Efeito do ácido linoléico conjugado TRANS-10, CIS-12 na regulação do acúmulo de lípides e expressão gênica em embriões produzidos in vitroBatista, Ribrio Ivan Tavares Pereira 25 February 2010 (has links)
Submitted by Renata Lopes (email@example.com) on 2016-09-16T11:53:05Z No. of bitstreams: 1 ribrioivantavarespereirabatista.pdf: 1023205 bytes, checksum: 9149da7ed1e431e5565e4816dc74a814 (MD5) / Approved for entry into archive by Diamantino Mayra (firstname.lastname@example.org) on 2016-09-26T20:21:30Z (GMT) No. of bitstreams: 1 ribrioivantavarespereirabatista.pdf: 1023205 bytes, checksum: 9149da7ed1e431e5565e4816dc74a814 (MD5) / Made available in DSpace on 2016-09-26T20:21:30Z (GMT). No. of bitstreams: 1 ribrioivantavarespereirabatista.pdf: 1023205 bytes, checksum: 9149da7ed1e431e5565e4816dc74a814 (MD5) Previous issue date: 2010-02-25 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A suplementação do ácido linoléico conjugado trans-10, cis-10 no meio de cultivo, representa uma importante alternativa para aumento da sobrevivência dos embriões após a criopreservação. No entanto este isômero de CLA no cultivo in vitro sem antioxidante pode aumentar a taxa apoptótica. O objetivo do presente estudo foi avaliar o efeito da adição CLA trans-10, cis-12 no cultivo in vitro de embriões sem antioxidante. Zigotos bovinos (n = 1.694) foram divididos em dois tratamentos: (T1) grupo controle, zigotos cultivados no meio CR2aa suplementado com soro fetal (n = 815); (T2) zigotos cultivados no meio CR2aa suplementado com soro fetal mais 100 µM CLA trans-10, cis-12. Os embriões foram avaliados quanto a desenvolvimento, quantidade de lípides e criotolerância. Transcritos dos RNA mensageiros (RNAm) dos genes selecionados foram mensurados pelo Real Time PCR. Suplementação de CLA trans-10, cis-12 não afeta significativamente a taxa de blastocisto (31,8% e 34,1% T1 e T2, respectivamente, p = 0,20) e nível dos RNAm dos genes relacionados com stress celular, apoptose e síntese de novo de ácido graxo. Quantidade de lípides e transcritos do RNAm do gene 1-acilglicerol-3-fosfato oaciltransferase 1 enzima relacionado a síntese de triglicérides foram significativamente reduzidos nos embriões cultivados na presença de CLA trans-10, cis-12 em comparação com o grupo controle. Teve aumento significativo na taxa de re-expansão dos blastocistos cultivados na presença de CLA trans-10, cis-12, após o descongelamento (34.4 e 56.3% para T1 e T2, respectivamente p = 0,002). Essa diferença não persistiu na taxa de eclosão (14,0% e 16,5% para T1 e T2, respectivamente, P = 0,62). Esses resultados mostram que o CLA trans-10, cis-12 reduz o acúmulo de lípides nos embriões pela redução nos níveis dos transcritos do gene 1-acilglicerol-3-fosfato o-aciltransferase 1 sem afetar a qualidade do embrião. Adicionalmente, este ácido graxo aumenta a taxa de re-expansão, no entanto, não melhora a taxa de eclosão. / Supplementation of conjugated linoleic acid trans-10, cis-10 in the culture medium, represents an important alternative to increasing the survival of embryos after cryopreservation. However the addition of culture media with CLA trans-10, cis-12 without antioxidant may increase the apoptotic rate. The aim of this study was to evaluate the effect of adding CLA trans-10, cis-12 in vitro culture of embryos without antioxidant. Bovine zygotes (n = 1,694) were divided into two treatments: (T1) control group, zygotes cultured in CR2aa medium supplemented with fetal calf serum (n = 815), (T2) zygotes cultured in CR2aa medium supplemented with fetal calf serum plus 100 µM CLA trans-10, cis-12. Embryos were evaluated for development, amount of lipids and cryotolerance. Transcripts of messenger RNA (mRNA) of selected genes were measured by real time PCR. Supplementation of CLA trans-10, cis-12 did not significantly affect the blastocyst rate (31.8% and 34.1% T1 and T2, respectively, p = 0,20) and the mRNA level of genes related to cell stress, apoptosis and de novo synthesis of fatty acid . Lipids and transcripts of the mRNA of the gene 1-acilglicerol-3-phosphate o-acyltransferase 1 enzyme related to the synthesis of triglycerides were significantly reduced in embryos cultured in the presence of CLA trans-10, cis-12 in comparison the control group. Had increased rate re-expansion of blastocysts cultured in the presence of CLA trans-10, cis-12, after thawing (34.4 and 56.3% for T1 and T2, respectively p = 0,002). This difference did not persist in the hatching rate (14.0 and 16.5% for T1 and T2, respectively, P = 0,62). These results show that the CLA trans-10, cis-12 reduces the accumulation of lipids in the embryos by reducing the levels of gene transcripts acilglicerol-1-3-phosphate oacyltransferase 1 without affecting the quality of the embryo. Additionally, this fatty acid increases the rate re-expansion, but does not improve the hatching rate.
