Thesis (M.S.)--University of Southern California, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Southern California, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Made available in DSpace on 2015-12-06T22:54:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As ações biológicas e os efeitos farmacológicos da bradicinina (BK) são mediados por dois receptores, um constitutivo (B2R) e outro induzido (B1R). No fígado, nós verificamos que a resposta hipertensiva portal à BK é mediada por receptores B2, mas não por receptores B1, tanto em ratos normais quanto naqueles submetidos a diferentes agentes de agressão hepática. Nesta última situação verificamos que há aumento da expressão de B1R com a progressão para a cirrose. O objetivo deste trabalho é verificar se o B1R está envolvido no processo de fibrogênese. Para tanto 3 modelos experimentais de fibrose foram utilizados em camundongos knockout de B1R (KOB1R) e selvagens: a) com injeções intraperitoniais (ip) de CCl4 (6 semanas); b) injeções ip de soro de porco (10 semanas) e c) ligadura do ducto biliar (BDL). Após uma semana da última injeção, os fígados foram exanguinados, retirados, pesados e processados. A fibrose foi avaliada por análise histológica (coloração com Picrus-Sirius), e quantificada pelo programa AxionVision 4.5 da Carl Zeiss. Avaliação da fibrogênese foi estudada pela expressão de “heat shock protein”-47 (HSP 47) analisada por Western Blotting. A indução da fibrose com CCl4 foi obtida com sucesso nos camundongos selvagens e KOB1R, com o tradicional padrão de formação de septos hexagonais. O modelo BDL apresentou fibrose com padrão característico de colestase em ambos animais. Pela análise morfométrica, a quantidade de colágeno (% da área total) nos animais KOB1R-CCl4 (1,9 ± 0,3, n=5) é significativamente maior (ANOVA, p = 0,006) que nos animais selvagensCCl4 (1,0 ± 0,2, n=7), bem como quando comparado com o respectivo controle (KOB1R–óleo) (0,4 ± 0,3%, n=3). Por outro lado, a quantidade de colágeno nos animais KOB1R-BDL (3,1 ± 1,4, n=5) é significativamente menor que nos selvagens (16,1 ± 3,1, n=6). A expressão de HSP 47 está significativamente (t-test, p = 0,04) aumentada nos animais KOB1R (10,6 ± 1,6 x 106 UA), comparada aos animais selvagens (6,1 ± 1,3 x 106 UA), ambos tratados com CCl4, confirmando o pior padrão de fibrose observado também na quantificação morfométrica. No modelo de BDL, houve também maior expressão de HSP 47 nos animais KOB1R que nos animais selvagens Em conclusão, nossos resultados demonstram a participação do sistema calicreína-cinina na fibrogênese hepática e sugere que a ativação do B1R protege o fígado da fibrose hepática no modelo de CCl4, mas não no modelo BDL. Estes dados são importantes considerando o potencial terapêutico com o uso de agonistas no tratamento e/ou prevenção da fibrose. / The biological and pharmacological effects of bradykinin (BK) are mediated by a constitutive B2 and induced B1R. In the liver, we verified that the portal hypertensive response to BK is mediated by B2R, but not by B1R, in normal rats and those submitted to different kind of hepatic injury. In the latter, we also verified an increased expression of B1R in relation to the progression of the cirrhosis. The aim of this work is to analyze the role of BK in the fibrogenesis in wild and knockout of B1R (KOB1R) mice. Three models of fibrosis were used: a) intraperitoneal (ip) injection of of CCl4 (6 weeks); b) ip injection of porcine serum (10 weeks) and c) bile duct