Made available in DSpace on 2015-03-26T13:42:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 209546 bytes, checksum: d5ff785c114b2b115999b7a4b5ade9bd (MD5) Previous issue date: 2007-07-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Among the productive traits, the number of the piglets is essential for success in swine production. The ovulation rate is one of the key factors that determine the litter size in pigs and the study of gene expression in the ovary follicles, more precisely in the follicular cells, might cause a big advance in the comprehension of this characteristic. Thus, the objective in this work was to identify the differential expression of the STAR (Steroidogenic Acute Regulator-STAR), GATA (GATA binding protein 4), PGF2α (Prostaglandin F2α), P4R (Progesterone Receptor), FSHR (Follicle Stimulating Hormone Receptor) and CYP19 (Cytochrome Aromatase P450) genes in follicular cells, colleted during the follicular phase in the estrous cycle of three sows with high and three sows with low litter size which comes from a tricross of the Landrace, Large White and Pietrain breeds, throughout the semi quantitative real time PCR (qPCR). In the hyperprolific sows, analyzed in this study, the average of the number of the piglets were 8,51 and for the hypoprolific sows the average was 12,03. For examination if any one related genes above were affecting the ovulation rate in the prolificity rate in the animals studied, the total RNA of the follicular liquid was extracted and than was made two pools with the total RNA of the hypo and hyperprolific sows groups. The total RNA of each pool was used for the first strand cDNA synthesis. The PCR reactions were accomplished using as an endogenous control the β-Actina gene. The primers were generated by sequences in the Pig ESTs data base. The major expression difference was observed for P4R gene, who express 7,73 turns mostly in the animals with low prolificity. The PGF2α gene displayed differential expression of 2,84 times as a large in the hypoprolific animals. The STAR gene expression, that codes for the protein regulating hormone steroids synthesis, was 2.32 turns mostly in the animals with low prolificity sows. The GATA gene expression was 2.31 times as a large in the hypoprolific sows. This gene codes for the GATA binding protein 4 belonging a transcription group factors that are responsible for too many gene expression and diverse cell differentiation types. The CYP19 gene expression taken the enzyme production that is the key factor in the estrogens biosynthesis, the Cytochrome Aromatase P450 (P450aro), for this gene was observed the relative expression of the 2.30 turns mostly in the sows with low prolificity. The FSHR gene expression hasn t achieved the threshold difference established in this study (1.5 times).The STAR, GATA, PGF2α, FSHR, P4R and CYP19 gene expression in the follicular cells has enabled identify difference of expression between sows with low and high prolificity, where the largest difference of expression in relation to the studied genes was verify in the hypoprolific sows, except for the FSHR gene that wasn t considerate differentially express in this work. With views the best agreement about the reproductive phenotypes in swine, it s necessary yet, studies to examine the expression in these and other genes related to female reproductive cycle, in as much as gene expression is dependent, in the midst of another factors, the stage of development of