A invertase comercial (Bioinvert®) foi imobilizada por adsorção em resinas aniônicas do tipo Dowex® [1x8:50-400, 1x4:50-400 e 1x2:100-400, todos copolímeros estireno-divinilbenzênicos, porém de granulometria (50-400 mesh) e quantidades de ligações cruzadas diferentes (2-8%)] em meio aquoso. A melhor percentagem de adsorção da invertase nas resinas foi observada em pH 5,5 a 32°C, tendo o complexo Dowex®1x4-200/lnvertase apresentado índice de adsorção e coeficiente de imobilização iguais a 100%. Os parâmetros cinéticos e termodinâmicos foram determinados para a invertase solúvel e insolúvel de Bioinvert® e também para a invertase purificada (Fluka®). O complexo Dowex®1 x4-200/Bioinvert® apresentou-se estável durante as reações sem desprendimento da enzima do suporte. Os parâmetros termodinâmicos da forma solúvel e insolúvel da Fluka® foram idênticos aos do Bioinvert®, no entanto, após a imobilização apresentou uma redução de 28% na sua atividade. O estudo da atividade transferásica de ambas as formas de Bioinvert® em diferentes concentrações de sacarose foram analisadas através da cromatografia de camada delgada. A estabilidade operacional e de estocagem foi também determinada para o complexo Dowex®1x4-200/Bioinvert®. / The invertase (trademarked as Bioinvert®) solubilized in deionized water was immobilized by adsorption on anion exchange resins, collectively named Dowex®, [1x8:50-400, 1x4:50-400 and 1x2:100-400, styrene-divinylbenzene copolymers, with different granulometry (50-400mesh) and different degrees of cross-linking (2-8%)]. The best percentage of adsorption of invertase on resins was observed in pH 5.5 at 32°C and the complex Dowex®1x4-200/invertase has shown a coupling yield and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrosehydrolysis for both soluble and insoluble enzyme were evaluated to Bioinvert® and purified invertase purchased from Fluka®. The complex Dowex®/Bioinvert® was stable without any desorption of enzyme from the support during the reaction and having the thermodynamic parameters equal to the soluble formo However, the loss of activity for immobilized Fluka® was found to be 28% when compared to the soluble one. The transfructosylating activity of Bioinvert® in both forms in different concentrations of sucrose was investigated through TLC. In regard to insoluble Bioinvert® its storage and operational stability were also determined.

A epidemia causada pelo vírus da imunodeficiência humana (HIV) é a mais importante das ultimas décadas. A despeito dos avanços no conhecimento da patogenia do vírus e da resposta imune à infecção, até o momento não existe uma vacina eficaz contra a aquisição do HIV. Diversas linhas de evidência indicam que anticorpos neutralizantes ou ligadores, linfócitos T CD4+ e T CD8+ desempenham um papel importante na imunidade contra o HIV. Os anticorpos que são capazes de neutralizar o HIV são direcionados principalmente à glicoproteína do envelope do vírus (env), mas os candidatos vacinais baseados na proteína de envelope gp120 monomérica testados até hoje falharam em induzir proteção nos ensaios de eficácia. Avanços no entendimento da estrutura e função da glicoproteína env tem facilitado o desenvolvimento de uma nova geração de imunógenos baseada em trímeros mais estáveis e solúveis da glicoproteína gp140. Em uma formulação vacinal além da escolha do antígeno, os adjuvantes desempenham um papel fundamental. Os adjuvantes são conhecidos por aumentar a imunogenicidade das vacinas, e nos últimos anos vários compostos, incluindo agonistas de receptores do tipo Toll (TLR) e NOD (NLR) têm demonstrado eficácia em ensaios clínicos. Em estudos prévios, nosso grupo demonstrou que a imunização de camundongos com uma vacina de DNA codificando 18 epítopos para linfócitos T CD4+ do HIV-1 (HIVBr18), foi capaz de induzir resposta específica e ampla de linfócitos T CD4+ e T CD8+. Devido ao importante papel do efeito auxiliar de linfócitos T CD4+ na resposta humoral nas imunizações assistidas por diversos adjuvantes, o objetivo central do trabalho foi verificar se a imunização inicial com a vacina de DNA HIVBr18 seria capaz de aumentar a magnitude/qualidade de resposta imune humoral e celular induzida pelo trímero de gp140 na presença de diferentes adjuvantes. Para tal, camundongos BALB/c foram imunizados inicialmente com a vacina HIVBr18 ou com o vetor vazio e posteriormente com a proteína gp140 na presença dos adjuvantes: completo de Freund (ACF), poly IC, CpG ODN 1826, Monofosforil lipídeo A (MPL), Muramildipeptídeo (MDP), Imiquimod (R837), e Resiquimod (R848). Observamos que a imunização inicial com HIVBr18 foi capaz de fornecer um auxílio cognato para a proliferação específica de linfócitos T CD4+ e T CD8+ e também para a produção da citocina IFNy. A análise da xx resposta humoral mostrou que a imunização inicial com a vacina HIVBr18, foi capaz de