The creation of stable mammalian cell lines for biopharmaceutical production often require several months, and is unfavourable for the rapid production of multiple drug candidates for screening in the early stages of development. Biopharmaceutical production by transient transfection provides a possible alternative of quickly producing these early stage drug candidates. The Epi-CHO transient expression system, which consists of a Chinese hamster ovary (CHO) cell line (CHO-T) expressing the murine polyomavirus Large T-Antigen (LT), emonstrated enhanced transient recombinant protein production. The aim of this study was to prolong transient recombinant protein prod.Jction of the Epi-CHO expression system by creating a CHO cell line expressing both LT and EBNA1 (ECHO-T). The pEBNA1-LT expression vector encoding LT and EBNA1 was constructed and transfected into CHO-K1. A total of 20 clones were isolated from the antibioticresistant pool and screened for the expression of functional LT and EBNA1. PCR analysis showed 16 of the 20 clones was positive for EBNA1 and LT DNA. Of the 16 clones, six were positive for EBNA1 and LT expression by RT-PCR. Detection of LT and EBNA1 by immunofluorescence showed positive staining for the P7-G3 clone. Western blotting suggested the P7-G3 clone was: positive for EBNA1, and clones P3-C7 and P7-E2 were positive for LT. A plasmid replication assay confirmed the expression of functional LT in all six clones. Plasmid maintenance assay confirmed clone P7-G3 as the ECHO-T clones to express functional EBNA1. The P7-G3 clone demonstrated prolonged and sustained transient recombinant protein expression when compared to CHO-T. The P7-G3 clone achieved sustained transient protein expression for 32 days in the absence of selection, the longest currently reported for CHO cells.

Thesis (Ph.D.)--Tufts University, 2004. / Adviser: Alice H. Lichtenstein. Submitted to the School of Nutrition Science and Policy. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph. D.)--University of Hong Kong, 1992.

Thesis (M. Phil.)--University of Hong Kong, 1993.

Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-03-14T18:58:18Z No. of bitstreams: 1 dissertação Ananda Isis Lima de Marinho.pdf: 1076899 bytes, checksum: 21e99adf683c19eda3cf6952ccc97d03 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-04-24T13:08:50Z (GMT) No. of bitstreams: 1 dissertação Ananda Isis Lima de Marinho.pdf: 1076899 bytes, checksum: 21e99adf683c19eda3cf6952ccc97d03 (MD5) / Made available in DSpace on 2017-04-24T13:08:50Z (GMT). No. of bitstreams: 1 dissertação Ananda Isis Lima de Marinho.pdf: 1076899 bytes, checksum: 21e99adf683c19eda3cf6952ccc97d03 (MD5) / CNPq / Nas infecções parasitárias alguns fatores estão associados com a susceptibilidade do hospedeiro, dentre eles o sexo, a idade, condição genética, fatores ambientais e o estado imunológico. Diferenças entre os sexos são observadas em diversas infecções. Acredita- se que isto ocorra devido a ações de hormônios sexuais, podendo afetar os diferentes tipos de resposta imune. A Leishmaniose é um grave problema de saúde pública apresentando aumento da prevalência e afetando grande parte da população mundial. No Brasil é endêmica e o Nordeste é a região mais afetada. A forma visceral é a mais grave podendo levar o indivíduo a óbito se não tratada. Objetivamos determinar se existe diferença entre sexos no modelo experimental de LV. Hamsters (Mesocricetus auratus) machos e fêmeas foram infectados com L. infantum/chagasi através da injeção intradérmica e comparados com animais controles, após 5 meses de infecção. Após eutanásia sangue, baço e fígado foram coletados para as análises de carga parasitária, histológicas e de parâmetros bioquímicos. As fêmeas infectadas apresentaram um maior tamanho de baço que os machos infectados. Porém, foram os machos que apresentaram maior carga parasitária. As alterações bioquímicas foram maiores nas fêmeas, podendo estar relacionadas às alterações histológicas observadas no fígado das mesmas. As fêmeas infectadas apresentaram concentrações séricas inferiores de estradiol quando comparadas com fêmeas não infectadas. Essa redução não foi detectada nos machos. Nossos resultados indicam que existem diferenças entre sexos na LV experimental. A determinação de diferenças entre os sexos nas respostas às infecções é de fundamental importância na escolha da melhor abordagem terapêutica para o tratamento das enfermidades, incluindo as leishmanioses. / In parasitic infections some factors are associated with the susceptibility of the host, including sex, age, genetic condition, environmental factors and immune status. Gender differences are observed in various infections. It is believed it occurs due to sex hormones, which may affect different types of immune responses. Leishmaniasis is a serious public health problem presenting increased prevalence and affecting much of the world population. In Brazil it is endemic and the Northeast is the most affected region. The visceral form is the most severe and may cause death if left untreated. We aimed to determine whether there is gender difference in the experimental model of LV. Hamsters (Mesocricetus auratus) males and females were infected with L. infantum / chagasi by intradermal injection and compared with control animals after 5 months of infection. After euthanasia, blood, spleen and liver were collected for parasite load, histological and biochemical parameters analysis. Infected females showed an increase in the size of spleen compared to infected males. However, males had higher parasite burden. Biochemical changes were higher in females, which may be related to the histological changes observed in their liver. Infected females had lower serum concentrations of estradiol compared to uninfected females. This reduction was not detected in males. Our results indicate that there are gender differences in experimental LV. The determination of sex differences in responses to infection is very important in choosing the best therapeutic approach for the treatment of diseases, including leishmaniasis.

