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1
Kennedy, Zachary C. "Optimizing CRISPR/Cas9 for Gene Silencing of SOD1 in Mouse Models of ALS." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1047.
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Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.
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Abens, Ryan. "GENE EXPRESSION OF CYTOKINES AND OXIDATIVE STRESS MARKERS IN CTRP3 TRANSGENIC MICE WITH CHRONIC ETHANOL EXPOSURE." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/asrf/2019/schedule/2.
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Oxidative stress and inflammation are often linked to the prognosis of diseases caused by chronic alcohol consumption. Chronic alcohol consumption plays a key role in brain tissue damage, often leading to the development of cognitive disorders and loss of brain function. In addition to the direct effects of alcohol on brain function, consumption of alcohol can lead to psychosocial stressors such as legal, financial, and interpersonal problems. It has been found that mice that overexpress C1q/Tumor Necrosis Factor-related protein-3 (CTRP3) and exposed to ethanol daily do not die like the mice who did not overexpress CTRP3 and fed the same diet. Although the specific physiological functions regulated by the CTRP family are largely unknown, there is evidence showing that they have diverse biological effects on inflammation, metabolism, and survival signaling in several different types of tissue. Postmortem brain tissue samples were collected from mice that were exposed to ethanol with transgenic overexpression of CTRP3 and from wild type mice that were only exposed to ethanol. Interestingly, previous immunoblotting of the cerebellum and the hippocampus using collected tissue demonstrated that glia activation was present in the CTRP3 overexpressing mice but not in the wild-type ethanol fed mice. This finding suggests that glia cells are either dying in the ethanol fed wild type mice or that CTRP3 protects and prolongs activated glia cells. The current study will determine if markers of oxidative stress and cell viability are altered in the CTRP3 overexpressing mice when compared to wild-type mice at the molecular level. RNA isolation using the Directzol system and cDNA synthesis using punch dissected homogenate tissue collected from the hippocampus was used for this investigation. Gene expression of BDNF, SOD1 and PARP1 in mouse tissue was determined using quantitative PCR. Immunoblotting of a small number of hippocampal tissue using PARP1 was performed. The mice that were CTRP3 overexpressed and fed ethanol will likely exhibit altered gene expression of cytokines and increased oxidative stress gene expression in postmortem hippocampal brain tissue when compared to wild-type ethanol fed mice. The current studies could contribute to the body of knowledge for the development of novel therapies that may alleviate the neuro-inflammatory effects of alcohol use.
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Baker, David. "Gene expression profiling and functional studies of astrocytes in SOD1-related amyotrophic lateral sclerosis." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/9173/.
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Amyotrophic Lateral Sclerosis (ALS) is the most common adult onset motor neuron (MN) disorder, characterised by muscle wasting due to MN death. Astrocytes play an important role in disease progression in the SOD1G93A transgenic mouse model and patients of ALS. Although astrocytes display a selective toxicity to MN, the toxic factor(s) have not been identified. We hypothesise that differential gene expression in SOD1-ALS astrocytes will reveal targets for therapeutic intervention. Microarray analysis was performed upon Laser Capture Microdissected astrocytes isolated from spinal cord of symptomatic (90 day) and late-stage (120 day) SOD1G93A mice and non-transgenic (NTg) littermates, and from post-mortem human SOD1-ALS and control spinal cord. Functional studies were performed using enzymatic activity assays and immunohistochemistry upon spinal cord and in vitro validation studies were performed using murine neonatal cultures of astrocytes, microglia and embryonic MNs and “i-astrocytes” directly converted from human ALS fibroblasts. In murine astrocytes annotation clustering analysis showed increased expression of lysosomal transcripts which were validated by qPCR . Using functional experiments, we have found significantly higher activity of the lysosomal enzyme β hexosamindase in the spinal cord of SOD1G93A mice. Immune response and phagocytic pathways are also enriched within both datasets, and phagocytosis assays using fluorescently labelled NSC34 cell debris show that SOD1G93A astrocytes engulf significantly higher amounts of neuronal debris compared to NTg controls, highlighting an increased reactivity of astrocytes at symptom onset. Human SOD1-astrocytes show down-regulation of transcripts involved in tight junction formation such as ZO-2, Claudin-5 and Occludin, and we hypothesise that ALS-astrocytes contribute to the breakdown in blood-brain-barrier (BBB) integrity seen in ALS. qPCR confirmed differential expression of BBB-influencing genes such as claudin-5, junctional adhesion molecule 2 and transforming growth factor beta-2. Model BBBs made with i-astrocytes from human patients co-cultured with endothelia show significantly lower transendothelial electrical resistance and dextran-permeability values pointing to an astrocyte role in increased BBB permeability during disease. These studies show the breadth of behaviours displayed by astrocytes during ALS disease progression and will provide an important guide for future therapeutic intervention.
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Likhite, Shibi B. "Therapeutic suppression of mutant SOD1 by AAV9-mediated gene therapy approach in Amyotrophic Lateral Sclerosis." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417394084.
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Stoica, Lorelei I. "Gene Therapy for Amyotrophic Lateral Sclerosis: An AAV Delivered Artifical MicroRNA Against Human SOD1 Increases Survival and Delays Disease Progression of the SOD1G93A Mouse Model: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/813.
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About ten percent of cases are inherited, of which twenty percent are due to mutations in the superoxide dismutase 1 (SOD1) gene. Since the only FDA approved ALS drug prolongs survival by just a few months, new therapies for this disease are needed. Experiments in transgenic ALS mouse models have shown that decreasing levels of mutant SOD1 protein alters and in some cases entirely prevents disease progression. We explored this potential therapeutic approach by using a single stranded AAV9 vector encoding an artificial microRNA against human SOD1 injected bilaterally into the cerebral lateral ventricles of neonatal SOD1G93A mice. This therapy extended median survival from 135 to 206 days (a 50% increase) and delayed hind limb paralysis. Animals remained ambulatory until endpoint, as defined by a sharp drop in body weight. Treated animals had a reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons. As compared to untreated SOD1G93A mice, the AAV9 treated mice also had significant improvements in multiple parameters including the number of motor neurons, diameter of ventral root axons, and degree of neuroinflammation in the spinal cord. These studies clearly show that an AAV9-delivered artificial microRNA is a translatable therapeutic approach for ALS.
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Scarrott, Joseph. "Investigating the specificity of RNAi molecules in human gene therapy for SOD1-linked familial amyotrophic lateral sclerosis." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/22558/.
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20% of familial amyotrophic lateral sclerosis (fALS) cases are caused by mutations in the gene encoding the cytosolic protein human Cu/Zn superoxide dismutase 1 (hSOD1). RNA interference (RNAi) technology offers the therapeutic potential for the treatment of SOD-linked fALS by reducing the burden of pathogenic mutant SOD1 protein. Translation of this gene therapy strategy to the clinic requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to determine treatment efficacy, successful target gene reduction, and correct dosing. Using self-complementary adeno-associated virus serotype 9 (scAAV9) to deliver RNAi against hSOD1 in the SOD1G93A mouse model, the work presented in this thesis demonstrates that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. Using a panel of purposefully designed RNAi constructs cloned into the scAAV9 backbone this is, to our knowledge, the first study to demonstrate no significant in vitro off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. This study also reports the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, this data provides further confidence in the safety of the clinical therapeutic vector.
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Oliveira, Yonara Monique da Costa. "Avalia??o do status antioxidante, express?o g?nica e polimorfismos dos genes SOD1, SOD2 e GPx1 em crian?as, adolescentes e adultos jovens com diabetes tipo 1." Universidade Federal do Rio Grande do Norte, 2012. http://repositorio.ufrn.br:8080/jspui/handle/123456789/13503.
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Previous issue date: 2012-02-27Studies report that the pathophysiological mechanism of diabetes complications is associated with increased production of Reactive Oxygen Species (ROS)-induced by hyperglycemia and changes in the capacity the antioxidant defense system. In this sense, the aim of this study was to evaluate changes in the capacity of antioxidant defense system, by evaluating antioxidant status, gene expression and polymorphisms in the genes of GPx1, SOD1 and SOD2 in children, adolescents and young adults with type 1 diabetes. We studied 101 individuals with type 1 diabetes (T1D) and 106 normoglycemic individuals (NG) aged between 6 and 20 years. Individuals with type 1 diabetes were evaluated as a whole group and subdivided according to glycemic control in DM1G good glycemic control and DM1P poor glycemic control. Glycemic and metabolic control was evaluate by serum glucose, glycated hemoglobin, triglycerides, total cholesterol and fractions (HDL and LDL). Renal function was assessed by measurement of serum urea and creatinine and albumin-to-creatinine ratio (ACR) in spot urine. Antioxidant status was evaluate by content of reduced glutathione (GSH) in whole blood and the activity of erythrocyte enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). We also analyzed gene expression and gene polymorphisms of GPx1 (rs1050450), SOD1 (rs17881135) and SOD2 (rs4880) by the technique of real-time PCR (Taqman?). Most individuals with DM1 (70.3%) had poor glycemic control (glycated hemoglobin> 8%). Regarding the lipid profile, individuals with type 1 diabetes had significantly elevated total cholesterol (p <0.001) and LDL (p <0.000) compared to NG; for triglycerides only DM1NC group showed significant increase compared to NG. There was an increase in serum urea and RAC of individuals with DM1 compared to NG. Nine individuals with type 1 diabetes showed microalbuminuria (ACR> 30 mg / mg). There was a decrease in GSH content (p = 0.006) and increased erythrocyte GPx activity (p <0.001) and SOD (p <0.001) in DM1 group compared to NG. There was no significant difference in the expression of GPx1 (p = 0.305), SOD1 (.365) and SOD2 (0.385) between NG and DM1. The allele and genotype frequencies of the polymorphisms studied showed no statistically significant difference between the groups DM1 and NG. However, the GPx1 polymorphism showed the influence of erythrocyte enzyme activity. There was a decrease in GPx activity in individuals with type 1 diabetes who had a polymorphic variant T (p = 0.012). DM1 patients with the polymorphic variant G (AG + GG) for polymorphism of SOD2 (rs4880) showed an increase in the RAC (p <0.05). The combined data suggest that glucose control seems to be the predominant factor for the emergence of changes in lipid profile, renal function and antioxidant system, but the presence of the polymorphisms studied may partly contribute to the onset of complications
Estudos relatam que o mecanismo fisiopatol?gico das complica??es do diabetes est? associado ao aumento na produ??o de Esp?cies Reativas de Oxig?nio (ERO) induzido pela hiperglicemia persistente e altera??es na capacidade de defesa do sistema antioxidante. Nesse sentido, o presente estudo teve como objetivo avaliar altera??es na capacidade de defesa do sistema antioxidante, atrav?s da avalia??o do status antioxidante, express?o g?nica e pesquisa de polimorfismos nos genes da GPx1, SOD1 e SOD2 de crian?as, adolescentes e adultos jovens com Diabetes Mellitus tipo 1 (DM1). Foram estudados 101 indiv?duos com diabetes tipo 1 (DM1) e 106 indiv?duos normoglic?micos (NG) com idade entre 6 e 20 anos. Os indiv?duos com DM1 foram avaliados como um grupo total e subdivididos de acordo com o controle glic?mico em DM1NC diab?tico n?o-compensado e DM1C diab?ticos compensados. Para avaliar o controle glic?mico e metab?lico foram realizadas as dosagens de glicose s?rica, hemoglobina glicada, triglicer?deos, colesterol total e fra??es (HDL e LDL). A fun??o renal foi avaliada pelas dosagens de ureia e creatinina s?ricas e a rela??o albumina/creatinina (RAC) urin?ria. Os par?metros antioxidantes avaliados foram o conte?do da glutationa reduzida (GSH) em sangue total e a atividade eritrocit?ria das enzimas glutationa peroxidase (GPx) e super?xido dismutase (SOD). Tamb?m foi avaliada a express?o g?nica e a pesquisa dos polimorfismos dos genes GPx1 (rs1050450), SOD1(rs17881135) e SOD2 (rs4880) pela t?cnica da PCR em tempo real (Taqman?). A maioria dos indiv?duos com DM1 (70,3%) apresentou controle glic?mico insatisfat?rio (hemoglobina glicada >8%). Em rela??o ao perfil lip?dico, indiv?duos com DM1 apresentaram valores significativamente elevados de colesterol total (p<0,001) e LDL (p<0,000) em rela??o ao NG; para os triglicer?deos s? o grupo DM1NC apresentou aumento significante em rela??o ao NG. Observou-se o aumento na ur?ia s?rica e na RAC dos indiv?duos com DM1 em rela??o ao NG. Nove dos indiv?duos com DM1 apresentaram microalbumin?ria (RAC> 30 μg/mg). Houve diminui??o no conte?do de GSH (p=0,006) e aumento na atividade eritrocit?ria da GPx (p<0,001) e SOD (p<0,001) do grupo DM1 em rela??o ao NG. N?o foi observada diferen?a significante na express?o de GPx1 (p=0,305), SOD1 (0,365) e SOD2 (0,385) entre NG e DM1. As freq??ncias genot?picas e al?licas dos polimorfismos estudados n?o mostraram diferen?a estatisticamente significante entre os grupos DM1 e NG. Por?m o polimorfismo da GPx1 mostrou influ?ncia na atividade eritrocit?ria da enzima, observando-se diminui??o da atividade nos indiv?duos com DM1 que possu?am a variante polim?rfica T (p=0,012). J? para o polimorfismo Ala16Val da SOD2 observou-se eleva??o da RAC para aqueles indiv?duos diab?ticos que possu?am o alelo G (p<0,05). O conjunto dos dados sugere que o controle glic?mico parece ser o fator predominante para o surgimento de altera??es no perfil lip?dico, fun??o renal e no sistema antioxidante, por?m a presen?a dos polimorfismos estudados possam, pelo menos em parte, contribuir para o aparecimento de complica??es
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Malaspina, Andrea. "Amyotrophic lateral sclerosis (ALS) : analysis of differential gene expression in spinal cord and study of the Cu/Zn superoxide dismutase gene (SOD1) in familial ALS cases (FALS) of Italian origins." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248425.
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Andrade, Alice Tavares Reis 1977. "Evolução do gene sodC nas bactérias naturalmente transformáveis Neisseria meningitidis e Haemophilus influenzae." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314453.