the tissue and the animal age. / Dentre as características produtivas, o número de leitões é fundamental para o sucesso na produção de suínos. A taxa de ovulação é um dos principais fatores que determinam o tamanho da leitegada. Deste modo, o estudo da expressão gênica nos folículos ovarianos, mais precisamente nas células foliculares, pode causar grande avanço no entendimento desta característica. Portanto o presente trabalho foi realizado com o objetivo de identificar a expressão diferencial dos genes STAR (Proteína esteroidogênica regulatória aguda), GATA (Proteína de ligação GATA-4), PGF2α (Prostaglandina F2α), P4R (Receptor de progesterona 4), FSHR (Receptor do hormônio folículo estimulante) e CYP19 (Citocromo aromatase P450) em células foliculares, coletadas durante a fase folicular do ciclo estral de três fêmeas suínas de alta e três de baixa prolificidade, provenientes de um tricross das raças Landrace, Large White e Pietrain, por meio da metodologia de PCR quantitativo em tempo real (qPCR). Nas fêmeas com alta prolificidade, utilizadas neste estudo, a média do número de leitões por leitegada foi 8,51 enquanto para as fêmeas com baixa prolificidade foi de 12,03. Para examinar se algum dos genes relacionados acima afetava as taxas de prolicifidade nos animais deste estudo, o RNA total do líquido folicular foi extraído e, em seguida, fez-se dois pools com o RNA total, sendo um do grupo de fêmeas com alta prolificidade e o outro do grupo com baixa prolificidade. O RNA total de cada um dos pools foi usado na confecção da primeira fita de cDNA. As reações de qPCR foram realizadas usando-se o gene β-Actina como controle endógeno. Os primers foram gerados a partir de seqüências obtidas nos bancos de ESTs de suínos. A maior diferença de expressão foi observada para o gene P4R, que foi expresso 7,73 vezes a mais nos animais com baixa prolificidade. O gene PGF2α apresentou expressão diferencial de 2,84 vezes a mais nas fêmeas hipoprolíficas. A expressão do gene STAR, que codifica para uma proteína reguladora da síntese de hormônios esteróides, foi 2,32 vezes maior nos animais com baixa prolificidade. A expressão do gene GATA foi 2,31 vezes maior nas fêmeas hipoprolíficas. Esse gene codifica para a proteína de ligação GATA-4, pertencente a um grupo de fatores de transcrição responsáveis pela expressão de vários genes e a diferenciação de diversos tipos celulares. A expressão do CYP19 leva à produção de uma enzima chave na biossíntese de estrogênio, a Citocromo aromatase P450 (P450aro), sendo que, para este gene, foi observada uma expressão relativa de 2,30 vezes a mais nas fêmeas de baixa prolificidade. O gene FSHR não atingiu o limiar de diferença de expressão, estabelecido neste estudo (1,5 vezes). A expressão dos genes STAR, GATA, PGF2α, FSHR, P4R e CYP19 nas células foliculares possibilitou a identificação de diferenças de expressão entre as fêmeas com alta e baixa prolificidade, sendo que a maior diferença de expressão para os genes estudados foi verificada nos animais de baixa prolificidade, exceto para o gene FSHR, que não foi considerado diferencialmente expresso nesse trabalho. Com vistas ao melhor entendimento dos fenótipos reprodutivos em suínos, é necessário que estudos sejam conduzifos no sentido de examinar a expressão desses e de outros genes relacionados, durante todo do ciclo reprodutivo das fêmeas, pois, a expressão de um gene depende, dentre outros fatores, do estádio do desenvolvimento do tecido e da idade do animal.