I. A phosphoglycerate kinase (PGK) deficient variant cell line, R1.1.7, derived from the Chinese hamster ovary cell line, CHO-K1, is used as a recipient in the development of a DNA-mediated transfection system designed for the study of in vivo expression of exogenous PGK gene sequences. II. Several PGK DNA sequences are detected in the Chinese hamster genome by hybridisation of genomic DNA digests with a human PGK cDNA probe. A number of these sequences are shown to be X-linked. Four of the PGK sequences observed in the blot hybridisations are isolated from a CHO-K1 DNA genomic library and analysed by probing with different regions of the PGK cDNA, heteroduplex mapping and DNA sequencing. One of these sequences is a 572 bp exon from the functional X-linked PGK gene (PGK-1) which is expressed in all somatic cells. The three remaining PGK sequences are intronless pseudogenes which were apparently derived independently, from an ancestral Chinese hamster PGK gene, within the last 50 million years. They exhibit features typical of 'processed' pseudogenes which are generated via an mRNA intermediate and integrated into breaks in the chromosomal DNA.

Orientador: Miguel Arcanjo Areas / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T01:48:46Z (GMT). No. of bitstreams: 1 Pinto_WagnerdeJesus_D.pdf: 9148387 bytes, checksum: 345d71d8c53d2ef44fc1086f9218b432 (MD5) Previous issue date: 2004 / Resumo: Estudos têm demonstrado que as fibras alimentares desempenham papéis protetores contra o impacto danoso da hiperlipidemia. A mistura polpa de laranja/goma-guar (110:40) foi utilizada como fonte de fibra alimentar e caracterizada físico-quimicamente apresentando a seguinte composição: fibra total, 76,12%; fibra alimentar solúvel, 43,54; fibra alimentar insolúvel, 32,58. Os efeitos hipolipidêmicos da mistura polpa de laranja/goma-guar (PLG) foram verificados em hamsters jovens adultos machos (3 meses) submetidos a dietas hipercolesterolêmicas sendo avaliados parâmetros bioquímicos, cardiovasculares, morfométricos, e ultraestruturais. A mistura PLG adicionada a dietas hipercolesterolêmicas foi eficaz em reduzir os níveis plasmáticos de triacilglicerol (78,8%), colesterol total (29,2%) o que se refletiu na incorporação hepática de lipídeos que foi reduzida em 28,6%. A análise fecal mostrou que os animais que consumiram a dieta hipercolesterolêmica acrescida da mistura PLG (grupo HF) apresentaram aumento de 37,89% na excreção fecal de colesterol. A esteatose cardíaca sofreu importante declínio nos animais hipercolesterolêmicos que consumiram a mistura PLG suplementada a dieta, de fato, verificou-se redução de 58% no volume das inclusões lipídicas presentes no citosol dos cardiomiócitos. A adição da PLG à dieta hipercolesterolêmica reduziu significativamente os valores da pressão arterial (23,2%) e da freqüência cardíaca (12,9%), em relação ao grupo hipercolesterolêmico. Tais resultados foram os responsáveis pela redução do desvio do eixo elétrico médio ventricular que, nos animais hipercolesterolêmicos apresentou-se dramaticamente deslocado para a esquerda (SÂQRS = 11º) sugerindo presença de hipertrofia ventricular, com intervalo QTc de longa duração (408,1 ms). Em nível ultraestrutural, os animais do grupo HF apresentaram maior responsividade dos receptores adrenérgicos às catecolaminas reduzindo o deslocamento de curva doseresposta para a direita. Em conclusão, os efeitos hipolipidêmicos da mistura PLG podem ser atribuídos a sua capacidade físico-química de adsorção de ácidos biliares e lipídeos da dieta formando géis que carreiam esses materiais para as fezes, mas também a formação fermentativa de ácidos graxos de cadeia curta, sobretudo o propionato que, reduzem a atividade da 3-OH-metil glutaril CoA redutase, enzima chave na biosíntese de colesterol. Estudos conduzidos em nosso laboratório mostraram que a polpa de laranja apresenta potencial antioxidante em hamsters alimentados com dieta hipercolesterolêmica. Desse modo, é possível que tal efeito antioxidante tenha colaborado para a redução da peroxidação de componentes da lipoproteína de baixa densidade (LDL), um evento capital na gênese do ateroma preservando ainda o óxido nítrico (NO), por bloquear a ação de espécies reativas do oxigênio, impedindo a formação de ácido peroxinitrítico (ONOOH) que por sua vez se decompõe em uma grande quantidade de produtos citotóxicos, como NO2 ¿, OH¿, e, também, o NO2 + / Abstract: Several studies have been