Made available in DSpace on 2014-12-17T14:16:25Z (GMT). No. of bitstreams: 1 ZamaMLS_DISSERT.pdf: 2983727 bytes, checksum: 7afd93b3940a02c3ef820577c21fb808 (MD5) Previous issue date: 2010-02-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Beta thalassemia arises as a consequence of the reduction (?+, ?++, ?silent) or absence (?0) of beta globin chain synthesis and results from a number of mechanisms that lead to genetic defects. The inheritance of beta thalassemia is characterized by the existence of heterozygous individuals, compound heterozygotes, homozygotes and those with coinheritance of beta thalassemia allele and other thalassemias and/or hemoglobin variants. The aim of this study was to perform molecular and laboratory characterization of beta thalassemia in heterozygous and homozygous individuals and in those with coinheritance of S beta thalassemia. A total of 48 individuals were included (35 heterozygotes, 4 homozygotes and 9 S beta thalessemia carriers) referred to the Integrated Laboratory of Clinical Analyses of the Federal University of Rio Grande do Norte (UFRN) and the Hematology Ambulatory Facility of the Dalton Barbosa Cunha Hemocenter (Hemonorte Natal, Brazil). Peripheral blood samples form each patient underwent the following laboratory examinations: erythrogram, hemoglobin electrophoresis at alkaline pH, measurements of Hb A2, Fetal Hb and serum ferritin. DNA was extracted using the illustra blood genomicPrep Mini Spin Kit and molecular characterization was performed by the PCR/RFLP technique, which involves digestion with specific restriction enzymes for IVS-1 nt 1 (G?A), IVS-1 nt 6 (T?C) and codon 39 (CAG?TAG) mutations. Of the 35 heterozygotes, 37.1% showed IVS-1 nt 6 mutation, 42.9% IVS-1 nt 1 and 20% were carriers of other mutations not identified by the technique used. The four homozygous patients presented with the IVS-1 nt 6 mutation, while 66.7% of the individuals with S beta thalassemia had the IVS-1 nt 1 mutation. Codon 39 was not detected in any of the patients investigated. Of the thallasemic alleles found, 40.4% were IVS- 1 nt 1, 40.4% IVS-1 nt 6 and 19.2% were not identified. Laboratory data showed that the heterozygotes exhibited microcytosis and hypochromia, evidenced by MCV ranging from 57 to 75fL and MCH from 15.9 to 23.6 pg. Hemoglobin A2 varied between 3.7 and 7.2%. The homogygotes also showed reduced MCV and MCH and elevated HbA2.. Comparison of laboratory data between heterozygous individuals with IVS-1 nt 1 and IVS-1 nt 6 mutations showed that heterozygotes for the IVS1-1 mutation had significantly lower mean MCV and MCH (p = 0.023 and 0.007, respectively) and significantly higher hemoglobin A2 (p < 0.001) when compared to heterozygotes for the IVS-1 nt 6 mutation. PCR/RFLP was useful in identifying the presence or absence of IVS-1 nt 6, IVS-1 nt 1 and codon 39 mutations in most of the patients investigated here. This is the first study conducted in the state of Rio Grande do Norte, Brazil aimed at identifying beta thalassemia mutations and represents an important contribution to the knowledge regarding the molecular profile of beta thalassemia in our country / A talassemia beta ocorre devido ? diminui??o (?+, ?++, ?silent) ou aus?ncia (?0) de s?ntese de cadeias beta de globina e ? decorrente de v?rios mecanismos que provocam o defeito gen?tico. A heran?a da talassemia beta ? caracterizada pela exist?ncia de indiv?duos heterozigotos, heterozigoto compostos, homozigotos e portadores de intera??o entre o alelo talass?mico beta e outras talassemias e/ou variantes de hemoglobina. O objetivo principal deste estudo foi realizar a caracteriza??o molecular e laboratorial em indiv?duos heterozigotos e homozigotos da talassemia beta e em portadores da intera??o hemoglobina S/talassemia