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Lommel, Silvia. "Conditional inactivation of N-WASP in the mouse analyses of N-WASP function /." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964801922.
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Dagälv, Anders. "Role of Heparan Sulfate N-sulfation in Mouse Embryonic Development." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-123474.
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Heparan sulfate (HS) is a sulfated glycosaminoglycan expressed by all cells in the body. It is found at the cell surface and in the extracellular matrix where it binds a large amount of various ligands including growth factors and morphogens. HS is important for building up morphogen gradients during embryonic development and to act as coreceptors for signaling molecules. Many different Golgi enzymes are involved in the biosynthesis of HS. It is known that some of these enzymes interact with each other but not how the whole biosynthesis machinery works or how the cell regulates the structure of the HS that it produces. In this thesis, cells and mice deficient in two of these biosynthetic enzymes, glucosaminyl N-deacetylase/N-sulfotransferase-1 (NDST1) and the isoform NDST2 have been studied. NDSTs perform the first modifications during biosynthesis where they replace N-acetyl groups on N-acetyl-glucosamine units with sulfate groups. It is known that deficiency of NDST1 is lethal, while lack of NDST2 only results in abnormal connective tissue type mast cells. Here it is shown that deficiency of both NDST1 and NDST2 is embryonically lethal. The embryonic stem (ES) cells extracted from the inner cell mass of double knockout blastocysts show in addition an impaired differentiation capacity compared to wild-type ES cells and fail completely to differentiate into cardiac muscle cells which NDST1-/-, NDST2-/- and wild-type ES cells all do. Cultured mast cells that lack NDST2 produce heparin that is low-sulfated compared to wild-type HS. To our surprise, we could show that mast cells deficient in NDST1 instead produce a more highly sulfated heparin than wild-type cells. We use a model that predicts that the biosynthesis enzymes work together in a multienzyme complex, the GAGosome, to explain our results. We hypothesize that NDST1 has a higher affinity for the GAGosome than NDST2 which only in the absence of NDST1 gets incorporated into the enzyme complex. When all GAGosomes contain NDST2, a more highly sulfated glycosaminoglycan chain will be synthesized. A splice variant of NDST1, NDST1S, has also been studied. We could show that NDST1S lacks enzyme activity but that it probably has the capacity to incorporate into GAGosomes. Overexpression of NDST1S results in altered structure of the HS produced by the cells. We speculate that expression of the splice variant during development may be one way to regulate HS structure.
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Mazza, Graziella. "Antibody responses to Schistosoma mansoni in the CBA/N mouse." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303138.
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Barron, Catherine Mary. "Effects of Trimethylamine N-Oxide on Mouse Embryonic Stem Cell Properties." Thesis, Virginia Tech, 2020. http://hdl.handle.net/10919/99602.
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Trimethylamine N-oxide (TMAO) is a metabolite derived from dietary choline, betaine, and carnitine via intestinal microbiota metabolism. In several recent studies, TMAO has been shown to directly induce inflammation and reactive oxygen species (ROS) generation in numerous cell types, resulting in cell dysfunction. However, whether TMAO will impact stem cell properties remains unknown. This project aims to explore the potential impact of TMAO on mouse embryonic stem cells (mESCs), which serve as an in vitro model of the early embryo and of other potent stem cell types. Briefly, mESCs were cultured in the absence (0mM) or presence of TMAO under two different sets of treatment conditions: long-term (21 days), low-dose (20µM, 200µM, and 1000µM) treatment or short-term (5 days), high-dose (5mM, 10mM, 15mM) treatment. Under these treatment conditions, mESC viability, proliferation, and stemness were analyzed. mESC properties were not negatively impacted under long-term, low-dose TMAO treatment; however, short-term, high-dose treatment resulted in significant reduction of mESC viability and proliferation. Additionally, mESC stemness was significantly reduced when high-dose treatment was extended to 21 days. To investigate an underlying cause for TMAO-induced loss in mESC stemness, metabolic activity of the mESCs under short-term, high-dose TMAO treatment was measured with a Seahorse XFe96 Analyzer. TMAO treatment significantly decreased the rate of glycolysis, and it increased the rate of compensatory glycolysis upon inhibition of oxidative phosphorylation (OxPHOS). It also significantly increased the rate of OxPHOS, maximal respiratory capacity, and respiratory reserve. These findings indicate that TMAO induced a metabolic switch of mESCs from high glycolytic activity to greater OxPHOS activity to promote mESC differentiation. Additionally, TMAO resulted in increased proton leak, indicating increased oxidative stress, and elucidating a potential underlying mechanism for TMAO-induced loss in mESC stemness. Altogether, these findings indicate that TMAO decreases stem cell potency potentially via modulation of metabolic activity.Master of Science
Trimethylamine N-oxide (TMAO) is a metabolite that is produced by the bacteria in the gut after the consumption of specific dietary ingredients (e.g., choline, carnitine, betaine). These ingredients are commonly found in meat and dairy products, and thus make up a large part of the average American diet. Recently, it was discovered that high TMAO levels in the bloodstream put people at an increased risk for heart disease, neurodegenerative diseases (e.g., Alzheimer's Disease), diabetes, stroke, and chronic kidney disease. At the cellular level, there is evidence that TMAO increases inflammation and the production of oxygen radicals, which causes cells to lose their function and promotes the onset of disease. TMAO has been well studied in adult cell types; however, no one has investigated whether TMAO will impact cells of the early embryo. This project aims to explore the impact of TMAO on mouse embryonic stem cells (mESCs), which are cells that represent the early stage of embryonic development and are critical for proper development of the final offspring. In addition, mESCs may also help to provide insight into how TMAO impacts other stem cell types, some of which are present throughout the entire human lifespan and play an important role in the body's ability to repair itself and maintain overall health. My project demonstrated that TMAO does not impact the overall health of mESCs under normal conditions, which signifies that TMAO generated by a pregnant mother may not directly impact the early embryonic stage of development. Further studies should be conducted to determine the potential impact of TMAO on late stages of embryonic and fetal development. Next, to simulate diseased conditions, the mESCs were treated with extremely high concentrations of TMAO in order to determine what concentration of TMAO will negatively impact these cells. It was found that at 5mM TMAO, mESCs begin to lose their basic properties and become dysfunctional. They are impaired in their viability, growth, ability to become other cell types, and in their metabolic activity. These mESC properties are shared with several types of adult stem cells, and therefore, these findings help to provide insight into how TMAO may impact stem cells found in the adult body which are exposed to a lifetime of high TMAO levels. In the future, we would like to further explore the impact of TMAO on mESCs at the molecular level as well as examine the direct impact of TMAO on other stem cell types.
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Ly, Lan H. "Immunosuppressive dietary n-3 polyunsaturated fatty acids differentially modulate costimulatory regulation of murine CD4+ T-cell function." Texas A&M University, 2004. http://hdl.handle.net/1969.1/1535.
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Consumption of fish oils (FO) enriched with the n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), is beneficial to a variety of inflammatory disorders due, in part, to the alteration of membrane composition of T-lymphocytes and other immune cells. We previously observed that down-regulation of proliferation and cytokine synthesis by CD4+ T-cells in mice fed diets rich in n-3 PUFA was dependent on the involvement of CD28, a co-stimulatory molecule necessary for T-cell activation. Since the co-receptor homologues, CD28 and CTLA-4, have opposing effects on T-cell activation, we hypothesized that the balance of costimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. A significant increase (p<0.05) in CD28 and CTLA-4 surface expression was observed in CD4+ T-cells post-stimulation with phorbol ester and calcium ionophore (PMA/Iono) or anti-CD3 and anti-CD28 (αCD3/CD28) antibodies in all diet groups. A significant increase (p<0.01; 20%) in the number of CD28 molecules was observed in n-3 PUFA vs. CO-fed mice after 48 h of in vitro CD4+ T-cell activation, and both CTLA-4 mRNA transcript and protein levels were upregulated by 50% at 72 h post-activation (p<0.01). Treatment with anti-CTLA-4 mAb in vivo in Mycobacterium bovis (BCG)-vaccinated mice did not alter the suppressive effects of dietary n-3 PUFA on antigen (PPD)-induced lymphocyte proliferation or delayed hypersensitivity reactions.
T-cells from both the C57BL/6 and IL-10mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with αCD3/CD28. CD4T-cells from C57BL/6 mice fed DHA produced significantly less IFNγ and IL-10, while CD4T-cells from IL-10Ligation of CD28 upregulates IL-10 receptor (IL-10R) expression on CD4+ T-cells. Therefore, we hypothesized that dietary n-3 PUFA would suppress T-cell function through the effects of IL-10. Surprisingly, the proliferation of purified splenic CD4+ T-cells activated in vitro with αCD3/CD28 was suppressed by dietary n-3 PUFA in both conventional mice (C57BL/6) and IL-10 gene knockout (IL-10(-/-)) mice. Furthermore, IL-10R cell surface expression was significantly down-regulated on CD4+ T-cells from both the C67BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA produced significantly more IFNγ compared to the CO-fed group.
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Munro, Sandra Bronwen. "Characterization of a composite cDNA clone encoding mouse testicular N-Cadherin and the mouse homologue of a human breast tumor autoantigen." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69645.
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A mouse testis cDNA library was screened with an oligonucleotide probe corresponding to a sequence in the 5$ sp prime$ region of mouse N-cadherin cDNA. A composite clone containing three individual cDNAs was isolated. These included a 711 bp cDNA encoding part of mouse testicular N-cadherin, an unidentified 392 bp cDNA, and a 1500 bp cDNA encoding the mouse homologue of a human breast tumor autoantigen.The cadherins, the influenza strain A hemagglutinins, and the fibroblast growth factor receptors are three different families of integral membrane glycoproteins that harbour the amino acid motif histidine-alanine-valine (HAV) in regions involved in protein-protein interactions. In order to identify other proteins that possess the HAV motif in functionally important regions, the SwissProt database was searched using a consensus sequence derived from the cadherins, influenza strain A hemagglutinins, and fibroblast growth factor receptors. This search identified the $ alpha$ chains of the HLA class I histocompatibility antigens as a fourth family of integral membrane glycoproteins with an HAV-containing region that is involved in a protein-protein interaction. (Abstract shortened by UMI.)
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Yu-Plant, Violeta Lynn. "The development of gene targeted mouse strains for studying arylamine N-acetyltransferase function." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ34082.pdf.
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Neale, Sondra-Ann. "Characterization of the neural cell adhesion molecule N-CAM in splotch mutant mouse embryos." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69646.
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Cell adhesion molecules are known to play crucial roles in a variety of developmental processes. The neural cell adhesion molecule N-CAM is strongly implicated in neurulation and neural crest cell (NCC) migration and was thus studied in splotch (Sp) neural tube defect mutant embryos. At the 20 somite-stage of gestation day 9, Sp N-CAM was found to contain polysialic acid (PSA) side chains which are normally only present beginning at gestational day 11. Younger embryos at 12 and 14 somites also showed the presence of PSA on N-CAM, which was absent in controls. Enzymatic removal of PSA from N-CAM resulted in isoforms which migrated identically to PSA-free N-CAM isoforms in SDS-polyacrylamide gels. The post-translational modification of N-CAM appears to be the primary target of the Sp gene. In view of N-CAM's importance during development, an alteration at a critical stage is likely to result in the cascade of abnormalities seen in Sp mutants.A new genotyping assay was also implemented for examination of N-CAM in Sp and other related wildtype strains.
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Rago, Luciano F. [Verfasser], and Rolf [Akademischer Betreuer] Kemler. "N-cadherin expression regulation by microRNAs in the mouse developing neocortex = N-Cadherin-Expressionsregulation von microRNAs in der Maus entwickelt Neokortex." Freiburg : Universität, 2014. http://d-nb.info/1122646674/34.
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Muraveika, Liudmila [Verfasser], and Daniela N. [Akademischer Betreuer] Männel. "Binding specificity of mouse ficolin to different bacterial strains / Liudmila Muraveika. Betreuer: Daniela N. Männel." Regensburg : Universitätsbibliothek Regensburg, 2012. http://d-nb.info/1028392583/34.