influenciar a produção das subclasses de imunoglobulinas, independente do adjuvante testado. No presente trabalho, também analisamos a influência dos adjuvantes na imunogenicidade da gp140. Os animais que receberam os adjuvantes MPL, poly IC e CpG ODN 1826 apresentaram títulos de anticorpos estatisticamente superiores quando comparados aos animais que receberam os adjuvantes Alum, MDP, R837 e R848. Observamos que os animais imunizados com a gp140 na presença dos diferentes adjuvantes desenvolveram células B do centro germinativo e células TCD4+ auxiliar foliculares. Estes resultados nos permitem concluir que a imunização inicial com HIVBr18 é capaz de alterar a qualidade da resposta humoral e celular gp140- específica. Assim, essa formulação poderia ser utilizada para auxiliar e/ou direcionar a resposta imune induzida por outros imunógenos como por exemplo o trímero de gp140. Podemos concluir também que diferentes formulações de adjuvantes que se encontram em ensaios clínicos como poly IC, CpG ODN e MPL podem ser capazes de induzir um resposta imune humoral e celular tão ou mais potente que aquela induzida pelo ACF / The epidemic caused by the human immunodeficiency virus (HIV) is the most important in the last decades. Despite advances in the knowledge about virus pathogenesis and immune response to infection, until now there is not an effective vaccine against HIV acquisition. Several evidences indicate that neutralizing or binding antibodies, CD4+ and CD8+ T lymphocytes play an important role in immunity against HIV. The antibodies that are able to neutralize HIV are primarily directed against the virus envelope glycoprotein (env), but the vaccine candidates based on monomeric gp120 envelope protein tested so far failed to induce protection in efficacy trials. Advances in understanding the structure and function of the env glycoprotein have facilitated the development of a new generation of immunogens based on trimers, a more stable and soluble form of gp140 glycoprotein. In a vaccine formulation, in addition to the antigen, adjuvants play a pivotal role. Adjuvants are known to increase the immunogenicity of vaccines and, in the last years, several compounds, including agonists of Toll-like receptors (TLR) and NOD (NLR), have presented efficacy in clinical trials. In previous work, our group demonstrated that immunization of mice with a DNA vaccine (HIVBr18) encoding 18 CD4+ T cells epitopes from HIV-1 was able to induce a broad CD4+ T and CD8+ T cells specific response.. Given the important role of CD4+ T cells in the humoral response after adjuvant-assisted immunization, the aim of the study was to verify whether an initial immunization with the DNA vaccine HIVBr18 could increase the magnitude/quality of humoral and cellular immune response induced by gp140 trimer in the presence of different adjuvants. Therefore, BALB/c mice were initially immunized with the vaccine HIVBr18 or empty vector and then with gp140 in the presence of the following adjuvants: Freund\'s complete (CFA), poly IC, CpG ODN 1826, monophosphoryl lipid A (MPL), Muramyl dipeptide (MDP), Imiquimod (R837), and Resiquimod (R848). We observed that initial immunization with HIVBr18 was able to provide cognate help for specific CD4+ and CD8+ T cells proliferation and also for IFN-y production. Analysis of humoral response showed that initial immunization with the HIVBr18 vaccine was able to alter the production of immunoglobulin subclasses independent of the adjuvant tested. This work also analyzed the influence of adjuvants on the immunogenicity of gp140. Mice that received the adjuvant MPL, poly IC and CpG ODN 1826 presented higher antibody titers when compared to animals that received Alum, MDP, R837 and R848. We observed that mice immunized with gp140 in the presence of all adjuvants tested developed germinal center B cells and follicular helper T cells (TFH). We conclude that initial immunization with HIVBr18 is able to alter the quality of specific humoral and cellular immune responses.. Therefore, this formulation could be used in combination with other immunogens, such as gp140, to help/redirect the immune response. We also conclude that the adjuvants that are in clinical trials such as poly IC, MPL and CpG ODN 1826 may be able to induce stronger humoral and cellular response than CFA

