The metabolism of vitamin D commences with exposure of the skin to sunlight. The growing recognition of its role in insulin resistance, autoimmune disorders, infections, cancer, as well as the health of cells that influence physical and mental function have profound implications on how we define vitamin D requirements and why we should care whether they are met or not. Most of the actions of vitamin D are mediated by the vitamin D receptor (VDR), a protein whose gene sequence can vary, giving rise to polymorphic forms which are potent enough to affect the binding capacity of this protein to vitamin D. Some of these polymorphic forms of VDR gene may be associated with reduced effectiveness of vitamin D and hence predispose individuals to diseases such as type 2 diabetes and insulin resistance. An earlier study, the Surya Study, looked at the responsiveness of the South-Asian women living in Auckland to vitamin D. The research described here is an extension of this study and its focus was to identify the associations/linkages between certain polymorphic forms of the VDR gene and the disease conditions and intervention responsiveness in the same women. The first objective was to compare two well known techniques for genotyping single nucleotide polymorphisms (SNPs) of the VDR gene at the 3’ end, namely BsmI, ApaI and TaqI: the newer real-time polymerase chain reaction (qPCR) and the traditional restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) techniques. This comparison was performed to evaluate alternative methods for genotyping which consumed less time than RFLP-PCR. When the presence of each polymorphism by both the techniques was compared in this cohort of South-Asian women, it was found that RFLP-PCR proved to be a more reliable technique than qPCR for genotyping the VDR gene. Another objective of this project was to investigate the prevalence of the above three polymorphisms along with Cdx-2 and FokI SNPs which are present at the 5’ end of the VDR gene, in the population under study and their possible association with phenotypes such as vitamin D responsiveness and insulin resistance. These women were screened and biochemical data was collected during the earlier Surya Study. Of these, eighty-one women were then selected for intervention based on them having high insulin resistance (HOMA-IR>1.93) and serum 25(OH)D<50 nmol/L. Out of these eighty-one women, forty-two were given vitamin D supplement and thirty-nine were given a placebo for six months. Baseline and endpoint measurements included insulin resistance (HOMA-IR), insulin sensitivity (HOMA2%S) etc. How each individual responded to treatment in the intervention group was analysed in the context of the polymorphisms that they had. An association of insulin resistance with BsmI, ApaI and TaqI SNPs was observed in this cohort of 239 women. The response to insulin resistance in the vitamin D supplemented group significantly differed for FokI genotype compared to other genotypes. This explained why certain women responded to treatment better than the others. When the frequencies of the genotypes of these five SNPs of the VDR gene were compared to other studies of different ethnicities, the results of this study were consistent with few studies but contradictory to others. The possible reasons for these differences could be because of small sample size and different ethnicities under study due to which the frequency of alleles and hence the genotypes differed.

The metabolism of vitamin D commences with exposure of the skin to sunlight. The growing recognition of its role in insulin resistance, autoimmune disorders, infections, cancer, as well as the health of cells that influence physical and mental function have profound implications on how we define vitamin D requirements and why we should care whether they are met or not. Most of the actions of vitamin D are mediated by the vitamin D receptor (VDR), a protein whose gene sequence can vary, giving rise to polymorphic forms which are potent enough to affect the binding capacity of this protein to vitamin D. Some of these polymorphic forms of VDR gene may be associated with reduced effectiveness of vitamin D and hence predispose individuals to diseases such as type 2 diabetes and insulin resistance. An earlier study, the Surya Study, looked at the responsiveness of the South-Asian women living in Auckland to vitamin D. The research described here is an extension of this study and its focus was to identify the associations/linkages between certain polymorphic forms of the VDR gene and the disease conditions and intervention responsiveness in the same women. The first objective was to compare two well known techniques for genotyping single nucleotide polymorphisms (SNPs) of the VDR gene at the 3’ end, namely BsmI, ApaI and TaqI: the newer real-time polymerase chain reaction (qPCR) and the traditional restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) techniques. This comparison was performed to evaluate alternative methods for genotyping which consumed less time than RFLP-PCR. When the presence of each polymorphism by both the techniques was compared in this cohort of South-Asian women, it was found that RFLP-PCR proved to be a more reliable technique than qPCR for genotyping the VDR gene. Another objective of this project was to investigate the prevalence of the above three polymorphisms along with Cdx-2 and FokI SNPs which are present at the 5’ end of the VDR gene, in the population under study and their possible association with phenotypes such as vitamin D responsiveness and insulin resistance. These women were screened and biochemical data was collected during the earlier Surya Study. Of these, eighty-one women were then selected for intervention based on them having high insulin resistance (HOMA-IR>1.93) and serum 25(OH)D<50 nmol/L. Out of these eighty-one women, forty-two were given vitamin D supplement and thirty-nine were given a placebo for six months. Baseline and endpoint measurements included insulin resistance (HOMA-IR), insulin sensitivity (HOMA2%S) etc. How each individual responded to treatment in the intervention group was analysed in the context of the polymorphisms that they had. An association of insulin resistance with BsmI, ApaI and TaqI SNPs was observed in this cohort of 239 women. The response to insulin resistance in the vitamin D supplemented group significantly differed for FokI genotype compared to other genotypes. This explained why certain women responded to treatment better than the others. When the frequencies of the genotypes of these five SNPs of the VDR gene were compared to other studies of different ethnicities, the results of this study were consistent with few studies but contradictory to others. The possible reasons for these differences could be because of small sample size and different ethnicities under study due to which the frequency of alleles and hence the genotypes differed.