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Orientador: Marcelo LancellottiDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Em 1998, foi relatada a transferência lateral do gene sodC do gênero Haemophilus para a espécie Neisseria meningitidis. Sabe-se que, nestes dois grupos a dinâmica deste gene é bastante distinta. Este trabalho tem por objetivo estimar árvores filogenéticas que possam apontar qual a espécie do gênero Haemophilus compartilhou o gene sodC com a espécie N. meningitidis. Testes de seleção positiva foram empregados no intuito de avaliar quais forças evolutivas estão subjacentes ao processo de diversificação molecular do gene nestas espécies ao longo do tempo. Além disso, foi realizada uma modelagem protéica computacional por homogia para avaliar quais substituições de aminoácidos tinham impacto no processo adaptativo da enzima nas espécies consideradas. Ao se reconstruir uma filogenia para o gene sodC, foi constatado que a origem deste gene na espécie H. influenzae é distinta. Um grupo de linhagens recebeu o gene, provavelmente por transferência lateral, da espécie H. haemolyticus, enquanto o outro grupo recebeu o gene da espécie H. parainfluenzae. Neste grupo, o gene sofreu pseudogeneização. Foi observado também que as sequências de N. meningitidis agrupam com as sequências que compartilham um ancestral comum com a espécie H. haemolyticus, porém as sequências do meningococo formam um ramo distinto dentro deste clado. Dada à alta clonalidade das sequências de N. meningitidis, foi constatado que o evento de transferência lateral de genes foi muito recente na escala do tempo. O teste de seleção positiva demonstrou que seleção positiva está atuando especificamente no ramo da árvore que compartilha um ancestral comum com a espécie H. haemolyticus, através da modificação de uma alanina por uma serina na posição 72, embora a nota geral da árvore tenha sido menor que 1. Sabe-se que pseudogenes, por não codificarem uma proteína ativa e, portanto, por não estarem sob nenhum tipo de restrição funcional, estão sob uma ação maior da deriva genética. Portanto, diferentes forças evolutivas estão governando a evolução deste gene nas espécies consideradas. A modelagem protéica concluiu que tal modificação contribuiu para o aumento do potencial redox do sítio ativo. Desta forma, a ação da seleção positiva sob um único resíduo de aminoácido foi benéfica para a função da enzima como um todo
Abstract: In 1998, it was reported the lateral transfer of the sodC gene from the genus Haemophilus to Neisseria meningitidis. It is known that this two groups show a quite distinct dynamics of this gene. This study aims to estimate phylogenetic trees that might point to which species of the genus Haemophilus shared the sodC gene with N. meningitidis. In addition, tests of positive selection were employed in order to assess which evolutionary forces are governing the process of molecular diversification of the gene in these species through time. Moreover, we performed a computational protein modeling by homology to asses which amino acids substitutions had an impact on the adaptative process of the enzyme in the species considered. A phylogeny of the sodC gene was reconstructed and it was found that this gene in H. influenzae has two different origins. A group of lineages has received the gene, probably by lateral transfer, from H. haemolyticus, whereas the other group has received the gene from H. parainfluenzae. In the latter, the gene has become a pseudogene. It was also observed that the sequences from N. meningitides group together with those sequences that share a common ancestor with H. haemolyticus, but they form a distinct branch within this clade. Given the high clonality of the sequences from N. meningitidis, it was found that the lateral gene transfer event is very recent in the time scale. A test of positive selection showed that positive selection is acting specifically in the branch that shares a common ancestor with H. haemolyticus through the substitution of an alanine to a serine at position 72, though the overall score of the tree is less than one. It is known that pseudogenes do not encode active proteins and therefore they are not under any kind of functional constraints, so they are under greater influence of genetic drift. Thus, it was concluded that different forces are driving the evolution of this gene in the species considered here. Protein modeling concluded that this modification contributed to the increase in the redox potencial of the active site. Thus the action of positive selection under a single amino acid residue was beneficial to the function of the enzyme as whole
Mestrado
Bioquimica
Mestra em Biologia Funcional e Molecular
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Turner, Taylor Brian. "Cloning and Characterization of the Salt Overly Sensitive 1 (SOS1) Gene in Chenopodium quinoa WILLD." BYU ScholarsArchive, 2007. https://scholarsarchive.byu.edu/etd/1023.
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Salt tolerance is a commercially important trait that affects plant species around the globe. Cellular response to saline environments is a well studied but complex system that is far from being completely understood. The SALT OVERLY SENSITIVE 1 (SOS1) gene is a critical component of salt tolerance in many species, encoding a plasma membrane Na+/H+ antiporter that plays an important role in germination and growth in saline environments. Here we report a preliminary investigation of salt tolerance in quinoa (Chenopodium quinoa Willd.). Quinoa is a halophytic grain crop of the Chenopodiaceae family with impressive nutritional content and an increasing world-wide market. Many quinoa varieties have impressive salt tolerance characteristics and research suggests quinoa may utilize novel mechanisms to confer salt tolerance. At this time there is no published data on the molecular characteristics of those mechanisms. We report the identification and sequencing of the SOS1 gene in quinoa, including a full length cDNA sequence of 3490 bp and a full length genomic clone of 21314 bp. Sequence analysis predicts the quinoa SOS1 homolog spans 23 exons and is comprised of 3474 bp of coding sequence (excluding the stop codon). Introns comprise 17840 bp of the genomic clone and range in size from 77 to 2123 bp. The predicted protein contains 1158 amino acid residues and aligns closely with SOS1 homologs of other species. The quinoa SOS1 homolog contains two putative domains, a Nhap cation-antiporter domain and a cyclic-nucleotide binding domain. Sequence analyses of both cDNA fragments and intron fragments suggest that two SOS1 loci are present in the quinoa genome that are likely orthologous loci derived from the ancestral diploid genomes of the modern allotetraploid quinoa genome. This report represents the first molecular characterization of a putative salt-tolerance gene in C. quinoa.
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Turner, Taylor B. "Cloning and characterization of the Salt Overly Sensitive 1 (SOS1) gene in Chenopodium quinoa WILLD /." Diss., CLICK HERE for online access, 2007. http://contentdm.lib.byu.edu/ETD/image/etd2005.pdf.
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Costa, Felipe. "INFLUÊNCIA DO POLIMORFISMO ALA16VAL DO GENE DA ENZIMA SUPERÓXIDO DISMUTASE (SOD2) NO EFEITO ANTIOXIDANTE IN VITRO DO CITRATO DE CLOMIFENO." Universidade Federal de Santa Maria, 2011. http://repositorio.ufsm.br/handle/1/8972.
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To investigate the in vitro antioxidant properties of the ovulation induction drug,
Citrate clomiphene (CC), and to access whether its effects are influenced by the
Ala16Val polymorphism in the SOD2 gene, which encodes mitochondrial manganese
superoxide dismutase (SOD), an in vitro experimental study testing the effect of different
concentrations of CC on antioxidant capacity, reactive oxygen species (ROS) production
and peripheral blood mononuclear cells (PBMC) culture viability was performed. A total
of 58 healthy adult women were genotyped for the Ala16Val SOD2 polymorphism, and
blood samples were collected to perform in vitro experiments analyzing the biological
antioxidant effects of CC. Free radical production and cytotoxicity assays were
performed on blood and peripheral blood mononuclear cells (PBMCs) with different
Ala16Val SOD2 genotypes. According to the observations described here, CC exhibited
antioxidant effects. Additionally, the CC treatments led to a decrease in ROS production,
with blood samples from the AA genotype displaying a more responsive antioxidant
effect from CC than other genotypes. AA and AV PBMCs showed an increase in
viability following treatment with 10 μM CC when compared with PMBCs from control
groups. In the VV PBMC group, only the 5 μM and 10 μM CC treatments presented a
significant positive viability effect. The CC exhibits antioxidant activity, similar to that
observed with other Selective Estrogen Receptor Modulators (SERMs), in the presence
of the Ala16Val-SOD2 polymorphism suggesting pharmacogenetic effect.Para investigar in vitro as propriedades antioxidantes de drogas que induzem a ovulação, como citrato de clomifeno (CC) e verificar se os efeitos são influenciados pelo polimorfismo Ala16Val do gene da SOD2, o qual codifica a superóxido dismutase (SOD) mitocondrial dependente de manganês. Um estudo experimental in vitro foi conduzido testando o efeito de diferentes concentrações de CC sobre a capacidade antioxidante, produção de espécies reativas de oxigênio (EROs) e na viabilidade de células mononucleadas de sangue periférico (CMSP) em cultura celular. Um total de 58 mulheres adultas saudáveis foram genotipadas para o polimorfismo Ala16Val do gene da SOD2, e sangue foi coletado para realizar os experimentos in vitro e analisar os efeitos antioxidantes biológicos do CC. A produção de radicais livres e análise de citotoxicidade foram conduzidas em sangue e CMSP com diferentes genótipos do Ala16Val SOD2. De acordo com as observações descritas aqui, o CC exibiu um efeito antioxidante. Adicionalmente, os tratamentos com CC levaram a um decréscimo na produção de EROs, com amostras de sangue o genótipo AA desenvolveu um efeito antioxidante mais responsivo para o CC do que os outros genótipos. A cultura de CMSP dos genótipos AA e AV mostraram um aumento na viabilidade seguida do tratamento com 10μM CC quando comparada com as culturas CMSP do grupo controle. No grupo de cultura CMSP do genótipo VV, somente os tratamentos com 5μM e 10μM de CC apresentaram efeito positivo na viabilidade. O CC apresentou uma atividade antioxidante similar à observada com outros moduladores seletivos dos receptores de estrogênio (SERMs). Entretanto, essa atividade foi influenciada pelos diferentes genótipos do polimorfismo Ala16Val SOD2 sugerindo efeito farmacogenético.
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Parikh, Suchi Vipin. "Ependymin peptide mimetics that assuage ischemic damage increase gene expression of the anti-oxidative enzyme SOD." Link to electronic thesis, 2003. http://www.wpi.edu/Pubs/ETD/Available/etd-0429103-132144.
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Alves, Aleandro Geraldo. "MUTAÇÕES DO GENE SOD-1 (SUPERÓXIDO DISMUTASE 1) NA FORMA FAMILIAR DA ESCLEROSE AMIOTRÓFICA LATERAL: REVISÃO SISTEMÁTICA." Pontifícia Universidade Católica de Goiás, 2011. http://localhost:8080/tede/handle/tede/2361.
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Previous issue date: 2011-08-11Amyotrophic lateral sclerosis (ALS) is a multifactorial disease that affects motor neurons. In most cases, the disease is sporadic, however, 5 to 10% of patients have a familial history (FALS). Among patients with FALS, 12 to 23% present with mutations in the SOD1 gene. Objectives: To present a systematic review about the mutations described in SOD1 gene in patients with FALS. Methods: The databases used in this study included PubMed, ISI Web of Science and Cochrane Library Virtual Health. After reading the abstracts, 71 articles were selected and systematically reviewed on this study. Results: The largest number of publications was found in 1997, and Japan was the country with the majority of published studies on the subject, with 23 articles. The majority of the mutations were described in éxons four and five of SOD1 gene, and A4V, I113T, I144F, D90A and L38V were the most commonly mutation described. More than 156 mutations in the SOD1 gene have been cataloged in patients with ALS-F and these data are deposited in ALS GENETICS ONLINE DATABASE, a database that contains specific information on mutations associated with amyotrophic lateral sclerosis. However, the articles reviewed in this study described 103 mutations. Conclusions: Several mutations in the SOD1 gene have been described in patients with ALS-F, however, the relationship between such mutations and the pathogenesis of ALS-F remains unclear, as well as the relationship between mutations and disease progression. Further studies are necessary in order to better explain such relationship.
A esclerose amiotrófica lateral (EAL) é uma doença multifatorial que afeta os neurônios motores. Na maioria dos casos, a doença é esporádica, entretanto, 5 a 10% dos pacientes apresentam história familiar (EAL-F). Dentre os pacientes com EAL-F, 12 a 23% apresentam mutações no gene SOD1. O objetivo deste trabalho foi realizar uma revisão sistemática acerca das mutações descritas no gene SOD1 em pacientes com EAL-F. As bases de dados consultadas incluíram Pubmed, ISI Web of Science e Cochrane Biblioteca Virtual em Saúde. Após a revisão dos resumos, 71 artigos foram selecionados descrevendo mutações no gene SOD1 em pacientes com EAL-F. O ano que apresentou o maior número de publicações foi 1997 e o Japão foi o país que mais publicou sobre o assunto, aparecendo em 23 artigos. O maior número de mutações foi descrito nos éxons 4 e 5 do gene SOD1 e as mutações A4V, I113T, I144F, D90A e L38V foram as mais comumente citadas. Até o momento 156 mutações no gene SOD1 já foram catalogadas em pacientes com EAL-F e esses dados encontram-se depositados no ALS ONLINE GENETICS DATABASE, um banco de dados que contém informações específicas sobre mutações associadas à esclerose amiotrófica lateral. Entretanto, os artigos revisados neste estudo descrevem 103 destas mutações. As causas relacionadas às mutações no gene SOD1 permanecem incertas, assim como a relação entre tais mutações e a evolução da doença, portanto, muito ainda deve ser estudado acerca desse tema.
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Jarvis, David. "Functional and Evolutionary Analysis of Cation/Proton Antiporter-1 Genes in Brassicaceae Adaptation to Salinity." Diss., The University of Arizona, 2013. http://hdl.handle.net/10150/312652.
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The accumulation of salts in soil is an important agricultural problem that limits crop productivity. Salts containing sodium (Na⁺) are particularly problematic, as cytosolic Na⁺ can interfere with cellular metabolism and lead to cell death. Maintaining low levels of cytosolic Na⁺, therefore, is critical for plant survival during growth in salt. Mechanisms to regulate Na⁺ accumulation in plant cells include extrusion of Na⁺ from the cell and sequestration of Na⁺ into intracellular compartments. Both of these processes are controlled in part through the action of Na⁺/H⁺ exchangers belonging to the Cation/Proton Antiporter-1 (CPA1) gene family. Genes belonging to this family have been identified in both salt-sensitive and salt-tolerant species, suggesting that salt-tolerant species may have evolved salt tolerance through modification of these existing pathways. The research presented here has focused on understanding how salt tolerance has evolved in Brassicaceae species, and particularly on the role that CPA1 genes have played in the adaptation to salinity of Eutrema salsugineum. Specific projects have sought to understand 1) how copy number variation and changes in coding sequences of CPA1 genes contribute to salt tolerance in E. salsugineum and its salt-tolerant relative Schrenkiella parvula, 2) whether functional or regulatory changes in Salt Overly Sensitive 1 (SOS1) from E. salsugineum (EsSOS1) contribute to its enhanced salt tolerance, and 3) whether accessions of Arabidopsis thaliana differ significantly in their response to salt stress.The results indicate that EsSOS1 and SOS1 from S. parvula (SpSOS1) both confer greater salt tolerance in yeast than SOS1 from A. thaliana (AtSOS1) when activated by the complex of the SOS2 kinase and SOS3 calcium-binding protein, whereas only EsSOS1 confers enhanced salt tolerance in the absence of activation. When expressed in A. thaliana, EsSOS1 also confers greater salt tolerance than AtSOS1 through regulatory changes that likely involve differences in expression pattern. Together, the results presented here suggest that mechanisms regulating cellular Na⁺ accumulation that exist in salt-sensitive crop species could be altered to enhance growth in salty soils. In addition, the 19 A. thaliana accessions used to create the MAGIC population were shown to differ significantly in their response to salt stress.