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Domke, Tanja Carolina Elisabeth. "O-linked N-acetylglucosamine in differentiation and gene expression of mouse and human pluripotent stem cells." Thesis, University of Dundee, 2016. https://discovery.dundee.ac.uk/en/studentTheses/e84ff9a1-a4ab-41d0-828c-60a191b42c69.
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Peptide posttranslational modifications have been shown to regulate multiple aspects of cell signalling, thereby influencing cellular functions. The addition of O-linked Nacetylglucosamine to serine or threonine residues (O-GlcNAcylation) of proteins has only recently been characterized and its overall role in cell signalling remains elusive to date. Recent studies suggest an essential role of O-GlcNAcylation on the viability and pluripotency of mouse and human embryonic stem (ES) cells. Here we show that increased levels of O-GlcNAcylation in response to specific inhibition of O-GlcNAc hydrolase (Oga) hinder mouse ES cell differentiation. In addition to these findings, I could also demonstrate that increased O-GlcNAcylation leads to expression of a gene set normally epigenetically repressed in mouse ES cells and associated with a subpopulation resembling cells in the 2-cell-stage embryo. I also extended our lab's investigations to human induced pluripotent stem (iPS) cells. While mesendodermal differentiation remains unaffected by high O-GlcNAc levels, neural differentiation is severely disrupted in these cells. Human iPS cells with elevated O-GlcNAcylation are unable to commit to the ectodermal lineage and fail to organize in neural tube-like structures, so-called neural rosettes. Following these observations we performed mRNA sequencing analysis on human iPS cells with high O-GlcNAc levels and found gene expression to be significantly altered. Genes affected by increased O-GlcNAcylation include modulators of key neural developmental processes, for example components of the bone morphogenic protein signalling cascade.
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Davis, Jada Leanne-Bittle. "The role of redox dysregulation in the effects of prenatal stress on the embryonic and adult mouse brain." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6562.
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Maternal stress during pregnancy is associated with increased risk of psychiatric disorders in offspring, but embryonic brain mechanisms disrupted by prenatal stress are not fully understood. Our lab has shown that prenatal stress delays inhibitory neural progenitor migration. Here, we investigated redox dysregulation as a mechanism for embryonic cortical interneuron migration delay, utilizing direct manipulation of pro- and anti-oxidants and a mouse model of maternal repetitive restraint stress starting on embryonic day 12. Time-lapse, live-imaging of migrating GABAergic interneurons showed that normal tangential migration of inhibitory progenitor cells was disrupted by the pro-oxidant, hydrogen peroxide. Interneuron migration was also delayed by in utero intracerebroventricular rotenone. Prenatal stress altered glutathione levels and induced changes in both activity of antioxidant enzymes and expression of redox-related genes in the embryonic forebrain. Assessment of dihydroethidium (DHE) fluorescence after prenatal stress in ganglionic eminence, the source of migrating interneurons, showed increased levels of DHE oxidation. Maternal antioxidants (N-acetylcysteine and astaxanthin) normalized levels of DHE oxidation in ganglionic eminence, and ameliorated the migration delay caused by prenatal stress.
In adult male offspring, prenatally-stressed mice exhibited anxiety-like behavior on the elevated plus maze, impaired motor learning on the rotarod, cognitive flexibility on the water T-maze task, and deficits in sensorimotor gating in the pre-pulse inhibition task. Prenatally-stressed adult female offspring showed anxiety-like behavior, deficits in sociability and impaired motor learning. Maternal antioxidants prevented anxiety-like behaviors and improved sensorimotor gating in both sexes, and improved habitual learning and cognitive flexibility in adult female mice. Lastly, prenatal stress led to increases in PV+/GAD67+ cell ratios in mFC in male mice, but decreases in female mice, and antioxidant treatments eliminated those differences. Hippocampal GAD67+ cell densities were reduced by prenatal stress and restored by astaxanthin in male mice, and PV+/GAD67+ cell ratio was reduced by prenatal stress and partially restored by N-acetylcysteine in female mice. GAD67+ cell densities across regions correlated significantly with anxiety-like behavior in both male and female mice and social behavior in female mice. Through convergent redox manipulations, delayed interneuron migration after prenatal stress was found to critically involve redox dysregulation. Redox biology during prenatal periods may be a target for protecting brain development.
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Lucey, Michelle M. "The expression of the High Mobility Group N (HMGN) family of proteins during development of the mouse eye." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 120 p, 2008. http://proquest.umi.com/pqdweb?did=1597629771&sid=19&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Berjanskii, Mark. "Structure and dynamics of the N-terminal J-domain of T antigens of murine polyomavirus." MU online access free, to others for fee Free online access, 2002. http://wwwlib.umi.com/cr/mo/preview?3052146.
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Ringvall, Maria. "Functions of Heparan Sulfate During Mouse Development : Studies of Mice with Genetically Altered Heparan Sulfate Biosynthesis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4244.
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Fernandes, Hugo. "A melatonina e seu efeito citoprotetor na maturação de oócitos murinos." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/74/74131/tde-03022016-153822/.
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Diversos são os fatores que estão envolvidos no controle da maturação oocitária e na qualidade e competência do oócito. A melatonina (MEL) é um hormônio que apresenta diversas funções, como atividade antioxidante e antiapoptótica, além de influenciar diferentes vias de sinalização celular. Ainda há poucos estudos sobre o papel da MEL na maturação de oócitos e o camundongo, por sua rápida reprodução e menor custo de manutenção, é um excelente modelo amplamente utilizado para estudos in vitro e, particularmente in vivo. O presente trabalho teve por objetivo avaliar o efeito da MEL sobre a maturação in vivo e in vitro e sua possível ação como protetor celular (antioxidante e/ou antiapoptótico) de complexos cumulus-oócitos (CCOs) murinos. No experimento 1, os animais receberam injeções de MEL nas concentrações de 0 (controle), 10 e 20 mg/kg/i.p./dia por 4 dias. Os CCOs foram maturados in vivo e recuperados 17 horas após a última injeção. No experimento 2, os animais receberam MEL nas mesmas dosagens do experimento anterior, porém por 3 dias. Os CCOs foram coletados 24 horas após a última injeção e maturados in vitro com hormônio folículo estimulante (0,5 µg/mL; FSH). No experimento 3, os CCOs foram maturados in vitro na presença de três diferentes doses de MEL (10-9, 10-6 e 10-3 M) ou em FSH (controle FSH). Por fim, no experimento 4, a melhor concentração de MEL (10-9 M) eleita no experimento 3, foi utilizada sozinha ou em associação com peróxido de hidrogênio (300µM; H2O2). Os CCOs foram maturados como no experimento anterior. Para os quatro experimentos foram avaliados a taxa de maturação mediante a extrusão do primeiro corpúsculo polar (1º CP) e a expressão de genes relacionados à apoptose (Bax e Bcl2l1) e enzimas antioxidantes (Gpx1, Sod1 e Sod2) por qPCR-RT tanto para as células do cumulus (CC), quanto para os oócitos (OO). No experimento 1, o tratamento com 20 mg/kg de MEL apresentou maior taxa de maturação in vivo de 80,3%, seguido do controle (69,4%) e 10 mg/kg de melatonina (62,4%; P>0,05). Para a expressão gênica não houve efeito de nenhum tratamento (P>0,05). No experimento 2, a taxa de maturação variou de 39 a 53,2% entre os tratamentos (P>0,05). Em CC, a expressão gênica foi diminuída de Bcl2l1 e aumentada de Gpx1 tratados com 20 mg/kg de MEL (P<0,05). Já para OO, somente houve aumento da expressão de Gpx1 para ambos os tratamentos com MEL (P<0,05). No experimento 3, a taxa de maturação foi de 48,9%, 53,7%, 56% e 57,3% para 10-3, 10-6, 10-9 M de MEL e FSH, respectivamente (P>0,05). As concentrações de 10-9 e 10-6 M de MEL aumentaram a expressão dos genes Gpx1 e Sod1 em CC (P<0,05). Em OO, somente houve aumento da expressão gênica de Bax na concentração de 10-6 M de MEL (P<0,05). No último experimento, não houve diferença significativa quanto à taxa de maturação, variando de 51,8% para o tratamento com H2O2 a 60% para o controle FSH (P>0,05). Em CC, Gpx1 e Sod1 tiveram suas expressões aumentadas em todos os tratamentos (P<0,05). De maneira contrária, o gene Bcl2l1 teve sua expressão diminuída (P<0,05). Com base nestes dados, conclui-se que a MEL aplicada in vivo não foi capaz de melhorar a taxa de maturação in vivo e in vitro, porém, em condições in vitro induziu a progressão da meiose em oócitos murinos. Além disso, em condições in vitro, genes antioxidantes como Gpx1 e Sod1 foram mais expressos em CC do que em OO em resposta ao tratamento com MEL, indicando a indução de um possível efeito protetor frente à condições de cultivo in vitro.Many factors are involved in the control of oocyte maturation and developmental competence. Melatonin (MEL) is a hormone showing varied functions including antioxidant and antiapoptotic activities, besides influencing many cell signaling pathways. There are few studies on the role of MEL in oocyte maturation and the mouse, due to its quick reproduction and lower maintencance cost, is an interesting model widely used for in vitro and particularly in vivo studies. The aim of this work was to study the effects of MEL on in vivo and in vitro maturation and its cytoprotective action (antioxidant/antiapoptotic) in murine cumulus-oocyte complexes (CCOs). In experiment one, mice received MEL injections at concentrations of 0 (control), 10 and 20 mg/kg/i.p./day for 4 days. CCOs were in vivo matured and recovered 17 hours after the last injection. In experiment 2, the animals received MEL in the same dosages of the previous experiment, but for 3 days. CCOs were collected 24 hours after the last injection and in vitro matured with follicle stimulating hormone (FSH). In experiment 3, CCOs were in vitro matured with three MEL concentrations (10-9, 10-6 e 10-3 M) or FSH (FSH control). Finally, in the fourth experiment, the best concentration of MEL (10-9 M) selected in experiment 3 was used alone or in association with hydrogen peroxide (300 µM; H2O2). CCOs were matured as in the previous experiment. For all four experiments maturation rates were evaluated by extrusion of the first polar body and the expression of genes related to apoptosis (Bax and Bcl2l1) and antioxidant enzymes (Gpx1, Sod1 and Sod2) by qPCR-RT both cumulus cells (CC), and for the oocytes (OO) were assessed as well. In experiment 1, treatment with 20 mg/kg MEL showed a higher rate of in vivo maturation of 80.3%, followed by the control (69.4%) and 10 mg/kg MEL (62.4%; P>0.05). No effect for gene expression treatments (P>0.05) was observed. In experiment 2, maturation rate ranged from 39 to 53.2% between treatments (P>0.05). In CC, the gene expression was reduced for Bcl2l1 and enhanced for Gpx1 in animals treated with 20 mg/kg MEL (P<0.05). For OO, only Gpx1 expression was increased for both MEL treatments (P<0.05). In experiment 3, the maturation rates were 48.9, 53.7, 56 e 57.3% for MEL 10-3, 10-6, 10-9 M and FSH, respectively (P>0.05). MEL concentrations of 10-6 and 10-9 M increased expression of Gpx1 and Sod1 genes in CC (P<0.05). For OO, only Bax increased the gene expression in 10-6 M MEL concentration (P<0.05). In the last experiment, there was no significant difference in maturation rate, ranging from 51.8 for H2O2 to 60% for FSH control (P>0.05). Gpx1 and Sod1 genes had their expression increased in all the treatments in CC (P<0.05). For Bcl2l1 gene, the expression was decreased in CC as well (P<0.05). Based on these data, we conclude that MEL treatment in vivo was unable to promote maturation rate in vivo and in vitro, but under in vitro conditions it induced meiosis progression in murine oocytes. In addition, Gpx1 and Sod1 antioxidant genes were more expressed in CC than OO in response to MEL treatments in vitro indicating induction of a possible protective effect against in vitro culture conditions.