Resumo:A leishmaniose representa um dos principais problemas de saúde pública do mundo. O protozoário do gênero Leishmania tem distribuição mundial e a epidemiologia da doença depende das características dos parasitas. As leishmanioses se dividem em leishmaniose visceral e tegumentar. O cão desempenha um papel fundamental na transmissão de L. infantum aos humanos e na epidemiologia da doença. Devido à adaptação da Leishmania a novos vetores ou hospedeiros é importante conhecer o agente etiológico circulante nos cães. As técnicas moleculares têm sido utilizadas para o diagnóstico das leishmanioses. A PCR-RFLP detecta e distingue as diferentes espécies do parasita. O objetivo do presente trabalho foi identificar as espécies de Leishmania encontradas em 103 amostras de sangue periférico de cães naturalmente infectados por esse protozoário, do município de Bauru - SP. Para o diagnóstico da leishmaniose foi realizado o exame parasitológico, ELISA e PCR. A determinação das espécies de Leishmania foi realizada pelo método de PCR-RFLP. Para a identificação das espécies, o DNA amplificado da região intergênica ITS1 foi digerido com a enzima de restrição HaeIII. As amostras positivas para Leishmania ssp, mostraram um perfil de restrição idêntico a L. amazonensis em 77/103 amostras, em 17/103 foram semelhantes a L. infantum, e em 09/103 apresentaram perfil misto. Em conclusão, identificamos L. amazonensis infectando maior número de cães do que a L. infantum em cães no município de Bauru, SP / Abstract:Leishmaniasis is a major public health problem in the world. The protozoa of the genus Leishmania has worldwide distribution and epidemiology of the disease depends on the characteristics of the parasites. Leishmaniasis are divided into visceral leishmaniasis and cutaneous. The dog plays a key role in the transmission of L. infantum to humans and in the epidemiology of the disease. Due to the adaptation of Leishmania to new hosts or vectors is important to know the current etiologic agent in dogs. Molecular techniques have been used for the diagnosis of leishmaniosis. The PCR-RFLP detects and distinguishes the different species of the parasite. The objective of this study was to identify the Leishmania species found in 103 samples of peripheral blood of dogs naturally infected with this protozoan, the city of Bauru - SP. For the diagnosis of leishmaniosis was determined by parasitological examination, indirect ELISA and PCR was performed. The determination of Leishmania species the DNA amplified intergenic region ITS1 was digested with the restriction enzyme HaeIII. Positive samples for Leishmania ssp. showed an identical restriction profile of L. amazonensis in 77/103 samples, 17/103 were similar to L. infantum, and 09/103 were mixed profile. In conclusion, we identified L. amazonensis greater number of dogs than L.infantum in Bauru city, SP / Orientador:Valéria Marçal Félix de Lima / Banca:Alex Akira Nakamura / Banca:José Eduardo Tolezano / Mestre

This work was focused on the study of variability DGAT1 gene in the pig population Czech Large White breed. Subsequently, association analysis was performed of the gene and the different production indicators of quality of pork meat. Examined polymorphism was found at position 103 of intron 2 of chromosome 4. DGAT1 gene has a major role in the synthesis tryacylglycerols and may affect the storage of fat in the body. Relative genotype frequencies were: AA = 0.4222; AG = 0.4889; GG = 0.0889. The values of the relative frequencies of alleles were as follows: A = 0.6666 and G = 0.3334. The association analysis didn't show any statistically significant differences.