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.

This diploma thesis is focused on optimalization and employing the PCR-RFLP method, based on the molecular biology principles, for an identification and taxonomy of the yeasts which colonize the leaf surfaces. Simultaneously the yeasts identification techniques based on physiological and morfphological attributes are compared and replaced. PCR-RFLP takes advantage of thermostable polymerases´ ability to amplify the specific segment in the rDNA, which can be split by restriction endonucleases to characteristical polymorphical fragments. Comparing these fragments and restriction´s positions which are for each species unique, demanded results were obtained. They´re summarized in the conclusion part. The theoretical part describes the morphology and cytology of the yeasts, taxonomy as a science, genuses of examined yeasts Cryptococcus, Rhodotorula a Saccharomyces are covered more thoroughly and the method of PCR-RFLP is described in detail.

This diploma thesis has analyzed the effect of organic wine-growing on the wine yeasts population. The wine yeasts were isolated from the Pinot Noir variety. They were identified by the molecular biological method PCR-RFLP. The theoretical research compiles basic information on yeasts, knowledge about the red wine production as well as information on molecular biological methods. The experimental part utilizes the 5,8S-ITS rDNA specific segment for analysis. The segment was amplified using the ITS1 and ITS4 primers and subjected to restriction analysis. The restriction analysis has used these restriction endonucleases - HaeIII, HinfI, Taq?I, AluI and MseI. The BioNumerics software was then used to compare genetic similarity between the isolated yeasts and these were taxonomically classified.

The aim of this diploma thesis is the identification of yeasts isolated during grape must fermentation. The must was obtained from Pinot Noir varieties grown in an integrated and organic production. The partner of this thesis was a winery Holánek. In the theoretical part of the work was the emphasis on information about the determinants quality of wine, yeasts and PCR-RFLP method. Physiological properties of yeasts were described and also the principles of the polymerase chain reaction (PCR) were explained. In the experimental part of the thesis was applied molecular biology method PCR-RFLP for identification of yeasts. The specific segment of DNA was amplified (5, 8S-ITS rDNA sequencing) with the help of ITS1 and ITS4 primers. The incurred amplicons were digested by applying restriction endonucleases: HaeIII, HinfI and TaqI. Subsequently the restriction fragments were analysed by using of electrophoresis. The yeasts were identified and classified by taxonomy on the level of genera and species.

This diploma thesis focuses on isolation and taxonomic classification of yeast species isolated during the red wine (Pinot noir) fermentation. Grapes were grown under organic and integrated farming in South Moravia wine region, Czech Republic. Processing was controlled – for inoculation was used strain Saccharomyces cerevisiae BS6. Polymerase chain reaction followed by restriction fragment lenght polymorphism of PCR-amplified fragments (PCR-RFLP) was used for yeast species identification. For DNA analysis we used coding region of 5.8S ITS rDNA which was amplified using ITS1-ITS4 primers. Amplicon was digested by three restriction endonucleases - HaeIII, HinfI and HhaI. Isolates were divided into eleven groups using UPGMA cluster analysis (software BioNumerics). We identified following yeast species: Candida valida, Candida vini, Issatchenkia occidentalis, Pichia fermentans, Saccharomyces cerevisiae and Zygosaccharomyces bailii. We were not able to identify some yeast species. Differences between organic and integrated farming were demonstrated with varying composition of yeast species.

The aim of this thesis is the isolation and identification of yeasts obtained from the wine berries and the characterization of the collection yeast by using processes of PCR - RFLP. The type yeasts were obtained from the collection of yeasts of CCY in Bratislava, yeasts from the wine berries were collected from the species of Hibernal wine from the wineries of Štěpán Maňák. Identification of individual yeast is then based on analysis of the DNA segment in the area of 5,8S - ITS using primers ITS1 and ITS4. The restriction analysis was performed using restriction endonucleases HaeIII, HinfI, HhaI a TaqI(a). Restriction analysis is used to chopp the DNA to specific sections that are characteristic for each microorganism. For the assesment of the genetic similarity analyzed yeasts the BioNumerics software has been used. BioNumerics processes the results using cluster analysis using Jaccard´s coefficients.

Lichens are defined as symbiotic associations composed of a fungal partner, the mycobiont, and one or more photosynthetic partners, the photobiont (1). A currently employed method for the identification of photobionts is the culture of photobiont from the lichen, but this method employs a labor intensive and long cultivation period, thus identification has been neglected. Out of the approximatelyl4,000 lichen described, only about 4% oflichen photobionts have been identified to species (1). In this study we investigated the feasibility of developing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) identification system for rapidly identifying lichen algae of the genus Trebouxia (In this study we consider Pseudo-Trebouxia part of the genus Trebouxia). DNA was isolated and purified from cultures of each Trebouxia species. A 1300 hp fragment of the 5' region of the nuclear-encoded large subunit (26S) ribosomal RNA genes was amplified by PCR (2). This 5'region of the 26S region is considered to be a byper-variable region because it differs amongst Trebouxia (2) making it a good candidate for RFLP. The sequences were then analyzed with restriction analysis software to determine restriction maps and individual virtual RFLP patterns. Patterns were constructed using the program SPR Opt (SNP and PCR-RFLP Optimization) allowing each Trebouxia species to be identified by a distinctive restriction pattern. We were unable to validate the key due to contamination of materials, which lead to inconclusive data. Future experiments aim to validate the key by comparing the virtual RFLP patterns to the actual patterns obtained for each type culture of each species.