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Pessoa, Carine Ribeiro. "Caracterização e análise da expressão de genes (sod3 e ccp) envolvidos no estresse oxidativo em Paracoccidioides brasiliensis." reponame:Repositório Institucional da UnB, 2006. http://repositorio.unb.br/handle/10482/3534.
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O fungo Paracoccidioides brasiliensis é dimórfico e patogênico ao homem. Ele é o agente etiológico da paracoccidioidomicose (PCM), a micose sistêmica de maior incidência da América Latina. O estabelecimento da infecção é dependente da transição de micélio para levedura e este processo in vitro é reversível e regulado pela mudança de temperatura de 22 °C para 36 °C. O projeto ¿Genoma funcional e diferencial do fungo Paracoccidioides brasiliensis¿ identificou 6.022 PbAEST, que correspondem a genes expressos no fungo, representando aproximadamente 80% do genoma do fungo. Na interação patógeno-hospedeiro, a resposta eficiente do patógeno contra o ataque oxidativo imposto pelas células fagocitárias do hospedeiro facilita a sua sobrevivência no hospedeiro. Os genes potencialmente envolvidos na resposta ao estresse oxidativo em P. brasiliensis, identificados no projeto transcriptoma, foram categorizados em 4 classes: enzimas antioxidantes (12 PbAESTs), biossíntese e metabolismo da glutationa e regeneração de NADPH (11 PbAESTs), homeostase de íons metálicos (3 PbAEST) e fatores transcricionais (7 PbAESTs). Entre as seqüências anotadas no transcriptoma, foram estudadas neste trabalho aquelas que codificam para a CuZn superóxido dismutase GPI-ancorada (PbSOD3) e a citocromo c peroxidase (PbCCP). A superóxido dismutase retira do ambiente radical superóxido produzindo peróxido de hidrogênio e por sua vez, a citocromo c peroxidase, uma das peroxidases existentes nas células, reduz o peróxido de hidrogênio a água. A seqüência de cDNA que codifica para a enzima CuZnSOD GPI-ancorada (PbSOD3) de P. brasiliensis foi completamente seqüenciada. Esta enzima possui massa molecular deduzida de 24,3 kDa e a assinatura característica da família CuZnSOD e da região de ancoragem a GPI na região carboxi-terminal. A análise comparativa das seqüências protéicas PbSOD3 e Sod5 de C. albicans indicou cerca de 40% de similaridade em toda a extensão da proteína, sendo mais conservadas as regiões da assinatura do sítio catalítico e de ancoragem a GPI. A análise no programa PSORT II indicou que a PbSOD3 deve estar localizada na parede celular (34,8% de probabilidade) ou com a mesma probabilidade, na membrana celular. A seqüência de cDNA que codifica para a citocromo c peroxidase (PbCCP) está parcial e a seqüência da proteína deduzida mostra a presença do sítio ativo da peroxidase, sendo bastante conservada quando comparada com CCPs de outros fungos. A análise de Southern blot indicou que os genes que codificam para as enzimas PbSOD3 e CCP estão presentes no genoma do fungo em cópia única. A análise da expressão dos genes que codificam para a PbSOD3 e PbCCP durante a transição dimórfica in vitro, mostrou que ocorreu uma oscilação; entretanto, não ocorreu uma alteração do padrão de expressão significativamente diferente entre as formas de micélio e levedura. Também não foi observada variação significativa da expressão para ambos os genes, em nível transcricional, durante o choque térmico a 42 °C. Os ensaios de medida de atividade enzimática extracelular para a enzima SOD de P. brasiliensis, utilizando o meio de cultura das células de levedura, não detectaram atividade desta enzima, sugerindo que a mesma deve estar associada à parede celular e/ou membrana celular. A atividade enzimática da CCP de P. brasiliensis durante o choque térmico a 42 °C diminuiu em função do tempo de incubação das células de levedura, entretanto os valores ainda eram altos no fim do choque térmico, indicando que provavelmente este patógeno é capaz de responder ao estresse oxidativo durante esta condição de drástica alteração fisiológica. Os dados sugerem fortemente que o P. brasiliensis está apto, desde a forma de micélio, a responder contra as injúrias provocadas pelo estresse oxidativo, uma vez que este patógeno apresenta níveis similares de expressão dos genes sod3 e ccp nas duas formas, micélio e levedura. Experimentos de RNAi ou nocaute dos genes sod3 e ccp de P. brasiliensis devem ser realizados para efetivamente demonstrar a função destas enzimas na proteção contra o estresse oxidativo. _______________________________________________________________________________ ABSTRACT
Paracoccidioides brasiliensis is a dimorphic and human pathogenic fungus. It is the causative agent of paracoccidiodoμycoses (PCM), an endemic disease widespread in Latin América. The establishment of infection depends on the transition from mycelium to yeast form and this process in vitro is reversible and dependent on temperature shifts from 22 C to 36 C. The transcriptome project identified 6.022 PbAEST that corresponds to expressed genes in fungus, representing approximately 80% of fungus genome. This data has allowed the identification of genes related in different biological processes of the fungus such as dimorphism, virulence and pathogenicity. In pathogen−host interaction, an efficient pathogen.s response against oxidative attack imposed from host phagocytic cells facilitates the pathogen survival in host. The genes potentially related in oxidative stress response in P. brasiliensis identified in transcriptoma project were categorized in 4 groups: antioxidant enzymes (12 PbAESTs); biosynthesis and metabolism of glutathione and NADPH regeneration (11 PbAESTs); ions homeostasis (3 PbAESTs) and transcription factors (7 PbAESTs). Among the annoted sequences in transcriptome, that ones coding for enzymes superoxide dismutase GPI−anchored (PbSOD3) and cytochrome c peroxidase (PbCCP) were analyzed in this study. The enzyme superoxide dismutase removes superoxide radical producing hydrogen peroxide and cytochroμe c peroxidase, one of peroxidases existing in cells, reduces hydrogen peroxide to water. The cDNA sequence coding for CuZnSOD GPI−anchored enzyμe from P. brasiliensis was completely sequenced. The deduced amino acid sequence of PbSOD3 has the molecular mass predicted of 24,3 kDa and pI of 6,05; furthermore, it has the protein signature of CuZnSOD family and a region for GPI anchoring in carboxyl−terminus. The comparative analysis of protein sequences of PbSOD3 and Sod5 of C. albicans indicated approximately 40% of similarity for the entire protein, where the most conserved regions are catalytic site of enzyme and GPI anchoring. The PSORTII program analysis revealed that PbSOD3 may be located in cell wall (34,8% of probability) or membrane plasmatic with the same probability. The cDNA sequence coding for cytochroμe c peroxidase is parcial and the protein deduced sequence has the active site of peroxidases and this region is very conserved when compared with CCPs of other fungi. The Southern blot analysis revealed that the genes coding for enzymes PbSOD3 and PbCCP are present as single copies in the fungus genome. The expression analysis of the same genes demonstrated an oscillation during dimorphic transition in vitro, however, do not occur any significantly difference in the gene expression pattern between mycelium and yeast forms. Also, it has not shown any significant difference in gene expression pattern during heat shock at 42 C, at transcriptional level, for both genes. The extracellular enzymatic activity assay, using yeast culture medium (or supernatant), was done to verify P. brasiliensis SOD activity with no activity detected. This result suggests that this enzyme may be associated to cell wall and/or plasmatic membrane. The enzymatic activity of P. brasiliensis CCP during heat shock at 42 C decreases in function of incubation time of yeast cells, but the values remained high at the end of heat shock, indicating that probably this pathogen is able to respond to oxidative stress during this drastic physiological change. These data strongly suggest that P. brasiliensis is capable, since mycelium form, to counteract injuries from oxidative stress, because this pathogen presents siμilar levels of gene expression for sod3 and ccp in mycelium and yeast forms. Experiments in vitro and in vivo through gene silencing (RNAi) or nocaute may be designed utilizing P. brasiliensis sod3 e ccp genes to efficiently determine if these enzimes are important to protect against oxidative stress.
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Algarve, Thaís Doeler. "EFEITO TOXICOGENÉTICO DO POLIMORFISMO ALA16VAL DO GENE MnSOD EM LEUCÓCITOS EXPOSTOS IN VITRO AO METIL MERCÚRIO." Universidade Federal de Santa Maria, 2012. http://repositorio.ufsm.br/handle/1/11191.
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Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorThe environmental contamination by methyl mercury (MeHg) is a great health public problem in some world regions like Amazonia. The MeHg toxic effects seem to be influenced by environmental and genetic factors. However, there are few studies evaluating the genetic influence on MeHg toxicity in humans. Therefore, the aim of this present study was to evaluate the genetic influence of Ala16Val superoxide dismutase manganese dependent gene polymorphism (Ala16Val-MnSOD) on cytotoxic effects of in vitro human leucocytes exposed to MeHg. Subjects were selected from 100 individuals genotyped to Ala16Val-MnSOD polymorphism (26,4±7,3 years) with different genotpypes (AA=08, VV=06 and AV=12) to perform the in vitro tests. The reactive oxygen species (ROS) production was measured using 2� 7�-dichlorofluorescein diacetate (DCFDA) fluorimetric assay and the cell viability was measured using the MTT assay were performed in leucocyte samples with the same subjects exposed and not exposed to MeHg (2,5μM for 6h). The results showed that AA and VVleucocytes exposed to MeHg did not increase the ROS levels when compared to the cells that were not exposed. However, the AV-leucocyte MeHg exposure increased the ROS levels. The cellular viability comparison among different genotypes exposed to MeHg showed lower AAleucocyte viability when compared to VV-leucocytes, whereas heterozygous cells (AV) presented intermediary values. This occurred probabibly due to the fact of AA-leucocytes present a higher basal H2O2 production than other genotypes. The whole of these results suggests toxicogenetic effects of Ala16Val-SOD polymorphism in human cells exposed to MeHg.
A contaminação ambiental por metilmercúrio (MeHg) é uma grande problema para a saúde pública em algumas regiões do mundo, como a Amazônia. Entretanto, os seus efeitos tóxicos parecem ser influenciados tanto por fatores ambientais como genéticos. Porém ainda há poucos estudos avaliando a influencia genética em humanos expostos ao MeHg. Assim, o presente estudo buscou avaliar a influência de um polimorfismo genético presente na enzima superóxido dismutase dependente de manganês (Ala16Val-MnSOD) sobre os efeitos citotóxicos relacionados a exposição ex vivo ao MeHg em leucócitos humanos. A partir da genotipagem de 100 indivíduos (26,4±7,3 anos) foram selecionados sujeitos com diferentes genótipos (AA=08, VV=06 e AV=12) para a realização dos testes. Foi avaliada a citotoxicidade (via ensaio MTT) e a produção de radicais livres RL (via ensaio da fluorescência do DCFDA) em amostras de leucócitos, dos mesmos sujeitos, expostas e não expostas ao MeHg (2,5μM por 6h). Os resultados mostraram que leucocitos AA e VV expostos ao MeHg não aumentaram os níveis de produção de ROS quando comparados ao grupo controle. Enquanto os leucocitos AV quando expostos ao MeHg aumentaram a produção de EROs. Contudo, leucocitos-AA expostos ao MeHg apresentaram menor viabilidade quando comparados aos leucócitos dos genótipos VV e AV sob as mesmas condições. Isto ocorre provavelmente pelo ao fato do genótipo AA apresentar maior produção basal de H2O2 do que os demais genótipos. Estes resultados sugerem efeito toxicogenético na resposta de células humanas expostas ao MeHg.
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Ribeiro, Alexsandra Christianne Malaquias de Moura. "Avaliação do padrão de crescimento na síndrome de Noonan em pacientes com mutações identificadas nos genes PTPN11, SOS1, RAF1 e KRAS." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-17062011-160529/.