A doença pulmonar obstrutiva crônica (DPOC) é caracterizada por inflamação crônica e alterações estruturais que levam a obstrução das pequenas vias aéreas e destruição do parênquima alveolar. A composição da matriz extracelular (MEC) nos pulmões tem um importante papel em prover e sustentar a arquitetura pulmonar. No entanto, não há uma descrição abrangente da composição da matriz extracelular no trato respiratório de indivíduos portadores de DPOC. No presente estudo investigou-se a composição da MEC das vias aéreas grandes (VAG), pequenas (VAP) e do parênquima pulmonar de pacientes com DPOC. Utilizando imunohistoquímica e análise de imagem analisou-se a área fracionada de fibras elásticas, colágenos I, III e IV, versicam, decorina, biglicano, lumicam, fibronectina e tenascina nas VAG, VAP e no parênquima peribrônquico e distal de 26 indivíduos com DPOC e comparou-se à área fracionada nos pulmões de 26 fumantes sem DPOC e 16 indivíduos não fumantes. A área fracionada de fibras elásticas foi significante maior no grupo de fumantes não obstruídos em comparação com os demais grupos, em todos os compartimentos analisados. Houve menor expressão de colágeno I na camada interna das VAG e nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC e na camada externa das VAP dos fumantes não obstruídos quando comparados ao grupo controle. A área fracionada de versicam mostrou-se menor apenas no parênquima distal do grupo DPOC comparado ao grupo controle. O estudo da matriz de glicoproteínas mostrou maior área fracionada de fibronectina nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC comparados aos demais grupos, assim como maior área fracionada de tenascina foi observado na membrana basal das VAG e na camada interna das VAP do grupo DPOC comparados aos controles. Além disso, a composição da MEC correlacionou-se com valores funcionais, como o VEF1 (% predito). A partir desses resultados, concluímos que a DPOC é caracterizada por complexas alterações nas principais proteínas estruturais nas pequenas e grandes vias aéreas. Tais alterações podem contribuir para a lesão tecidual persistente e com a obstrução ao fluxo aéreo observado na DPOC / COPD is characterized by chronic inflammation and structural alterations leading to small airway obstruction and to destruction of the lung parenchyma. The extracellular matrix (ECM) composition of the lungs has an important role in determining airway structure. However, there are no comprehensive descriptions of the ECM composition along the respiratory tract in COPD patients. We postulated that the ECM composition in large and small airways and in lung parenchyma of COPD patients differs from that observed in smoking and non-smoking controls. Using immunohistochemistry and image analysis, fractional areas of elastic fibers, type-I, -III and IV collagen, the proteoglycans versican, decorin, biglycan and lumican; fibronectin and tenascin were quantified in the large (LA) and small airways (SA), in peribronchiolar (PP) and distal parenchyma (DP) of 26 COPD patients and compared to 26 smokers without COPD and 16 non-smoking controls. The fractional area of elastic fibers was higher in non-obstructed smokers than in COPD and non-smoking controls subjects, in all lung compartments. Type-I collagen fractional area was lower in the inner layer of LA and in the inner, muscle and outer layer (OL) of SA of COPD patients and in the OL of SA of non-obstructed smokers when compared to non-smoking controls. The versican fractional area was lower in DP of COPD patients than non-smokers. Fibronectin fractional área was higher in the inner, muscle and outer layer of SA of COPD patients compared to non-smokers. Tenascin fractional area was higher in the subepithelial area of LA and inner layer of SA of COPD when compared to non-smoking controls. Furthermore, ECM composition correlated with FEV1% predicted. Architectural alterations due to an altered ECM composition in COPD are likely to contribute to the persistent tissue injury and to the airflow obstruction characteristic of this disease

A invertase comercial (Bioinvert®) foi imobilizada por adsorção em resinas aniônicas do tipo Dowex® [1x8:50-400, 1x4:50-400 e 1x2:100-400, todos copolímeros estireno-divinilbenzênicos, porém de granulometria (50-400 mesh) e quantidades de ligações cruzadas diferentes (2-8%)] em meio aquoso. A melhor percentagem de adsorção da invertase nas resinas foi observada em pH 5,5 a 32°C, tendo o complexo Dowex®1x4-200/lnvertase apresentado índice de adsorção e coeficiente de imobilização iguais a 100%. Os parâmetros cinéticos e termodinâmicos foram determinados para a invertase solúvel e insolúvel de Bioinvert® e também para a invertase purificada (Fluka®). O complexo Dowex®1 x4-200/Bioinvert® apresentou-se estável durante as reações sem desprendimento da enzima do suporte. Os parâmetros termodinâmicos da forma solúvel e insolúvel da Fluka® foram idênticos aos do Bioinvert®, no entanto, após a imobilização apresentou uma redução de 28% na sua atividade. O estudo da atividade transferásica de ambas as formas de Bioinvert® em diferentes concentrações de sacarose foram analisadas através da cromatografia de camada delgada. A estabilidade operacional e de estocagem foi também determinada para o complexo Dowex®1x4-200/Bioinvert®. / The invertase (trademarked as Bioinvert®) solubilized in deionized water was immobilized by adsorption on anion