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...

Teak tree (Tectona grandis L.f.) has a high value in the timber trade for fabrication of woody products due to its extraordinary qualities of color, density and durability. Despite the importance of this species, genetic and molecular studies available are limited. Also, the lack of molecular information about secondary xylem and tree maturation has hindered genetic exploration of teak. Therefore, gene expression studies and transcriptomic profiling are essential to explore wood formation and lignin biosynthesis through the development and aging of vascular plants. Aiming the gene expression studies, it was essential to identify and clone reference genes for teak. Eight genes were tested, commonly used in qRT-PCR, including TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ and TgEF1a. Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem). Stability validation by NormFinder, BestKeeper, geNorm and Delta Ct programs showed that TgUBQ and TgEF1a are the most stable genes to use as qRT-PCR reference genes in teak in the conditions tested. Due to the availability of 12- and 60-year-old teak trees, RNA-seq was performed in diferent organs (seedlings, leaves, flowers, root, stem and branch secondary xylem). A total of 462,260 transcripts were obtained by assembling with \"Trinity\" software. Also, 1,502 and 931 genes differentially expressed were identified for stem and branch secondary xylem, respectively, using DESeq program, and MYB transcription factors, which were characterized. TgMYB1 amino acid sequence displayed a predicted coiled-coil (CC) motif while TgMYB2, TgMYB3 and TgMYB4 showed R2R3-MYB domain. All of them were phylogenetically grouped with several gymnosperms and flowering plants. High expression of TgMYB1 and TgMYB4 in lignified tissues of 60-year-old trees was observed. In this work, the Cinnamyl Alcohol Dehydrogenase (CAD) gene family was also studied. One complete (TgCAD1) and three partial (TgCAD2 to TgCAD4) members were characterized. The four enzymes presented residues for catalytic and structural zinc action, NADPH binding and substrate specificity, consistent with the mechanism of alcohol dehydrogenases. TgCAD3 and TgCAD4 were highly expressed in young and mature sapwood and seem to be duplicated and highly related with lignin biosynthesis. Tree genetic improvement, marker-assisted selection and plant transformation seem to be promising lines of research for the data obtained from this research. This is the first study addressing gene characterization and expression, phylogeny and transcriptomic profiling in teak. / A árvore de teca (Tectona grandis L.f.) tem alto valor no comércio de madeira para a fabricação de produtos lenhosos, devido às suas qualidades extraordinárias de cor, densidade e durabilidade. Apesar da importância desta espécie, são poucos os estudos genéticos e moleculares disponíveis. Também, a falta de informação molecular sobre xilema secundário e maturação da árvore tem dificultado a exploração genética de teca. Assim, estudos de expressão gênica e perfis transcricionais são relevantes para explorar a formação da madeira e a biossíntese de lignina durante o desenvolvimento e envelhecimento das plantas vasculares. Visando os estudos de expressão gênica, foi essencial identificar e clonar genes de referencia para a teca. Foram testados oito genes comumente usados em qRT-PCR, TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ e TgEF1a. Perfis de expressão destes genes foram avaliados por qRT-PCR em seis amostras de tecidos e órgãos (folhas, flores, plântulas, raiz, xilema secundário de caule e ramo). A validação da estabilidade pelos programas NormFinder, BestKeeper, geNorm e Delta CT mostrou que TgUBQ e TgEF1a são os genes mais estáveis para usar como genes de referência em teca nas condições testadas. Em virtude da disponibilidade de árvores de teca de diferentes idades, entre 12 e 60 anos, foi realizado o RNAseq de diferentes órgãos (plântulas, folhas, flores, raiz, ramos e caules de árvores de 12 e 60 anos). Obteve-se um total de 462.260 transcritos pela montagem com o software \"Trinity\". Foram identificados 1.502 e 931 genes diferencialmente expressos para xilema secundário de caule e ramo, respectivamente, utilizando o programa DESeq e fatores de transcrição MYB, que foram posteriormente caracterizados. A sequência de aminoácidos do TgMYB1 exibiu um motivo \"coiled-coil\" (CC), enquanto TgMYB2, TgMYB3 e TgMYB4 mostraram domínio R2R3-MYB. Todos eles foram filogeneticamente agrupados com várias gimnospermas e angiospermas. Observou-se alta expressão do TgMYB1 e TgMYB4 em tecidos lignificados de árvores de 60 anos de idade. Neste trabalho também foi estudada a família gênica Cinamil álcool desidrogenase (CAD). Foi caracterizado um membro completo (TgCAD1) e três parciais (TgCAD2 a TgCAD4). As quatro enzimas apresentaram resíduos de ação catalítica e estrutural de zinco, de ligação ao NADPH e de especificidade de substrato, em conformidade com o mecanismo conservado de álcool desidrogenases. TgCAD3 e TgCAD4 foram altamente expressos no alburno jovem e maduro e parecem estar duplicados e relacionados com a biossíntese de lignina. O melhoramento genético de árvores, a seleção assistida utilizando marcadores moleculares e a transformação de plantas parecem ser linhas promissoras de pesquisa, a partir dos dados obtidos nesta pesquisa. Este é o primeiro estudo sobre caracterização e expressão gênica, filogenia e perfis transcricionais em teca.

No description available.

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...