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A Síndrome de Noonan (SN) é caracterizada por baixa estatura proporcionada de início pós-natal, dismorfismos faciais, cardiopatia congênita e deformidade torácica. A frequência da SN é estimada entre 1:1000 e 1:2500 nascidos vivos, com distribuição semelhante em ambos os sexos. A herança é autossômica dominante com penetrância completa, porém a maioria dos casos é esporádica. Até o momento, mutações em genes da via RAS-MAPK (PTPN11, KRAS, SOS1, RAF1, MEK1, NRAS e SHOC2) foram identificadas em aproximadamente 70% dos pacientes. Uma das principais características fenotípicas da SN é a baixa estatura pós-natal, embora o mecanismo fisiopatológico do déficit de crescimento nesta síndrome ainda não esteja totalmente esclarecido. Estudos que avaliaram o padrão de crescimento linear em crianças com SN foram realizados anteriormente ao conhecimento do diagnóstico molecular dessa síndrome. No presente estudo, avaliamos a frequência de mutação nos genes PTPN11, SOS1, RAF1 e KRAS em 152 pacientes com SN e o padrão de crescimento linear (altura) e ponderal [índice de massa corpórea (IMC)] dos pacientes com mutação identificada. No total, mutações nos genes relacionados foram encontradas em 99 pacientes (65%) do nosso estudo, com predominância do gene PTPN11 (47%), seguido do SOS1 (9%), RAF1 (7%) e KRAS (3%). Foram construídas curvas específicas para SN de Altura e IMC para idade e sexo utilizando o método LMS. Os pacientes com SN apresentaram crescimento pré-natal preservado, porém o comprometimento do crescimento pós-natal foi observado desde o primeiro ano de vida, atingindo uma altura final de -2,5 e -2,2 desvios-padrão da média para população brasileira em homens e mulheres, respectivamente. O prejuízo da altura foi maior nos pacientes com mutação no gene RAF1 em comparação com os genes PTPN11 e SOS1. O IMC dos pacientes com SN apresentou queda de 1 desvio-padrão em relação à média da população brasileira normal. O comprometimento do IMC foi menor nos pacientes carreadores de mutação no RAF1. Pacientes com mutação nos genes PTPN11 e SOS1 apresentaram maior frequência de estenose de valva pulmonar, enquanto a miocardiopatia hipertrófica foi mais frequente nos pacientes com mutação no gene RAF1. A variabilidade fenotípica observada nos pacientes com mutação no PTPN11 não pode ser explicada pelo grau que estas mutações influenciam a atividade tirosina fosfatase da SHP-2 nem pela presença de polimorfismos no gene KRAS. Com a análise dos éxons 3, 8 e 13 do PTPN11, seguido dos éxons 6 e 10 do SOS1 e éxon 7 do RAF1 identificamos 86% dos pacientes carreadores de mutações nos genes relacionados, propondo uma forma mais eficiente de avaliação molecular na SN. Acreditamos que a variabilidade fenotípica presente nessa síndrome esteja diretamente ligada aos diferentes papéis exercidos pelas proteínas que participam da via RAS/MAPK. Entretanto, mais estudos em relação à via RAS/MAPK serão necessários para esclarecer as questões relacionadas ao crescimento e outras características fenotípicas da SNNoonan Syndrome (NS) is characterized by distinctive facial features, short stature and congenital heart defects. The estimated prevalence is 1:1000 to 1:2500 live births, affecting equally both sexes. It is an autosomal dominant disorder with complete penetrance, but most cases are sporadic. To date, mutations in the RAS/MAPK pathway genes (PTPN11, KRAS, SOS1, RAF1, MEK1, NRAS and SHOC2) were identified in approximately 70% of patients. One of the cardinal signs of NS is proportional postnatal short stature although the physiopathological mechanism of growth impairment remains unclear. The current knowledge about the natural history of growth associated with NS was described before molecular diagnosis era. In this study, we performed PTPN11, SOS1, RAF1, and KRAS mutation analysis in a cohort of 152 NS patients and studied the natural linear (height) and ponderal growth [body mass index (BMI)] of NS patients with related mutations. Mutations in NS-causative genes were found in 99 patients (65%) of our cohort. The most common mutated gene was PTPN11 (47%), followed by SOS1 (9%), RAF1 (7%) and KRAS (3%). Sex-specific percentile curves for height and BMI were constructed using the LMS method. NS patients had birth weight and length within normal ranges but the postnatal growth impairment was observed during the first year of life, reaching a final height of -2.3 and -2.2 standard deviations from the mean for Brazilian healthy men and women, respectively. Postnatal growth impairment was higher in RAF1 mutation patients than in patients with SOS1 and PTPN11 mutations. BMI values in NS patients were lower in comparison with normal Brazilian population. BMI values were higher in patients with RAF1 mutations than in patients with other genotypes. Patients with mutations in PTPN11 and SOS1 genes were more likely to have pulmonary valve stenosis, whereas hypertrophic cardiomyopathy was more common in patients with mutations in the gene RAF1. The intensity of constitutive tyrosine phosphatase activity of SHP-2 due to PTPN11 mutations, as well as the presence of polymorphisms in KRAS gene did not influence the phenotype of NS patients with mutation in PTPN11 gene. Analysis of exons 3, 8 and 13 of PTPN11 gene, followed by exons 6 and 10 of SOS1 gene and exon 7of RAF1 gene identified 86% of patients harboring mutations in related genes, suggesting a more efficient evaluation of NS molecular diagnosis. We believe that the phenotypic variability in this syndrome is directly linked to the different roles played by proteins that participate in RAS/MAPK pathway. However, further studies in RAS/MAPK pathway are needed to clarify issues related to growth and other phenotypic characteristics of SN
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Tarradas, Pou Anna. "Els factors GATA4 i GATA5 en la regulació transcripcional del gen que codifica pel canal de sodi cardíac (SCN5A)." Doctoral thesis, Universitat de Girona, 2017. http://hdl.handle.net/10803/405732.
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The SCN5A gene encodes the alpha subunit of the cardiac sodium channel (NaV1.5), which is responsible for the influx of sodium ions through membrane of cardiomyocytes. Different evidences suggest that an aberrant expression of the SCN5A gene may cause cardiac arrhythmias. However, the mechanisms that control SCN5A expression regulation are largely unknown. This thesis proposes a new mechanism of SCN5A transcriptional regulation in the adult human heart: transcription factors GATA4 and GATA5 synergize in the activation of the SCN5A expression. In addition, it has been proposed that GATA4 activity on the SCN5A is regulated by acetylation/deacetylation via the acetyltransferase p300 and the deacetylase HDAC2. It has been identified three lysines of GATA4 that are targets of p300 and HDAC2. In summary, this study contributes to further understand the molecular basis of the cardiac arrhythmias associated with alteration of sodium currentsEl gen SCN5A codifica per la subunitat alfa del canal de sodi cardíac dependent de voltatge (NaV1.5), el qual permet l’entrada de ions sodi a través de la membrana dels cardiomiòcits. Diverses evidències suggereixen que una expressió anòmala del gen SCN5A pot donar lloc a arítmies cardíaques. Malauradament, els mecanismes que regulen l’expressió d’SCN5A són molt poc coneguts. Aquesta tesi proposa un nou mecanisme de regulació transcripcional del gen SCN5A en el cor humà adult: els factors de transcripció GATA4 i GATA5 activen sinèrgicament l’expressió del gen SCN5A. També s’ha proposat que l’activitat de GATA4 sobre SCN5A està regulada per un mecanisme d’acetilació/desacetilació a on hi participen l’acetiltransferasa p300 i la desacetilasa HDAC2. S’han identificat tres residus de lisines de GATA4 que són dianes de p300 i HDAC2. En resum, aquest estudi permet entendre millor les bases moleculars de les arítmies cardíaques associades amb alteracions del corrent de sodi
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Deus, Karinne Evaristo de. "Atividade enzimática e expressão diferencial da Superóxido Dismutase (SOD) em plantas de arroz de terras altas sob deficiência hídrica." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/8640.
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Drought is a major cause for reduced productivity in the cultivation of upland rice farming in many regions of the world. One of the consequences of the drought is the production in excess of reactive oxygen species (ROS), causing a series of oxidative damage to various biomolecules with subsequent cell death. With the function of protecting structures and functioning of cells from the damaging effects of ROS, a complex antioxidant system is activated in plants. This system consists of: (1) lipid soluble membrane-associated and tocopherols; (2) reducing water soluble compounds such as ascorbate (ASA) and glutathione (GSH) and (3) antioxidative enzymes, and superoxide dismutase (SOD) considered as a major enzymes of the antioxidant defense system. The present study aimed to evaluate the SOD in activity level via spectrophotometric method and level of gene expression via qPCR in two genotypes of upland rice (Oryza sativa japonica), Douradão and BRS Primavera, with contrasting for drought tolerance characteristics, watching the leaf and root tissue, two stages of plant development (vegetative and reproductive), grown under optimal water conditions and water deficit (100% and 50% water in the vessels), respectively. The results revealed a differential pattern of SOD activity in different tissues and developmental stages in tolerant and sensitive genotypes, and for the tolerant genotype that activity was increased only in leaf / root and vegetative/reproductive tissue, as was sensitive the leaf / reproductive and reproductive/ root. Regarding gene expression, we also observed a very different pattern of regulation in tolerant and sensitive genotypes. The CuZnSOD1, CuZnSOD4, and MnSOD genes, expression was significantly (p≤ 0.05) increased in the tolerant, the first in leaves and roots off the reproductive stage, the second vegetative stage only in leaves and the third gene in the two tissues and plant developmental stages. As for the sensitive only FeSOD1 gene had highlighted with increased expression in roots at the reproductive stage. Certainly, the different patterns of induction level of activity and / or gene expression of SOD in plants of upland rice, should be strongly considered to elucidate the cellular mechanisms of drought tolerance, aiming to support improvement programs to develop cultivars more efficient and better suited to prone areas with water deficiency.
A seca é uma das principais causas para redução da produtividade na cultura do arroz de terras altas em muitas regiões agrícolas do mundo. Uma das consequências da seca é a produção, em excesso, de espécies reativas de oxigênio (EROs), podendo causar uma série de danos oxidativos a diversas biomoléculas com consequente morte celular. Com a função de proteger estruturas e funcionamento das células dos efeitos prejudiciais das EROs, um complexo sistema antioxidativo é ativado nas plantas. Esse sistema é constituído de: (1) lipídeos solúveis e tocoferóis associados à membrana; (2) compostos redutores solúveis em água, tais como ascorbato (ASA) e glutationa (GSH), e (3) enzimas antioxidativas, sendo a superóxido dismutase (SOD) considerada como uma das principais enzimas do sistema de defesa antioxidativo. O presente estudo teve como objetivo avaliar a SOD, em nível de atividade via método espectrofotométrico e em nível de expressão gênica via qPCR, em dois genótipos de arroz de terras altas (Oryza sativa japonica), Douradão e BRS Primavera, com características contrastantes para tolerância à deficiência hídrica, contemplando parte aérea e tecido radicular, dois estádios de desenvolvimento das plantas (vegetativo e reprodutivo), cultivadas sob condição hídrica ótima e de deficiência hídrica (100 % e 50 % de água nos vasos), respectivamente. Os resultados revelaram um padrão diferencial de atividade da SOD nos diferentes tecidos e estádios de desenvolvimento nos genótipos tolerante e sensível, sendo que para o genótipo tolerante essa atividade foi aumentada somente em tecido foliar fase vegetativa e radicular fase reprodutiva, enquanto no sensível foi foliar e radicular estádio reprodutivo. Quanto à expressão gênica, também observou um padrão bastante diferenciado de regulação nos genótipos tolerante e sensível. Os genes Cu/ZnSOD1, Cu/ZnSOD4 e MnSOD apresentaram expressão significativamente (p ≤ 0,05) aumentada no tolerante, sendo o primeiro em folhas e raízes do estádio reprodutivo, o segundo estádio vegetativo somente em folhas e para o terceiro gene nos dois tecidos e estádios de desenvolvimento da planta. Já para o genótipo sensível somente o gene FeSOD1 apresentou destaque com aumento da expressão em raízes no estádio reprodutivo. Certamente, os diferentes padrões de indução em nível de atividade e/ou expressão gênica da SOD, em plantas de arroz de terras altas, devem ser fortemente considerados para elucidar os mecanismos celulares de tolerância à seca, objetivando subsidiar programas de melhoramento para desenvolvimento de cultivares mais eficiente e mais bem adaptada às áreas propensas à deficiência hídrica.
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LIMA, Maria da Graça de Souza. "Detecção de genes e expressão enzimática em cultivares de arroz (Oryza sativa L.) crescidas sob estresse salino." Universidade Federal de Pelotas, 2008. http://repositorio.ufpel.edu.br/handle/ri/2015.
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Previous issue date: 2008-07-18In Rio Grande do Sul State, the main system for irrigation of rice cultivation is by flooding, can lead to the salinization of soils with inadequate drainage, especially at the coastal region where crops using water from the Laguna dos Patos, which is subject to the salinization by sea water. This is a major environmental problem in the rice production. This survey aimed to examine the expression of enzyme in Oryza sativa L. ssp. indica S. Kato e Oryza sativa L. ssp. japonica S. Kato, grown in different levels of salinity, in order to identify genes involved in tolerance to salinity, based on the assumption that the second subspecies show greater tolerance to salinity. In the experiment were used Oryza sativa L. ssp. japonica S. Kato (BRS Bojuru, IAS 12-9 Formosa and Goyakuman) and Oryza sativa L. ssp. indica S. Kato (BRS-7 Taim, BRS Querência and BRS Atalanta). The seedling was done in plastic trays, containing sand washed as substrate. The seedlings were transferred to greenhouse with 10 days of emergency under temperature 25 °C and humidity 85 % controlled and placed in basins of 15 L containing nutrient solution of Hoagland half strength increased of 0, 25, 50, 75 and 100 mM NaCl. Seedlings were collected at 14, 28, 42 and 56 days after the transfer and immediately stored in ultrafreezer to -70 °C to subsequent analyses. The plant tissues were macerated and placed in tubes eppendorf with extractor solution of Scandálios. The electrophoresis was performed in 7% of polyacrylamide gels placed in vertical vats. The bands were revealed for several enzymes systems: superoxide dismutase, peroxidase, catalase, esterase, xvi glutamate dehydrogenase, alcohol dehydrogenase, fosfoglucose isomerase, malate dehydrogenase, málica enzyme, alpha amylase and glucose-6-phosphate dehydrogenase. Through the search in silico, conducted with the National Center for Biotechnology Information identified the genes AY785147 - SOS and AF319481 - CK1 involved in the salinity tolerance. The detection of the gene was the extraction of DNA using the method CTAB 2%, followed by reactions of PCR thermocycler held on through the use of primers also drawn in silico. The products of amplification were detected by agarose gel electrophoresis of 1.5%. The view of the DNA stained with bromide etídio was made on ultraviolet light and scanned images. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica. Fragment of SOS1 gene was found in all cultivars, except for BRS Atalanta. CK1 gene is present in all cultivars evaluated. It allows to conclude that enzyme systems were more expressed in cultivars O. sativa ssp. japonica, in the leaves and the 14 DAT, featuring bands more intense as the increase of salinity. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica and the genes studied are present in both subspecies.