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Tomazini, Ana Paula Inoe. "Ação do N-acetilmuramil-L-alanil-D-isoglutamina (MDP) na regeneração nervosa periférica. Estudo experimental em camundongos." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/10/10137/tde-21092007-170257/.
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Avaliou-se o efeito do N-acetilmuramil-L-alanil-D-isoglutamina (MDP) na regeneração de neurônios periféricos em animais adultos. O nervo ciático esquerdo de oito camundongos machos adultos da linhagem C57BL/6J foi seccionado e o coto proximal e distal foram ancorados no interior de um tubo de polietileno (TP) com diâmetro interno de 0,76 mm, mantendo-se uma distância de quatro mm entre os mesmos. Os animais foram divididos aleatória e eqüitativamente em dois grupos e receberam 2µl de solução purificada de colágeno (Vitrogen®2,4 mg/ml) (COL) ou colágeno e MDP na proporção de 1:1 obtendo-se uma concentração de 1 mM (COL/MDP). Outros quatro animais não operados serviram como controle de normalidade (NOR). Após quatro semanas, os TP com os cabos de regeneração (CR) no seu interior, foram coletados para determinação do número de axônios mielínicos e da média do diâmetro das fibras mielínicas regeneradas. Os resultados revelaram diferença significativa no número de axônios entre os grupos NOR (4355 ± 32), COL (1869 ± 289) e COL/MDP (2430 ± 223). Houve redução significativa no diâmetro das fibras mielínicas nos grupos que receberam as próteses tubulares (COL= 3,38 µm ± 1,16 e COL/MDP= 3,54 µm ± 1,16) quando comparados ao grupo NOR (6,19 µm± 2,45). O gânglio da raiz dorsal L5 também foi removido e seccionado em série para a contagem e mensuração dos neurônios sensitivos. O número de neurônios não diferiu entre os grupos experimentais (COL = 564 ± 51 e COL/MDP = 514 ± 56), os quais apresentaram menor número de neurônios sensitivos em relação ao grupo não operado (NOR = 1097 ± 142). Os resultados indicam que a aplicação local do MDP estimula a regeneração de nervos em camundongos.N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) was evaluated for its effect on the regenerating peripheral neurons in adult animals. The left sciatic nerve of nine male adult mice C57BL/6J was cut, the proximal and distal nerve stumps were inserted into a polyethylene tube (PT) with 0,76 mm inner diameter, leaving a four mm distance gap. Animals were randomly distributed into two groups and received 2µl of purified preparation of collagen (Vitrogen® 2,4 mg/ml) (COL) or collagen and MDP made up in 1:1 volume ratio, obtaining a 1 mM (COL/MDP) concentration. Other four non-operated animals served as controls (NOR). After four weeks, PT with the regenerated nerve cables (RC), were processed for total myelinated axon counts and myelinated fiber diameter. The results revealed significant difference (p<0.05) in the axons count among the groups NOR (4355 ± 32), COL (1869 ± 289) and COL/MDP (2430 ± 223). There was a significant reduction in the diameter of myelinated fibers in the operated groups (COL = 3,38 µm ± 1,16 and COL/MDP = 3,54 µm ± 1,16) when compared to non-operated animals (6,19 µm ± 2,45). The L5 dorsal root ganglion (DRG) was also removed form the same mice and serially sectioned for sensory neurons count and measurement. No difference was found in the number of DRG neurons among the experimental groups (COL = 564 ± 51 and COL/MDP = 514 ± 56), which presented fewer sensory neurons compared to non-operated group (NOR = 1097 ± 142). The results indicate that the locally applied MDP stimulates peripheral nerve regeneration in mice.
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Ferreira, Larissa Domenegueti. "Efeito do S-nitroso-N-acetilpenicilamina sobre receptores vaniloide de potencial transitório 1 em córnea de camundongos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17150/tde-05012017-093309/.
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Introdução: Receptores TRPV1 são canais catiônicos nociceptivos não seletivos. Eles desempenham um papel na sensação de dor na córnea. No presente trabalho, descreveremos a resposta do TRPV1 na córnea de camundongos e TRPV1 -/- a estímulos químicos e o efeito do SNAP sobre este receptor. Métodos: camundongos C57BL/6 e TRPV1 -/- foram comparados. As observações incluíram o influxo de cálcio na cultura, observação direta e o comportamento de limpeza do olho após o desafio na córnea com 1µM da capsaicina (CAP) com ou sem 1mM do colírio SNAP. Resultados: As características da superfície ocular não eram diferentes entre ambos os genótipos, exceto que a sensibilidade para CAP é inferior em TRPV1 -/- (p = 0,0019 e 0,0095, respectivamente). A imunocoloração revelou que TRPV1 é expresso na camada basal do epitélio da córnea, apenas nos camundongos C57BL/6. CAP estimula o influxo de cálcio em cultura de células epiteliais e a sensibilidade corneana em C57BL/6 in vivo foi reduzida na presença do SNAP (p = 0,0329 e 0,01, respectivamente). Conclusão: A ausência dos receptores TRPV1 no epitélio da córnea não afeta o fenótipo da superfície ocular de camundongos. A resposta para tirar colírios revelou que ela poderia funcionar como uma terapia analgésica devido ao seu antagonismo com o TRPV1.TRPV-1 receptors are non-selective nocioceptive cation channels. They play a role in cornea sensation. In the present work we describe the response of corneas of TRPV1-/- and mice to chemical stimuli and the effect of SNAP on this receptor. TRPV1-/- and C57BL/6 mice were compared. The observations included the calcium influx in culture, direct observation and the behavior of eye wipe after corneal challenge with 1?M Capsaicin (CAP) with or not 1mM SNAP eye drops. Ocular surface characteristics were not different between both genotypes, except that the sensitivity to CAP is lower in TRPV1-/- (p=0.0019 and 0.0095, respectively). Immunostaining revealed that TRPV 1 is expressed in the basal layer of the cornea epithelia, only in the C57 mice. CAP stimulated calcium influx in epithelial cells in culture and cornea sensitivity in C57 mice in vivo was reduced in the presence of SNAP (p=0.0329 and 0.01, respectively). The absence of TRPV-1 receptors in the cornea epithelia did not affect the ocular surface phenotype in mice. The response to SNAP eye drops revealed that it could work as an analgesic therapy due to its antagonism to the TRPV1.
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Bharadwaj, Rahul. "Regulation of Higher Order Chromatin at GRIN2B and GAD1 Genetic Loci in Human and Mouse Brain: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/651.
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Little is known about higher order chromatin structures in the human brain and their function in transcription regulation. We employed chromosome conformation capture (3C) to analyze chromatin architecture within 700 Kb surrounding the transcription start site (TSS) of the NMDA receptor and schizophrenia susceptibility gene, GRIN2B, in human and mouse cerebral cortex. Remarkably, both species showed a higher interaction between the TSS and an intronic sequence, enriched for (KRAB) Krueppel associated Box domain binding sites and selectively targeted by the (H3K9) histone 3 lysine 9 specific methyltransferase ESET/SETDB1. Transgenic mice brain cortical nuclei over-expressing Setdb1 showed increased heterochromatin-protein 1 signal at the interacting regions coupled with decreased Grin2b expression. 3C further revealed three long distant chromatin loop interactions enriched with functional enhancer specific (H3K27Ac) histone 3 lysine 27 acetylation signal in GRIN2B expressing tissue (human cortical nuclei and Human Embryonic Kidney - HEK cells). Doxycycline-induced SETDB1 over-expression decreased 2 out of 3 loop interaction frequencies suggesting a possible SETDB1-mediated transcription repression. We also report a specific looping interaction between a region 50Kb upstream of the (GAD1) Glutamic Acid Decarboxylase – 1 gene TSS and the GAD1 TSS in human brain nuclei. GAD1 catalyzes the rate limiting step in (GABA) gamma amino-butyric acid synthesis and is quintessential for inhibitory signaling in the human brain. Clinical studies in schizophrenia brain samples reveal a decreased looping interaction frequency in correspondence with a decrease in gene expression. Our findings provide evidence for the existence of transcription relevant higher order chromatin structures in human brain.
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Bharadwaj, Rahul. "Regulation of Higher Order Chromatin at GRIN2B and GAD1 Genetic Loci in Human and Mouse Brain: A Dissertation." eScholarship@UMMS, 2002. http://escholarship.umassmed.edu/gsbs_diss/651.
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Little is known about higher order chromatin structures in the human brain and their function in transcription regulation. We employed chromosome conformation capture (3C) to analyze chromatin architecture within 700 Kb surrounding the transcription start site (TSS) of the NMDA receptor and schizophrenia susceptibility gene, GRIN2B, in human and mouse cerebral cortex. Remarkably, both species showed a higher interaction between the TSS and an intronic sequence, enriched for (KRAB) Krueppel associated Box domain binding sites and selectively targeted by the (H3K9) histone 3 lysine 9 specific methyltransferase ESET/SETDB1. Transgenic mice brain cortical nuclei over-expressing Setdb1 showed increased heterochromatin-protein 1 signal at the interacting regions coupled with decreased Grin2b expression. 3C further revealed three long distant chromatin loop interactions enriched with functional enhancer specific (H3K27Ac) histone 3 lysine 27 acetylation signal in GRIN2B expressing tissue (human cortical nuclei and Human Embryonic Kidney - HEK cells). Doxycycline-induced SETDB1 over-expression decreased 2 out of 3 loop interaction frequencies suggesting a possible SETDB1-mediated transcription repression. We also report a specific looping interaction between a region 50Kb upstream of the (GAD1) Glutamic Acid Decarboxylase – 1 gene TSS and the GAD1 TSS in human brain nuclei. GAD1 catalyzes the rate limiting step in (GABA) gamma amino-butyric acid synthesis and is quintessential for inhibitory signaling in the human brain. Clinical studies in schizophrenia brain samples reveal a decreased looping interaction frequency in correspondence with a decrease in gene expression. Our findings provide evidence for the existence of transcription relevant higher order chromatin structures in human brain.
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SIMON, Caroline Ferreira. "Avaliação da histotoxicidade e de alterações metabólicas após o uso do etil-cianoacrilato e n-butil cianoacrilato em camundongos." Universidade Federal de Pelotas, 2011. http://repositorio.ufpel.edu.br/handle/ri/2571.
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Previous issue date: 2011-05-16The cyanoacrylate derivatives have been extensively used as tissue adhesives in wound. Among the most widely used adhesives are the n-butyl cyanoacrylate , which is a compound of long chain and the ethyl-cyanoacrylate which is an acid cyanoacrilic ester of short chain. The objective of this study was evaluated the hepatic, renal and cutaneous toxicity of ethyl-cyanoacrylate and n-butyl cyanoacrylate in mouse. 84 mouse of swiss albino strain were used and divided in three groups, each with 28 animals. A longitudinal incision was made to three centimeters on the back of each mouse. In these animals, the syntheses was realized with ethyl-cyanoacrylate (group A), with n-butyl cyanoacrylate (group B) and suture with náilon monofilament yarn (group C), the control group. Sample were collected at 7, 15, 30 and 45 days after treatment for biochemical examination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, creatinine, urea and for histopathological examination was collected liver, kidneys and animals skin. During the experiment was significant difference between groups in mean of ALT at 30 (p=0,039) and 45 days (p=0,0391), AST at 45 days (p=0,0000) and creatinine at 15 (p=0,0169) and 45 days (p=0,0062). In the histopathological examination were not observed changes in toxicity indicatives in the kidneys, liver, skin. In the studied condition not been demonstrated kidney or liver toxicity in the use of ethyl-cyanoacrylate and n-butyl cyanoacrylate for skin syntheses.