exchange resins, collectively named Dowex®, [1x8:50-400, 1x4:50-400 and 1x2:100-400, styrene-divinylbenzene copolymers, with different granulometry (50-400mesh) and different degrees of cross-linking (2-8%)]. The best percentage of adsorption of invertase on resins was observed in pH 5.5 at 32°C and the complex Dowex®1x4-200/invertase has shown a coupling yield and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrosehydrolysis for both soluble and insoluble enzyme were evaluated to Bioinvert® and purified invertase purchased from Fluka®. The complex Dowex®/Bioinvert® was stable without any desorption of enzyme from the support during the reaction and having the thermodynamic parameters equal to the soluble formo However, the loss of activity for immobilized Fluka® was found to be 28% when compared to the soluble one. The transfructosylating activity of Bioinvert® in both forms in different concentrations of sucrose was investigated through TLC. In regard to insoluble Bioinvert® its storage and operational stability were also determined.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2018-04-11T13:17:41Z No. of bitstreams: 1 2010 - Bruno Leite dos Anjos.pdf: 12893665 bytes, checksum: 99b4dd0f8ecdebb1f227aa0522ac061f (MD5) / Made available in DSpace on 2018-04-11T13:17:42Z (GMT). No. of bitstreams: 1 2010 - Bruno Leite dos Anjos.pdf: 12893665 bytes, checksum: 99b4dd0f8ecdebb1f227aa0522ac061f (MD5) Previous issue date: 2010-08-17 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq, Brasil. / Cases of diseases induced by toxic plants in domestic herbivores are well reported throughout the world and have been studied also in Brazil. However, not much is known about the epidemiological and pathological aspectos of these conditions in free-living wildlife or bred in captivity. The risk for developing the toxicoses in captivity has been increasing, since natural habitats are destroyed by human action, and more centers of wildlife conservation and zoological comes are created. This study describes the epidemiological, biological and clinicopathological, lectin-histochemical and ultrastructural aspects of an outbreak of lysosomal storage disease of oligosaccharides induced by ingestion of Sida carpinifolia in young Sambar deer (Cervus unicolor) in the Rio-Zoo Foundation in the State of Rio de Janeiro, Brazil. Nine deer showed neurological signs characterized by motor and proprioceptive deficits. Then neurological signs were mainly depression, incoordination, dysmetria, ataxia, broad-based members, muscle tremors, loss of tongue tone, frequent falls and death. Grossly hematomas were observed secondary to trauma caused by dominant males of the flock, and whitish striations, especially in the renal cortex. Histologic changes included marked swelling/cytoplasmic vacuolization especially in neurons, progressing to neuronal lysis and axonal spheroids, in exocrine pancreas, thyroid follicular cells and renal tubular epithelial cells. In the lectin-histochemical examination the vacuoles were formed by the accumulation of oligosaccharides specially marked by the lectins WGA, WGA and Con-A. Ultrastructurally, the swelling/vacuolation corresponded to intense cytoplasmic distention of lysosomes, formation of residual bodies or dense granular fragments of membranes and mielinoides bodies. The study has shown the susceptibility of Cervus unicolor to swainsonine by ingestion of S. carpinifolia. Possibly poisoning the animals in this study was conducted by food restriction by the hierarchy among males in the group. It might also determine the marked similarity between clinical and pathological aspects in Sambar deer with the one presented by other herbivores. / Casos de doen?as induzidas por plantas t?xicas em herb?voros dom?sticos s?o bastante relatados por todo o mundo e v?m sendo estudados tamb?m no Brasil. Pouco se sabe, contudo, sobre os aspectos epidemiol?gicos e patol?gicos dessas condi??es em animais selvagens de vida livre ou criados em cativeiro. Os riscos de desenvolvimento dessas toxicoses em cativeiros v?m aumentando, conforme os habitats naturais s?o destru?dos pela a??o humana, e mais centros de conserva??o de vida silvestre e zool?gicos s?o criados. Nesse estudo s?o descritos os aspectos epidemiol?gicos, biol?gicos e clinicopatol?gicos, lectinohistoqu?micos e ultraestruturais de um surto de doen?a do armazenamento lisossomal de oligossacar?deos induzido pela ingest?o de Sida carpinifolia em cervos Sambar jovens (Cervus unicolor) no zool?gico da Funda??o Rio-Zoo no Estado do Rio de Janeiro. Nove cervos apresentaram sinais cl?nicos neurol?gicos caracterizados por d?ficits proprioceptivo e motor. Os sinais neurol?gicos inclu?ram principalmente depress?o, incoordena??o, dismetria, ataxia, membros em base ampla, tremores musculares, perda do t?nus lingual, quedas frequentes e morte. Macroscopicamente