Stadienspezifische Expression und Lokalisation Kalzium-abhängiger Proteinkinasen (CDPK) von Cryptosporidium parvum in der In-vitro-KulturEtzold, Manja 28 September 2015 (has links) (PDF)
Die Kryptosporidiose stellt aufgrund ihres zoonotischen Charakters und der Entwicklung chronischer Durchfälle bei Immunsupprimierten ein hohes Gesundheitsrisiko für den Menschen, aber ebenso für Tiere dar. Derzeit verfügbare Therapeutika ermöglichen keine zuverlässige Bekämpfung klinischer Symptome oder eine Erregerelimination, daher ist die Erforschung neuer Therapieansätze dringend notwendig. CDPK stellen in diesem Zusammenhang interessante Zielmoleküle dar, da sie zwar in Pflanzen und Protisten einschließlich Apikomplexa, jedoch nicht in Pilzen und Säugetieren vorkommen. Trotz der Entdeckung vielversprechender neuer Wirkstoffe gegen CpCDPK1 in den letzten Jahren ist zur Lokalisation und Funktion von CDPK in C. parvum wenig bekannt.Diese Arbeit belegt die Transkription von sechs CpCDPK in vitro und beschreibt erstmals die Länge der 3’UTR von CpCDPK. Die Translation wurde durch den Nachweis spezifischen Proteins in Sporozoiten im Immunoblot sowie die Lokalisation von CpCDPK1 mit Hilfe der Immunfluoreszenz belegt. Möglicherweise wird die CpCDPK1 durch N-Myristoylierung an Membranen gebunden, an die Oberfläche von Zoiten gebracht und sezerniert. Eine Rolle des Enzyms im Invasions- und Egressmechanismus des Parasiten wird diskutiert.
DEVELOPMENT OF A NEW ALLELIC DISCRIMINATION REAL-TIME PCR ASSAY FOR THE DIAGNOSIS OF EQUINE HERPESVIRUS-1 AND CHARACTERIZATION OF THE VIRULENCE DETERMINANTS OF THE VIRUSSmith, Kathryn L 01 January 2013 (has links)
Equine herpesvirus-1 (EHV-1) can cause acute upper respiratory tract disease, abortion, neonatal death and neurological disease in horses. Rapid, accurate and timely diagnosis of EHV-1 infection in horses is important to curtail the spread of this pathogen. It has been reported that the neuropathogenic phenotype of EHV-1 can result from a single non-synonymous nucleotide substitution at position 2254 (A→G2254) in open reading frame 30 (ORF30). This was the basis for the development of an allelic discrimination, real-time PCR assay to distinguish between potential neuropathogenic and non-neuropathogenic EHV-1 strains. However, PCR analysis of a panel of EHV-1 abortion isolates revealed that other point mutations within ORF30 could produce false negative results with this previously described assay. Therefore, one of the objectives of this dissertation project was to develop a more sensitive and specific allelic discrimination real-time PCR assay for the detection of EHV-1. This was achieved by redesigning the primers and probes targeting ORF30. The new assay was ten times more sensitive than the original assay, with a lower detection limit of 10 infectious virus particles. While mutations within EHV-1’s genome can hinder diagnosis, they can also impact the virulence of the virus. Objective two, therefore, was to determine if sequential cell passage of T953 would induce sufficient attenuation of the EHV-1 genome to produce a low virulence phenotype. Two separate groups of 28 BALB/c mice were inoculated with either the parental strain or passage 135 (T953 P135) of EHV-1 strain T953. The animals were observed for fourteen days, euthanized and their tissues analyzed for the presence of EHV-1. At the conclusion of the fourteen day observation period, all of the mice infected with T953 P135 survived and regained their pre-inoculation body condition. Furthermore, there were significant differences in virus titer and viral DNA concentrations between T953 P135 and the parental strain, further confirming the attenuated phenotype of the virus. Taken together, data from this study clearly demonstrates that sequential cell culture passage of the neuropathogenic T953 strain of EHV-1 results in attenuation for young adult BALB/C mice.