Os derivados dos cianoacrilatos têm sido muito utilizados como adesivos teciduais em feridas. Dentre os adesivos mais utilizados encontram-se o n-butil-cianocrilato , o qual é um composto de cadeia longa e o etil-cianoacrilato que é um éster do ácido cianoacrílico de cadeia curta. O objetivo deste estudo foi avaliar a toxicidade hepática, renal e cutânea dos adesivos etil-cianoacrilato e n-butil cianoacrilato, em camundongos. Foram utilizados 84 camundongos da linhagem swiss albino e divididos em três grupos, cada um com 28 animais. Foi efetuada uma incisão longitudinal de três centímetros no dorso de cada camundongo. Nestes animais, foi depositado no tecido subcutâneo o adesivo etil-cianoacrilato (grupo A), n-butil cianoacrilato (grupo B). No grupo C, considerado grupo controle, foi realizada apenas sutura com fio de náilon monofilamentar. Foram coletadas amostras aos 7, 15, 30 e 45 dias após os tratamentos. Para análise bioquímica, foram avaliados os níveis séricos de Alanina aminotranferase (ALT), Aspartato aminotransferase (AST), fosfatase alcalina, creatinina e uréia. Para o exame histopatológico foi coletado fígado, rins e pele dos animais, os quais foram processados e analisados em microscopia. Durante o experimento houve diferença significativa entre os grupos nos valores médios de ALT aos 30 (p=0,039) e 45 dias (p=0,0391), AST aos 45 dias (p=0,0000) e creatinina aos 15 (p=0,0169) e 45 dias (p=0,0062). No exame histopatológico, não foram observadas alterações indicativas de toxicidade a nível renal, hepático e cutâneo. Nas condições estudadas não foi demonstrado toxicidade renal ou hepática no uso do etil-cianoacrilato e n-butil cianoacrilato para síntese cutânea.
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Jiang, Yan. "Chromatin Remodeling in Transgenic Mouse Brain: Implications for the Neurobiology of Depression: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/423.
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Histone lysine methylation is an important epigenetic mark for regulation of gene expression and chromatin organization. Setdb1 (Set domain, bifurcate 1), one of the histone lysine methyltransferases, specifically methylates histone H3 at lysine 9 (H3K9) and participates in transcriptional repression and heterochromatin formation. The major task of my thesis work was to investigate the epigenetic roles of Setdb1 in regulating brain functions. I started my thesis work by examining Setdb1 expression pattern during mouse brain development. The most robust signal of Setdb1 was detected in the fetal brains at embryonic day 12.5, with a ubiquitous distribution in all the proliferative zones, as well as the cortical plate and other regions comprised of postmitotic neurons. The expression of Setdb1 decreased as the brain developed, and this down-regulation profile was correlated to neuronal maturation as examined in a primary culture model of mouse cortical neurons. I then generated CK-Setdb1 transgenic mice, in which a myc-tagged full length mouse Setdb1 was constantly expressed in postmitotic neurons under the control of the CaMK II alpha promoter (CK). The expression of mycSetdb1 was detected in NeuN positive cells throughout most forebrain regions including cerebral cortex, striatum and hippocampus. A sustained increase of Setdb1 in CK-Setdb1 transgenics was verified at both mRNA and protein levels. Furthermore, an increase of H3K9 trimethylation was detected at major satellite DNA repeats in CK-Setdb1forebrains, which indicated that transgene-expressed mycSetdb1 was functionally active in adult brains.
The behavioral phenotype of CK-Setdb1 transgenics was examined by using two separate founder lines. Gross neurological functions including body weight, locomotion activity, motor coordination, and breeding behavior were generally normal in CK-Setdb1 mice. CK-Setdb1 mice were further subjected to behavioral paradigms related to mood and cognitive functions. Intriguingly, as compared to the littermate controls, CK-Setdb1 mice represent a lower level of depression as indicated by decreased total immobility in two different behavioral despair tests. Moreover, CK-Setdb1 mice showed an accelerated extinction in the learned helplessness paradigm after a delayed interval (7 days), indicating a faster recovery from an established status of despair. The potential confounding factors, like memory deficits, were ruled out as CK-Setdb1 mice showed normal or even improved performances in different memory-related paradigms. Anxiety scores and stimulant drug response were normal in CK-Setdb1mice. Taken together, these findings suggested that a specific antidepressant-like phenotype was elicited by the over-expression of Setdb1 in adult mice forebrains.
To further study the molecular mechanism underlying Setdb1-associated antidepressant-like behavioral changes, I screened for Setdb1-binding sites in a genome-scale by ChIP-on-chip using a tiling microarray from Affymetrix. Unexpectedly, Setdb1 showed a very restricted binding profile with a high specificity towards ionotropic glutamate receptor genes including the NMDA receptor 2B subunit gene Grin2b, which is a new target for the treatment for major depression. An increase of H3K9 dimethylation at Setdb1-binding site on Grin2b locus was detected in CK-Setdb1 hippocampus, which was correlated to a decrease of Grin2b expression as well as an accelerated desensitization of NMDA receptor. Furthermore, Chromosome Conformation Capture (3C) on Grin2b locus revealed a repressive chromatin loop structure, which tethered the distal Setdb1-binding site (~ 32 Kb downstream of transcriptional start site (TSS)) to a proximal intronic region (~12 Kb downstream of TSS) that is enriched for the binding of KAP1, a well-studied Setdb1-interacting transcriptional corepressor. Taken together, our data indicated that Setdb1 repressed Grin2b expression via H3K9 hypermethylation and higher-order chromatin loop formation, which may contribute to the antidepressant-like phenotype we observed in CK-Setdb1mice.
The second part of my thesis work was to investigate the role of Setdb1 in the animal model of a neurodevelopmental disorder - Rett syndrome (RTT). Loss-of-function mutations of the gene encoding methyl-CpG binding protein 2 (MECP2) is the primary cause of RTT. There is an overlap between Setdb1- and Mecp2-associated repressive chromatin machineries, which both include histone deacetylase complex, H3K9 methyltransferase, DNA methyltransferase and heterochromatin protein 1 (HP1). Moreover, in contrast to Setdb1, which is downregulated during the cortical neuronal differentiation, Mecp2 is upregulated and the expression level is positively correlated to neuronal maturation. Therefore, we hypothesized that there is a functional redundancy between Setdb1 and Mecp2, and the up-regulation of Setdb1 in mature neurons will compensate for brain deficiency due to the loss of Mecp2. To test this hypothesis, I crossed CK-Setdb1 transgenic mice with nestincre-Mecp2 conditional knockout mice (Mecp2-/y). The behavior changes of CK-Setdb1/Mecp2-/y mice, including body weight, locomotion, motor coordination, and life span, were then compared to Mecp2-/y mice. No significant improvements in behaviors or survival were observed from CK-Setdb1/Mecp2-/y mice. Because the activation of CK promoter is limited to defined population of postmitotic neurons in forebrain, I tested our hypothesis by generating another strain of Setdb1 overexpression mice – tauSetdb1, in which the expression of mycSetdb1 is under the control of an endogenous pan-neuronal active promoter Tau. However, the introduction of tauSetdb1 also failed to rescue Mecp2 deficiency. The life span of tauSetdb1/ Mecp2-/y was even shorter as compared to Mecp2-/y mice (Kaplan-Meier, p=0.07). In conclusion, up-regulation of Setdb1 in adult brain was not sufficient to rescue Mecp2deficiency in the mouse model of RTT.
One of the most challenges to study neuronal dysfunctions in brain diseases is the cellular heterogeneity of central nervous system. Current techniques for chromatin studies, including chromatin immunoprecipitation (ChIP) assays, usually lack of single cell resolution and are unable to examine the neurobiological changes in defined cell populations. In the third part of my thesis work, I developed a modified protocol to isolate neuronal nuclei from brain homogenates via Fluorescence-Activated Cell Sorting (FACS). In general, total nuclei was extracted from frozen brains, neuronal nuclei were then immuno-tagged with NeuN and sorted via FACS. Besides the NeuN labeling-FACS protocol, I also generated CK-H2BeGFP transgenic mice, in which a histone H2B-eGFP (enhanced green fluorescent protein) fusion protein was expressed in the nuclei of postmitotic neurons in mouse forebrain. Nuclei extracted from CKH2BeGFP brain were directly applied for FACS sorting. By using this protocol, we routinely got around 6-8 x106neuronal nuclei from one adult mouse forebrain, which was sufficient for ChIP applications followed by single gene PCR and microarray studies. In conclusion, our protocol permits large-scale studies of chromatin modifications or any other nuclei events in defined cell populations from distinct brain regions.
Taken together, my dissertation work will lead to a better understanding of the epigenetic roles of histone H3K9 methyltransferase Setdb1 in brain functions, and may provide new targets for the therapeutic treatment of major depression.
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Nguyen, Nguyen M. "Transgenerational inheritance of increased breast cancer risk in mouse offspring of dams exposed to high fat N-6 polyunsaturated fatty acid diet during pregnancy." Thesis, Georgetown University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10256175.
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Maternal high fat (HF) intake before and/or during pregnancy increases female offsprings’ mammary cancer risk in several preclinical models. Here I studied if maternal HF intake during pregnancy cause transgenerational increase in mammary cancer risk, and if the increase is reversible by treating adult offspring with inhibitors of histone deacetylases (HDAC) or DNA methyltransferases (DNMT).
Pregnant C57BL/6NTac mice were fed either a diet high in n-6 polyunsaturated fatty acids (HF) or control diet (CON). HF diet was given from gestational day (GD) 10 – 20 to target fetal primordial germ cell formation and differentiation to germ cells. Offspring in subsequent F1-F3 generations were only fed CON diet. Mammary tumor incidence, induced by 7,12-dimethylbenz[a]anthracene (DMBA), was significantly higher in F1 and F3 HF offspring, than in the controls. Tumor latency was shorter and burden higher in F1 HF, with similar trends, though not statistically significant, in F3 HF.
RNA-sequencing of normal mammary glands revealed 1587 and 4423 differentially expressed genes between HF and CON offspring in F1 and F3, respectively, of which 48 genes were similarly altered in both generations. Ingenuity Pathway Analysis identified genes associated with Notch signaling as key alterations in HF mammary glands. Knowledge-fused Differential Dependency Network analysis identified 10 node genes in HF offspring uniquely connected to genes linked to increased cancer risk, therapy resistance, poor prognosis, and impaired anti-cancer immunity.
Next, I studied whether HDAC and DNMT inhibitor treatment in adulthood of the offspring, prior to tumor formation, could reverse the increased mammary cancer risk caused by in utero HF exposure. CON and HF offspring were given valproic acid and hydralazine in drinking water (epi-treatment), starting one week after tumor initiation by DMBA. Epi-treatment significantly decreased tumor burden in HF offspring, potentially through reactivation of silenced tumor suppressors CLCA1 and CDKN2A, but adversely affected CON offspring. These adverse effects were linked to upregulation of PERK, p62 and HIF-1α in CON.
In summary, maternal HF intake during pregnancy induced transgenerational increase in offsprings’ mammary cancer risk, causes persistent changes in the expression of genes linked to increased breast cancer risk, and epi-treatment in adulthood may reduce this risk.
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Richard, Bernhard Clemens [Verfasser], Thomas A. [Akademischer Betreuer] Bayer, and Tiago Fleming [Akademischer Betreuer] Outeiro. "In Vitro and In Vivo Studies on Antibodies: N-terminally Truncated Abeta in the 5XFAD Mouse Model / Bernhard Clemens Richard. Gutachter: Thomas A. Bayer ; Tiago Fleming Outeiro. Betreuer: Thomas A. Bayer." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1071991639/34.
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Zalavadia, Ankit. "QUANTITATIVE ANALYSIS OF 5-CHLORO-2-METHOXY-N-[2-(4-SULFAMOYLPHENYL)ETHYL]BENZAMIDE (GLYBURIDE ANALOGUE, GA) IN MOUSE PLASMA AND WHOLE BLOOD USING A MICRO-EXTRACTION AND LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4279.
