Hepatic and intestinal endocrine cells are potentially helpful targets for recombinant insulin expression. As the two cell types exhibit different secretion kinetics,it has been hypothesized that a combination of the two would better approximate insulin secretion kinetics from normal, functioning beta-cells than either cell type alone. This hypothesis was tested using two hepatic cell lines transiently transduced with one of three adenoviruses for insulin expression along with a stably transfected recombinant intestinal L cell line. The insulin secretion kinetics were analyzed for both the hepatic and intestinal cells to determine the potential of combining them to reproduce the insulin secretion kinetics of a normal, functioning beta-cell. It was observed that the two recombinant hepatic cell lines secreted insulin in a more sustained manner exhibiting slower release kinetics. They also exhibited an increase in insulin secretion when stimulated by the cocktail of nutrient secretagogues (glucose and meat hydrolysate) versus stimulating with only glucose. The cells transduced with the adenovirus containing an additional cytomegalovirus (CMV) promoter and green fluorescent protein (GFP) exhibited the highest insulin secretion after stimulation, whereas the cells transduced with an adenovirus encoding for destabilized preproinsulin mRNA exhibited the lowest secretion rates. The recombinant intestinal cell line (GLUTag-INS) secreted insulin with rapid kinetics upon stimulation, apparently due to the presence of secretory granules containing pre-synthesized insulin. The experiments demonstrated that the cells stimulated with medium containing only meat hydrolysate exhibited a significantly higher insulin secretion relative to secretagogue-free controls. The insulin secretion was not further enhanced when meat hydrolysate was combined with glucose.

A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated. In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype. A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4. However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes. In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes. Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins. In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies. In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.

Thesis (Ph.D.). / Written for the Dept. of Mathematics and Statistics. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 134-146). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 209-236). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 219-242). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 133-143). Online version available via ProQuest Digital Dissertations.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-22Bitstream added on 2014-06-13T18:44:27Z : No. of bitstreams: 1 caetano_ag_dr_jabo.pdf: 1343619 bytes, checksum: 387f2f16ea3939c118f65585ece520a6 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O vírus da Bronquite infecciosa (VBI) é um Coronavírus aviário que infecta aves domésticas de corte e postura, ocasionando grandes perdas econômicas na indústria avícola. Dada a natureza altamente contagiosa e aguda da doença, há uma grande necessidade do desenvolvimento de métodos diagnósticos que possam ajudar na detecção e/ou caracterização de estirpes variantes do VBI. Sendo assim, para auxiliar no diagnostico laboratorial da infecção, foi construída uma biblioteca de fragmentos de anticorpos monoclonais pela técnica de Phage-display. Para tanto, após a imunização de galinhas com a estirpe vacinal H120, foi extraído o RNA total do baço das aves imunizadas e amplificadas as cadeias variáveis leve e pesada que foram unidas por linker, originando o fragmento gênico de cadeia única scFv. Após a realização de três ciclos de seleção foram obtidos 400 clones que foram avaliados em ensaios de ELISA e Western blotting para averiguação da especificidade dos mesmos frente às proteínas da estirpe H120. Após realização dos testes foram selecionados dois clones, um que apresentou grande reatividade para com a proteína de nucleocapsídeo (N) (scFv-N) e o outro com reatividade para com a subunidade 1 da glicoproteína de superfície (S) (scFv-S1). O anticorpo scFv-S1 quando utilizado em ensaio de vírus-neutralização em ovos embrionados mostrou titulo significativo de proteção. Já em testes de ELISA utilizando estirpes de referência e isolados brasileiros de campo do VBI, o anticorpo scFv-N foi capaz de detectar todas as estirpes (H120, M41, Arkansas, IBVPR05, IBVPR02, IBVPR01, IBVSC01), enquanto que o scFv-S1 pode discriminar as estirpes pertencentes ao sorotipo Massachusetts (H120, M41 e IBVSC01) das demais estirpes variantes avaliadas. Os fragmentos de anticorpos scFv-N e scFv-S1 também mostraram bons resultados quando utilizados na técnica... / Infectious bronchitis virus (IBV), the coronavirus of the chicken, is one of the main causes of economic loss within the poultry industry, affecting the performance of meattype and egg-laying domestic fowls. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect and/or characterize IBV strains. In order to improve the laboratory diagnosis of IBV infection, phage-displayed recombinant antibody library derived from splenic mRNA of chickens immunized with H120 vaccine strain of infectious bronchitis virus (IBV) was constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction (PCR) of the individual heavy (VH) and light (VL) chain variable gene segments. After three rounds of panning selection, ten scFv phage display antibodies of 400 randomly chosen clones were demonstrated to react with IBV antigens by ELISA. The western blot analysis selected two scFv antibodies reacting strongly with nucleocapsid (N) (scFv-N) protein or subunit 1 of spike glycoprotein (S1) (scFv-S1) of IBV. The anti-S1 scFv antibody showed a significant neutralization titre in embryonating chicken egg test. In ELISA analysis using reference IBV strains and Brazilian field isolates, the anti-N scFv antibody was able to detect all strains (H120, M41, ARKANSAS, IBVPR05, IBVPR02, IBVPR01, IBVSC01), while the anti-S1 could discriminate Massachusetts serotype (H120, M41 and IBVSC01) between variant strains. A scFv-based indirect immunoperoxidase (IP) procedure was also applied to detect infectious bronchitis virus (IBV) antigens in formalin-fixed tracheal tissue sections. Thus, the results showed that scFv-N and scFv-S1 antibodies can be used for the detection and differentiation of IBV strains.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-20Bitstream added on 2014-06-13T18:44:31Z : No. of bitstreams: 1 gibertoni_am_dr_jabo.pdf: 1047291 bytes, checksum: 9be7492afd065b969b043d3a6d16e4be (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foram realizadas a clonagem e expressão do gene da nucleoproteína (N) de uma estirpe vacinal de referência M41 do vírus da bronquite infecciosa (VBI), como proteína recombinante de fusão, contendo uma cauda de poli-histidina na extremidade carboxi-terminal, em 2 sistemas hospedeiros; na levedura metilotrófica, Pichia pastoris e na bactéria Escherichia coli. A proteína N derivada de um isolado variante do VBI de surtos a campo no Brasil, também foi expressa em E. coli. As características bioquímicas e imunoquímicas de tais proteínas recombinantes, foram determinadas, tendo sido evidenciado maior eficiência de produção no sistema hospedeiro constituído por E. coli, comparativamente ao sistema composto por P. pastoris. Uma vez obtidas, caracterizadas e purificadas, através da técnica de cromatografia de afinidade em resina de níquel-sepharose, as preparações de proteína N recombinante expressas em E. coli e derivadas ou da estirpe de referência M41 ou do novo isolado de campo no Brasil, foram utilizadas de forma bem sucedida, como antígenos alvo de ensaios indiretos de ELISA, que foram aplicados na detecção e mensuração de anticorpos dos isótipos IgG e IgM em aves infectadas com estirpes homóloga ou variantes do VBI. Foi, também, investigada a atividade imunogênica da proteína N recombinante em aves, que depois de imunizadas e re-imunizadas com essas proteínas recombinantes, produziram no soro sanguíneo e na secreção lacrimal quantidades elevadas de anticorpos anti-VBI específicos, mas não desenvolveram proteção efetiva contra o desafio com a estirpe homóloga desse vírus. Concluindo, a proteína N recombinante do VBI expressa pela E. coli possui elevada imunogenicidade, no sentido de induzir altos níveis de anticorpos específicos, e reatividade cruzada com proteínas N de outras variantes desse vírus, tendo um grande potencial de ser aplicada em... / Two host systems, represented by Escherichia coli and Pichia pastoris were used for cloning and protein expression of the nucleoprotein (N) gene of M41 strain of infectious bronchitis virus (IBV) as a fusion recombinant protein containing a poli-histidine tag. The N protein from a new variant Brazilian field isolate was also cloned and expressed by E. coli system. The biochemical and immunochemical properties of these recombinant N proteins were determined and higher efficiency on protein production was achieved by using the E. coli expression system. Both recombinant N proteins expressed by E. coli were purified in nickel-sepharose resin and used as antigen in indirect ELISA methods for the detection of IgG and IgM antibodies in birds infected with homologous and variant IBVs. The immunogenicity of N recombinant protein was also evaluated by immunizing and re-immunizing birds and high antibody levels were generated in lachrymal secretion and serum, but no effective protection against challenge with homologous virulent stain of IBV was induced. Concluding, the recombinant N IBV protein expressed by E. coli is highly immunogenic for inducing specific and crossreactive antibodies, and can be applied in the immuno-diagnosis of IB