demonstrating that the dietary fibers exercise protecting effects against the harmful impact of the hyperlipidemie. The orange pulp/guar-gum mix (110:40) was used as source of dietary fiber and characterized presenting the following physical-chemically composition: total fiber, 76,12%; soluble fiber, 43,54; insoluble fiber, 32,58. The hypolipidemics effects of the orange pulp/guar-gum mix (PLG) were verified in young male adults hamsters submitted to hypercholesterolemic diets being appraised biochemical, cardiovascular, morphometric, and ultrastructurals parameters. The PLG mix added to hypercholesterolemic diets was effective in reducing the plasma triacylglicerol levels (78,8%), total cholesterol (29,2%) what was reflected in the hepatic lipid incorporation that was reduced in 28,6%. The fecal analysis showed that the animals that consumed the hypercholesterolemic diet enriched of the PLG mix (group HF) presented increase of 37,89% in the fecal cholesterol excretion. The heart steatosis were reduced in the hypercholesterolemic animals fed PLG mix, reduction of 58% was verified in the lipids droplet in the citosol of the cardiac cells. PLG added in the hypercholerolemic diet reduced blood pressure significantly (23,2%) and heart rate (12,9%), in relation to the hypercholesterolemic group without PLG added. These results reflected in the deviation of the ventricular eletric axis that in hypercholesterolemic animals moved for the left (SÂQRS = 11º) suggesting ventricular hypertrophy, with long QTc interval (408,1 ms). In ultraestrutural response, the animals of the HF group presented larger responsivity of the catecholamines adrenergic response reducing the displacement dose-response curve for the right. In conclusion, the hipocholesterolemic effects of the PLG mix can be attribuid of the physico-chemical capacity biliary acid of adsorption and the lipids of the diet that brings those materials to the feces, but also the short chain fatty acid formation in the large intestine that, reduces the activity of the 3-OH-metil glutaril CoA reductase, key enzyme in the cholesterol biosynthesis. In our laboratory we identify that the orange pulp presents antioxidant potential in hamsters fed with hypercholesterolemic diet. This possible that antioxidant effect can collaborate for the reduction of LDL components oxidation, a decisive event in the atherosclerosis genesis and preserving the nitric oxide (NO) against oxidation by oxigen reactive species (free radicals) that convert it in peroxinitrite acid (ONOOH) that can decomposed in a great amount of toxic products as NO2 ¿, OH¿, and also NO2 + / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The hamster model of experimental chronic bronchitis comprises a persistent increase in the proportion of bronchial granulated secretory cells after a single intratracheal instillation of elastase. This granulated secretory cell increase, which does not occur in the trachea, has been termed secretory cell metaplasia (SCM). Susceptibility of the bronchial epithelium may be due to a large population of elastase-responsive cells specific to this region. Three dimensional reconstruction of the major form of bronchial secretory cells revealed very little or no rough endoplasmic reticulum (RER), thus demonstrating significant regional heterogeneity since all epithelial secretory cells in the trachea have abundant RER. Animals with bronchial SCM were stimulated with pilocarpine to determine whether the cells subsequent to discharge would re-accumulate granules, thus indicating a permanent phenotypic change. However, bronchial secretory cells failed to discharge at doses equal to and greater than those claimed to be effective in rats. Elastase instilled intratracheally was immuno-localized in the hamster airways to assess the possibility of regional differences in cellular uptake of the enzyme. Elastase was not seen intracellularly in trachea or bronchus suggesting that initiation of bronchial SCM results from a cell surface effect, possibly because of elastase-specific sites on bronchial but not tracheal cells. Tracheal resistance was tested by challenging the epithelial cells in vivo and in vitro with very high doses of elastase. Light and electron microscopy revealed no evidence of a stimulation of the mucus synthetic apparatus, suggesting that tracheal epithelial cells are inherently resistant to proteolytic up-regulation. / 2031-01-01

No description available.