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Pharmacokinetic evaluation of 5-chloro-2-methoxy-N-[2-(4- sulfamoylphenyl)ethyl]benzamide in mouse plasma demanded for a suitable bioanalytical method. No reported bioanalytical method exists to-date that can quantify concentration of this compound in any biological matrix. The purpose of this study was 1) to develop and validate a new bioanalytical method using a micro-extraction and LC-MS/MS to quantify the target analyte in mouse plasma and 2) to partially validate the method in whole blood. A bioanalytical method was developed and validated in both matrices for a linear concentration range of 2-1000 ng/ml. For both matrices, the reverse predicted concentration of calibration standards (-8.95% to 12.16% and -9.54% to 12.90% respectively) and precision and accuracy (QCs) were within ±15% (%RSD and %BIAS). Four-hour bench top stability and post preparative stability results for plasma and whole blood matrices were within ±15% and ±20% respectively. Blood –plasma concentration correlation co-efficient was 0.9956 with a slope value of 1.018.
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Kvist, A. P. (Ari-Pekka). "Type XIII collagen:organization and chromosomal localization of the mouse gene, distance between human COL13A1 and prolyl 4-hydroxylase α-subunit genes, and generation of mice expressing an N-terminally altered type XIII collagen." Doctoral thesis, Oulun yliopisto, 1999. http://urn.fi/urn:isbn:9514253949.
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Abstract
The complete exon-intron organization of the gene coding for the mouse α1(XIII) collagen chain, Col13a1, was characterized from genomic clones and multiple transcription initiation points were determined. Detailed comparison of the human and mouse genes showed that the exon-intron structures are completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly conserved putative promoter region. The chromosomal location of the mouse gene was determined to be at chromosome 10, band B4, between markers D10Mit5 – (2.3 ± 1.6 cM) – Col13a1 – (3.4 ± 1.9 cM) – D10Mit15.
The location of the genes for both the catalytically important α-subunit of prolyl 4-hydroxylase (P4HA) and human type XIII collagen (COL13A1) were previously mapped to 10q21.3-23.1. Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of peptide-bound proline and plays a crucial role in the synthesis of these proteins. The order and transcriptional orientation of the COL13A1 and P4HA was determined. These two genes were found to lie at tail to tail orientation on chromosome 10 and the distance between these genes was determined to be about 550 kbp.
To study the function of type XIII collagen we used gene targeting in ES cells to generate a mouse line that carries a mutated type XIII collagen gene. Instead of normal protein, mutant mice express type XIII collagen with an altered amino-terminus in which the cytosolic and the transmembrane domains have been replaced with an unrelated sequence. The homozygous mice are fertile and viable but they show alterations in skeletal muscles, mainly wavy sarcolemma and increased variation in muscle fiber diameter. Ultrastructural studies revealed additional abnormalities such as streaming of z-disks, accumulation and enlargement of mitochondria, and disorganized myofilaments. The basement membranes of the muscle cells showed areas of detachment from the plasma membrane and the fibrillar matrix of the cells was less compact than in control animals. Fibroblasts cultured from mutant mice had normal levels of type XIII collagen but exhibited decreased adhesion to substratum which might be explained by a reduced anchoring strength of the altered protein.
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Dietrich, Katharina [Verfasser], Thomas A. [Akademischer Betreuer] Bayer, and Uwe-Karsten [Akademischer Betreuer] Hanisch. "Impact of N-terminally truncated Aß4-42 on memory and synaptic plasticity - Tg4-42 a new mouse model of Alzheimer's disease / Katharina Dietrich. Gutachter: Thomas A. Bayer ; Uwe-Karsten Hanisch. Betreuer: Thomas A. Bayer." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/106504481X/34.
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Villain, Nicolas. "Rôle de la plasticité comportementale dans l'adaptation aux variations nutritionnelles chez un primate malgache." Thesis, Paris, Muséum national d'histoire naturelle, 2017. http://www.theses.fr/2017MNHN0006/document.
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Afin de se maintenir au sein d'un environnement changeant, les individus doivent mettre en place une réponse adéquate. Il est connu que les animaux ont la capacité d'ajuster leur comportement à leur environnement. Cette plasticité comportementale, permet une réponse adaptée et relativement rapide aux variations de l'environnement, maximisant ainsi les chances de survie et de transmission des gènes. Elle met en jeu des processus cérébraux couteux en énergie rendant ces adaptations particulièrement sensibles à des changements alimentaires. Le but de cette thèse a été de mieux comprendre les facteurs qui contraignent ces réponses chez une espèce à laquelle s'applique une forte pression de sélection. Pour ce faire, nous avons étudié les réponses comportementales d'un primate malgache, le microcèbe gris (Microcebus murinus) soumis à des changements dans la quantité ou la qualité des ressources alimentaires disponibles. La première partie de ce travail s'est intéressée aux effets à court terme d'une restriction alimentaire sans malnutrition. Cette partie comprenait deux études. La première s'intéressant aux effets d'une restriction alimentaire à 60% sans malnutrition sur la plasticité comportementale innée via l’étude de l'horloge biologique. Les résultats de cette étude montrent une diminution de la capacité à se resynchroniser après un décalage horaire en lien avec la perte de poids. Ainsi, les individus perdant le plus de poids sont le moins à même de se resynchroniser sur les cycles lumineux après un décalage horaire de 6 heures. La seconde s'intéressait aux effets d'une restriction alimentaire de 40% sans malnutrition sur la plasticité comportementale acquise et montre une diminution de la capacité d'apprentissage des individus restreints après 19 jours de traitement alimentaire sans influence sur la mémoire à long terme. La moindre capacité d’apprentissage chez les individus en restriction calorique est corrélée à la perte de poids, les individus perdant le plus de poids étant les moins performants. Dans une seconde partie j’ai étudié l'effet de modifications qualitatives de l'alimentation à travers une supplémentation à long terme des individus en acides gras polyinsaturés n-3. Cette partie m’a permis de mettre en évidence une amélioration des performances d'apprentissage chez les individus supplémentés après 18 mois de traitement alimentaire accompagnée d'une diminution de l'anxiété et d'une augmentation de la neurogenèse adulte dans trois zones cérébrales.Ces travaux démontrent que les variations nutritionnelles, qu’elles soient quantitatives ou qualitatives sont capables d’influencer les différentes formes de plasticités comportementales et donc les grandes fonctions cérébrales et constituent ainsi un paramètre clé dans l’adaptation et la survie des individusIn order to survive in a changing environment, individuals have to express an appropriate response. It is known that animals have the ability to adjust their behaviour to their environment. This behavioural plasticity allows a quick and adapted response to environmental variations, maximizing the individual'ssurvival and gene transmission. This plasticity relies on costly brain processes making these adaptations particularly dependent of food availability and maybe quality.This thesis project aimed at better understanding the constraints of these responses in a species under a strong selection pressure. To investigate this problematic, we studied the behavioural responses of a small Malagasy primate, the grey mouse lemur (Microcebus murinus), to both quantitative and qualitative changes in food resources. The first part of this work investigated the effect of a short-term caloric restriction without malnutrition over two studies. In the first one, we studied the effects of a 60% caloric restriction without malnutrition on innate behavioural plasticity via the study of the biological clock. The results show a decrease in the ability to resynchronize on a light/dark cycle following a time-shift. This difficulty to resynchronize was linked to body mass loss, the individuals loosing the more weight being the one unable to resynchronize after the 6-hours time shift. In the second study, we investigated the effect of a 40% caloric restriction without malnutrition on acquired behavioural plasticity. This study show a decrease in learning abilities of the restricted individuals after 19 days of dietary treatment and no influence on long term memory. This decrease in learning abilities was also linked with body mass loss, with the individuals loosing the more weight being the one with the worst success rate during this task. The second part focused on the effects of a qualitative variation in food supply via a long-term supplementation with n-3 polyunsaturated fatty acids. This part allowed us to show an increase in learning abilities associated with increased neurogenesis in three brain zones for supplemented animals after 18 month of treatment as well as a decrease of their anxiety level.This thesis work show that both quantitative and qualitative nutritional variations are able to influence different forms of behavioural plasticity and their cerebral basis and are of particular importance in the adaptation and survival of individuals
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Allouche, Ahmad. "Validation fonctionnelle d'approches nutritionnelles à allégation "Bien veillir", capables de prévenir le vieillissement cérébral et les maladies neurodégénératives." Thesis, Université de Lorraine, 2012. http://www.theses.fr/2012LORR0316/document.
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La maladie d'Alzheimer (MA) est une démence neurodégénérative caractérisée par des pertes de la mémoire et des troubles cognitifs. La toxicité des oligomères solubles du peptide [bêta]-amyloïde (A[bêta]Os) est centrale dans la phase précoce de la maladie. L'absence de traitements curatifs, l'aspect chronique des mécanismes pathogéniques et le partage de facteurs de risque communs avec les pathologies cardiovasculaires, notamment les paramètres alimentaires et le métabolisme lipidique, doivent inciter à considérer l'intérêt de méthodes préventives permettant d'empêcher l'apparition de la maladie. Dès lors, l'approche nutritionnelle apparaît comme une stratégie capable de limiter sa prévalence. Une souris modèle des stades précoces de MA nous a permit d'évaluer le potentiel préventif d'une alimentation enrichie en acide docosahexaénoïque (DHA, C22:6, n-3). Les résultats obtenus montrent qu'un apport alimentaire suffisant en DHA conduit à en enrichir les membranes dans les différentes structures cérébrales. En conséquence, les fonctions synaptiques sont préservées, même après exposition aigüe aux A[bêta]Os, ce qui maintient les capacités cognitives des souris exposées. Nous avons ensuite développé des stratégies nutritionnelles permettant d'évaluer les effets bénéfiques de DHA contre la dyslipidémie induite par l'alimentation et les déficits cognitifs associés au vieillissement normal ou pathologique. Les résultats obtenus à l'issue de notre étude révèlent qu'un apport alimentaire en DHA est capable de prévenir la dyslipidémie provoquée par un régime riche en lipides et de retarder le déclin cognitif lié au vieillissement cérébral normal ou pathologiqueAlzheimer's disease is a neurodegenerative aging-related dementia that is characterized by loss of memory and cognitive disorders. Toxicity of soluble oligomers of [beta]-amyloid peptide (sOA[beta]) is a key element in early stages of the disease. Absence of curative therapies, chronic aspects of the pathogenic mechanisms implicated and influence of common risk factors shared with the cardiovascular diseases, including dietary parameters and lipid metabolism, should widely encourage considering the interest of preventive interventions allowing slowing the progression and delaying the clinical onset of Alzheimer's-related troubles. Therefore, nutritional approaches could appear as a strategy able to reduce the prevalence of this disease. Early Alzheimer's mouse model allowed us to assess the preventive potential of diets supplemented in docosahexaenoic acid (DHA, C22:6 n-3). Our results show that adequate dietary intake of DHA lead to increased levels in different brain structures. Consequently, hippocampal and cortical synaptic functions were preserved, even upon acute exposure to A[beta] oligomers, maintaining or improving the cognitive capacities of A[beta]-exposed mice. These improvements were positively correlated with DHA-enrichment associated in hippocampus and with preserved synaptic integrity. We also designed nutritional strategies in order to evaluate the beneficial effects of DHA on diet-induced dyslipidemia as well as on cognitive impairment and neurodegenerative processes associated with normal or pathologic aging. Our results show that dietary DHA can prevent high-fat diet-induced dyslipidemia and delay cognitive decline related with normal or pathological aging
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Kakarla, Raghavi. "LIQUID CHROMATOGRAPHY - MASS SPECTROMETRIC ANALYSIS OF CLINICALLY AND PHARMACOLOGICALLY RELEVANT MOLECULES." Cleveland State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu1576196451854014.
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Royo, Julie. "Performances cognitives et neurogenèse au cours du vieillissement chez un primate non-humain." Thesis, Paris, Muséum national d'histoire naturelle, 2020. http://www.theses.fr/2020MNHN0001.
