobulin G (IgG), total protein (TP) and insulin like growth factor type I (IGF-I). Samples of the duodenum, jejunum and ileum were collected at 18, 36 and 96 hours of life for analysis using optical microscopy and scanning and transmission electron microscopy. The activity of extracellular enzymes, quantification of total protein, DNA and RNA and expression of the IGF-IR were also determined. Samples from an additional group (0 h), not suckling, were collected. In LBC group, serum IgG concentrations at 14, 18, 24 and 48 hours were higher than at 0 and 7 hours, while in the GC group, only at 18 hours serum concentration was higher than at 0 and 7 hours (P<0.05). The capacity of IgG absorption decreased between 7 and 14 hours in GC (P<0.05), while in LBC, remained unchanged at these times (P>0.05). The variables TP and IGF-I were not influenced by ingestion of lyophilized bovine colostrum (P>0.05). The indicators of cellular activity, total protein, DNA and RNA concentration, protein/DNA, protein/RNA and RNA/DNA, and the enzyme activity of aminopeptidase N, dipeptidyl peptidase IV, aminopeptidase A, lactase, maltase and sucrase differed in sampling times and in relation to the additional group (P<0.05). Only the rations protein/DNA, protein/RNA in the jejunum and aminopeptidase A activity in the ileum were higher in the LBC group (P<0.05). The acid phosphatase activity was observed in the first hours of life, especially in the duodenum. Regarding the intestinal epithelium morphology and villi density, the supply of lyophilized bovine colostrum did not determine alterations. Changes in enterocytes ultrastructure revealed the presence in the jejunum segment of absorbed material and apical endocytic complex at 0, 18 and 36 hours. In the duodenum, IGF-IR gene expression at birth was higher than at 18 and 36 hours in the GC and LBC (P<0.05). While the expression of this bioactive peptide receptor in the jejunum of the LBC group was higher at 18 and 36 hours relative to 0 h (P<0.05). The results of this work indicate that the alternative source of immunoglobulins, lyophilized bovine colostrum, can be used as a substitute for goat colostrum without significant changes in the acquisition of passive protection and histophysiology characteristics of the small intestine epithelium of goat kids.

Na imunodeficiência comum variável (ICV) têm sido descritas alterações de linfócitos T, incluindo a produção diminuída da interleucina-2 (IL-2). Desde que a IL-2 pode promover a produção de imunoglobulinas in vitro, nosso principal objetivo foi investigar os efeitos in vivo do tratamento com IL-2 recombinante (IL-2r) em pacientes com ICV. Foram selecionados 4 pacientes que apesar de tratamento adequado com imunoglobulina EV apresentavam infecções recorrentes. Após um período de observação de 12 meses, os pacientes receberam doses crescentes de IL-2r durante 16 semanas com reposição de imunoglobulina apenas se a IgG sérica atingisse níveis menores do que 400mg/dL. A seguir, permaneceram em observação por mais 12 meses recebendo imunoglobulina . A gravidade das infecções foi avaliada segundo um \"score\" numa escala de 3 a 10. A avaliação in vitro incluiu: quantificação dos níveis de IgG, IgA e IgM séricas; resposta linfoproliferativa à PHA; populações linfocitárias CD4+, CD8+, CD19+ e CD25+ no sangue periférico. As reações adversas à IL-2r foram leves e localizadas. Houve redução aparente do número e gravidade das infecções durante os 12 meses subseqüentes ao término da IL-2r. Os níveis da IgG sérica e das células CD4+, CD8+ e CD19+ mantiveram-se estáveis durante todo o estudo. Em 3 pacientes houve relação entre melhora clínica e aumento da proporção de linfócitos T CD25+. Isto permite supor que a remissão de infecções em alguns pacientes com ICV , sob terapêutica com IL-2r associada ou não à imunoglobulina EV, esteja parcialmente relacionada à melhora da imunidade celular. Adicionalmente, nossos dados indicam que a IL-2r pode ser utilizada de modo seguro nas dosagens e período utilizados como terapêutica adjuvante em alguns pacientes com ICV que apresentam infecções recorrentes e má resposta terapêutica à imunoglobulina endovenosa / In Common Variable Immunodeficiency (CVID) T cell function may be impaired and interleukin-2 (IL-2) production diminished. Since IL-2 stimulates immunoglobulin production in vitro, the aim of this study was to determine the in vivo effects of recombinant interleukin-2 (rIL-2) in patients with CVID. We selected four CVID patients, who despite intravenous immunoglobulin infusion (IVIG) had recurrent infections. After a twelve-month run-in period, escalating dosages of rIL-2 were administered during 16 weeks, during which rescue IVIG treatment was performed whenever serum IgG levels dropped below 400 mg/dL. During follow-up (12 months), patients were observed and treated with IVIG. Infection severity was assessed using a 3 to 10 infection score. In vitro analysis included: measurement of serum levels of IgG, IgA and IgM; lymphocyte proliferative responses to phytohaemaglutinin (PHA); CD4+, CD8+,CD19+ and CD25+ lymphocyte populations in peripheral blood. Few local side-effects were observed in 2 patients. In the follow-up period after rIL-2 treatment, patients experienced reduction of the number and severity of infections. Levels of serum IgG, CD4+, CD8+ and CD19+ were stable throughout the study. In 3 patients we observed a relation between improvement of clinical parameters and number of T CD25+ cells. These findings suggest that remission of infections in some CVID patients treated with rIL-2, in combination or not with IVIG is, in part, associated with the improvement of cell immunity. Additionally, our results indicate that rIL-2 administration is safe and may serve as adjuvant therapy in some CVID patients with recurrent infections and poor response to IVIG treatment