foram observados hematomas, secund?rios a traumas provocados por machos dominantes do rebanho, e estria??es esbranqui?adas, principalmente no c?rtex renal. As les?es histol?gicas inclu?am acentuada tumefa??o/vacuoliza??o citoplasm?tica especialmente em neur?nios, p?ncreas ex?crino, c?lulas foliculares da tireoides e do epit?lio renal, necrose neuronal com evolu??o para lise e esferoides axonais. Pelo exame lectino-histoqu?mico os vac?olos eram formados por ac?mulo de oligossacar?deos marcado especialmente pelas lectinas S-WGA, WGA e Con-A. Ultraestruturalmente, a tumefa??o/vacuoliza??o citoplasm?tica correspondeu ? intensa distens?o de lisossomos, forma??o de corpos residuais densos ou granulares, fragmentos de membranas e corpos mielinoides. O estudo demonstrou a suscetibilidade de Cervus unicolor ? swainsonina contida na S. carpinifolia. Possivelmente, a intoxica??o nos animais deste estudo ocorreu pela restri??o alimentar sofrida pelos animais mais jovens, decorrente da hierarquia entre machos no grupo. P?de-se determinar ainda a marcada similaridade do quadro cl?nico e patol?gico entre os cervos descritos nesse trabalho e outros herb?voros dom?sticos.

A doença pulmonar obstrutiva crônica (DPOC) é caracterizada por inflamação crônica e alterações estruturais que levam a obstrução das pequenas vias aéreas e destruição do parênquima alveolar. A composição da matriz extracelular (MEC) nos pulmões tem um importante papel em prover e sustentar a arquitetura pulmonar. No entanto, não há uma descrição abrangente da composição da matriz extracelular no trato respiratório de indivíduos portadores de DPOC. No presente estudo investigou-se a composição da MEC das vias aéreas grandes (VAG), pequenas (VAP) e do parênquima pulmonar de pacientes com DPOC. Utilizando imunohistoquímica e análise de imagem analisou-se a área fracionada de fibras elásticas, colágenos I, III e IV, versicam, decorina, biglicano, lumicam, fibronectina e tenascina nas VAG, VAP e no parênquima peribrônquico e distal de 26 indivíduos com DPOC e comparou-se à área fracionada nos pulmões de 26 fumantes sem DPOC e 16 indivíduos não fumantes. A área fracionada de fibras elásticas foi significante maior no grupo de fumantes não obstruídos em comparação com os demais grupos, em todos os compartimentos analisados. Houve menor expressão de colágeno I na camada interna das VAG e nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC e na camada externa das VAP dos fumantes não obstruídos quando comparados ao grupo controle. A área fracionada de versicam mostrou-se menor apenas no parênquima distal do grupo DPOC comparado ao grupo controle. O estudo da matriz de glicoproteínas mostrou maior área fracionada de fibronectina nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC comparados aos demais grupos, assim como maior área fracionada de tenascina foi observado na membrana basal das VAG e na camada interna das VAP do grupo DPOC comparados aos controles. Além disso, a composição da MEC correlacionou-se com valores funcionais, como o VEF1 (% predito). A partir desses resultados, concluímos que a DPOC é caracterizada por complexas alterações nas principais proteínas estruturais nas pequenas e grandes vias aéreas. Tais alterações podem contribuir para a lesão tecidual persistente e com a obstrução ao fluxo aéreo observado na DPOC / COPD is characterized by chronic inflammation and structural alterations leading to small airway obstruction and to destruction of the lung parenchyma. The extracellular matrix (ECM) composition of the lungs has an important role in determining airway structure. However, there are no comprehensive descriptions of the ECM composition along the respiratory tract in COPD patients. We postulated that the ECM composition in large and small airways and in lung parenchyma of COPD patients differs from that observed in smoking and non-smoking controls. Using immunohistochemistry and image analysis, fractional areas of elastic fibers, type-I, -III and IV collagen, the proteoglycans versican, decorin, biglycan and lumican; fibronectin and tenascin were quantified in the large (LA) and small airways (SA), in peribronchiolar (PP) and distal parenchyma (DP) of 26 COPD patients and compared to 26 smokers without COPD and 16 non-smoking controls. The fractional area of elastic fibers was higher in non-obstructed smokers than in COPD and non-smoking controls subjects, in all lung compartments. Type-I collagen fractional area was lower in the inner layer of LA and in the inner, muscle and outer layer (OL) of SA of COPD patients and in the OL of SA of non-obstructed smokers when compared to non-smoking controls. The versican fractional area was lower in DP of COPD patients than non-smokers. Fibronectin fractional área was higher in the inner, muscle and outer layer of SA of COPD patients compared to non-smokers. Tenascin fractional area was higher in the subepithelial area of LA and inner layer of SA of COPD when compared to non-smoking controls. Furthermore, ECM composition correlated with FEV1% predicted. Architectural alterations due to an altered ECM composition in COPD are likely to contribute to the persistent tissue injury and to the airflow obstruction characteristic of this disease