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La neurogenèse correspond à la capacité du cerveau à former de nouveaux neurones. Ce mécanisme permet d’induire des changements structurels et fonctionnels dans le cerveau pouvant atténuer le déclin cognitif observé avec l’âge. Cette neuroplasticité persiste à l’âge adulte mais diminue au cours de la vie. Au cours de ce travail, nous avons caractérisé l’évolution des fonctions cognitives et de la neurogenèse avec l’âge chez le Microcèbe (Microcebus murinus) qui présente des changements morphologiques, comportementaux et physiologiques similaires à ceux observés chez l’Homme au cours du vieillissement. Nous avons pu montrer qu’une partie des animaux âgés présentaient une diminution de leurs capacités cognitives tandis que d’autres ont conservé des performances à un niveau équivalent à celui des individus jeunes. Ce maintien des fonctions cognitives avec l’âge pourrait être dû en partie au processus de neurogenèse. En effet, au niveau de la zone sous-ventriculaire, la balance neurone/glie serait en faveur de la neurogenèse dans la partie dorsale tandis que l’oligodendrogenèse serait favorisée dans la corne. La stimulation de la neurogenèse pourrait permettre de remplacer les neurones lésés avec l’âge ou détruits par un traumatisme. Parmi les stratégies possibles pour stimuler ce mécanisme, l’alimentation et l’activité physique apparaissent être des interventions pertinentes. Au cours de ce travail de thèse, nous nous sommes intéressés, en particulier, à l’impact d’une supplémentation en acides gras polyinsaturés n-3 ainsi qu’à la combinaison de la restriction calorique et de l’activité physique à l’âge adulte. Ces interventions ont induit une amélioration des fonctions cognitives associée à une hausse du nombre de nouveaux neurones. Ces différentes approches constituent donc une stratégie non médicamenteuse prometteuse afin de lutter contre le déclin des fonctions cognitives au cours du vieillissement en participant à la plasticité cérébraleNeurogenesis is the ability of the adult brain to build new neurons. This process induces structural and functional changes in the brain that can reduce cognitive decline during aging. This neuroplasticity exists throughout life but it gradually decreases with aging. In this study, we characterized the evolution of cognitive functions and neurogenesis during aging in the grey mouse lemur (Microcebus murinus) that shares morphological, behavioural and physiological changes with aged humans. We observed that some aged animals presented a specific deficit in learning and memory whereas others had cognitive performances equivalent or better than young animals. It might be due to the neurogenesis process that would preserve cognitive functions during aging. Indeed, in the subventricular zone, the balance between neurons and glial cells would be in favour of neurogenesis in the dorsal part while oligodendrogenesis would be favoured in the horn. Stimulation of neurogenesis could help replace neurons lost due to injury or aging. Among the possible strategies to stimulate neurogenesis, food and physical activity seem pertinent. During this thesis project, we studied, in particular, the impact of n-3 polyunsaturated fatty acid supplementation and the combination of caloric restriction and physical activity in adulthood. These interventions induced an improvement of cognitive functions associated with an increase in the number of new neurons. These different approaches constitute a promising strategy without drugs against cognitive decline during aging by participating in brain plasticity
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Rosales, Gerpe María Carla. "The Role of APOBEC3 in Controlling Retroviral Spread and Zoonoses." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31484.
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APOBEC3 (A3) proteins are a family of host-encoded cytidine deaminases that protect against retroviruses and other viral intruders. Retroviruses, unlike other viruses, are able to integrate their genomic proviral DNA within hours of entering host cells. A3 proteins hinder retroviral infectivity by editing retroviral replication intermediates, as well as by inhibiting retroviral replication and integration through deamination-independent methods. These proteins thus constitute the first line of immune defense against endogenous and exogenous retroviral pathogens. The overall goal of my Master's project was to better understand the critical role A3 proteins play in restricting inter- and intra-host transmission of retroviruses. There are two specific aspects that I focused on: first, investigating the role of mouse APOBEC3 (mA3) in limiting the zoonotic transmission of murine leukemia retroviruses (MLVs) in a rural environment; second, to identify the molecular features in MLVs that confer susceptibility or resistance to deamination by mA3. For the first part of my project, we collected blood samples from dairy and production cattle from four different geographical locations across Canada. We then designed a novel PCR screening strategy targeting conserved genetic regions in MLVs and Mouse Mammary Tumor Virus (MMTV) and MMTV-like betaretroviruses. Our results indicate that 4% of animals were positive for MLV and 2% were positive for MMTV. Despite crossing the species barrier by gaining entry into bovine cells, our study also demonstrates that the bovine A3 protein is able to potently inhibit the spread of these murine retroviruses in vitro. The next question we asked was whether mA3 could also mutate and restrict murine endogenous retroviruses and thereby partake in limiting zoonotic transmission. Moloney MLV and AKV MLV are two highly homologous murine gammaretroviruses with opposite sensitivities to restriction by mA3: MoMLV is resistant to restriction and deamination while AKV is sensitive to both. Design of MoMLV/AKV hybrid viruses enabled us to map the region of mA3 resistance to the region encoding the glyco-Gag accessory protein. Site-directed mutagenesis then allowed us to correlate the number of N-linked glycosylation sites with the level of resistance to deamination by mA3. Our results suggest that Gag glycosylation is a possible viral defence mechanism that arose to counteract the evolutionary pressure imposed by mA3. Overall, my projects show the important role A3 proteins play in intrinsic immunity, whether defending the host from foreign retroviral invaders or endogenous retroviral foes.
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Lloyd, Gemma. "Morse theory for invariant functions and its application to the n-body problem." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506273.
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Ruiz, Camila Mariana. "Sobre a topologia das singularidades de Morin." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/55/55135/tde-12012016-155424/.
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Neste trabalho, nós abordamos alguns resultados de T. Fukuda e de N. Dutertre e T. Fukui sobre a topologia das singularidades de Morin. Em particular, apresentamos uma nova prova para o Teorema de Dutertre-Fukui [2, Theorem 6.2], para o caso em que N = Rn, usando a Teoria de Morse para variedades com bordo. Baseados nas propriedades de um n-campo de vetores gradiente (∇ f1; : : : ∇fn) de uma aplicação de Morin f : M → Rn, com dim M ≥ n, na segunda parte deste trabalho, nós introduzimos o conceito de n-campos de Morin para n-campos de vetores que não são necessariamente gradientes. Nós também generalizamos o resultado de T. Fukuda [3, Theorem 1], que estabelece uma equivalência módulo 2 entre a característica de Euler de uma variedade diferenciável M e a característica de Euler dos conjuntos singulares de uma aplicação de Morin definida sobre M, para o contexto dos n-campos de Morin.In this work, we revisit results of T. Fukuda and N. Dutertre and T. Fukui on the topology of Morin maps. In particular, we give a new proof for Dutertre-Fukui\'s Theorem [2, Theorem 6.2] when N = Rn, using Morse Theory for manifolds with boundary. Based on the properties of a gradient n-vector field (∇ f1; : : : ∇ fn) of a Morin map f : M → Rn, where dim M ≥ n, in the second part of this work, we introduce the concept of Morin n-vector field for n-vector fields V = (V1; : : : ; Vn) that are not necessarily gradients. We also generalize the result of T. Fukuda [3, Theorem 1], which establishes a module 2 equivalence between Euler\'s characteristic of a manifold M and Euler\'s characteristic of the singular sets of a Morin map defined on M, to the context of Morin n-vector fields.
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Rodriguez, Rodriguez Marisol. "Feminismo e innovaci��n en la narrativa gallega de autor��a femenina: Xohana Torres, Mar��a Xos�� Queiz��n, Carmen Blanco y Teresa Moure." Thesis, University of Auckland, 2009. http://hdl.handle.net/2292/5836.
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This study examines the importance and impact of the literary careers and works of four Galician women writers from two different generations within specific cultural and historical contexts: the last years of the Franco regime and Spain's ensuing democracy. Its purpose is to unravel the way in which their corpus, from deeply seated feminist convictions, contributes to the renovation of the genre of narrative. This genre has always been characterized in Galicia by its slow and anomalous development, given that there are very few female narrative writers until the 1980s. However, as will be seen, the evolution of the genre will bear fruit in a qualitative and quantitative increase in female narrative writers in the new millennium. The first generation consists of Xohana Torres (Santiago de Compostela, 1931-) and Mari��a Xose�� Queiza��n (Vigo, 1939-), who begin to publish under the Franco regime. The second is that composed by Carmen Blanco (Lugo, 1954-) and Teresa Moure (c. 1969-), who initiate their literary careers in the democratic period, publishing their narrative works from 2004 onwards. An analysis of their narrative production will reveal how innovative and original it was at the time of publication. This innovation is evident in different aspects, such as the introduction of subversive themes and characters, original structures or the use of other literary genres, such as the fairy tale and feminized quest romance. I will examine how Torres, who publishes her only novel in 1971, articulates a strong critique of Francoism and its basic tenets through her adolescent protagonist, Maxa, to reclaim a rural Galicia destroyed by capitalist exploitation. In Queiza��n's novels, controversial themes, such as motherhood, paedophilia and alternative sexualities, have led to the silencing of her works. As for Blanco's texts, they rewrite traditional fairy tales to effect a penetrating analysis of Galicia, reviewing its past and present. Such recreations proclaim the deep yearning for a libertarian dream in which Galicia might be an egalitarian place for all men and women. Finally, Moure's narrative defends the 'nature' of women, in keeping with her ecofeminist stance. For all these feminist writers, the equation of women with an autonomous Galicia is the fundamental thread woven through the entirety of their work.
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Divín, Jan. "Měření směrových charakteristik antén." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2011. http://www.nusl.cz/ntk/nusl-218898.
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This Master’s Thesis is dealing with measuring antennas in far field. Especially with automation this measure, direction sensors made by optical mouse, Integrated Hall ICs for Linear and Off-Axis Rotary Motion Detection and remote unit this workplace, which can made communication with PC by USB. It describes the making control program for the PC.
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Athamena, Ahmed. "N-méthylation de la Phosphatidyléthanolamine, une voie métabolique aux fonctions énigmatiques : caractérisation de la voie dans la moule Mytilus galloprovincialis et rôle physiologique au cours de l’osmorégulation chez les crustacés marins." Thesis, Lyon 1, 2011. http://www.theses.fr/2011LYO10115.
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Les fonctions physiologiques spécifiques de la voie de N-méthylation de la phosphatidyléthanolamine (PE), une des deux voies de biosynthèse de la phosphatidylcholine (PC), restent relativement énigmatiques. Il a été démontré chez les poissons euryhalins qu’un stress hyperosmotique induisait une activation de cette voie métabolique au niveau hépatique. L’objectif de notre travail était de vérifier si ce phénomène se produit aussi chez d’autres animaux euryhalins. Les études réalisées in vivo sur deux espèces de crâbes, Eriocheir sinensis et Carcinus maenas, nous ont permis de montrer que l’acclimatation en eau de mer de ces animaux active la synthèse de PC par N-méthylation de la PE dans l’hépatopancréas. Les marquages radioisotopiques montrent aussi que cette PC est échangée avec le plasma et que ce phénomène est amplifié chez les animaux en eau de mer. Ce pool de PC est utilisé comme précurseur de la bétaïne, un osmoeffecteur organique important chez ces animaux. Nous avons ensuite caractérisé la voie de N-méthylation de la PE chez un animal osmoconformeur, la moule Mytilus galloprovincialis. Les résultats, obtenus in vivo et in vitro sur les tissus isolés, démontrent qu’une activité de N-méthylation de la PE en PC est exprimée dans la glande digestive et les hémocytes circulant de M. galloprovincialis. La PC ainsi synthétisée dans ces tissus est échangée avec l’hémolymphe de l’animal. De l’ensemble de ces observations, nous pouvons conclure que la synthèse de PC par N-méthylation est largement exprimée chez les animaux marins euryhalins et qu’une des fonctions physiologiques de cette voie métabolique est de synthétisée des osmolytes organiques comme la bétaïneThe specific physiological functions of the N-methylation of phosphatidylethanolamine (PE), one of the two biosynthetic pathways of phosphatidylcholine (PC), remain relatively mysterious. It has been demonstrated in euryhaline fish that hyperosmotic stress induced activation of this pathway in the liver. The aim of our work was to verify whether this phenomenon also occurs in other euryhaline animals. In vivo studies on two species of crabs, Eriocheir sinensis and Carcinus maenas, showed that seawater acclimation activates PC synthesis by N-methylation of PE in the hepatopancreas. Radioisotopic labelling also showed that PC is exchanged with the plasma and that this phenomenon is amplified in animals in seawater. This pool of PC is used as a precursor of betaine, an important organic osmoeffector in these animals. We then characterized the process of PE N-methylation in an osmoconforming animal, the mussel Mytilus galloprovincialis. The results, obtained in vivo and in vitro on isolated tissues, show that N-methylation of PE to PC is expressed in the digestive gland and circulating haemocytes in M. galloprovincialis. The PC synthesized in these tissues is exchanged with hemolymph of the animal. From all these observations, we conclude that the synthesis of PC by N-methylation is widely expressed in marine euryhaline animals and that a physiological function of this pathway is to provide organic osmolytes such as betaine
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Derrien, Danièle. "Modulateur de la réponse immune : ciblage par des polymères glycosyles reconnus par les lectines membranaires des macrophages murins." Orléans, 1988. http://www.theses.fr/1988ORLE2037.