Biodegradace 17alfa-ethinylestradiolu enzymy ligninolytických hub / Biodegradation of 17alfa-ethinylestradiol by enzymes of ligninolytic fungiPřenosilová, Lenka January 2012 (has links)
This work is aimed at the study of the effect of 17α-ethinylestradiol (EE2) on the production and characteristics of ligninolytic enzymes (laccase, Mn-dependent peroxidase and lignin peroxidase) in I. lacteus, T. versicolor, P. chrysosporium and P. ostreatus cultures grown on two types of liquid media. Enzyme activity production in fungal cultures was affected by the composition of culture medium. In the case of P. chrysosporium, the addition of EE2 to the complex- medium cultures led to a MnP activity stimulation and simultaneously LiP production was partially repressed in these cultures. In the mineral MM medium, no effect of EE2 on enzyme production by P. chrysosporium was observed. In EE2 treated MM cultures of P. ostreatus lower MnP activities were found when compared to biotic controls. In the case of T. versicolor cultures, the addition of EE2 to the complex medium caused laccase and LiP stimulation in the cultures. In the MM medium, however, only laccase production was affected by EE2. I. lacteus MnP production was partially repressed by EE2 in MM medium. In contrast to that, significantly higher MnP activities were detected in complex- medium I. lacteus cultures after the treatment with EE2. Further EE2 degradation by the fungal cultures was studied. The highest degradation effeciency was...
Análise da expressão gênica induzida por Phakopsora pachyrhizi em soja / Analysis of gene expression induced for Phakopsora pachyrhizi in soybeanBrito Júnior, Salvador Lima 26 July 2007 (has links)
Made available in DSpace on 2015-03-26T13:42:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 790827 bytes, checksum: 31229e606f6ff3a38ad36a37dcb0cbb9 (MD5) Previous issue date: 2007-07-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Soybean Asian Rust, a disease caused by the fungi Phakopsora pachyrhizi, has caused serious economic damages to the Brazilian and world-wide soybean crop. The development of soybean lines resistant to this fungi has been a challenge, and currently the measures of control this disease are based on the handling of the crop and application of fungicides. With the objective to advance the knowledge on the molecular mechanism of defense of the plant to the Soybean Asian Rust, it was evaluated, by PCR in real time, the expression of genes that supposedly are involved in the defense mechanism. Two genotypes had been selected for the conduction of the experiment, PI 230970, that it possess allele Rpp2 and develops type RB type injuries, when in presence of P. pachyrhizi, and Embrapa-48, susceptible, which develops injuries of Tan type. The experiment was carried out in Fitotron and for the analysis of the gene expression was done using RNAs from leaves of the soybean inoculated either with pathogen or mock-inoculated with water. Amongst the evaluated genes, chitinase presented an expression 117 times higher in the resistant genotype (PI 230970), when compared with the calibrator (PI 230970 mock-inoculated) possibly acting as a barrier of defense of the plant. In the susceptible genotype the expression of this gene was 1.75 time higher that your calibrator (Embrapa-48 mock-inoculated). It was observed a differential expression of related genes with reactive species oxygen, production of fitoalexinas as well as involved genes in the release of elicitors. These genes can be acting in the mechanism of defense against P. pachyrhizi in the resistant genotype; however its expression in the susceptible genotype can be related to a bigger settling of the pathogens in the tissues of the host or to a delayed response the infection. The identification of a defense route should be farther investigated, with the objective to identify more specific genes in the process of cellular defense and to point out new alternatives for the development of resistant cultivars. / Ferrugem asiática da soja, uma doença causada pelo fungo Phakopsora pachyrhizi, tem provocado sérios danos econômicos à sojicultura brasileira e mundial. A obtenção de linhagens de soja resistentes ao fungo tem sido um desafio, e atualmente as medidas de controle da doença são baseadas no manejo da cultura e aplicação de fungicidas. Com o objetivo de avançar o conhecimento sobre o mecanismo molecular de defesa da planta frente a ferrugem asiática da soja, avaliou-se, através da técnica de PCR em tempo real, a expressão de genes que supostamente estão envolvidos no mecanismo