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Protein folding in the endoplasmic reticulum (ER) is essential for proper cellular function. However, the sensitive environment in the ER can be perturbed by both pathological processes as well as by physiological processes such as a large biosynthetic load placed on the ER. ER stress is a specific type of intracellular stress caused by the accumulation of immature or abnormal misfolded or unfolded proteins in the ER. Simply defined, ER stress is a disequilibrium between ER load and folding capacity. Cells have an adaptive response that counteracts ER stress called the "Unfolded Protein Response” (UPR). The ability to adapt to physiological levels of ER stress is especially important for maintaining ER homeostasis in secretory cells. This also holds true for pancreatic β-cells, which must fold and process large amounts of the hormone insulin. Pancreatic β-cells minimize abnormal levels of glycemia through adaptive changes in the production and regulated secretion of insulin. This process is highly sensitive, so that small degrees of hypo- or hyperglycemia result in altered insulin release. The frequent fluctuation of blood glucose levels in humans requires that β-cells control proinsulin folding in the ER with exquisite sensitivity. Any imbalance between the load of insulin translation into the ER and the actual capacity of the ER to properly fold and process the insulin negatively affects the homeostasis of β-cells and causes ER stress. In this dissertation, we show that Inositol Requiring 1 (IRE1), an ER-resident kinase/endoribonuclease and a central regulator of ER stress signaling, is essential for maintaining ER homeostasis in pancreatic β-cells. Importantly, IRE1 has a crucial function in the body’s normal production of insulin in response to high glucose. Phosphorylation and subsequent activation of IRE1 by transient exposure to high glucose is coupled to insulin biosynthesis, while inactivation of IRE1 by siRNA or inhibition of IRE1 phosphorylation abolishes insulin biosynthesis. IRE1 signaling under these physiological ER stress conditions utilizes a unique subset of downstream components of IRE1 and has a beneficial effect on pancreatic β-cell homeostasis. In contrast, we show that chronic exposure of β-cells to high glucose causes pathological levels of ER stress and hyperactivation of IRE1, leading to the degradation of insulin mRNA. The term “glucose toxicity” refers to impaired insulin secretion by β-cells in response to chronic stimulation by glucose and is characterized by a sharp decline in insulin gene expression. However, the molecular mechanisms of glucose toxicity are not well understood. We show that hyperactivation of IRE1 caused by chronic high glucose treatment or IRE1 overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1 signaling using a dominant negative form of the protein prevents insulin mRNA degradation in β-cells. Additionally, islets from mice heterozygous for IRE1 retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates. This work suggests that the rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism may represent an essential element in the adaptation of β-cells to chronic hyperglycemia. This adaptation is crucial for the maintenance of β-cell homeostasis and may explain in part why the β-cells of diabetic patients with chronic hyperglycemia stop producing insulin without simply undergoing apoptosis. This work implies that prolonged activation of IRE1 signaling is involved in the molecular mechanisms underlying glucose toxicity. This work therefore reveals two distinct activities elicited by IRE1 in pancreatic β-cells. IRE1 signaling activated by transient exposure to high glucose enhances proinsulin biosynthesis, while chronic exposure of β-cells to high glucose causes hyperactivation of IRE1, leading to the degradation of insulin mRNA. Physiological IRE1 activation by transient high glucose levels in pancreatic β cells has a beneficial effect on insulin biosynthesis. However, pathological IRE1 activation by chronic high glucose or experimental drugs negatively affects insulin gene expression. In the future, a system to induce a physiological level of IRE1 activation, and/or reduce the pathological level of IRE1 activation could be used to enhance insulin biosynthesis and secretion in people with diabetes, and may lead to the development of new and more effective clinical approaches to the treatment of this disorder.