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Realisation d'un vecteur reconnu par les lectrines membranaires des macrophages et destine a transporter le n-acetylmuramyl dipeptide vers ces cellules. L'efficacite de ce vecteur est etudiee in vitro et in vivo sur des modeles murins. L'activation du macrophages a ete evaluee par la cytostase exercee vis-a-vis des cellules tumorales et par la secretion d'interleukine 1 et de facteur de necrose tumorale
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Strehl, Britta Katharina. "Struktur und Funktion der 20S Proteasomen aus Organen Listeria monocytogenes infizierter Mäuse." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2005. http://dx.doi.org/10.18452/15276.
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Das Proteasomensystem der Zelle ist für die Degradation von Proteinen verantwortlich und spielt eine zentrale Rolle bei der Generierung von Epitopen, die auf MHC-Klasse-I Molekülen den cytotoxischen T-Lymphozyten (CTLs) präsentiert werden. Die Stimulation von Zellen mit Interferon-gamma (IFNgamma) führt zu der Bildung von Immunoproteasomen, die im Vergleich zu den konstitutiven Proteasomen eine verbesserte Generierung vieler MHC-Klasse-I Epitope aufweisen. In gesunden Mäusen werden Immunoproteasomen vorwiegend in den lymphatischen Geweben exprimiert, wohingegen nicht-lymphatische Gewebe hauptsächlich konstitutive Proteasomen enthalten. In der vorliegenden Arbeit wurde der Einfluss der Listeria monocytogenes Infektion auf die aus der Leber, der Milz, dem Dünndarm und dem Colon stammenden murinen 20S Proteasomen untersucht. Die Struktur der isolierten 20S Proteasomen wurde mittels zweidimensionaler Gelelektrophorese und Westernblot ermittelt, während die Funktion durch in vitro Prozessierung von drei oligomeren Peptidsubstraten analysiert wurde. Die Prozessierungsprodukte wurden mittels HPLC-ESI-Ionenfalle massenspektrometrisch identifiziert sowie quantifiziert. Die vorliegende Arbeit zeigt zum ersten Mal, dass nach einer Infektion die aus den nicht-lymphatischen Organen und Zellen isolierten 20S Proteasomen eine strukturelle und funktionelle Plastizität aufweisen: Nach der Infektion wurde die Bildung von Immunoproteasomen induziert, was mit der gesteigerten Generierung der immunrelevanten Fragmente korreliert werden konnte. Dies verlief unabhängig von der direkten Präsenz von Listeria monocytogenes in den Organen und wurde ausschließlich durch das Cytokin IFNgamma reguliert. Es konnte außerdem eine Zunahme der posttranslationalen Modifikation von Leberproteasomen mit dem Monosaccharid N-Acetylglucosamin nach der Infektion nachgewiesen werden. Des Weiteren wurde eine detaillierte Analyse der massenspektrometrischen Daten hinsichtlich des Schnittverhaltens der konstitutiven und Immunoproteasomen etabliert. Die Auswertung ergab, dass die Immunoproteasomen nach der Infektion durch schnellere und veränderte Nutzung bestehender Spaltstellen an der verbesserten Epitoppräsentation beteiligt sind.The proteasome system of the cell is responsible for the degradation of proteins and plays a central role in the generation of epitopes which are presented to cytotoxic T-lymphocytes (CTLs) on MHC-class-I molecules. The stimulation of cells by interferon-gamma (IFNgamma) leads to the formation of immunoproteasomes that show an improved generation of many MHC-class-I epitopes compared to constitutive proteasomes. In healthy mice, immunoproteasomes are mainly expressed in the lymphatic tissues, whereas the non-lymphatic organs predominantly contain constitutive proteasomes. In this project the effect of Listeria monocytogenes infection on murine 20S proteasomes derived from the liver, spleen, small intestine and colon were investigated. The structure of the isolated proteasomes was analyzed by two-dimensional gel electrophoresis and western blots while the function was studied by in vitro processing of three oligomeric peptide substrates. Identification and quantification of the processing products was performed by HPLC-ESI-ion trap mass spectrometry. The project showed for the first time, that after infection 20S proteasomes isolated from non-lymphatic organs as well as from non-lymphatic cells displayed structural and functional plasticity: immunoproteasomes were induced post infection which could be correlated with the enhanced generation of immuno-relevant fragments. This was independent of the direct presence of Listeria monocytogenes in the organs and solely controlled by the cytokine IFNgamma. In addition, an increased posttranslational modification with the monosaccharide N-acetylglucosamine could be detected in liver-derived proteasomes after infection. Furthermore, a detailed analysis of the mass spectrometry data was established according to the cleavage site usage of constitutive and immunoproteasomes. The result was that immunoproteasomes are involved in improved generation of the immuno-relevant fragments by the faster cleavage and the changed usage of existing cleavage sites after infection.
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Jean, Bruno. "Un polymere thermosensible a l'interface eau-air : interaction avec les tensioactifs et stabilisation de films minces." Paris 6, 2000. http://www.theses.fr/2000PA066230.
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Ce travail concerne l'etude de l'interface entre l'air et une solution aqueuse contenant a la fois des chaines d'un polymere neutre thermosensible qui precipite au-dela de tc 32 \c, le poly(n-isopropylacrylamide) (pnipam), et des molecules tensioactives de dodecyl sulfate de sodium (sds). Separement, ces deux especes s'adsorbent de maniere forte et spontanee aux interfaces hydrophile/hydrophobe. De plus, en volume, a partir d'une concentration critique en sds appelee cac, elles interagissent entre elles pour former des complexes polymere-tensioactif. Grace a la reflectivite des neutrons, les differentes structures adoptees par ce systeme mixte a l'interface air-solution en fonction de la temperature et de la concentration en sds ont ete mises en evidence. En particulier, il a ete montre que pour le polymere seul et a des concentrations en sds inferieures a la cac, la densite d'adsorption du polymere augmente fortement avec la temperature et n'est pas affectee par la presence de tensioactifs. Au-dela de la cac, l'adsorption du polymere diminue avec la concentration en sds. Cette desorption progressive du polymere est due a l'interaction en volume entre le pnipam et le sds. Dans le cadre general de la comprehension de la stabilite des mousses, des films minces de mousses formes a partir de solutions aqueuses de pnipam avec et sans sds ont ete etudies par la technique de thin film balance. Les resultats montrent que, sous certaines conditions, il est possible de former des films metastables de tres grande epaisseur et remarquablement homogenes a partir de solutions de pnipam. Ces films sont stabilises par les interactions repulsives entre queues de polymere adsorbees sur les interfaces du film. L'addition de sds entraine un diminution importante de l'epaisseur des films. Dans tous les cas, avec et sans tensioactifs, il existe une correlation directe entre l'epaisseur des films de mousse et la quantite de pnipam adsorbee a l'interface libre.
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Hsu, Ren-Jun, and 許仁駿. "Establishment and analysis of (CAG)n transgenic mouse model." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/06769567736631251094.
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碩士中山醫學大學
醫學研究所
91
Among human trinucleotide repeat disorders, the most frequent triplets found to be expanded are CAG and its complementary sequence, CTG. CAG repeats are almost always found in coding regions whereas CTG expansions are located in untranslated regions (UTRs). Previously we showed that expanded CTG and CAG repeat in the 3’-UTR of GFP both had pathogenic effect in transgenic C. elegans. To investigate the possible pathogenic effect of untranslated (CAG)n trinucleotide repeat in mammals, we made transgenic mice expressing a muscle-specific transcript with (CAG)200 inserted in the 3’-UTR of EGFP gene. The long tract of CAG repeat was found to cause reduced protein expression of EGFP as evidenced by direct fluorescence and western blotting. Histological analysis of the muscle sections revealed atypical muscle cell morphology as well as abnormal staining patterns of succinate dehydrogenase and NADH activity in (CAG)200 mice. Furthermore, mice expressing expanded CAG repeat exhibited a slight deviation in muscle tension recording, suggesting a sign of low muscle activity. By RNA fluorescent in situ hybridization, the muscle cells of (CAG)200 mice were shown to contain nuclear retentions of transcripts, or nuclear foci. Further analysis demonstrated that the expression of muscle differentiation markers, MyoD, Myf5, myogenine and CHCR, were upregulated in the muscle cells of adult (CAG)200 mice. These effects were specific to CAG repeat because mice expressing EGFP RNA without CAG repeat did not differ from the nontransgenic mice in any aspects as mentioned above. This result is the first to show that the expanded CAG repeat in UTR could cause pathogenic effects in mice and suggested that disorders linked to untranslated CAG repeat expansion may exist in human.
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Lommel, Silvia [Verfasser]. "Conditional inactivation of N-WASP in the mouse : analyses of N-WASP function / von Silvia Lommel." 2002. http://d-nb.info/964801922/34.
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Li, Pei-Tzu, and 李倍慈. "The physiological role of mouse b1,4-N-acetylgalactosaminyl transferase in uterus." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/76441207496126264289.
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博士國立清華大學
生物資訊與結構生物研究所
100
Glycosylation is a fundamentally important modification in reproductive physiology, including embryo implantation. The B4galnt2 encodes an enzyme, the Sda β-1,4-N-acetylgalactosaminyltransferase II (β4GalNAcT-II, B4galnt2), which catalyzes the GalNAc linking to the Gal of the NeuAcα2-3Galβ terminal structure via the β-1,4 linkage and forms the sda antigen. In the reproductive system, sda antigen has been found in association with glycoproteins, which are specifically related with pregnancy, such as bovine pregnancy-associated glycoproteins (PAGs), zona pellucida glycoprotein 3 (Zp-3) and glycodelin (Gd). Via hormone treatment in vitro and promoter reporter assay, this study realized that B4galnt2 was positively regulated by progesterone (P4) and negatively regulated by estrogen (E2) in the uteri. These results coincided with in vivo observation. During pregnancy, B4galnt2 is significantly expressed on the intracellular portion of uterine tissue for high levels of P4 at E3.5 and E10.5. This study suggested that the uteri could provide Sda modified protein(s) or lipid(s) on the surface of uterine epithelia at these moments. Using siRNA assay to reduce the B4galnt2 expression in vivo, the reduction of embryonic numbers in uteri revealed the importance of this gene in embryo implantation and development. Furthermore, Bgalnt2 was found to locate on the surface and intracellular portion of E3.5 blastocysts, via confocal microscopy observation. The suppression of blastocyst adhesion by the anti-B4galnt2 antibody and lectin, Dolichos biflous agglutinin (DBA), in vitro and in vivo, was demonstrated by the involvement of B4galnt2 in embryonic early implantation. These results suggested that B4galnt2 on the blastocyst membrane surface may be associated with the sda antigen containing protein(s) or lipid(s) on the uterine epithelium. In addition, the sda antigen on the blastocyst membrane surface bound the receptor/acceptor on the endometrial surface to permit early embryo implantation jointly.
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Lin, Shih-Wei, and 林仕韋. "Functional characterization of β-1,4-N-acetyl-galactosaminyl transferase Ⅱ protein in mouse ovary." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/24249444616208476461.