ligation (BDL). After one week of injection, the liver were exsanguinated, removed, weighted and processed. Fibrosis was evaluated by histological analysis (Picrus-Sirius staining) and quantified by AxionVision 4.5 software (Carl Zeiss). Fibrogenesis was evaluated by heat shock protein-47 (HSP 47) expression by Western Blotting. Porcine serum did not induce fibrosis in all animals. CCl4 induced fibrosis in wild and KOB1R in the traditional pattern with hexagonal septa. BDL resulted in fibrosis with the characteristic pattern of colestasis. By morphometric analysis, hepatic collagen (% of total area) of KOB1R-CCl4 (1,9 ± 0,3, n=5) was significantly higher (ANOVA, p = 0,006) than in wild-CCl4 (1,0 ± 0,2, n=7). On the other hand, hepatic collagen in KOB1R-BDL (3,1 ± 1,4, n=5) is lower than in wild mice (16,1 ± 3,1, n=6). The liver HSP 47 expression in CCl4 model is significantly (t-test, p = 0,04) higher in KOB1R (10,6 ± 1,6 x 106 UA), compared to wild mice (6,1 ± 1,3 x 106 UA). Interestingly, hepatic HSP 47 expression in KOB1R was also higher than in wild mice. In conclusion, our results suggested that bradykinin participates in the fibrogenesis, and probably activation of B1R protects liver from hepatic fibrosis in the CCl4 model, but not in BDL model. This data is important considering the potential therapeutic with the use of agonist in the threatment and/or prevention of liver fibrosis. / FAPESP: 02/05260-6 / FAPESP: 06/58533-0 / FAPESP: 08/55928-0 / BV UNIFESP: Teses e dissertações

In the developing mouse oocyte, as well as in the preimplantation embryo, betaine (N,N,N-trimethylglycine) plays an important role first as a mechanism for cell volume regulation and second as a major methyl donor. Thus, the presence of betaine has implications both during development, and throughout the lifespan. It has previously been observed that betaine accumulates in the mouse oocyte as it matures, however its origin in the egg is unknown. Here I explore the enzyme choline dehydrogenase (CHDH; EC 1.199.1) as a method by which the mouse oocyte synthesizes the betaine that we observe prior to initiation transport activity in the preimplantation embryo. I carefully monitored betaine transport throughout meiotic maturation to confirm that no other previously unobserved membrane transport existed in the maturing oocyte. However, no betaine transport into oocytes was detected during meiotic maturation suggesting de novo synthesis. Previous data suggests that the enzyme is expressed (at the transcript level) in the developing oocyte, and becomes active during meiotic maturation. I demonstrated the presence of CHDH protein in the oocyte and preimplantation embryo. I then examined whether the mouse oocyte synthesizes betaine autonomously and addressed whether CHDH is a requirement for this process. Chdh knockout oocytes did not accumulate betaine in vivo, while normal betaine levels were observed in Chdh wildtype oocytes. CHDH-mediated synthesis of betaine was directly confirmed by detection of increased betaine in oocytes matured in vitro in the presence of choline. Chdh-/- oocytes failed to produce betaine when similarly cultured in choline. This establishes the production of betaine as an autonomous process in maturing oocytes. Overall, I have built upon previous data to demonstrate that betaine accumulation is a feature of meiotic maturation that occurs by de novo synthesis of the molecule, a process that requires transient activation of the enzyme choline dehydrogenase.