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-07-18T12:44:19Z No. of bitstreams: 1 texto completo.PDF: 331413 bytes, checksum: a46035999a9f1c27325bb8517e1b3b42 (MD5) / Made available in DSpace on 2017-07-18T12:44:19Z (GMT). No. of bitstreams: 1 texto completo.PDF: 331413 bytes, checksum: a46035999a9f1c27325bb8517e1b3b42 (MD5) Previous issue date: 2001-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A lagarta elasmo, Elasmopalpus lignosellus (Lepidoptera: Pyralidae), e a lagarta do cartucho, Spodoptera frugiperda (Lepidoptera: Noctuidae), são pragas relevantes, particularmente da cultura de milho. O presente estudo objetivou estudar regiões do DNA de E. lignosellus e avaliar milhos transgênicos, expressando as toxinas Cry 1 A(b), Cry 9C e Cry 1F, como potencial de controle desta lagarta, além de estudos de tolerância às toxinas de Cry 1 A(b) em S. frugiperda avaliando a herdabilidade desta tolerância e diferenciação, por marcadores AFLP, das populações de S. frugiperda que apresentaram tolerância e susceptibilidade à toxina avaliada. É possível diferenciar as populações brasileira e americana de E. lignosellus com PCR-RFLP e sequenciamento do gene mitocondrial COI. Milhos Bt, expressando as toxinas Cry 1 A(b), Cry 9C e Cry 1F, estão protegidos contra o ataque da lagarta elasmo. S. frugiperda mostrou aumento na tolerância à toxina Cry 1A(b) após seleção por quatro gerações e registrou-se um componente hereditário nesse fenômeno. Os dados de AFLP mostram haver diferença entre as populações tolerantes e susceptíveis. / The lesser cornstalk borer (LCB), Elasmopalpus lignosellus (Lepidoptera: Pyralidae) and the fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae) are important insect pest. To contribute to managing programs of these insects we propose to study mtDNA region in LCB allowing differentiation on populations from Brazil and USA, and evaluate the efficiency of Bt corn to control this pest. Also, to determine the FAW tolerance to Cry1A(b) toxin and to study the genetic polymorphism of survival of fall armyworm using AFLP technique. The results show that is possible to distinguish LCB populations from Brazil and USA using COI mitochondrial gene by PCR-RFLP and sequencing, and that Bt transgenic maize were able to protect the plants against LCSB damage. Increased tolerance to Cry 1A(b) protein was found in populations of fall armyworm after selection for four generations with Cry 1A(b)toxin. Tolerance of FAW to Cry1 A(b) had a heritage component in the studied generations. The following work, also, describes efforts to detect genetic polymorphisms between survivors.

Made available in DSpace on 2016-08-29T15:35:05Z (GMT). No. of bitstreams: 1 tese_7779_TESE LBF-2014.pdf: 10712510 bytes, checksum: 8a20db56f2fe6ca735be70a109f4ef61 (MD5) Previous issue date: 2014-06-04 / O câncer de colo uterino (CCU), cujo agente etiológico é o papilomavírus humano (HPV), é um dos tipos de câncer mais frequentes em mulheres em todo o mundo, não só em incidência como também em mortalidade. Alguns genótipos de HPV, denominados de alto risco (HR-HPV), e suas variantes gênicas, estão mais associados à indução de lesões malignas, sendo HPV16 e 18 os mais frequentes. Algumas infecções do trato genital podem atuar como cofatores da progressão carcinogênica do CCU, porém a infecção por vírus adeno-associado (AAV) parece estar inversamente relacionada, o que pode refletir em um papel protetor no desenvolvimento do CCU induzido pelo HPV. Portanto, este estudo objetivou investigar o papel da infecção mista AAV-HPV e das variantes oncogênicas de HPV na progressão das lesões intraepiteliais de colo de útero e acompanhar a eliminação /persistência viral em relação à progressão / regressão das lesões cervicouterinas. Exames citológicos foram realizados em amostras de espécime cervical, coletadas em dois momentos, de mulheres atendidas no Hospital Universitário Cassiano Antonio Moraes HUCAM e seguiram para tratamento conforme preconizado. DNA foi extraído pelo kit comercial QIAamp® DNA Mini Kit, seguindo instruções do fabricante. DNA de AAV foi investigado por PCR e nPCR e, de HPV, por PCR e Captura Híbrida® (CH). Genotipagem de AAV e HPV foram realizadas por RFLP e RLB, respectivamente. Dos casos encaminhados ao ambulatório de colposcopia, 57,3% tiveram citologia normal, 23,1% lesões de baixo grau e 19,6% lesões de alto grau. Dos casos com citologia normal, 78% permaneceram normais, enquanto 22% progrediram à lesão; dos casos com lesão de baixo grau, 74% regrediram para citologia normal, enquanto 78,6% dos casos com lesão de alto grau apresentaram lesão de baixo grau ou citologia normal na segunda coleta. Foram positivas para HPV, 56% e 36,5% das amostras da primeira e segunda coletas, respectivamente. Foi observada boa correlação (kappa= 0,66) entre os testes de PCR e CH para detecção de HPV. Os HR-HPV foram detectados em mais de 90% das amostras de ambas as coletas, sendo os mais frequentes os HPV16, 58, 51, 52 e 53. Variante não-europeia esteve associada ao desenvolvimento de lesão cervical de alto grau, enquanto a presença de AAV foi inversamente relacionada à progressão da lesão cervical induzida por HPV.