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碩士國立臺灣大學
生化科學研究所
101
Sda β-1,4-N-acetyl-galactosaminyl transferase Ⅱ(β4galNAcTⅡ,B4GALNT2 in human and B4galnt2 in mouse) catalyze the addition of GalNAc to the Gal of the [Neu5Acα2-3]Galβ1-4GlcNAcβ1 -3Gal terminal structure via the β-1,4 linkage to form Sda antigen. The Sda antigen was first reported as a human blood surface antigen, and in the later researches, it was found in several tissue types, including stomach, colon, kidney, and oocytes. Its presence was also demonstrated in various body fluids, such as saliva, milk, serum and urine. Current studies revealed that Sda antigen reduced remarkably in cancer lesions of the gastrointestinal tract. It indicated the significance of this antigen in biosystem and also associated it is importance to B4galnt2. In 2003, Dell’s group found Sda antigen in reproductive system firstly, however, the significance of Sda is still unclear. My research intends to approach biological characteristics of B4galnt2, and on it, can investigate physiological role of Sda in reproductive system. Female ICR mouse is selected as an animal model. B4galnt2 expression was detected in ovary, and Sda antigen was further confirmed in oocyte but not in cumulus cell. Based on that, Immunohistochemical analysis was used to investigate B4galnt2 distribution in whole cumulus-oocyte complexes (COCs) , and the B4galnt2 existing on plasma membrane of cumulus cell was observed. Blocking the COC cultivation with B4galnt2 antibody resulted in disturbing of cumulus expansion, and it indicated that B4galnt2 protein is essential for cumulus expansion, which is a critical step during oogenesis. Cumulus expansion depends on both FSH in the environment and B4galnt2 protein that exist on the cell membrane of cumulus cell, and may be associated with cell migration ability. B4GALNT2 silencing of HCT116 with transfecting the plasmid contained B4GALNT2-siRNA caused the cell migration slower, but blocking with antibody made no difference, and the similar result was obtained from the two other colonic cell-CaCo2 and SW480 in antibody blocking test.
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Lu, Po-Hsun, and 呂柏勳. "Functional test of β-1,4-N-acetyl-galactosaminyl transferase 2 in mouse sertoli cell." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/40824461573505875544.
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碩士國立臺灣大學
生化科學研究所
101
Sda β-1,4-N-acetyl-galactosaminyl transferaseⅡis a kind of carbohydrate transferases, whose main function is to catalyze and transfer GalNAc to Gal of [Neu5Acα2-3]Galβ1-4GlcNAcβ1-3Gal through β-1,4 bond, so that carbohydrate is linked to form the Sda structure. The structure was earliest found in the antigen structure on the blood corpuscle surface; in fact, however, it is widely distributed in the tissues and body fluids of the mammals, including stomach, intestine, kidney, serum, urine, and milk. Recent studies showed that the B4GALNT2 expression was obviously reduced in the gastric cancer and colon cancer cells of human being, causing decrease in the Sda antigen, and the said reduction was closely correlated with the metastasis of the cancer cells; this indicated the importance of B4GALNT2 and its correlation with the bio-system. However, very few studies have been conducted on its physiological functions. In 2003, the research team led by Dell first found the Sda structure in the female reproductive system. Before the physiological functions of the Sda structure was learned about, its invertase B4galnt2 needed to be studied. The research showed that B4galnt2 was correlated with the mouse’s embryo implantation and the maturation of its oocytes. It was presumed that the Sda structure played an important role in the female reproductive system. Therefore, to infer the role of the Sda structure, the regulation and control by the Sda invertase should be learned. The laboratory has found B4galnt2 in the cell strains (TM4) of the Sertoli cells, so it was supposed that B4galnt2 might play a certain role in the male reproductive system, but its functions in the male reproductive system remained unknown yet. In the experiment, a male mouse was taken as the subject. The Quantitative Real-Time PCR and immune fluorescent staining method were used to confirm the existence of B4galnt2 on the cell membranes of the primary Sertoli cells and Sertoli cell strains. The observation indicated that whether the proliferation of the Sertoli cells was declined or enhanced after the cell strains were treated with follicle stimulating hormone, the B4galnt2 gene expression showed no marked difference. In this way, it was presumed that B4galnt2 did not participate in the proliferation of the Sertoli cells. By using hormone to treat the Sertoli cell strains, the Sertoli cells might be induced into the state of maturity. The maturity of the Sertoli cells could be confirmed by the mature marker genes and post-maturation physiological features. In the process of maturation, the B4galnt2 gene expression was increased. The RNAi suppress gene expression experiment showed that when the B4galnt2 expression was reduced, the maturation marker gene expression decreased accordingly. This showed that the Sertoli cells could not enter the state of maturity. To sum up, it was presumed that B4galnt2 participated in the maturation of the Sertoli cells. In addition, the experiment on the primary Sertoli cells showed that the B4galnt2 expression in the mouse coming close to the puberty was higher than that when it was born. When the immature primary Sertoli cells were induced to be mature, the B4galnt2 gene expression in them was raised. Thus, it was presumed that B4galnt2 might participate in the maturation of the Sertoli cells in the mouse.
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Anderson, Nicole Marie. "Generation of Mouse Models of Human Hematopoietic Disease and their Use to Analyze Hematopoietic Development and Function." Thesis, 2012. http://hdl.handle.net/1807/33872.
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Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen.
The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS.
The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder.
A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.
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Li, Ying Shiuan, and 李映萱. "Use a Glycine N-Methyltransferase knockout mouse model to study the hepatocarcinogenesis of Aflatoxin B1." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/34479176690172174867.
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碩士國立陽明大學
公共衛生研究所
95
Primary hepatocellular carcinoma (HCC) is one of the common malignancies in the world. Its risk factors include hepatitis viruses B and C infections, dietary exposure to aflatoxins and alcoholism. Glycine N methyltransferase (GNMT) is an enzyme with multiple functions. It not only regulates the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine, but also serves as a folate-binding protein in the liver cytosol. Besides, we found that it can bind benzo (a) pyrene as well as aflatoxin B1 (AFB1) and reduces DNA adducts formation (Cancer Res.64:3617-3623, 2004; manuscript in preparation). Since its expression was down-regulated in HCC, the goals of this study were 1) to study the tumorigenesis effect of AFB1 in a Gnmt knockout mouse model; 2) to compare the gender differences of susceptibility to AFB1 treatment using a Gnmt knockout mouse model; 3) to analyze the gene expression profiles of the liver from animals treated with AFB1. A Gnmt knockout (KO) mouse model, developed in our laboratory was used for this study. Both male and female wildtype (Wt) and homozygous KO (Gnmt -/-) mice were challenged twice with AFB1 on the 7th day (10ug per body weight in gram) and at the 9th weeks (40ug) of age intraperitoneally. Another two groups of mice were challenged with solvent-tricaprylin. The liver tumorigenesis of all the experimental groups were regularly monitored using ultrasound (US) and magnetic resonance imaging (MRI) examinations. All eight groups of mice were sacrificed at the age of 3 and 6 months old and their liver were used for pathological studies and gene profiling analysis. The results showed that hepatomegaly was observed in the male Wt mice treated with AFB1 at 3 and 6 months of age. All Gnmt -/- mice treated with solvent had hepatomegaly at 3 and 6 months of age while only 50% (2/4) of Gnmt -/- mice treated with AFB1 had hepatomegaly at 3 months of age. For the female mice, AFB1 treatment did not induce hepatomegaly in Wt mice while both AFB1 and solvent groups of Gnmt -/- mice developed hepatomegaly at 3 and 6 months of age. Elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels correlate with the hepatomegaly in different groups. At 3 months, no obvious pathological changes were observed in Wt mice in the solvent group. In animals treated with AFB1, inflammation, enlarged nuclei and hyper-chromatic staining (anisonucleosis) were observed both in Wt and Gnmt -/- mice. In addition, fatty degenerative changes were found in 5 of 6 female Gnmt -/- mice at 6 months old which was less obvious in male Gnmt -/- mice and none was observed in wildtype mice of both gender. Through using ultrasound and magnetic resonance imaging examinations, small nodules were detected in all (5/5) female and 50.0% (4/8) male Gnmt -/- mice in the treatment groups at the age of 13 -14 month. In contrast, no nodules were observed in Wt of both genders at the same age. All mice carrying nodules larger than 0.6cm were sacrificed for pathological examinations and RNA analysis. Four markers; AFP, glypican-3, survivin, and LYVE1 were also applied to confirm pathology definitions. We will continue monitoring the tumorigenesis of all the mice. In addition, the gene expression profiles of the liver from animals treated with AFB1 will be analyzed using microarray and real-time PCR in the near future.
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Richard, Bernhard Clemens. "In Vitro and In Vivo Studies on Antibodies - N-terminally Truncated Abeta in the 5XFAD Mouse Model." Doctoral thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-0022-6000-1.
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Chen, Shih-Hui, and 陳詩蕙. "Persistent Hepatitis B Viral Replication in an FVB/N Mouse Model: Impact of Host and Viral Factors." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/68204592483401377584.
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博士國立臺灣大學
微生物學研究所
100
The mechanism underlying the chronicity of hepatitis B virus (HBV) infection has long eluded researchers. The mechanism has remained unclear largely due to the lack of an animal model that can support persistent HBV replication and allow for the investigation of the relevant immune responses. In this study, we used hydrodynamic injection to introduce HBV replicon DNA into the livers of three different mouse strains, BALB/c, C57BL/6, and FVB/N. Interestingly, we found that an HBV clone persistently replicated in the livers of FVB/N mice for up to 50 weeks but was rapidly cleared from the livers of BALB/c and C57BL/6 mice. Flow cytometric analysis and quantitative reverse transcription PCR analysis of the mouse livers indicated that after DNA injection, FVB/N mice had few intrahepatic activated cytotoxic T lymphocytes (CTLs) and produced low levels of alanine aminotransferase, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and the CXCL9 and CXCL10 chemokines. These responses were in sharp contrast to those observed in BALB/c and C57BL/6 mice, reflecting a strong correlation between the degree of liver inflammation and viral clearance. Mutational analysis further demonstrated that a change from Asn-214 to Ser-214 in the HBV surface antigen permitted the clearance of the persistent HBV clone in FVB/N mice, and the response was accompanied by increased levels of activated CTLs and upregulated liver expression of IFN-γ, CXCL9, and CXCL10. The model was demonstrated to be useful for the in vivo evaluation of the efficacies of various anti-HBV drugs. Supplementary expression of CXCL9, CXCL10 and C5a in the carrier mice enhanced the clearance of the chronic infection. These results indicate that the heterogeneity of the host factors and viral sequences may influence the immune responses against HBV. An inadequate activation of immune or inflammatory responses can lead to persistent HBV replication in vivo.
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Jao, Jo-Hui, and 饒若卉. "Purification and Crystallization of the N-terminal domain of Mouse Tumor-necrosis factor receptor associated factor 6." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/d9s2qr.
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碩士國立臺北科技大學
有機高分子研究所
96
Tumor-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is an adaptor protein involved in interleukin-1 receptor (IL-1) and toll-like receptor (TLR) induced activation of nuclear factor κB (NF-kB). TRAF6 contains two domains, C- and N-terminal domains, which associate with other adaptor proteins of IL-1R/TLR pathway. Crystal structure of TRAF6 C-domain has been reported but lack of N-domain which contains a RING finger domain and several zinc finger motifs. In order to understand the protein-protein interaction, we plan to determine crystal structures of the TRAF6 N-domain residues 1~350 (TRAF6N), and the full-length TRAF6. The aim of this study was to clone DNA, protein expression, purification, and crystallization. Our results show that the E. coli expression of TRAF6N and TRAF are in suspension (70%) and inclusion body, respectively. Recombinant TRAF6N was purified by 30% ammonium sulfate salt-out method and followed a glutathione-S-transferase (GST)-affinity chromatography. TRAF6N protein was concentrated to 5.2 mg/ml and crystallized by the hanging-drop vapor diffusion method.