It is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is obtained after interrogation of appropriate DNA databases, the DNA profile per se presently provides no meaningful information to investigators, with the notable exception of gender determination. In these situations it would be advantageous to the investigation, if additional probative information could be obtained from the biological stain. A useful biometric that could provide important probative information, and one that may be amenable to molecular genetic analysis, is the biological age of an individual. The ability to provide investigators with information as to whether a DNA donor is a newborn, infant, toddler, child, adolescent, adult, middle-aged or elderly individual could be useful in certain cases, particularly those involving young children such as kidnappings or in providing additional intelligence during terrorist investigations. Currently no validated molecular assays exist for age determination. Biological human ageing can be defined by two distinct processes, degenerative and developmental ageing. The degenerative process of ageing is based on theories which identify an increase or decrease in physiological conditions with increasing age. In contrast, the developmental process of ageing is based on the theory that as individuals increase in chronological age, there will be subtle corresponding molecular based biological changes, each requiring genes to be expressed or silenced, indicative of that particular stage of life. We investigated the degenerative process of chromosomal telomere shortening, as well as the developmental process of gene expression profiling analysis, in an attempt to identify biomarkers of biological age in a self-renewing tissue such as blood. While telomere length analysis was an ineffective method for age determination; gene expression analysis revealed three gene transcripts expressed in an age-dependent physiological manner. These species namely- COL1A2, HBE1 and IGFBP3, were found to be expressed at elevated levels in younger individuals, newborns, or post-pubertal individuals, respectively. The biological process of hemoglobin switching was also investigated for the possibility of determining human age. While experimenting with the potential of using the gamma-hemoglobin chains, as newborn specific gene candidates, we serendipitously discovered four novel truncated transcripts, which we have termed HBG1n1, HBG1n2, HBG2n2 and HBG2n3; whose expression was restricted to whole-blood newborn samples and specific fetal tissues. The molecular origin of these transcripts appears to be at the RNA level, being produced by specific rearrangement events occurring in the standard gamma hemoglobin transcripts (HBG1 and HBG2), which yield these new isoforms that are expressed in a highly regulated tissue specific manner.

Philadelphia (Ph) chromosome, as a result of reciprocal translocation, is in majority of cases connected to two types of leukaemia - chronic myelogenous (CML) and acute lymphoblastic (ALL). The translocation occurs within large intronic sequences of BCR and ABL genes. The breakpoints are specific for individual patient and may be used as a target for monitoring of leukemic burden (MRD, minimal residual disease) during the treatment. In general, MRD is an important prognostic factor, which influences the treatment intensity. Two standardized methods are currently used for its monitoring. The first one is based on the detection of clonal specific Immunoglobulin and/or T-cell receptor genes rearrangements (and thus cannot be used for CML cases) at the DNA level, the second one utilizes detection of the BCR/ABL fusion gene at the mRNA level. Our aim was to optimize and standardize the process to find individual patient breakpoints on Ph chromosome and to use it for MRD quantification. We found the breakpoint in 80 % cases. The MRD data from 15 patients obtained by our method were compared to the levels obtained by standard methods (Ig/TCR and BCR/ABL transcript quantification). In all but 1 patient we found significant discrepancies, raising the questions about leukemic origin and the most accurate method for...

Tick saliva contains a lot of biological active substances helping them to succesfully complete their feeding which is neccesary for their next development. Both proteinaceous and non-proteinaceous molecules including protease inhibitors are present in tick saliva. The biggest family of these proteases are serpins. Serpins are involved in many biological processes as blood coagulation, fibrinolysis, apoptosis or inflammation. The aim of this diploma work was to determine expression profiles of 10 serpins from nymphs of Ixodes ricinus fed for different times using quantitative real time PCR. For chosen genes (IRS 10, IRS 20) dsRNA for silencing of the gene was prepared and using RNA interference the role of these genes during tick (I. ricinus nymphs) feeding and transmission of Borrelia afzelii spirochetes, a vector of Lyme borreliosis, was evaluated.Tick saliva contains a lot of biological active substances helping them to succesfully complete their feeding which is neccesary for their next development. Both proteinaceous and non-proteinaceous molecules including protease inhibitors are present in tick saliva. The biggest family of these proteases are serpins. Serpins are involved in many biological processes as blood coagulation, fibrinolysis, apoptosis or inflammation. The aim of this diploma work was to determine expression profiles of 10 serpins from nymphs of Ixodes ricinus fed for different times using quantitative real time PCR. For chosen genes (IRS 10, IRS 20) dsRNA for silencing of the gene was prepared and using RNA interference the role of these genes during tick (I. ricinus nymphs) feeding and transmission of Borrelia afzelii spirochetes, a vector of Lyme borreliosis, was evaluated.