de defesa. Dois genótipos foram selecionados para a condução do experimento, PI 230970, que possui o alelo Rpp2 e desenvolve lesões do tipo RB, quando em presença de P. pachyrhizi, e Embrapa-48, suscetível, desenvolvendo lesões tipo TAN. O experimento foi conduzido em sala climatizada (Fitotron) e para a análise de expressão gênica foram extraídos RNAs de folhas da soja inoculadas com o patógeno e falso-inoculadas (água). Dentre os genes avaliados, quitinase apresentou uma expressão 117 vezes maior no genótipo resistente (PI 230970), quando comparado com o calibrador (PI 230970 falso-inoculado), atuando possivelmente como uma barreira de defesa da planta. No genótipo suscetível a expressão deste gene foi 1,75 vezes maior que o seu calibrador (Embrapa-48 falsoinoculado). Foi observada uma expressão diferencial de genes relacionados a espécies reativas de oxigênio, produção de fitoalexinas bem como genes envolvidos na liberação de elicitores. Esses genes podem estar atuando no mecanismo de defesa contra P. pachyrhizi no genótipo resistente, porem sua expressão também no genótipo suscetível pode estar relacionado a uma maior colonização do patógeno no tecido hospedeiro ou uma resposta tardia à infecção. A identificação de uma rota de defesa pode possibilitar o estudo mais aprofundado da mesma, com o objetivo de identificar genes mais específicos no processo de defesa celular e apontar novas alternativas para obtenção de cultivares resistente.
BK-polyomavirová infekce u pacientů po kombinované transplantaci ledviny a pankreatu / BK-polyomavirus infection in patients after simultaneous pancreas and kidney transplantationMindlová, Martina January 2011 (has links)
Introduction. The aim of the study was to introduce a new BKV PCR protocol in our centre and to verify its accuracy as well as to assess the prevalence, risk factors of BK virus replication, course of BKV infection and therapeutic approaches in simultaneous pancreas and kidney (SPK) recipients in order to design a screening protocol. Methods. The results analysed by both Affigene® and Transplantation Virology, Basel PCR protocols were compared. Thereafter 183 SPK patients were examined to assess the prevalence of BK viremia, viruria and BKVN and to identify the risk factors of BKV replication. The cases of retransplantation after a graft loss due to BKVN were retrospectively described. Results. 100 of results were analysed according to the Affigene ® and Transplantation Virology, Basel PCR protocols with the accordance of 95%, Rho = 0,946, 95% CI: 0.920 - 0.963, P<0,0001, Bland-Altman plot analyses: bias Basel PCR protocol/Affigene® BKV trender: -0,1 (mean) *±1.96 SD: -1,6 - 1,3] for both methods. Point-prevalence was assessed in 183 patients; Viruria found in 17,3 %, viremia in 3.8% of patients. High-level viruria >107 copies/mL detected in 3,7% of patiets, high-level virémia >104 in 1,6% of patients simultaneously with high-level viruria. BKVN was found in 0,5% of patients. Diabetes duration...
01 September 2004
(has links) (PDF)
In recent years, foods produced by genetic engineering technology have been on the world food market. The biosafety aspects, regulations, and labelling of these foods are still contentious issues in most countries. It is necessary to have approval for the use of GMOs in the production of food. Thus, detection and quantification of GMOs play crucial role for developing regulations on GM foods. In this study, raw and processed maize samples were analysed for genetic modification using a DNA based detection method, the Polymerase Chain Reaction. Ten raw food and 18 processed maize food including maize flour, starch, corn flakes, maize chips were collected from different markets located in different places in Turkey. The samples were examined for the presence of genetic elements located in the majority of transgenic crops such as NOS terminator, CaMV 35S promoter, kanamycin resistance (KanR) gene, using conventional PCR with oligonucleotide sets targeting to novel genes. Furthermore screening was conducted via Real-Time PCR assay for NOS terminator and 35S promoter. For confirming the presence of Bt11 maize lines event specific primers were utilised. Quantification of Bt11 maize lines were performed via Real-Time PCR. The result indicates that foreign genetic elements were found in all analysed raw material. In six out of 10 raw material, presence of Bt11 gene were identified. GMO detection was also possible for maize flour and starch, however in processed material as corn starch, corn flakes, corn chips and pop corn, transgenes were not detected.