No Rio Grande do Sul, o principal sistema de irrigação da cultura do arroz é por inundação, podendo conduzir à salinização os solos com drenagem inadequada, especialmente as lavouras da região litorânea que utilizam a água da Laguna dos Patos, que está sujeita à salinização pela entrada do mar quando é baixo o nível da referida Laguna, tornando-se uma das maiores limitações ambientais na produção de arroz. Esta pesquisa teve como objetivos analisar a expressão enzimática de cultivares de Oryza sativa L. ssp. indica S. Kato e Oryza sativa L. ssp. japonica S. Kato, crescidas em diferentes níveis de salinidade e detectar genes envolvidos com a tolerância à salinidade, com base na hipótese de que as cultivares da Segunda subespécie apresentam maior tolerância à salinidade. No experimento foram utilizadas as cultivares BRS Bojuru, IAS 12-9 Formosa e Goyakuman pertencentes à O. sativa ssp. japonica e as cultivares de O. sativa ssp. indica BRS-7 Taim, BRS Querência e BRS Atalanta. As plântulas de arroz com 10 dias após a emergência (DAE) foram transferidas para casa de vegetação com temperatura e umidade controlada e crescidas em bacias de 15 L, contendo solução nutritiva de Hoagland meia força acrescida de 0, 25, 50, 75 e 100 mM de NaCl. As plântulas foram coletadas aos 14, 28, 42 e 56 dias após a transferência (DAT) e imediatamente armazenadas em ultrafreezer à -70 °C para posterior anáxiv -lises. Os tecidos vegetais foram macerados e colocados em tubos eppendorf com solução extratora de Scandálios. A eletroforese foi realizada em géis de poliacrilamida 7% colocados em cubas eletroforéticas verticais. As bandas foram reveladas para os sistemas enzimáticos superóxido dismutase, peroxidase, catalase, esterase, glutamato desidrogenase, álcool desidrogenase, fosfoglucose isomerase, malato desidrogenase, enzima málica, alfa amilase e glucose-6-fosfato desidrogenase. Por intermédio de pesquisa in silico, realizada junto ao National Center for Biotechnology Information foram identificados os genes AY785147 SOS e AF319481 - CK1, envolvidos na tolerância a salinidade. A detecção dos genes consistiu da extração de DNA genômico segundo o método CTAB 2%, seguido de reações de PCR realizadas em termociclador mediante a utilização dos primers desenhados também in silico. Os produtos da amplificação foram detectados por eletroforese em gel de agarose 1,5%. A visualização do DNA corado com brometo de etídio foi feita sobre iluminação ultravioleta e as imagens digitalizadas. A expressão das enzimas envolvidas nos mecanismos de tolerância ao estresse salino é maior em O. sativa ssp. japonica. Fragmento do gene SOS1 foi encontrado em todas cultivares, com exceção da BRS Atalanta e o gene CK1 está presente em todas as cultivares avaliadas. Conclui-se que os sistemas enzimáticos são mais expressos nas cultivares de O. sativa ssp. japonica, nas folhas e aos 14 DAT, apresentando bandas mais intensas conforme o aumento da salinidade. A expressão das enzimas envolvidas nos mecanismos de tolerância ao estresse salino é maior em O. sativa ssp. japonica e os genes estudados estão presentes nas duas subespécies.
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Montagner, Greice Franciele Feyh dos Santos. "Efeito in vitro do polimorfismo ala16val do gene da superóxido dismutase dependente de manganês no metabolismo oxidativo de linfócitos." Universidade Federal de Santa Maria, 2010. http://repositorio.ufsm.br/handle/1/11126.
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Conselho Nacional de Desenvolvimento Científico e TecnológicoEpidemiological studies suggest that the imbalance in antioxidant enzymes activity genetically caused, increases the risk of metabolic disorders and chronic noncommunicable diseases. This is the case of polymorphism that occurs in an substitution of Alanine (A) by a Valine (V) at codon 16 of the gene for superoxide dismutase manganese-dependent (Ala16Val-SOD2). The A allele has a higher efficiency of catalysis than allele V. However, this efficiency increases the levels of H2O2 leading to a consequent oxidative imbalance that, in turn, increases the risk of cancer. Furthermore, the V allele that presents a smaller efficiecy SOD2 is associated with endothelial dysfunction and cardiometabolic. Therefore, Ala16VALSOD2 polymorphism serves as a model for in vitro analysis of differential responses to pro and antioxidants factors. The objective of this study was to investigate the in vitro response of lymphocytes culture with different genotypes (AA, VV and AV) from healthy subjects exposed to ultraviolet (UV) is a pro-oxidant factor. In lymphocyte not exposed and UV exposed it was analyzed the cytotoxic variables (cell viability, mitotic index), biomarkers of oxidative metabolism (lipid peroxidation, enzymatic activity of SOD, catalase, thiol groups, protein carbonylation, total polyphenols and ascorbic acid) and genotoxic effects (by Comet Assay and chromosomal instability). The results showed that cells carrying the AA genotype had higher cell viability and mitotic index. However, after UV exposure such viability was similar between genotypes. At baseline levels higher SOD and total polyphenol levels were observed in AA genotype when compared to the VV genotype. However, lymphocytes AA had higher rates of DNA damage in the presence of UV radiation. These results suggest that the polymorphism Ala16Val-SOD2 may differentially affect the toxicity factors such as UV radiation.
Estudos epidemiológicos sugerem que o desbalanço na atividade de enzimas antioxidantes, geneticamente causado, aumenta o risco de disfunções metabólicas e doenças crônicas não-transmissíveis. Este é o caso do polimorfismo em que ocorre uma troca da Alanina (A) por uma Valina (V) no códon 16 do gene da enzima superóxido dismutase dependente de manganês (Ala16Val-SOD2). O alelo A possui uma eficiência de catálise maior do que o alelo V. Entretanto, esta maior eficiência aumenta os níveis de H2O2 levando a um conseqüente desbalanço oxidativo que, por sua vez, aumenta o risco de neoplasias. Por outro lado, o alelo V que apresenta uma SOD2 menos eficiente está associado a disfunções endoteliais e cardiometabólicas. Portanto, este polimorfismo serve como modelo in vitro para análises de respostas diferenciais a agentes pró e antioxidantes. Sendo assim, o objetivo desse estudo foi investigar a resposta in vitro de cultura de linfócitos com diferentes genótipos (AA, VV e AV), provenientes de indivíduos saudáveis, expostas a radiação ultravioleta (UV) que é um fator pró-oxidante. Antes e após exposição, foram analisados os efeitos citotóxicos (viabilidade celular e índice mitótico), indicadores do metabolismo oxidativo (peroxidação lipídica, atividade enzimática da SOD, catalase, grupos tióis, polifenóis totais e de ácido ascórbico) e efeitos genotóxicos (dano de DNA pelo Ensaio Cometa e instabilidade cromossômica,). Os resultados mostraram que as células portadoras do genótipo AA apresentaram maior viabilidade celular e índice mitótico. Entretanto, após a exposição UV tal viabilidade foi similar entre os genótipos. Em condições basais níveis elevados de SOD e polifenóis totais plasmáticos foram observados no genótipo AA em relação ao genótipo VV. Porém, linfócitos AA apresentaram maior índice de danos no DNA na presença da radiação UV. Estes resultados sugerem que o polimorfismo Ala16Val- SOD2 pode afetar diferencialmente a toxicidade a fatores como a radiação UV.
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Jackson, Mandy. "Screening of familial and sporadic amyotrophic lateral sclerosis patients for mutations in CuZn superoxide dismutase (SOD-1) and other candidate genes." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363787.
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Derda, Katharina [Verfasser], and Annette [Akademischer Betreuer] Raabe. "Bedeutung von Einzelnukleotidpolymorphismen in den Genen ATM, GSTP1, SOD2, TGFB1, XPD und XRCC1 bei Brustkrebspatientinnen für die Erythementstehung als Akutreaktion nach Strahlentherapie / Katharina Derda. Betreuer: Annette Raabe." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2011. http://d-nb.info/1020383828/34.
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Niewinski, Desi. "Water column oxygen respiration dynamics and quantification of nitrogen cycling genes insediment of Lake Erie." Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1547330489488682.
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Ramos, Gonçalo Luis Monteiro 1988. "A1 and A2A Adenosine receptors expression in ALS transgenic mice for the human gene SOD1." Master's thesis, 2012. http://hdl.handle.net/10451/7946.
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Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012A Esclerose Lateral Amiotrópica (ELA) é uma doença progressiva e fatal caracterizada pela degeneração selectiva dos neurónios motores do córtex motor, tronco cerebral e medula espinal, que provoca atrofia muscular, paralesia e morte por falha respiratória. A etiologia da doença continua desconhecida, mas com um consenso de que o dano dos neurónios motores é causado por uma rede de processos patológicos complexos. Os mecanismos envolvidos na degeneração dos neurónios motores são melhor conhecidos num subtipo da doença causada por mutações na enzima superóxido dismutase 1 (SOD1). Esta enzima actua na eliminação de radicais livres de oxigénio e na ELA o processo de degeneração neuronal deve-se a um ganho de função da SOD1. A adenosina tem uma função importante na modulação da transmissão sináptica no SNC e SNP, actuando a dois níveis: inibitório, modulado pelos receptores do subtipo A1 e excitatório, mediado pelos receptores do subtipo A2A. É conhecido que a expressão dos receptores A1 e A2A da adenosina está alterada nalgumas doenças neurodegenerativas, mas o seu papel na ELA é ainda muito pouco conhecido. O objectivo deste trabalho foi determinar o efeito da ELA na expressão proteica e de mRNA dos receptors A1 e A2A da adenosina no decurso da doença. O modelo de murganhos transgénicos para o gene SOD1 humano com a mutação G93A foi usado neste trabalho. Os níveis proteicos e de mRNA de ambos os receptores foram quantificados através das técnicas de immunoblotting e PCR quantitativo em tempo real, respectivamente. Foram estudados diferentes tecidos do SNC e SNP, nomeadamente, córtex e medula espinal (apenas immunoblotting) e nervo frénico-diafragama, de animais selvagens e portadores da doença nas fases pre-sintomática (4-6 semanas) e sintomática (13-14 semanas). Resultados deste estudo indicaram níveis proteicos não alterados nos SNC e SNP do receptor A1 ao longo da progressão da doença. No entanto, observou-se uma sobreexpressão dos receptores A2A no córtex na fase pre-sintomática e um decréscimo na fase sintomática. Os outros tecidos mantiveram-se inalterados no que se refere aos receptores A2A em ambas as fases da doença. A avaliação da expressão de mRNA no diafragma não revelou quaisquer alterações em ambos os receptores da adenosina durante a progressão da doença. Assim, no que se refere aos receptores da adenosina em ELA, as primeiras alterações parecem ocorrer logo no início da doença nos receptores A2A do SNC.
Amyothrophic Lateral Sclerosis (ALS) is a progressive and fatal disease categorized by a selective degeneration of motor neurons from the cerebral cortex, brainstem and spinal cord that provokes muscle atrophy, progressive paralysis and death due to respiratory failure. The etiology of most ALS cases remains unknown but there is a current consensus that motor neuron degeneration is caused by a complex interaction between multiple pathogenic processes. The mechanisms of motor neuron degeneration are best understood in the subtype of disease caused by mutations in the enzyme superoxide dismutase 1. This enzyme is enrolled in the degradation of free oxygen radicals and in ALS neuronal damage is due to its gain-of-function. Adenosine has a central role as a neuromodulator of the CNS and PNS synaptic transmission. Adenosine acts at two levels: inhibitory through the subtype A1 receptor and excitatory through the subtype A2A receptor. Variation on the expression of A1 and A2A receptors has been identified in some neurodegenerative diseases, but their role in ALS is not yet understood. The objective of this work was to determine the effect of ALS on the protein and mRNA expression of A1 and A2A adenosine receptors through disease progression. The transgenic model of mice carrying the human SOD1 gene with the G93A mutation was used in this work. Protein and mRNA levels of both receptors were quantified through immunblotting and quantitative real time PCR, respectively. Different tissues of the CNS and PNS, namely cortex and spinal cord (immunoblotting only) and phrenic nerve-diaphragm were studied in wild-type and transgenic mice in the pre-symptomatic (4-6 weeks) and symptomatic (13-14 weeks) phases of the disease. Results from this study indicate unaltered A1 receptor protein levels at the CNS and PNS through disease progression. However, there is an overexpression of A2A receptors in the cortex of pre-symptomatic mice and a decrease in the symptomatic phase. The A2A receptors are unaltered in the other tissues in both phases of the disease. The mRNA evaluation does not reveal significant alterations in both adenosine receptors during disease progression. Thus, regarding adenosine receptors in ALS, the first changes seem to occur early in the disease at the CNS in A2A receptors.
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Hsiao, Yu-Feng, and 蕭郁丰. "An Investigation on MMP-2、MMP-9、TIMP-2 and SOD1 Gene Polymorphisms in Taiwan (Han ethnicity) Patients with Chronic /Generalized Aggressive Periodontitis." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/31247529967517848160.
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碩士弘光科技大學
營養醫學研究所
103
1. Introduction： Both aggressive periodontitis (AgP) and chronic periodontitis (CP) have genetic basses. Matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) have been shown to play an important role in the pathogenesis of tissue destruction of periodontitis. The production of reactive oxygen species (ROS) has been found during the process of periodontitis. Superoxide dismutase (SOD), an antioxidant can effectively reduce cell damage caused by ROS. The interactions of ROS, MMPs and cytokines can lead to destruction of periodontal tissues. The associations between single nucleotide polymorphisms (SNPs) in the promoter region of MMP-2, MMP-9 ,TIMP-2 and SOD1 genes and the risk of AgP and CP were investigated in a Taiwanese (Han ethnicity) population. 2.Material and Methods： MMP-2 -1306C/T, MMP-2 -735C/T, MMP-2 -790T/G, MMP-9 -1562C/T ,TIMP-2 -418G/C and SOD1 -267C/T SNPs were genotyped by polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP) and ABI StepOnePlus ™ Real-Time PCR Systems analysis in 69 and 129 patients with AgP and CP respectively and 117 periodontal healthy individuals as control (HC). Health questionnaires were used to collect data of smoking, alcohol drinking and betal-quid chewing habits.Chi-square test, logistic regression and t-test analysis were used to investigate the possible association of the genotypes with the periodontitis. 3. Results： No significant differences in the distributions of the -1306C/T and -735C/T variants of MMP-2, -418G/C of TIMP-2 ,-1562C/T of MMP-9 and -267C/T of SOD1 between periodontitis and control groups were detected. Only MMP-2-790G/T had a trend of significant (p=0.08) different in the allele frequencies among the studied groups. The logistic analysis showed that only carriers of the T allele of MMP-2-790G/T had crude OR of 0.56 (95%CI=0.31-0.98) and adjusted OR of 0.56 (95%CI=0.29-1.07). The CC genotype of MMP-2-735C/T had increased risk for CP in betel-quid chewer group (adj. OR=10.56,95%CI=1.40-224.49) and in alcohol drinker group (adj. OR=3.26, 95%CI=1.09-10.66).Only the frequencies of TIMP-2-418G/C gene polymorphisms in non-smokers were near to statistically significant different among AgP, CP and HC groups (p=0.07) and a crude OR of 2.21 (95%CI=1.14-4.4) and adjusted OR of 1.95 (95%CI=0.95-4.11) for CP. The non-alcohol drinking subjects with T allele of MMP-9 -1562C/T as compared to C allele, were less susceptible to AgP (adj. OR = 0.39, 95%CI = 0.18-0.90). 4. Conclusions： It is suggested thatMMP-2 -735C/T,MMP-2 -790T/G,TIMP-2 -418 G/C and MMP-9 -1562C/T gene polymorphisms may be associated with periodontitis in the Taiwanese Han population with the influence of personal habits (e.g. betel-quid chewing, smoking and alcohol drinking).