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-03-15T13:03:38Z No. of bitstreams: 1 PGCA16MA207.pdf: 1011799 bytes, checksum: cc3f945283e9dbd2cb016b09c05e7f70 (MD5) / Made available in DSpace on 2018-03-15T13:03:38Z (GMT). No. of bitstreams: 1 PGCA16MA207.pdf: 1011799 bytes, checksum: cc3f945283e9dbd2cb016b09c05e7f70 (MD5) Previous issue date: 2016-02-26 / Capes / Yersinina enterocolitica is a Gram-negative bacteria with zoonotic potential. It is associated with the occurrence of enteric diseases in humans. Pigs are considered the main source of Y. enterocolitica and the bacteria is mainly found in the pig’s palatine tonsils. The objective of this study was to evaluate the occurrence of pathogenic Y. enterocolitica in palatine tonsils of healthy pigs from Santa Catarina, during the slaughter process. In order to achieve this goal, a multiplex PCR technique was performed so as to detect the presence of virulence genes (ail, yadA and virF). This technique was compared to quantitative real time PCR (qPCR), only for the ail gene. Palatine tonsils were randomly collected from 400 pigs from four federally inspected slaughterhouses of the state of Santa Catarina. One positive sample was found for the three studied virulence genes, which were confirmed by DNA sequencing. The analysis of partial sequences of the three virulence genes identified three unique amino acid changes, one in the virF gene and two in YadA gene. This sample had 11.058.398 molecules/μL detected by qPCR. By comparing the two techniques, qPCR was 100 times more sensitive than standard PCR. This result shows low occurrence of pathogenic Y. enterocolitica in healthy pigs from federally inspected slaughterhouses in Santa Catarina / Yersinia enterocolitica é uma bactéria Gram-negativa emergente que possui potencial zoonótico e está associada a quadros de infecção alimentar em humanos. Os suínos são considerados o principal reservatório de Y. enterocolitica, abrigando-a principalmente nas tonsilas. Tendo em vista a carne suína como uma das mais consumidas no mundo e a importância deste agente zoonótico, o objetivo deste trabalho foi avaliar a ocorrência de Y. enterocolitica patogênica em tonsila de suínos no momento do abate no estado de Santa Catarina. Para isto, foi utilizada uma PCR convencional multiplex que detecta a presença de genes de virulência (ail, yadA e virF) e comparou-se esta técnica com a PCR quantitativa em tempo real (qPCR), somente para o gene ail. Foram coletadas aleatoriamente tonsilas de 400 suínos provenientes de quatro frigoríficos com inspeção federal em diferentes regiões do estado. Foi realizado o sequenciamento do DNA dos genes amplificados das amostras positivas na cPCR e posteriormente foi feita a análise filogenética. Apenas uma amostra foi positiva para os três genes pesquisados na PCR convencional, os quais foram confirmados por sequenciamento. A análise das sequências parciais dos três genes de virulência identificou três mudanças de aminoácidos exclusivas, sendo uma no gene virF e duas no gene yadA. Na qPCR esta amostra apresentou 11.058.398 moléculas/μL. Ao comparar as duas técnicas, a qPCR foi 100 vezes mais sensível que a PCR convencional. Isso demonstra uma baixa ocorrência de Y. enterocolitica patogênica em suínos sadios ao abate em frigoríficos com inspeção federal em Santa Catarina

MicroRNA (miRNA) are small non-coding 21-23 nucleotides long one strand RNAs. They are among the major posttranscriptional regulators of gene expression that regulate both physiological and pathological processes. Some of microRNAs, amount of their expression respectively, are specific only for certain type of tissue or pathological condition. The hypothesis for my diploma thesis was that gene expression of 28 cardiovascular disease associated microRNAs (miR-1-3p, miR-16-5p, miR-17-5p, miR- 20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-92a-3p, miR-100-5p, miR-103a-3p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181-5p, miR-195-5p, miR- 199a-5p, miR-210-3p, miR-221-3p, miR-342-3p, miR-499a-5p, miR-574-3p) would differ in umbilical cord blood between groups of women with physiological pregnancies (FG), gestational hypertension (GH), preeclampsia (PE) and fetal growth restriciton (FGR). The studied cohort consisted of 184 pregnant women involving 44 controls, 47 GH pregnancies, 56 PE pregnancies and 37 FGR pregnancies. Relative quantification of microRNAs was performed by quantitative real-time PCR. Results showed a trend to miR-195-5p down-regulation in umbilical cord blood of GH patients. On the other hand, mild PE...