O presente projeto teve como proposta estudar a utilização do colostro bovino liofilizado como manejo alternativo para caprinos recém-nascidos. Estudos relacionados com a aquisição de proteção passiva e o desenvolvimento do epitélio intestinal, como aspectos citológicos e histológicos, atividade celular e expressão do receptor específico para o IGF-I (IGF-IR), foram realizados. Às 0, 7 e 14 horas de vida, 15 machos recém-nascidos receberam 5% do peso inicial de colostro bovino liofilizado (CBL) e 14 colostro caprino (CC), ambas as refeições com 55 mg/mL de IgG. Amostras de sangue foram coletadas às 0, 7, 14, 18, 24, 36, 48, 72 e 96 horas de vida para a quantificação da imunoglobulina G (IgG), proteína total (PT) e do fator de crescimento semelhante à insulina tipo I (IGF-I). Amostras do duodeno, jejuno e íleo foram coletadas às 18, 36 e 96 horas de vida para análises utilizando microscopia óptica e microscopia eletrônica de varredura e transmissão. A atividade de enzimas extracelulares, quantificação da proteína, DNA e RNA total e expressão do IGF-IR também foram determinadas. Amostras de um grupo adicional (0 h), que não ingeriu colostro, foram coletadas. No grupo CBL, as concentrações séricas de IgG às 14, 18, 24 e 48 horas foram maiores que às 0 e 7 horas, enquanto no grupo CC, apenas às 18 horas a concentração sérica foi maior que às 0 e 7 horas (P<0,05). A capacidade de absorção de IgG diminuiu entre 7 e 14 horas em CC (P<0,05), enquanto em CBL, manteve-se sem alteração nestes horários (P>0,05). As variáveis PT e IGF-I não foram influenciadas pela ingestão de colostro bovino liofilizado (P>0.05). Os indicativos de atividade celular, concentração de proteína, DNA e RNA total, proteína/DNA, proteína/RNA e RNA/DNA, e a atividade das enzimas aminopeptidase N, dipeptidil peptidase IV, aminopeptidase A, lactase, maltase e sacarase diferiram nos horários experimentais e em relação ao grupo adicional (P<0,05). Apenas as razões proteína/DNA, proteína/RNA no jejuno e a atividade da aminopeptidase A no íleo foram maiores no grupo CBL (P<0,05). A atividade da fosfatase ácida foi observada nas primeiras horas de vida, especialmente no duodeno. Com relação à morfologia do epitélio intestinal e densidade das vilosidades, o fornecimento de colostro bovino liofilizado não determinou alterações. As variações na ultraestrutura dos enterócitos revelaram a presença, no segmento jejuno, de material absorvido e do complexo endocítico apical às 0, 18 e 36 horas. No duodeno, a expressão gênica do IGF-IR ao nascimento foi maior que às 18 e 36 horas nos grupos CC e CBL (P<0,05). Enquanto, a expressão do receptor deste peptídeo bioativo no jejuno do grupo CBL foi maior às 18 e 36 horas em relação a 0 h (P<0,05). Os resultados obtidos do presente trabalho contribuem para que a fonte alternativa de imunoglobulinas, colostro bovino liofilizado, seja utilizada como um substituto para o colostro caprino sem alterações significativas na aquisição de proteção passiva e nas características histofisiológicas do tecido epitelial do intestino delgado de cabritos. / This project was proposed to study the use of lyophilized colostrum as an alternative management for newborn goat kids. Studies related to the acquisition of passive protection and development of the intestinal epithelium, as cytological and histological aspects, cellular activity and expression of the receptor specific for IGF-I (IGF-IR), were performed. At 0, 7 and 14 hours of life, 15 newborn males received 5% of initial weight of lyophilized bovine colostrum (LBC) and 14 goat colostrum (GC), both meals with 55 mg/mL of IgG. Blood samples were collected at 0, 7, 14, 18, 24, 36, 48, 72 and 96 hours of life to quantification of immunoglobulin G (IgG), total protein (TP) and insulin like growth factor type I (IGF-I). Samples of the