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Kluge, Friedrich. "Oxidativer Stress als Biomarker für die (Neben-) Wirkungen von Strahlentherapie: Bestimmung von Isoprostanspiegeln und Genexpressionsprofilen in Patientenproben." Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-0006-B2A0-E.
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Yang, Miin-Jou, and 楊敏洲. "Stuidies on Molecular Cloning, Selection and sod Gene of ced and sod Gene in Corn (Zea mays L.)." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/17661376026965768342.
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Chang, Keng-Hao, and 張耿豪. "Molecular Cloning, Characterization of the Vibrio cholerae sodA, Gene Fusion with Growth Hormone Gene from Epinephelus awoara and sodA Gene Probe Specificity for Vibrio cholerae Strain Identification." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/92818854045367027886.
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碩士國立海洋大學
水產生物技術研究所
88
The sodA genes coding fore manganese superoxide dismutase from Vibrio cholerae O1 Inaba(-), Inaba(+) and O139 were cloned by PCR techniques using the degenerate primers designed from Mn-SOD conserved amino acid regions and random primed gene walking PCR techniques. Vibrio cholerae O1 Inaba(-) sodA gene was over-expressed in the heterologous host E. coli. BL21(DE3)pLysS using the pET20b(+) expression vector. The full-length gene was 615 base pairs long and encoded 205 amino acid residues. The recombinant protein was efficiently purified from the host cell lysate by His-tag affinity chromatography. The recombinant VCI(-)Mn-SOD protein was active and insensitive to inhibitors such as NaN3, H2O2, KCN and DDC. Several gene specific primer pairs designed from nucleotide sequences of V. cholerae O1 Inaba(-) sodA were used to analyze the sodA gene probe specificity for Vibrio cholerae strain identification by cross-species PCR. Results indicated the specificity of the gene probe based on the upstream and downstream nucleotide sequences of V. cholerae sodA was very high. Vibrio cholerae O1 Inaba(+), Inaba(-), Ogawa(+), Ogawa(-), Non-O1 and O139 strains could be further distinguished from each other by randomly amplified polymorphic DNA analysis (RAPD). The VCI(-)sodA -Eagh gene fusion was constructed by Vibrio cholerae O1 Inaba(-) sodA and growth hormone gene from Epinephelus awoara. The fusion gene was over-expressed in E. coli. host BL21(DE3)pLysS using the pET20b(+) expression vector. The over-expressed recombinant fusion protein was insoluble, but could be purified from the inclusion bodies by His-tag affinity chromatography under the denatured conditions.
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Lin, I.-Ling, and 林奕伶. "Characterization of sodA genes in Stenotrophomonas maltophilia." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/37473236651109756977.
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Tsang-pai, Liu, and 劉滄柏. "The Cloning/Transformation and Expression of Human Mn-SOD Gene." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/68900982492529240825.
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碩士台北醫學院
醫學研究所
90
Reactive oxygen species and other oxidants cause DNA damage, lipid peroxidation, protein denaturation, cell death and resultant organ injury. Substantial evidence suggests that oxygen free radicals are associated with not only aging process but also degenerative disorders, such as Parkinson’s disease, Amyotrophic Lateral Sclerosis, atherosclerosis and Rheumatoid Arthritis. Free radicals are also reported to be associated with malignancy, ischemia/reperfusion and inflammation. Manganese superoxide dismutase (Mn-SOD), an endogenous superoxide radical-scavenging mitochondrial enzyme, is crucial for protecting cells and tissues from oxidant injury and hyperoxia. Enhancement of endogenous cellular anti-oxidation capacity by introduction of Mn-SOD cDNA or full-length active Mn-SOD protein into cells may, therefore, offers attractive therapeutic strategy for diseases associated with oxidative stress. We therefore sought to sequence the human Mn-SOD gene and purify the active human Mn-SOD protein. Human Mn-SOD gene library was established by reverse transcription and polymerase chain reaction (RT-PCR) of the total mRNA extracted from human liver tissue. Agarose gel electrophoresis indicated that Mn-SOD gene is a 667 base pairs DNA which encodes a 20 kDa protein. SOD expression vector was thus constructed and transformed into Escherichia coli (E. Coli) for gene expression. Western blotting analysis indicated that Mn-SOD could be over-expressed in the presence of IPTG. After isolation and purification, the enzyme activity of recombinant human Mn-SOD was assayed by using pyrogallo method. Our data indicated that Mn-SOD enzyme activity was Mn2+ ion concentration dependent. Baculovirus expression system was also employed to test the expression of Mn-SOD gene. Similar results were observed. We, therefore, concluded that sequencing of human Mn-SOD gene and synthesis of active recombinant human Mn-SOD protein are feasible and can contribute to future therapeutic strategies against diseases associated with oxidative stress.
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Hsieh, Feng-Ken, and 謝豐懇. "Cloning of Human CAT, GPX1, and SOD1 Genes and Expression in Methylotropic Yeast Pichia pastoris." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/43320634299899899375.
Full textAbstract:
碩士國立中興大學
畜產學系
90
The objective of this experiment is to clone the cDNA sequence of human CAT, GPX1, and SOD1. After constructing and electroporating，the cDNAs were introduced into the methylotropic yeast Pichia pastoris, and then we may assay for the expression of these human antioxidant cDNAs. First, in the part of human CAT gene, the primers were designed according to the hCAT cDNA sequence published on NCBI GeneBank. And a 1609 bp fragment was amplified by PCR using these primers and the human kidney cDNA bank as template. The fragment was ligated into the vector pcDNA3.1/V5/His-TOPO to form the plasmid pCMVCATns, and then was used for sequencing. After comparing with this sequence with the published sequence, we found that there are two nucleotides in this cDNA sequence are different form. And this difference would cause two amino acid residues change after translation. The hCAT cDNA was then cloned into the pPICZB vector that was named as pPICZBCATns and was then introduced into the methylotropic yeast Pichia pastoris to generate the KM71HCATns transformant. Been induced by methanol, we found that the cell lysate of it were showed a fragment around the 60 kDa marker band on the gel assayed by SDS-PAGE and western-blot。And after the activity assay, we also found that the specific activity of the small-scaled induced sample was 666.7 ± 10.893 U/mg protein, while it was only 8.4 ± 1.814 U/mg protein and 15.3 ± 1.706 U/mg protein in non-induced and control samples. Meanwhile, it was 1387.8 ± 38.349 U/mg protein when the transformant were incubated by large-scaled fermentor. Second, the hGPX1 cDNA, the procedures were the same as above. The products pCMVGPX1ns and the pCMVGPX1UTR were sequenced and the results were compared with the published sequence. The former was different from the report by seven nucleotides changes and it would cause the six changes in amino acid sequence. The later had only two difference but they caused no change in the polypeptide chain. Again as above, these two cDNA were ligased into the pPICZB, and the pPICZBGPX1ns and pPICZBGPX1UTR were made. After introducing they into the Pichia pastoris, we have gained both the KM71HGPX1ns and the KM71HGPX1UTR transformants, and they were induced by methanol either. The results were showed no any difference between control and them. So it might be caused by unsuccessed transformation. Finally, the hSOD1 cDNA was cloned and constructed as above, and the sequencing result showed that there was no any change in the cloned sequence. There was an obvious different band between the induced Pichia pastoris transformant KM71HSOD1ns and the control, but it is too large (about 50 ~ 60 kDa) to be the correct recombinant hSOD1. After it being large-induced, a band was appeared at about 20 kDa on western-blot, suggested that the difference might be the level of expression.
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Yang, Pao-Ying, and 楊寶英. "The role of Sox1 gene in neural differentiation of Human Embryonic Stem cells." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/25213038503313178533.
Full textAbstract:
碩士國立臺灣海洋大學
生物科技研究所
96
The generation of functional neuronal subtypes in the vertebrate central nervous system involves several steps. Early vertebrate neural development started from epiblast which is the pluripotent descendents of inner cell mass(ICM) of the blastocyst. The epiblast cells give rise to three embryonic germ layers(ectoderm,mesoderm and endoderm). Subsequently, ectodermal precursors forms early neuroepithelium containing very early neural stem cells. Then, neuroectoderm forms the neural plate and neural tube which become the source of the central nervous system. Recently, it has been demonstrated that Pax6 , Neurogenin, Sox1 and Nestin genes play an important role in early development of murine neuroectoderm. Among these genes, Sox1 is the earliest gene to be expressed in neuroectoderm and plays essential role in neural specification. However, the function of Sox1 gene in the development of human is still unknown. In order to understand the functional role of Sox1 in human neural specification, I generated lenti-viral Sox1 RNAi clones with GFP reporter and transducted them into hESCs to knowdown the expression of Sox1. In addition, heterogeneity among progenitor cells in the vertebrate nervous system has been demonstrated and it is believed that celluar heterogeneity is an important step toward developmental diversity. Therefore, I also investigated the expression profile of Oct4,Pax6 and Sox1 gene of single hESCs and their differentiated derivatives. My results show Sox1 siRNA knockdown has no obvious effect on altering characteristics of hESCs and their potentials in neural differentiation. Furthermore, results of the single cell analysis indicated the expression pattern of Oct4,Pax6 and Sox1 genes are not uniform in individual cells of early neural differentiation. This may imply a diversified neural cell types are emerging at these developmental stages.
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Wang, Kuei-Ling, and 王貴靈. "Molecular cloning and characterization of the sodA gene from Streptococcus gordonii ATCC 10588." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/55669111834158359944.
Full textAbstract:
碩士國立臺灣大學
微生物學研究所
89
Streptococcus gordonii is a species of the viridans group streptococci, and is the major pathogen of dental caries. Superoxide dismutase, SOD, is an antioxidant enzyme, which scavenges O2-to protect cells from damage. In this study, we characterized the SOD of S. gordonii as an MnSOD by H2O2 and diethyldithiocarbamate inhibition test. Furthermore, we also found that the SOD has activity as a cambialistic enzyme, which can accept either iron or manganese as the cofactor to present enzyme activity. The activity of manganese —containing SOD was higher than that iron-containing SOD. The SOD activity of S. gordonii was variable in the growth phase of the organism, and was found three to four folds increasing from the exponential phase to the stationary phase. The sodA gene of S. gordonii was cloned in this study. The gene contains 603 bp nucleotides. The nucleotide sequence of sodA has 81.51﹪identity to that of S. pneumoniae, and 76.4﹪to that of S. agalactiae. The amino acid sequence of this gene has 89.05﹪identity to that of S. pneumoniae, and 78.6﹪to that of S. mutans. It also has 90.54﹪similarity to that of S. pneumoniae, and 80.59﹪to that of S. mutans. The coding region of gene was recombined with native expression vector pDESTTM15 and was transformed into E. coli BL21-SI strain. After induction, the transformant expressed about 23 kD protein, which showed SOD activity by native PAGE gel analysis.
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Wen-Chung, Tseng, and 曾文仲. "Characterization and gene expression of Mn-SOD from zebrafish (Danio rerio)." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/15139556103165985698.
Full textAbstract:
碩士國立彰化師範大學
生物技術研究所
95
A full-length cDNA clone encoding a Mn-SOD was amplified by RACE-PCR from zebrafish cDNA library. The deduced amino acid sequence of Mn-SOD showed high similarity with the sequences of Mn-SOD from human (91.3%), mouse (86.6%), rat (86.3%), chicken (87.1%), fruit fly (78.5%), nematode (76.4%). Furthermore, the coding region of zebrafish Mn-SOD was introduced into an expression vector, pGEM-T EASY by modified primer, and transformed into sodA and sosB mutant Escherichia coli. A 26-kDa active Mn-SOD protein was expressed and detected by activity staining on polyacrylamide gel followed electrophoresis. The recombinant Mn-SOD stability was characterized by reaction to temperature, pH, and detergent treatment. The recombinant MnSOD facilitating the reduction reaction of superoxide anion retained 50% of its original activity after the enzyme was heated at 80℃ for 10 min. The recombinant Mn-SOD has a half life of deactivation of 48 min at 70 °C. Its thermal inactivation rate constant Kd was 0.0154 min-1 at 70℃. The enzyme was stable in a broad pH range from 2.2 to 12.44 at 37℃ for 3hours. In pH 9, the recombinant MnSOD facilitating the reduction reaction of superoxide anion retained 86% of its original activity after 8 hours at 37℃. In pH 2.2, the recombinant MnSOD facilitating the reduction reaction of superoxide anion retained 39% of its original activity after 8 hours at 37℃. The presence of sodium dodecylsulfate (up to 4%) had no effect on the enzyme’s activity at 37℃ for 1hours. The Mn-SOD, Cu/Zn-SOD, Catalase and Prx6 mRNA expression was analysis by real-time PCR during development of zebrafish embryos. These anti-oxidant mRNA was highly expressed between the one-cell stage and blastula which is about maternal effect. Immerged paraquat for harvested embryos inhibits Mn-SOD and Cu/Zn-SOD mRNA expression after 72hr. Immerged paraquat after hatched inhibits Mn-SOD, Cu/Zn-SOD and Catalase mRNA expression after 48hr. To realize the mechanism of Mn-SOD gene regulation, a 4Kb Mn-SOD promoter region was amplified by long-term PCR technique. An IRE site (insulin-response element) and three DBE site (DNA binding element) were shown. Both response elements are binding site for forkhead transcription factor Foxo5 and possibly involved in insulin signaling pathway. We also cloned forkhead transcription factor Foxo5 to future study.
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Kuo, Wen-Shuo, and 郭文碩. "Gene Expression And Physiological Analysis Of Sponge Gourd APX Gene And Winter Squash SOD Gene Under Respective Flooding And Chilling Stresses." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/85478413517520782019.