Profound lymphopenia has been observed during many acute viral infections, and our laboratory has previously documented a type 1 IFN-dependent loss of most memory (CD44hi) and some naïve (CD44lo) CD8 T cells immediately preceding the development of the antiviral T cell response at days 2-4 following lymphocytic choriomeningitis virus (LCMV) infection. In this thesis, I will examine additional mechanisms involved in the early attrition of CD8 T cells and evaluate whether antigen-specific and non-specific CD8 T cells are equally susceptible. Lastly, I will examine whether the early attrition of CD8 T cells contributes to the generation of an effective immune response. Poly(I:C), a potent inducer of type 1 IFN, was previously shown to cause the attrition and apoptosis of CD8α+CD44hi cells in normal mice, but not in type 1 IFN receptor–deficient mice (IFN1-R KO). I questioned whether additional molecule(s) might contribute to the type 1 IFN-induced apoptosis of CD8α+CD44hi cells. I used a PCR array to determine the expression of 84 apoptosis-related genes at 6 hours post-poly(I:C) treatment, relative to an untreated control. There was an 11-fold increase in CD40 RNA expression in CD8α+CD44hi cells isolated from poly(I:C)-treated mice. CD40 protein expression was also increased on CD8α+CD44hi cells, peaking between 9 and 12 hours following poly(I:C) treatment, before declining thereafter. This increase in CD40 protein expression directly correlated with an increase in Annexin V reactivity, an indicator of early apoptosis. Nevertheless, CD40 was not required for the loss of CD8α+CD44hi cells, as both wildtype and CD40-deficient mice were equally susceptible to the poly(I:C)-induced attrition. Upon further characterization, I found this population of CD40+CD8α+CD44hi cells to be CD11c+B220-Thy1.2- MHCIIhi, which is consistent with a “lymphoid” CD8α+ DC phenotype. Kinetic analysis revealed a type 1 IFN-dependent increase in this CD8α+ DC population at 12 hours post-poly(I:C) treatment. This increase was only observed in the spleen, as no increase in percentage was observed in the peritoneal cavity (PEC), lungs, inguinal lymph nodes (iLN), or peripheral blood. Collectively, these results suggest that the type 1 IFN-dependent increase in splenic CD8α+DCs accounts for the observed increase in Annexin V reactive cells following poly(I:C) treatment. These findings required a re-evaluation of the type 1 IFN-induced attrition of CD8+CD44hi T cells with an anti-CD8β antibody, which is a more exclusive marker for T cells than the anti-CD8α antibody. Kinetic analysis revealed a significant decrease in splenic CD8β+CD44hi T cells at 12 hours post-poly(I:C) treatment. This reduction in splenic CD8β+CD44hi T cells was not due to trafficking to other organs, as the PECs, lungs, iLN, lungs, and peripheral blood all exhibited significant, although varying, decreases in the percentage of CD8β+CD44hi T cells at 12 hour following poly(I:C) treatment. These data support the notion that the type 1 IFN-induced attrition of CD8β+CD44hiT cells was a “global” phenomenon and could not be completely due to migration out of the spleen. The attrition of CD8β+CD44hi T cells was also dependent upon type 1 IFN at 3 days post-LCMV infection, as there was no significant reduction of this population in IFN1-R KO mice. The loss of wildtype CD8β+CD44hi T cells correlated with an increased activation of caspases 3 and 8, which are enzymes that play essential roles in apoptosis and inflammation. A significant loss of CD4+CD44hi T cells, which also correlated with an increased activation of caspases 3 and 8, was observed at 3 days post-LCMV infection. Collectively, these results suggest that attrition of both CD4+CD44hi and CD8β+CD44hiT cell populations is type 1 IFN-dependent and associated with the activation of caspases following LCMV infection. At 3 days post-LCMV infection, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had a higher frequency of cells with fragmented DNA, a hallmark characteristic of the late stages of apoptosis, as revealed by terminal transferase dUTP nick end labeling (TUNEL), relative to uninfected controls. This suggests that the loss of both populations was due to apoptosis. Therefore, I questioned whether the LCMV-induced apoptosis of both CD4+CD44hi and CD8β+CD44hi T cell populations occurred through a mitochondrial-induced pathway involving the pro-apoptotic molecule Bim. The attrition of both CD4+CD44hi and CD8β+CD44hi T cells was significantly higher in wildtype mice compared to Bim KO mice at 3 days post-LCMV infection. Moreover, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had