Submitted by Barroso Patrícia (barroso.p2010@gmail.com) on 2013-04-04T19:59:26Z No. of bitstreams: 1 Dissertação milton galdino 05062012 - DEFINITIVA.pdf: 2594051 bytes, checksum: 1b7dc66215e94866d37e7c0e7c6430df (MD5) / Made available in DSpace on 2013-04-04T19:59:26Z (GMT). No. of bitstreams: 1 Dissertação milton galdino 05062012 - DEFINITIVA.pdf: 2594051 bytes, checksum: 1b7dc66215e94866d37e7c0e7c6430df (MD5) Previous issue date: 2011 / A Linfadenite Caseosa, causada por Corynebacterium pseudotuberculosis (C. pseudotuberculosis, é uma enfermidade de alta prevalência nos rebanhos de pequenos ruminantes da região semi-árida brasileira, trazendo grandes prejuízos particularmente para os pequenos produtores, pois a criação desses animais é a principal atividade de subsistência nesta região. O presente trabalho teve como objetivo avaliar aspectos da resposta imune de camundongos da linhagem C57/Black 6 selvagem e C57/Black 6 Knockout para o óxido nítrico (KO-NO), durante a infecção experimental por C. pseudotuberculosis. A utilização de animais Knockout possibilitou a compreensão da atuação de alguns tipos celulares do sistema imune frente ao patógeno, já que se sabe que o óxido nítrico é um importante agente na eliminação de microrganismos intracelulares. Para tanto, foram utilizados e eutanasiados 28 animais em dois tempos (7 e 14 dias), divididos em quatro grupos, a saber: grupo 1 – controle – com 4 animais selvagens; grupo 2 - infectado por C. pseudotuberculosis – com 10 animais selvagens; grupo 3 – controle – com 4 animais KO-NO; e grupo 4 – infectado por C. pseudotuberculosis – com 10 animais KO-NO. Na metodologia avaliamos a suscetibilidade de camundongos KO-NO, na infecção, verificando que animais C57/Black 6 KO-NO são susceptíveis à infecção por C. pseudotuberculosis com mortalidade observada a partir do 4º dia, o que indica a importância dessa molécula na defesa inata contra essa bactéria. No estudo de células do lavado peritoneal os neutrófilos foram as células encontradas em maior número no infiltrado dos animais KO-NO, o que pode indicar um mecanismo compensatório pela ausência do óxido nítrico. Com o estudo do baço observou-se a presença de linfócitos, com predomínio de TCD8 entre as células esplênicas de ambos os grupos, o que sugere a importância dessa sub-população na resposta imune contra esse microorganismo. A partir da presença de granulomas verificou-se a maior presença nos animais KO-NO infectados com 7 dias, sendo os linfonodos os mais afetados, o que aponta também para uma tentativa compensatória de conter a infecção; constatou-se uma maior recuperação bacteriana de linfonodo mesentério nos camundongos KO-NO, provavelmente devido a sua maior capacidade de escape. Foi realizado também o estudo de anticorpos séricos, através da detecção de IgG total e subclasses, o qual foi observado uma produção 14 dias pós infecção, tanto nos animais selvagens como nos KO-NO, similar aos animais controle, o que indica uma resposta humoral tardia por esse tipo de anticorpo para esse microorganismo. Os resultados obtidos sugerem a importância do óxido nítrico no processo de combate à infecção por este microrganismo, posto que os animais KO-NO para esta substância foram os mais afetados pelo processo infeccioso instalado. / Universidade Federal da Bahia, Instituto de Ciências da Saúde

The transcription factor DEAF-1 is the mammalian homologue of a critical Drosophila developmental gene and is essential for neonatal survival in mice. Haploinsufficiency of Deaf-1 in the testis of adult mice was initially thought to cause loss of spermatogenesis and disrupted morphology of the seminiferous tubules, and this heterozygosity was thought to be sufficient to disrupt epigenetic programming in the developing sperm to produce inheritance of testicular defects in both heterozygous and genotypically normal offspring. Although Deaf-1 knockout mice do display disrupted testis structure, infertility at advanced age, hyperproliferation of early germ cells, and abnormal staging of seminiferous tubules, this phenotype was also observed in normal mouse strains that were born in the SIUC vivarium. Mice ordered from a vendor and raised at SIUC did not show testicular defects. This suggests an environmental factor at the SIUC vivarium may act as an endocrine disruptor during embryonic testicular development. Deaf-1-/- mice die soon after birth, often as the result of exencephaly, a gross neural tube defect (NTD). Unlike many mouse models, exencephalic Deaf-1-/- mice do not display a higher incidence of NTDs in females as compared to their male littermates. DEAF-1 promotes Bax-mediated apoptosis; studies using terminal UTP nick-end labeling (TUNEL) suggest a global increase in apoptosis in both exencephalic and normal Deaf-1-/- fetuses during neurulation as compared to their Deaf-1+/+ littermates. This indicates that Deaf-1 is crucial for correct apoptotic patterning during development, which, in turn, is essential for neural tube closure. Finally, cDNA microarray comparison of e14.5 Deaf-1 knockout and wildtype fetuses reveals expression of translation initiation factor 4g3 (Eif4g3) to be downregulated in Deaf-1-/- fetuses. Electrophoretic mobility shift assay using recombinant DEAF-1, and chromatin immunoprecipitation assay of a human cell line confirmed DEAF-1 could bind the eIF4G3 promoter both in vitro and in vivo. Additionally, transcription of the Deaf-1 Antisense Transcript (Das) was found to be significantly downregulated in both e14.5 fetuses and e18.5 fetal brains from Deaf-1-/- mice, suggesting that either lack of Deaf-1 protein or lack of exons 2 through 5 in Deaf-1 knockout mice causes changes in levels of the noncoding RNA that shares Deaf-1's promoter in the mouse.