<p>Base excision repair (BER) is a DNA repair mechanism that involves the removal of single damaged bases from DNA and their excision as free bases. Another DNA repair mechanism known as nucleotide excision repair (NER) involves the removal of bulky lesions from DNA. Previous published reports have suggested a role for certain NER proteins in BER. Methylene blue (MB) is a type II photosensitizer, which upon excitation by visible light (VL) produces singlet oxygen that reacts with DNA to form 8hydroxyguanine (8-oxoG) lesions. The BER mechanism has been shown to preferentially remove 8-oxoG lesions from DNA. In the current work, the role of NER proteins in the BER of [MB+VL]-induced DNA damage was examined using a reporter gene assay. AdMCMVlacZ and AdHCMVlacZ are non-replicating adenoviruses that express the bacterial β-galactosidase ( β-gal) reporter gene under the control of the murine or human cytomegalovirus immediate early promoter, respectively. Host cell reactivation (HCR) of β-gal activity for a [MB+VL]-treated AdMCMVlacZ was examined in two repair-proficient normal Chinese hamster ovary (CHO) cell lines, NER-deficient mutants from rodent complementation groups (RCGs) 1 through 6, and the HER-deficient cell line EM9. Results show a decreased HCR capacity of [MB+VL]-induced DNA damage in the UV61 (RCG-6; CSB) cell line when compared to the repair-proficient normal AA8. This suggests an involvement of CSB in BER of [MB+VL]-induced damage. In contrast, the XRCCl-deficient EM9 showed an increased HCR capacity when compared to the parental AA8. This suggests a beneficial role for an XRCCl deficiency or for the specific gain-of-function gene mutation in the XRCCl gene for the repair of [MB+VL]induced damage. Similarly, the ERCCl-deficient UV20 (RCG-1) showed an increased HCR capacity when compared to the parental AA8. In contrast, another ERCCJdeficient cell line, 30PV, and the ERCCJ knock-out cell line, CH0-7-27, showed no significant increase in β-gal activity when compared to the parental CHO-Kl. The ability to induce BER of [MB+VL]-induced DNA damage was also examined. HCR of β-gal activity for [MB+VL]-treated AdHCMVlacZ or AdMCMVlacZ was examined in several CHO cell lines that were either untreated or treated with low levels of UVC or MB plus VL. Pre-treatment of NER-deficient UV61 and repair-proficient AA8 cells with UVC resulted in an enhanced HCR for [MB+VL]-treated AdHCMVlacZ, although the results were only significant for UV61. Similarly, an enhanced HCR of [MB+VL]-treated AdMCMVlacZ was observed in AA8 cells, suggesting that the repair of the [MB+VL]treated reporter gene is inducible by UVC. A significant enhancement in HCR of [MB+VL]-treated AdMCMV lacZ was not observed in AA8 cells following pre-treatment with MB, VL, or both, indicating that the detection of any induction in the repair of DNA damage from MB plus VL could not be made with the conditions of the HCR assay employed. As investigations into the effects of MB plus VL on whole cells have been limited, clonogenic survival of BER-and NER-deficient CHO cell lines was also observed following treatment with MB alone or MB plus VL. Results showed that the sensitivities of the NER-deficient CHO cell lines from RCGs 2, 4 and 5 and the BERdeficient EM9 cell line to MB alone were within the range obtained for the two repairproficient CHO normal cell lines, AA8 and K 1. In contrast, the UV20 cell line was more sensitive to MB alone compared to the normal cell lines. Additionally, both UV24 (RCG-3; XPB) and UV61 showed a decreased sensitivity toMB alone when compared to the normal AA8 cell line. The sensitivity of cells to MB plus VL was greater in the UV24, UV135 (RCG-5; XPG) and the XRCCl-deficient EM9 cell lines compared to that of the range obtained for the two repair-proficient normal cell lines, AA8 and Kl. Taken together with the results from the HCR assays, these results suggest that [MB+VL]induced DNA damage to cells and its repair do not play a major role in the survival of cells following treatment with MB plus VL. It appears likely that damage to cellular components other than DNA, such as protein, lipid and biological membranes, play a more important role in cell killing by MB plus VL.</p> / Thesis / Master of Science (MSc)