Full textAbstract:
碩士中國文化大學
生物科技研究所
100
Bitter gourd is one of the most important economical vegetables in Taiwan. However, the growth and development of the plants are strongly influenced by flooding and chilling stresses that can be initiated by oxidative stress. Bitter gourds have been grafted onto the sponge gourd and winter squash to cope with flooding and chilling stresses due to its susceptible to both conditions. These scions have developed a series of defense mechanisms and detoxification systems including antioxidants and enzymes to scavenge the reactive oxidative species (ROS). The objectives of our work were to study the changes of physiological parameters in bitter gourd, sponge gourd and winter squash, and identify any antioxidant enzyme under flooding and chilling stresses. The phenotypic traits, physiological parameters (variable fluorescence/maximum fluorescence, electrolyte leakage, malondialdehyde), and antioxidant enzymes (APX, CAT, SOD, and GR) were measured during different time periods of stress. The results of the study show that the physiological damages include chlorophyll breakdown, membrane permeability increased, and caused lipid peroxidation. The value of Fv/Fm was decreased under flooding and chilling stresses. The APX activity between sponge gourd and bitter gourd was found to be significant difference under flooding stress. APX mRNA transcripts of sponge gourd were relatively higher than bitter gourd based on real-time PCR analysis using actin as internal control.SOD activity between bitter gourd and winter squash was significantly different under chilling stress. Therefore, APX gene from the flooding-tolerant sponge gourd was identified and isolated using RACE technology. Both APX and SOD genes were cloned into E. Coli using transformation methodology. The results may be helpful to those bitter gourd farmers to reduce damages of the plants, and also be informative to screen for suitable scions and for stress tolerant breeding as well.
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Wang, Shih-Hsin, and 王詩欣. "studies on the sodF, gapA, and pgk genes of Bacillus subtilis." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/60988010835838328035.
Full textAbstract:
碩士國立陽明大學
生物化學研究所
91
In Bacillus subtilis, two genes, sodA and sodF, might produce superoxide dismutase (SOD), which catalyzes the dismutation of superoxide radicals. However, only SodA was detected in both vegetative cells and spores. In this study, the features of SodF were investigated. The results showed that the SodF protein was insoluble when overproduced in Escherichia coli strain JM109. Furthermore, the SodF protein was not detectable in the whole-cell extract from the E. coli sodA sodB mutant bearing the sodF-overexpressing plasmid. Based on these two facts, the SOD activity of SodF could not be determined by the currently available methods. However, when the N-terminal 70 amino acids of SodF were deleted, the solubility of SodF in E. coli JM109 was increased. This truncated form of SodF exhibited very low SOD activity. Moreover, sodF was found to be not constitutively expressed. On the other hand, the genes gapA, pgk, tpi, pgm, and eno, which encode the enzymes required for the interconversion of triose phosphates in glycolysis pathway, are transcribed in a hexacistronic operon. cggR, the first gene of this gapA operon, encodes a repressor of this operon. Site-directed mutagenesis and gel mobility shift assays demonstrated that a 25 base-pair palindromic sequence located downstream of the cggR transcriptional start site is the CggR binding site. Another transcriptional unit was previously proposed to be initiated from a promoter located upstream of pgk. The pgk promoter is located immediately upstream of a palindromic structure, which is presumed to be a rho-independent transcriptional terminator. However, our data revealed that this palindromic structure plays somewhat different roles in pgk expression.
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Yiu, Jin-Chin, and 尤進欽. "Transformation and cloning of superoxide dismutase (sod) and catalase (cat) genes." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/54148579167681685829.
Full textAbstract:
博士國立中興大學
園藝學系
88
Based on the enhanced activities of the antioxidative enzymes providing plants with additional stress-defense capability, attempts had been made to increase activities of superoxide dismutase (SOD) and catalase (CAT) by gene transfer technology. The purposes of this study were to examine the effects of transferring sod and/or cat genes into a plant on the oxygen stress-resistance, and to study the possibility of mutigene transfer via Agrobacterium mediated co-transformation. Sod and cat genes were cloned and selected from Chinese cabbage. Structure, composition, and the regulation of selected sod and cat genes were also analyzed. Based on the enhanced activities of the antioxidative enzymes providing plants with additional stress-defense capability, attempts had been made to increase activities of superoxide dismutase (SOD) and catalase (CAT) by gene transfer technology. The purposes of this study were to examine the effects of transferring sod and/or cat genes into a plant on the oxygen stress-resistance, and to study the possibility of mutigene transfer via Agrobacterium mediated co-transformation. The sod and cat genes were isolated from E. coli and corn and cloned into the plant transformation vectors driven by CaMV 35S or rbcS promoter. In addition, the DNA fragment of soybean chloroplast transit peptide was inserted into the constructed chimeras. Hypocotyl and cotyledon of Chinese cabbage (Brassica campestris L. ssp. Pekinensis cv. Tropical Pride and cv. New No. 38) and broccoli (Brassica oleracea var. italica Plenck. cv. Green King and Early Value) were infected with an Agrobacterium strain (LBA4404) carrying a distinct disarmed T-DNA containing E. coli sod and/or cat gene. Regenerated rates of hypocotyl explants of Chinese cabbage and hypocotyl and cotyledon explants of broccoli were 1.17~2.5% and 0.67~3.67%, respectively. The results of PCR, Southern, and Northern bolt hybridization indicated that the sod and/or cat genes had been transferred into Chinese cabbage and broccoli, inserted into their genomes, and transcribed. Isozyme profiles of SOD and CAT showed that the E. coli Mn-SOD and CAT were present in the transformed plants. The co-transformation rates of sod and cat genes into a single cabbage or broccoli plant were 52.5%. Severely water-soaked appearances (80~90%) were found in the controls after 3 days of 800 ppb SO2 treatments. Transformants contained both sod and cat genes showed higher resistance (10~20%) to the SO2 treatment than those containing sod or cat gene (50~60%). Our results clearly indicate that alterations in the expression of E. coli sod and/or cat gene had significant stress-resistance and suggested the co-transformation system can be used to develop plants with increasing stress tolerance. The purposes of this study are to explore the possibility of enhanced SOD and/or CAT activities in the cytosol or chloroplast by gene transfer, and to establish the transformation system for improving crop stress-resistance. The superoxide dismutase (SOD) and catalase (CAT) genes of corn were isolated by RT-PCR and cloned into the plant transformation vectors driven by CaMV 35S or rbcS promoter. In addition, the DNA fragment of soybean chloroplast transit peptide was inserted into the constructed chimeras. Hypocotyls and cotyledons of Chinese cabbage and broccoli were infected with a single type Agrobacterium LBA4404 strain carrying a distinct disarmed T-DNA containing maize sod and/or cat gene. The SOD activities were 270 to 400% higher in transformants contained either maize sod and cat genes or sod gene only than the controls. The CAT activities were 80 to 150% higher in transformants contained either maize sod and cat genes or cat gene only than the controls. In the transformants contained soybean transit peptide sequence, more than 85% of maize SOD and/or CAT activities were found in chloroplast. Chloroplast enzyme activities in transformants were 8 to 20 times higher in SOD and 7 to 20 times higher in CAT than that of controls. Transgenic Chinese cabbage plants that overexpress SOD and CAT either in the cytosolic or chloroplastic compartments showed less damage caused by exposure to 400 ppb SO2 for 3 days. Transformants contained both transformed sod and cat genes were more resistant to the SO2 treatment than those contained either sod or cat gene. Increases in SOD and CAT activities were found in transformants that overexpress SOD or/and CAT either in the cytosol or chloroplastic compartments, but only transgenic plants that express increased levels of SOD and CAT had elevated AP and GR activity. Only slight decreases (1~5%) in chlorophyll fluorescence parameter (Fv/Fm) were found in transgenic Chinese cabbage plants that overexpress SOD and CAT either in the cytosolic or chloroplastic compartments after 3 days of 400 ppb SO2 treatments, while 25% decreases were found in controls and 10~25% decreases in transformants that overexpress SOD or CAT either in the cytosol or chloroplast. Overexpression of sod or cat RNA in the mesophyll and vascular tissue of transformants were detected by in situ hybridization. Genetic analysis confirmed Mendelian segregation of the transgenes in R1 generations. Furthermore, co-integration frequency of 50% for the sod or cat genes carried on separate plasmids was found. Only the R1 transformants that overexpress SOD and CAT showed no damage caused by exposure to 200 ppb SO2 for 7 days. There were no differences in appearances between transformants that overexpress SOD and CAT in the cytosol and that in chloroplast upon exposure to SO2 . A cDNA library was constructed using polyA RNA from 800 ppb sulfur-dioxide-fumigated Chinese cabbage leaves for 7 hours. Contained cDNA was cloned into a λZAPII system was screened using a maize sod4 or cat1 as probe. The inset of pSOD62 (sod62) has 69 untranslated bases at the 5'' end preceding the open reading frame 456 bp and then a 228-bp 3'' untranslated region that contains one characteristic polyadenylation signal (AAUAAA) at the nucleotide position 671 and 676 followed by a polyA tail. The open reading frame deduces 152 amino acids and encodes a 15.1 kD protein. Either the nucleic acid or amino acid sequence of sod62 shows above 90% homology with the rice cytosolic Cu/Zn-SOD, and 70~90% with other plants SOD. Six histidine and one aspartic acid of conserved Cu/Zn binding sites, and two cysteine of disulfate bond sites are found in sod62. The inset of pCAT78 (cat78) has 1694 bp that contains an open reading frame of 492 amino acids with an estimated molecular mass of 56.9 kD. There is 98% of homology in the sequences of nucleotide or amino acid between cat78 and cat4 of Brassica juncea, above 80% of homology in comparison with other plants CAT. The open reading frame of cat78 extends from an ATG start codon at position 43 to a TAA stop codon at position 1521 and contains one polyadenylation signal (AAUAAA) at the nucleotide position 1652 to 1657 followed by polyA tail. The sod62 and cat78 protein and specific activity could be detected using SDS and native PAGE by introduction of sod62 and cat78 into E. coli expression vector. High levels of sod62 and cat78 RNA could be detected in Chinese cabbage after 1 hour of 200μM of MV treatment, and remained prominent thereafter till 24 hours.
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Leu, Kuen Lin, and 呂昆霖. "The programmed disintegration and SOD gene cloning of Chlorella / study of chlorophyll polyphasic fluorescence rise." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/6xt7f3.
Full textAbstract:
博士國立清華大學
生命科學系
93
The small vegetative cells of a synchronous Chlorella pyrenoidosa culture subjected to a heat pre-treatment (46.5ºC for 1 h in the dark) and cultured again under continuous illumination thereafter started to disintegrate within hours. Chlorophylls were degraded via photooxidation, started after a 2-h-delay and completed within 8 h. However, no free radicals were released from the sites of photooxidation, but a FeSOD gene from Chlorella pyrenoidosa 211-8b was cloned. The full length of cDNA is 1063 base pairs, with an open reading frame (ORF) of 609 base pairs (203 amino acids), and compared with NCBI gene databank. It revealed 73.5% identity in nucleic sequence and 70.5% identity in amino acids sequence, respectively, with the FeSOD sequence of Chlamydomonas reinhardtii, an eukaryotic unicellular green alga. The nucleic sequence of FeSOD (609 bps ORF) along with the expression vector was transformed into E. coli expression host. An over-expressed protein of about 22.3 kDa was detected. The activity of the expressed protein was assayed by using a method of gel activity staining, and a clear band was observed at about 44 kDa, likely a dimer form of FeSOD. Besides, DNA in nucleus and chloroplast disappeared along with chlorophyll pigments, but mitochondrial DNA appeared to decay at a much slower rate. In addition, a novel nuclease activity was detected in heat-treated cells undergoing DNA degradation. The decomposition of DNA rendered the disintegration process irreversible. Contrary to the programmed cell death of higher plants, these heat-treated Chlorella cells failed to exhibit DNA laddering and massive protein degradation, but retained their cell membrane integrity. Thus, it might be in a way very different from the programmed cell death observed in many higher plants. However, study of chlorophyll fluorescence induction of higher plants, Ficus microcapa L. f. cv. Golden-leaves. The rise of the chlorophyll fluorescence of a whole leaf as induced by high intensity actinic light comprises three distinct phases, termed O-J-I-P polyphasic rise. The initial rise (the O-J phase) was found to be the most sensitive to light intensity, being slower and smaller with decreasing irradiation. The leaf was also found to be transparent for chlorophyll fluorescence to a considerable extent, so that the fluorescence originating from deep inside the sample could still be detected. In contrast, the actinic light used to induce fluorescence was strongly absorbed by chlorophylls, so that a steep light gradient was created along the light path. The fluorescence transient of a leaf thus was always a mixture of the fluorescence from the surface of the sample as well as that from the inside of the sample, whose O-J phase is slower as it is induced by a weaker actinic light. We have provided evidences suggesting that, in an intact leaf, the middle phase of the measured polyphasic fluorescence transient (the J-I phase) might actually reflect the initial rise of the transient coming from the abaxial layer of the leaf.
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Chiu, Ting-Ting, and 邱亭亭. "Study on the Cu,Zn-SOD gene of the Tsou’s osteoarthritis and the possibile effect from environmental toxic metals to the Cu,Zn-SOD molecule." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/66130359182641748921.
Full textAbstract:
碩士臺北醫學大學
醫學研究所
94
Osteoarthritis is characterized by focal loss of cartilage due to an up-regulation of catabolic pathways, induced mainly by pro-inflammatory cytokines. Reactive oxygen species (ROS) have been proposed to involved in this extracellular-matrix-degrading activity, chondrocyte oxidative status responsible for cartilage damage occurring in primarily degenerative joint disease. We presumed that osteoarthritis is associated with sod1 gene. SOD for parenteral administration is in clinical used in several European countries, where it is prescribed principally for treatment of musculoskeletal inflammation, especially osteoarthritis. We hypothesized that the prevalence of osteoarthritis was high in Tsou was thought associated to the SOD activity of articular fluid. It may be accounted by the abnormality of gene or protein activity. In order to explore the relationship of osteoarthritis and sod1 gene, we sequenced 56 Tsou, 48 Taiwanese and 20 Atayal. We hadn’t found any polymorphism or point mutation of sod1 gene. We demonstrated that sod1 gene is unusually stable, so it was hard to find any polymorphism. It is possible that environment population or toxic metals of soil induced abnormality of protein activity. Copper/Zinc-superoxide dismutase (Cu,Zn-SOD) is a homodimer antioxidant enzyme. The reduce in enzyme activity of Cu,Zn-SOD (SOD1) is one of the reasons that caused the familial amyotrophic lateral sclerosis (FALS). The pollution of heavy metal in the general environment may interfere metal binding site of the SOD protein, change its conformation, and lead to a reduced activity of Cu,Zn-SOD in the living being. Among the heavy metals, arsenate, cadmium, chromium, mercury and lead are the most toxic and widely polluted. Therefore, they have been selected to interact with Cu,Zn-SOD in this study. The activities of purified Cu,Zn-SOD from E.coli overexpression system in LB broths containing various concentrations of toxic metals were compared. In addition, the activities of purified Cu,Zn-SOD proteins, from E.coli cultured in higher concentrations of toxic metals, decreased dose-dependently. By ICP-AES, we demonstrated that adding of toxic metals significantly increased the content of toxic metals, but reduced its zinc content of the Cu,Zn-SOD protein. In conclusion, the presence toxic metals have affected the expression of Cu,Zn-SOD in E.coli, but they decreased the enzyme activity probably by replacing the binding of zinc to the metal binding sites. Therefore, the presence of heavy metals in the culture broth might cause incorrect binding of metal ions to the SOD protein, and result in decreased the enzyme activity of SOD, which is a pathogenic reason of osteoarthritis. The obtained information may offer some directions to the studies on pathogenic mechanism of sporadic osteoarthritis.