Abstract The carbonic anhydrases (CAs) catalyze the reversible hydration of carbon dioxide. In mammals, there are 13 active isoenzymes, which clearly differ in their cell localisation, tissue distributions and functions. CA IX, a unique transmembrane member of the CA gene family, is a tumour-associated protein which is thought to be involved in malignant cell invasion, adhesion and the regulation of cell proliferation. The main focus in the present study was on elucidating the function and expression of CA IX in normal and malignant tissues, especially in the alimentary tract. The functional studies also included CA II, which is regarded as another important CA isoenzyme in the alimentary tract. CA IX immunostaining showed a decrease in the staining intensity of gastric adenomas with increasing dysplasia grade. Well differentiated carcinomas of the intestinal type showed expression comparable to that in the normal mucosa, while expression was decreased in the less differentiated tumours. CA IX deficiency (Car9-/-) genotype and C57/BL6 strain were the main factors which increased the susceptibility of CA IX deficient mice fed on either a normal or high-salt diet to histological abnormalities, including foveolar hyperplasia and glandular atrophy in the gastric body mucosa, while CA II deficiency was associated with only minor histological abnormalities. In a physiological analysis, CA IX played only a minor role in duodenal bicarbonate secretion (DBS), whereas absence of CA II in mice completely abolished the stimulatory effect of E-type prostaglandin 2 (PGE2) on duodenal alkalisation. The results demonstrate that CA IX expression is diminished in most gastric tumours. The variations observed in its expression support the concept that gastric adenomas and carcinomas do not emerge as progressive steps on a single pathway but may instead represent distinct entities with heterogenic genetic backgrounds. In the stomach, CA IX is mainly involved in the regulation of tissue morphogenesis in the body mucosa, while CA II has a major role in maintaining the gastroduodenal acid/base balance.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22318号 / 医博第4559号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 林 康紀, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Iron is extremely important for many organisms. Despite its abundancy, it exists in insoluble forms that makes its usability difficult. Some organisms secrete siderophores, low molecular weight compounds, that can chelate iron and convert it into usable forms for cells. One such organism, Rhizobium leguminosarum, is a nitrogen fixing symbiont proteobacteria that infects leguminous plants. The genome of Rhizobium leguminosarum ATCC 14479, which infects the red clover, Trifoli pratense, has previously been completely sequenced in our lab. Our lab has identified several genes in this strain involved in the biosynthesis of a siderophore, vicibactin. The protein product of one of those genes, VbsS, is hypothesized to be a non-ribosomal peptide synthase. It has been attempted to knockout the VbsS gene utilizing the ‘splicing by overlap extension’ method. Additionally, an in-silico analysis of the genome revealed the Vbs genes in R. leguminosarum ATCC 14479 strain were similar to genes in found in the proteobacterium Phyllobacterium sp. 628 and the fungi Aspergillus fumigatus Af293.