higher frequency of TUNEL+ cells, relative to Bim KO populations. These results suggest that the apoptosis of CD8β+CD44hi and CD4+CD44hiT cells, following LCMV infection, might occur through a mitochondrial-induced pathway involving Bim. Studies have shown “lymphoid” CD8α+ DCs to be involved in the phagocytosis of apoptotic lymphocytes. Therefore, I evaluated whether host CD8α+ DCs are capable of phagocytosing apoptotic lymphocytes by adoptively transferring CFSE-labeled wildtype donor splenocytes (Ly5.1) into congenic wildtype hosts (Ly5.2), followed by inoculation with poly(I:C). There was an increased frequency of donor cells (Ly5.1, CFSE+) within the host CD8α+CD11c+ gate at 9 and 12 hours post-poly(I:C) treatment. The results suggest that type 1 IFN-activated CD8α+DCs might aid in the rapid clearance of apoptotic cells during the type 1 IFN-induced attrition associated with viral infections. I next questioned whether TCR engagement by antigen would render CD8 T cells resistant to attrition. I tested whether a high concentration of antigen (GP33 peptide) would protect LCMV-specific naïve TCR transgenic P14 cells specific for the GP33 epitope of LCMV and GP33-specific LCMV-immune cells from depletion. Both naïve P14 and memory GP33-specific donor CD8 T cells decreased substantially 16 hours after inoculation poly(I:C), regardless of whether a high concentration of GP33 peptide was administered to host mice beforehand. The increased activation status of naïve antigen-specific cells via peptide inoculation did not confer resistance to type 1 IFN-induced depletion. Donor naïve P14 and LCMV-specific memory cells were also depleted from day 2 LCMV-infected (Clone 13) hosts by 16 hours post-transfer. These results indicate that antigen engagement does not protect CD8 T cells from the type 1 IFN-induced attrition associated with viral infections. Computer models indicated that early depletion of memory T cells may allow for the generation for a more diverse T cell response to infection by reducing the immunodomination caused by cross-reactive T cells. To test this in a biological system, I questioned whether the reduced apoptosis of the crossreactive memory CD8 population (NP205), in aged LCMV-immune mice (18-22 months), following heterologous virus challenge (PV), would allow it to dominate the immune response. At day 8 post-PV infection, the cross-reactive memory CD8 T cell response (NP205) was more immunodominating in aged LCMV-immune mice relative to younger LCMV-immune mice. This was indicated by the increased ratio of the cross-reactive NP205 response to the newly arising noncross-reactive, PV-specific NP38 response in older LCMV-mice relative to younger LCMV immune-mice, at day 8 post-PV infection. These data suggest that the early attrition of T cells allows for the generation of a more diverse T cell response to infection by reducing the immunodomination caused by crossreactive T cells. Collectively, these findings offer further insight into the early attrition of T cells associated with viral infections.

I characterized several potential glycoproteins in salivary gland extracts from unfed and partially fed females of ticks Ixodes ricinus using enzyme deglycosylation and lectin labeling. Affinity-based (chromatografic) analysis was applied for isolations of glycoproteins with specificity for GNA (mannose), HPA (N-acetylgalactosamine) and MAA II (sialic acid) lectins. GNA specific 120 kDa glycoprotein was isolated from partially fed females and is modified with N-linked glycans containing {$\alpha$}1,3-mannose. Mass spectrometry analyses confirmed the presence carboxypeptidase M in elution fraction gain with GNA affinity chromatography. GNA specific proteins were purified from unfed female salivary gland extracts. MS analyses identified them as proteins similar to arylsulfatase B and cytoskeletal Sojo protein. Proteins (85 and 56 kDa) isolated with HPA affinity chromatography were characterized as Trappin 12, which is a host protein. MAA II lectin was used for labelling and isolation of 100 kDa protein. N-terminal sequence of the MAA II specific protein predicted similarity with a host protein, Siglec 1. Fucose in salivary gland extract was detected with the labelling of AAA, AAL, UEA I and LTL lectins. Results showed that salivary gland extracts contain {$\alpha$}1,2-; {$\alpha$}1,3- and {$\alpha$}1,6- N-linked fucose and O-linked fucose probably as well. GNA specific proteins were detected in partially fed salivary glands acini type II and III using electron transmission microscopy. Fucose was detected on gut and salivary gland structures using fucose-specific lectin AAL.