duodenum, jejunum and ileum were collected at 18, 36 and 96 hours of life for analysis using optical microscopy and scanning and transmission electron microscopy. The activity of extracellular enzymes, quantification of total protein, DNA and RNA and expression of the IGF-IR were also determined. Samples from an additional group (0 h), not suckling, were collected. In LBC group, serum IgG concentrations at 14, 18, 24 and 48 hours were higher than at 0 and 7 hours, while in the GC group, only at 18 hours serum concentration was higher than at 0 and 7 hours (P<0.05). The capacity of IgG absorption decreased between 7 and 14 hours in GC (P<0.05), while in LBC, remained unchanged at these times (P>0.05). The variables TP and IGF-I were not influenced by ingestion of lyophilized bovine colostrum (P>0.05). The indicators of cellular activity, total protein, DNA and RNA concentration, protein/DNA, protein/RNA and RNA/DNA, and the enzyme activity of aminopeptidase N, dipeptidyl peptidase IV, aminopeptidase A, lactase, maltase and sucrase differed in sampling times and in relation to the additional group (P<0.05). Only the rations protein/DNA, protein/RNA in the jejunum and aminopeptidase A activity in the ileum were higher in the LBC group (P<0.05). The acid phosphatase activity was observed in the first hours of life, especially in the duodenum. Regarding the intestinal epithelium morphology and villi density, the supply of lyophilized bovine colostrum did not determine alterations. Changes in enterocytes ultrastructure revealed the presence in the jejunum segment of absorbed material and apical endocytic complex at 0, 18 and 36 hours. In the duodenum, IGF-IR gene expression at birth was higher than at 18 and 36 hours in the GC and LBC (P<0.05). While the expression of this bioactive peptide receptor in the jejunum of the LBC group was higher at 18 and 36 hours relative to 0 h (P<0.05). The results of this work indicate that the alternative source of immunoglobulins, lyophilized bovine colostrum, can be used as a substitute for goat colostrum without significant changes in the acquisition of passive protection and histophysiology characteristics of the small intestine epithelium of goat kids.

<p>More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has&nbsp / been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term&nbsp / delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in&nbsp / Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother&rsquo / s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were&nbsp / collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of&nbsp / periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association&nbsp / between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant&nbsp / associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM&nbsp / against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to&nbsp / PLBW. </p>

<p>More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has&nbsp / been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term&nbsp / delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in&nbsp / Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother&rsquo / s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were&nbsp / collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of&nbsp / periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association&nbsp / between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant&nbsp / associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM&nbsp / against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to&nbsp / PLBW. </p>

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2001. / Härtill 5 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains x, 202 p. : ill. (some col.). Includes abstract. Includes bibliographical references.