Made available in DSpace on 2015-10-06T13:03:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-06-16. Added 1 bitstream(s) on 2015-10-06T13:18:30Z : No. of bitstreams: 1 000849239.pdf: 306516 bytes, checksum: cab162ea96c4456d9e47531798b2c3eb (MD5) / Leishmaniasis is a major public health problem in the world. The protozoa of the genus Leishmania has worldwide distribution and epidemiology of the disease depends on the characteristics of the parasites. Leishmaniasis are divided into visceral leishmaniasis and cutaneous. The dog plays a key role in the transmission of L. infantum to humans and in the epidemiology of the disease. Due to the adaptation of Leishmania to new hosts or vectors is important to know the current etiologic agent in dogs. Molecular techniques have been used for the diagnosis of leishmaniosis. The PCR-RFLP detects and distinguishes the different species of the parasite. The objective of this study was to identify the Leishmania species found in 103 samples of peripheral blood of dogs naturally infected with this protozoan, the city of Bauru - SP. For the diagnosis of leishmaniosis was determined by parasitological examination, indirect ELISA and PCR was performed. The determination of Leishmania species the DNA amplified intergenic region ITS1 was digested with the restriction enzyme HaeIII. Positive samples for Leishmania ssp. showed an identical restriction profile of L. amazonensis in 77/103 samples, 17/103 were similar to L. infantum, and 09/103 were mixed profile. In conclusion, we identified L. amazonensis greater number of dogs than L.infantum in Bauru city, SP

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-08-29T19:15:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-08-29T19:16:10Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2017-08-29T19:16:10Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2011-08-31 / Dermatophytes comprise a group of filamentous fungi of great interest on public health because of their ability to parasitize keratinized tissues, such as skin, hair and nails, and for their wide distribution in the world. As a consequence of this parasitism, an infectious process of dermatophytosis is established, from which a variety of clinical manifestations can occur, affecting people of both genders and all age groups. Laboratory methods for mycological diagnosis do not always allow a clear an especific definition of the agent. In this study, different strategies for extraction of DNA, and molecular typing by PCR-RFLP, of seven dermatophyte species were assessed. Two target regions: ITS/rDNA and the topoisomerase II gene were evaluated, by testing three PCR protocols and three restriction enzymes (DdeI, HinfI, HaeIII). For the DNA extraction, the glass bead shaking technique for cell lysis, followed by Gustincich (1991) based mehod for DNA separation, demonstrated more advantages. Our results has demonstrated that the topoisomerase II gene is a suitable target region for identification of the seven major pathogenic dermatophyte fungal species, reinforcing previous studies, and pointed to a new PCR-RFLP protocol, which is based on a PCR of this gene using dPsD2 primer, followed by digestion of PCR products with HaeIII restriction enzyme. / Os dermatófitos compreendem um grupo de fungos filamentosos de grande interesse na área da saúde, devido à sua capacidade de parasitar os tecidos queratinizados, como a pele, pêlos e unhas, e à sua ampla distribuição no mundo. Como conseqüência desse parasitismo instala-se um processo infeccioso de dermatofitose, a partir do qual pode ocorrer uma diversidade de manifestações clínicas, acometendo pessoas de ambos os gêneros e de todos os grupos