Transcriptional analysis of chicken immune cells following exposure to 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD)Puebla-Osorio, Nahum 12 April 2006 (has links)
In the present investigation, microarray analysis was used to identify potential TCDD gene targets. Three microarray experiments were performed to study the effect of TCDD in an established chicken B-cell line (DT40), in a chicken macrophage cell line (HD11), and in the bursa of Fabricius from embryos exposed in ovo at 6 days of incubation. From the DT40 microarray analyses, clones with sequence similarity to the apoptotic genes caspase 8 and caspase 9, and the transcription factor NFΜB, among others, were identified. Real-time quantitative polymerase chain reaction (RT-PCR) revealed that TCDD elicits aryl hydrocarbon receptor (AhR)-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3 (see chapter III). During the course of the HD11 microarray analyses, a consistent down-regulation of the matrix metalloprotease MMP-2 was observed. This finding was the basis for the hypothesis that TCDD has an effect on the gene expression of the MMP-2 and MMP-9 in macrophages. Then, gene expression analysis and functional zymography showed that TCDD impairs the MMP-2 and MMP-9 response to LPS stimulation in HD11 chicken macrophages (see chapter V). The microarray analyses of the embryonic bursa of Fabricius provided the basis to further study of the effect of TCDD in the chicken embryo. The shifted genes were classified according to their function. The down-regulated genes included: precursor of matrix metalloprotease-inhibitor, histone acyl-transferase 1, homeobox protein CUX-2, Death Associated Protein Kinase, and UDPglucosyl transferase, among others. The up-regulated genes included: phosphoinositidespecific phospholipase, acyl Co-A oxidase, and protein effector of Cdc42, among others. Together, these microarray analyses produced a database of genes of interest that will provide sufficient hypotheses to inspire multiple investigations aimed at confirming and refining the gene expression alterations as a consequence of TCDD exposure.
Investigating the role and activity of CC-Type glutaredoxins in the redox regulation of TGA1/TGA4 in <i>Arabidopsis thaliana</i>Hahn, Kristen Rae 07 July 2009 (has links)
Plants respond to and defend themselves against a wide range of disease-causing microbes. In order to do so, massive reprogramming of cellular protein expression patterns, which underpin various defense pathways, must occur. A family of basic leucine zipper transcription factors, called TGA factors, has been implicated in mediating this response. The TGA factors themselves are subject to complex regulation; of note, TGA1 and TGA4 are regulated via a reduction of conserved cysteines after treatment with the phenolic signaling molecular salicylic acid, which accumulates following pathogen challenge. Previous studies indicate that TGA factors physically interact in the yeast two-hybrid system with the plant-specific CC-type of glutaredoxin (Grx)-like proteins. Grx are a family of oxidoreductases that are important for maintaining the cellular redox status and often are required to modulate protein activity. The goal of this study was to ascertain the role of these Grx-like proteins in regulating TGA1 redox state. To this end, the expression patterns of several Grx genes were analyzed.<p> Quantitative-reverse-transcriptase PCR (q-RT-PCR) experiments indicated that TGA1 and TGA4 may be involved in down-regulating levels Grx-like gene transcripts after exposure to pathogens or salicylic acid (SA). Furthermore, qRT-PCR experiments also indicated that expression of some Grx-like genes is induced by SA, jasmonic acid (JA), and <i>Pseudomonas syringae</i>. Overexpression of the Grx-like protein, CXXC9, in <i>Arabidopsis thaliana</i> revealed that it is a regulatory factor in the cross-talk between vi theSA/JA pathways as it is able to suppress expression of PDF1.2, a marker for the JA defense pathway, as determined by qRT-PCR. The â-hydroxy ethyl disulfide (HED) assay was utilized to determine if the CC-type of Grx-like proteins have oxidoreductase activity <i>in vitro</i>. These studies revealed that that the Grx-like proteins do not exhibit oxidoreductase activity in this assay.
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