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林冠宇. "Characterization of zebrafish extracellular superoxide dismutase (ES-SOD) and its developmental gene expression under oxidative stress." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/62998123141472078709.
Full textAbstract:
碩士國立彰化師範大學
生物技術研究所
97
Extracellular superoxide dismutase (EC-SOD) is one of the important antioxidant enzyme that can scavenge superoxide(O2−•) radical in extracellular matrix. In this study, we cloned zebrafish EC-SOD cDNA sequence by RACE-PCR, the deduced amino acid sequence shows higher conservation with more than 60% similarity when compared with other species. The gene was expressed and purified from E. coli. BL21 (DE3) pLysS, and the yield was 170 μg from 200 mL E. coli. culture. The recombinant zEC-SOD can retain about 60% activity at 70℃ for 10 minutes, but lost its activity at 90℃ treatment. The half-life of the enzyme is approximately 72 min and its thermal inactivation rate constant kd is 0.0078 min-1 at 70ºC. The enzyme was active between pH 5-10, and with higher activity at pH 9. The enzyme was SDS and trypsin sensitive. RT-PCR and Real-time RT-PCR were used to analyze this gene expression during zebrafish embryonic development. The results showed zEC-SOD mRNA was first detected at 1-cell stage, and with higher expression between 24-36 h post fertilization (hpf). The gene was major expressed in head, heart and blood vessel by whole mount in situ hybridization. The embryos were treated with Cu2+ (2.5 ppm) and LPS (10 μg/mL) for 48 h to induce oxidative stress, the expression of zEC-SOD was not increase but was inhibited more than 10-40 folds. This result indicated the oxidative gene regulation of zEC-SOD is puzzled. The Antioxidant-1 (Atox1) is a copper chaperone and a new transcription factor that can regulate EC-SOD gene expression. We also cloned the zebrafish Atox1 cDNA sequence, and the deduced amino acid sequence is about 70% similarity when compare with other species. In gene expression profile analysis, showed atox1 mRNA can detect at 1-cell stage and a lot of expression after 12 hpf. In whole mount in situ hybridization assay, this gene was major expressed in head, heart and blood vessel, just the same as the expression of zEC-SOD. Under copper or LPS induced oxidative stress, the gene expression of Atox1 was increased rather than zEC-SOD that was inhibited. The results showed Atox1 was not responsible for oxidative regulation of zEC-SOD gene expression during embryonic development. To analyze zebrafish antioxidant enzyme Cu/Zn-SOD、Mn-SOD、Calatase、Prx-2、Prx-6 gene expression under LPS treated. Gene expression assay showed Cu/Zn-SOD、Prx-2 and Prx-6 mRNA was no variation , but Mn-SOD and Calatase mRNA was inhibited. The results showed that LPS-induced oxidative stress can regulate Mn-SOD and calatase gene expression.
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Chen, Yi-Ping, and 陳奕平. "The Molecular Action Mechanism of Human Cu,Zn-SOD Gene Deficiency-induced Cell Death in Amylotrophic Lateral Sclerosis." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/24312327188321119641.
Full textAbstract:
碩士台北醫學院
醫學研究所
89
The most frequent genetic causes of amylotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene coding for copper/zinc superoxide dismutase (Cu, Zn-SOD).The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SOD protein. We found that sequence of genomic sod1 gene from a sporadic ALS patient has two point mutations.In order to investigate the possible roles on genetic causes of ALS, SOD cDNA and mutants were constructed and transduced into PC12 cell to observe the cellular consequences. RT-PCR from human liver extract generated the SOD gene (sod1). Wild-type sod1 was constructed into a transcription-translation expression vector to examine the SOD1 production in vitro. Wild-type SOD1 was highly expressed in Sf-9 cells infected by Baculovirus and in E. coli. Active SOD1 was expressed by IPTG induction and in a metal-dependent matter.SDS-PAGE and Western blot analysis illustrated a 23kDa monomer product.To study the cellular effect of SOD1, Tat-tag was used as a cellular transduction delivery vehicle. Denatured Tat-sod1 was observed to be successfully transduced into undifferentiated PC12 or differentiated PC12 neural cells and retained its activity via protein refolding. Site-directed mutagenesis created a genetic deficiency in tat-tag wild-type with three point mutations as E21K, D90V and D101G. Tat-tag mutants expressed in E. coli completely lost SOD activity.In undifferentiated PC12 cells, Tat-SOD avoided DNA fragmentation from superoxide attack generated by 70mM paraquat; however, mutant Tat-D101G enhanced cell death.
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Lu, Chiao-Hua, and 盧俏樺. "The association of the adult asthma with the activity and gene polymorphism of the antioxidant SOD and Catalase." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/86727058835336948980.
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碩士高雄醫學大學
公共衛生學研究所
96
Background： Adult asthma is a multifactorial disorder caused by interaction effects of genetic and environmental factors. Antioxidant enzyme like SOD or catalase may contribute to the pathogenesis of asthma. It may play an important role in the development of inflammatory airway diseases such as asthma.In the present study, we evaluated the activities and polymorphisms of SOD and catalase gene between adult asthmatic cases and normal controls. Materials and Methods： This is a matched case-control study. We enrolled 296 adult asthmatic cases from Chung-Ho memorial hospital, Kaohsiung Medical University and Chang-Gung memorial hospital in Taiwan. And 251 community-based controls who matched age(±7 years), sex and race to asthmatic adults. All subjects performed questionnaire, blood sample collection and lung function test, and also conducted DNA genotyping (ABI-7900HT real-time PCR). 33 incident cases and 65 matched normal controls who are non-smokers or quit smoking at least 1 year were selected to test the SOD and catalase activity (Spectrophotometer). Results and Discussion： The less frequent variant of SOD gene(Ala16Val, AV/VV) was found to be dangerous from the development of asthma(AOR＝1.98, 95％CI：1.08-3.65). The less frequent variant of CAT gene(C-262T, CT/TT) was found to be protective from the development of asthma(AOR＝0.34, 95％CI：0.13-0.88). The activity of SOD in new onset asthma patients was significantly lower than that in control subjects(412.49±82.89 U/ml，475.40±117.55 U/ml；P value＝0.012). The activity of CAT in new onset asthma patients was significantly higher than that in control subjects(83.40±59.58 nmol/min/ml，35.57±34.90 nmol/min/ml；P value<0.0005) . Conclusion： The difference of SOD and CAT activity is related to the presence of asthma. The measure of SOD activity can be a tool to differentiate the possible asthma population or can be considered a marker to disease severity of asthma.
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"Role of the vascular endothelium in erectile physiology and disease: Influence of in vivo gene transfer ofeNOS and EC-SOD." Tulane University, 2004.
Find full textAbstract:
Erectile dysfunction (ED) is predominately a disease of vascular origin. It is well recognized that the incidence of ED dramatically increases in men with advancing age and diabetes mellitus. Endothelial-derived nitric oxide (NO) is an important factor in cardiovascular homeostasis and erectile function. Given the impact of endothelial-derived NO in vascular biology, we hypothesized that impairment in NO synthesis from the penile endothelium cause age- and diabetes-associated-ED. Neurogenic- and endothelium-dependent erectile function was significantly impaired with advancing age and after diabetes induction with streptozotocin in Brown Norway and Sprague Dawley rats. Corporal endothelial nitric oxide synthase (eNOS) and the second messenger, cGMP, were significantly decreased in the penile vascular bed of the aged and diabetic rat suggesting reductions in erectile function are a result of impairments in the NO/cGMP system. Adenoviral gene transfer of endothelial NOS (eNOS) to the aged and diabetic rat penis restored corporal eNOS expression/activity and cGMP levels thus improving neurogenic- and endothelium-dependent erectile responses. In another series of experiments, superoxide anion formation and total superoxide dismutase (SOD) were measured in the aged and diabetic penile vascular bed. Superoxide anion formation was significantly increased in the penis of the aged and diabetic rat with no change in the antioxidant SOD. Adenoviral gene transfer of extracellular SOD (EC-SOD) to the aged and diabetic rat penis reduced corporal superoxide anion formation, peroxynitrite, and improved cGMP levels thus improving neurogenic- and endothelium-dependent erectile responses. These data suggest eNOS and EC-SOD gene transfer can improve endogenous NO production in the penis and restore erectile function In another series of experiments, RhoA/Rho-kinase expression was measured in the penile vascular bed of the diabetic rat. RhoA/Rho-kinase expression/activity were upregulated in the diabetic corpora. Adeno-associated viral gene transfer of the dominant negative RhoA mutant (T19NRhoA) reduced RhoA/Rho-kinase expression and improved eNOS expression/activity and cGMP levels thus improving neurogenic-mediated erectile function. These data imply that inhibition of RhoA/Rho-kinase improves eNOS expression/activity thus restoring erectile function in diabetes. Putative gene therapy interventions to restore eNOS expression, endothelial NO bioavailability and subsequent endothelial function may represent an exciting new therapeutic strategy for the future treatment of EDacase@tulane.edu
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Nguyen, Linda. "The effects of photosymbiosis on gene expression in the facultatively symbiotic coral Astrangia poculata, with a focus on NF-kappaB signaling and antioxidant enzymes." Thesis, 2020. https://hdl.handle.net/2144/41678.
Full textAbstract:
Corals are critical to marine biodiversity and human welfare. Coral reefs cover <1% of the seafloor but support ~1/3 of all marine species. Approximately 1.5 billion people live within 100 km of coral reefs, relying upon them for food, income from tourism, and protection from storms. Their economic value has been estimated at $375 billion annually.
The foundation of coral reefs is the intracellular symbiosis between corals and photosynthetic dinoflagellates of the family Symbiodiniaceae. Tropical corals satisfy up to 95% of their nutritional requirements through photosynthesis, and their ability to construct reefs is biochemically coupled to photosynthesis.
While permitting corals to thrive, photosymbiosis also increases their exposure to environmental stressors and vulnerability to climate change. Reliance on photosynthesis restricts reef-building corals to shallow, clear, tropical waters, where they experience higher temperatures and UV exposure. The generation of reactive oxygen species by the symbiont also exposes corals to greater oxidative stress. The symbiosis is particularly sensitive to climate change: all of the mass coral bleaching events have occurred since 1982, driven by elevated ocean temperatures.
Molecular cross-talk between host and symbiont impacts resilience of the coral holobiont and resistance to bleaching. Unfortunately, we know little about how photosymbiosis impacts expression or activity of coral genes. Tropical corals engage in an obligate symbiosis with Symbiodiniaceae, so we cannot study their gene expression in a stable aposymbiotic state. However, the northern star coral, Astrangia poculata, engages in a facultative symbiosis with Symbiodiniaceae.
I used RNA sequencing to investigate how symbiosis impacts gene expression in A. poculata, focusing on genes implicated in photosymbiosis: antioxidant enzymes (specifically superoxide dismutases) and the NF-κB signaling pathway. From an improved transcriptome assembly, I recovered core elements of a primitively simple NF-κB signaling pathway and a rich complement of SOD proteins. 273 coral transcripts—many associated with protein metabolism and vesicle-mediated transport— were differentially expressed in symbiotic versus aposymbiotic corals. Unlike in the facultatively symbiotic sea anemone Exaiptasia, symbiosis was not associated with depressed NF-κB transcript levels. IKKε, a potential positive regulator of NF-κB activity, was strongly up-regulated, as was one particular superoxide dismutase.
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Borstelmann, Sonko [Verfasser]. "Bedeutung von Einzelnukleotidpolymorphismen in den Genen TGFβ [TGF-Beta] und SOD2 bei Brustkrebspatientinnen für die Fibrose nach Strahlentherapie / Sonko Borstelmann." 2010. http://d-nb.info/1000346153/34.
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Wang, Vinchi, and 王文奇. "Studies on signal transduction pathway of NO-induced prion protein expression and the genetic polymorphisms of sod2 and prnp genes in Taiwanese population." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/17798884090410516087.
Full textAbstract:
博士國防醫學院
醫學科學研究所
94
The neurodegenerative disorders, such as Parkinson’s disease and human Creutzfeldt-Jakob disease, may involve nitric oxide signaling. Nitric oxide (NO) acts as a signaling gas molecule and also plays a role under stress conditions in neurodegeneration. The prion protein (PrP) is the key pathogen in the transmissible spongiform encephalopathy. We proposed that the NO might mediate the induction of PrP. The lipopolysaccharide and NO donor, sodium nitroprusside, were used as the NO stimuli in cellular studies. MEK and p38 MAPK signaling were documented by the pharmacological blockers on the induction of PrP by NO. The NO during stress conditions in neurodegeneration lays prime importance on further investigation about the pathogenesis and therapeutic approach. Besides, due to the importance of oxidative stress in neurodegeneration, the genetic evaluation about the general population in Mn-bound superoxide dismutase (MnSOD) and PrP may be a new molecular epidemiological study for the public health. The results showed that the Taiwanese genetic polymorphisms in MnSOD and PrP had different distributive pattern as compared with those in the Caucasians. It is interesting for further monitoring the disease incidence and prevalence in the population, especially the neurodegenerative disorders.
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吳佳春. "Transformation of superoxide dismutase(sod) and catalase(cat) genes of chinese cabbage into cabbage(brassica oleracea L. var. capitata L.)and chinese cabbage(Brassica campestris L. ssp. Pekinensis." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/83187369563311763743.
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Spieler, Derek. "Molekulare Analyse eines Homöobox-Gen-Promotors in der Gehirnanlage von Wirbeltierembryonen." Doctoral thesis, 2002. http://hdl.handle.net/11858/00-1735-0000-0006-B325-D.
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