etários. Os métodos laboratoriais para o diagnóstico micológico nem sempre permitem uma clara definição do agente em nível de espécie. No presente estudo foram analisadas diferentes estratégias para a extração de DNA e para a identificação molecular por PCR-RFLP das principais espécies de dermatófitos. Duas regiões alvo, a região ITS/DNAr e o gene da topoisomerase II foram analisadas, testando-se três protocolos de PCR e três enzimas de restrição (DdeI, HinfI, HaeIII). Na extração do DNA, o método de lise utilizando pérolas de vidro e a separação do DNA com base no método de Gustincich (1991) demonstrou importantes vantagens. Nossos resultados demonstraram que o gene da topoisomerase II é uma região alvo adequada para identificação das sete principais espécies de fungos dermatófitos patogênicos, reforçando estudos anteriores, e apontaram para um novo protocolo de RFLP-PCR, que se baseia em uma PCR desse gene utilizando o primer dPsD2, seguido da digestão dos produtos obtidos com a enzima de restrição HaeIII.

Made available in DSpace on 2014-06-12T23:13:47Z (GMT). No. of bitstreams: 2 arquivo8_1.pdf: 1293352 bytes, checksum: 746673d69958e017a467483837256871 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco / A caça predatória ilegal é o segundo maior fator de impacto em populações de animais silvestres no Brasil, ficando atrás apenas da perda de habitat por desmatamento. Apesar de ser proibida no Brasil (Leis n° 5.197/1967 e n° 9.605/1998), o poder público ainda não dispõe de recursos eficientes e cientificamente testados que possam ser utilizados em análises forenses visando comprovar o ato da caça, seja ela desportiva ou com finalidade comercial. A análise baseada no DNA tem sido utilizada em várias situações, com a finalidade de detectar fraudes comerciais e outras ilegalidades. Diante do exposto, o objetivo do presente trabalho foi à identificação e a utilização de perfis de PCR/RFLP espécieespecíficos para diagnóstico forense de 15 espécies da fauna brasileira e sua diferenciação de quatro espécies domésticas (bovino, suíno, caprino, ovino), totalizando 19 espécies. Para tanto foram realizadas análises de seqüências de nucleotídeos com os programas Sequencher 4.9, BioEdit 6.0.7, CLEAVER, pDRAW e Gene Runner 3.0.5, tendo sido identificados vários SNPs associados à criação de sítios de enzimas de restrição discriminantes. Com apenas 9 enzimas foram obtidos os perfis de PCR/RFLP discriminantes para as 19 espécies. A validação do protocolo in vitro foi realizada com amostras biológicas de 6 espécies silvestres (Agouti paca, Cebus apella, Dasyprocta leporina, Dasypus novemcinctus, Euphractus sexcinctus, Tayassu tajacu) juntamente com 4 espécies domésticas (Bos taurus, Capra hircus, Ovis aries and Sus scrofa), e os perfis detectados na analise in silico foram confirmados em gel de agarose 2%. O presente estudo reforçou o potencial de polimorfismos do gene Citocromo b como poderosos marcadores para identificação de espécies. Os dados produzidos aqui podem ser úteis como ferramentas em conservação no combate a caça predatória e monitoramento do comércio ilegal de carne de caça e de seus produtos

This thesis explores the optimizing of the methods relevant to the cultivation, isolation, and identification of individual yeast strains from selected matrices, using the molecular biology methods. Grape berries, vine-leaves and soil from vineyard have been used as matrices. As yeasts are an integral part of fermentation processes, they are used for making of wine, the organoleptic charakteristics of which they influence. For identification of yeast strains the PCR-RFLP method has been used. Specifity of yeast DNA sequences of certain species has been used for this analysis. These spacers have been amplificated through the use of PCR and consequently they have been subjected to a restrictive analysis with specific restrictive endonucleases. These fragments have been detectioned through horizontal electrophoresis. In the literature background research section the basic informations about viticulture, yeasts, their cultivation and separation, PCR, restrictive analysis and horizontal electrophoresis is presented.