beta. Foram inclu?dos 48 indiv?duos (35 heterozigotos, 4 homozigotos e 9 com intera??o HbS/talassemia beta) atendidos no Laborat?rio Integrado de An?lises Cl?nicas (UFRN) e no Ambulat?rio de Hematologia do Hemocentro Dalton Barbosa Cunha (Hemonorte Natal/RN). As amostras de sangue perif?rico de cada paciente foram submetidas aos seguintes exames laboratoriais: eritrograma, eletroforese de hemoglobina em pH alcalino, dosagem das hemoglobinas A2 e Fetal, e ferritina s?rica. O DNA foi extra?do utilizando-se o kit blood genomicPrep Mini Spin e a caracteriza??o molecular foi realizada atrav?s da t?cnica PCR/RFLP mediante digest?o com enzimas de restri??o espec?ficas para as muta??es IVS-1 nt 1 (G?A), IVS-1 nt 6 (T?C) e nonsense c?don 39 (CAG?TAG). Dentre os 35 heterozigotos, 37,1% apresentaram a muta??o IVS-1 nt 6, 42,9% a IVS-1 nt 1 e 20% eram portadores de outras muta??es n?o identificadas com a t?cnica utilizada. Os quatro pacientes homozigotos apresentaram a muta??o IVS-1 nt 6, enquanto 66,7% dos indiv?duos com intera??o HbS/talassemia beta tinham a muta??o IVS-1 nt 1. A c?don 39 n?o foi detectada em nenhum dos pacientes investigados. Dentre os alelos talass?micos encontrados, 40,4% eram IVS-1 nt 1, 40,4% eram IVS-1 nt 6 e 19,2% n?o foram identificados. Em rela??o aos dados laboratoriais, os heterozigotos apresentaram microcitose e hipocromia evidenciada pelo VCM variando de 57 a 75fL, e o HCM, entre 15,9 a 23,6 pg. A hemoglobina A2 variou de 3,7 a 7,2%. Os homozigotos tamb?m apresentaram VCM e HCM reduzidos e HbA2 elevada. Ao comparar os dados laboratoriais entre os indiv?duos heterozigotos para as muta??es IVS-1 nt 1 e IVS-1 nt 6 observou-se que os heterozigotos da muta??o IVS1-1 apresentaram valores m?dios de VCM e HCM significativamente menores (p = 0,023 e 0,007, respectivamente) e hemoglobina A2 significativamente mais elevados (p < 0,001) quando comparados aos heterozigotos da muta??o IVS-1 nt 6. A t?cnica de PCR/RFLP se mostrou ?til para a identifica??o da presen?a ou aus?ncia das muta??es IVS-1 nt 6, IVS-1 nt 1 e ?0 c?don 39 na maioria dos pacientes investigados na pesquisa. O presente estudo ? o primeiro trabalho realizado no estado do Rio Grande do Norte para identifica??o das muta??es da talassemia beta, e constitui importante contribui??o para o conhecimento do perfil molecular da talassemia beta em nosso pa?s

Made available in DSpace on 2014-12-17T15:34:45Z (GMT). No. of bitstreams: 1 MiguelASFC_DISSERT.pdf: 664410 bytes, checksum: ea1412fbbc90b4e28d5ec902a12a2ff5 (MD5) Previous issue date: 2011-08-29 / Universidade Federal do Rio Grande do Norte / The objective of this study was to identify DNA polymorphisms at the genes leptin, &#946;-lactoglobulin and pituitary-specific transcription factor in three genetic groups of Holstein x Guzerat dairy cows and investigate the relationship between their genotypes and the composition and quality of milk of dairy cows. Samples were collected in August 2009, being 113 blood samples from lactating crossbred cows and 58 milk samples. For analysis of DNA polymorphisms blood samples were collected, analyzed later in the Genetic Laboratory affiliated to the Zootechny Institute of S?o Paulo and individual milk samples were collected according to standards established by the laboratory of Management Program of Northeast Dairy Herds (PROGEN), at Federal Rural University of Pernambuco (UFRPE) for analysis of milk composition and quality. The characterization of genotypes was performed by PCR-RFLP, for which were designed specific primers for each studied gene and restriction enzymes Kpn2I, HaeIII and HinfI that cut the DNA of the following genes: leptin, &#946;-lactoglobulin and a PIT, respectively. The leptin estimate genotypic frequence were CC 0.112, TT 0.225 and CT 0.661, for &#946;-lactoglobulin were AA 0.136, AB 0.323 and BB 0.539, and for PIT were ++ 0.655, -- 0.311 and +- 0.032. The results show that the population is in Hardy-Weinberg disequilibrium for leptin, &#946;-lactoglobulin and a PIT due to excess of heterozygotes in the population, however, as these genes are associated with the milk production it is considered that the animals have genetic potential for milk production in the Brazilian semi-arid conditions. Through the characterization of the studied herd there were not found implications of the polymorphism of leptin, &#946;-lactoglobulin and PIT in the composition and quality of milk from cows in the different genetic groups 1/2, 3/4 and 7/8 Holstein x Guzerat. Key words: &#946;-lactoglobulin, crossbred cows, leptin, PCR-RFLP, PIT1, semi-arid. / O objetivo do presente estudo ? identificar os polimorfismos de DNA nos genes leptina, &#946;-lactoglobulina e fator de transcri??o pituit?rio espec?fico, em tr?s grupos gen?ticos de vacas leiteiras Holand?s x Guzer? e verificar a rela??o entre seus gen?tipos com a composi??o e qualidade do leite de vacas leiteiras. As amostras foram coletadas em agosto de 2009, sendo 113 amostras de sangue de vacas mesti?as em lacta??o e 58 amostras de leite. Para a an?lise do polimorfismo de DNA foram coletadas amostras de sangue, analisadas posteriormente no Laborat?rio de Gen?tica do Instituto de Zootecnia do Estado de S?o Paulo. As amostras individuais de leite foram coletadas segundo os padr?es estabelecidos pelo laborat?rio do Programa de Gerenciamento de Rebanhos Leiteiros do Nordeste (PROGENE), da Universidade Federal Rural de Pernambuco (UFRPE) para as an?lises de composi??o e qualidade do leite. A caracteriza??o dos gen?tipos foi realizada atrav?s da t?cnica de PCR-RFLP, em que foram utilizados primers espec?ficos para cada gene estudado e enzimas de restri??o Kpn2I, HaeIII e HinfI, promotoras de cortes nos genes leptina, &#946;- lactoglobulina e PIT1, respectivamente. A freq??ncia genot?pica estimada da leptina foi CC 0,112 , TT 0,225 e CT 0,661, da &#946;-lactoglobulina foi AA 0,136, BB 0,323 e AB 0,539 e PIT1 ++ 0,655, -- 0,311 e +- 0,032. Os resultados mostram que a popula??o se encontra em desequil?brio de Hardy-Weinberg para a leptina, &#946;-lactoglobulina e PIT1 devido ao excesso de heterozigotos presentes na popula??o, entretanto, como estes genes est?o associados ? produ??o de leite, considera-se que os animais t?m potencial gen?tico para a produ??o leiteira nas condi??es do semi?rido brasileiro. Atrav?s da caracteriza??o do rebanho estudado n?o foram encontradas implica??es referentes ao polimorfismo da leptina, &#946; lactoglobulina e PIT1 na composi??o e qualidade do leite de vacas nos grupos gen?ticos 1/2, 3/4 e 7/8 Holand?s x Guzer?

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the genus Melipona, a bee Melipona quadrifasciata is one of the best known species and is divided into two subspecies M. q. quadrifasciata and M. q. anthidioides. This study aimed to evaluate the identity of bees mandaçaia the northern coast of Sergipe through morphometric and molecular techniques, and verify the possible geographic barrier represented by the Rio São Francisco. The survey was conducted in a total area of 30ha subdivided into three fragments in the municipalities of Piaçabuçu - AL, Brejo Grande SE, Ilha da Criminosa - SE. Were recorded in 14 colonies mandaçaia Brejo Grande and collected 10 specimens of each colony. No nest was located on the Ilha da Criminosa and Piaçabuçu, which reinforces the hypothesis that the Rio São Francisco could're representing a geographical barrier to the spread of the colonies. The spatial distribution of nests on a scale of 10ha, can be considered as random. Morphometry were used in the wings of the specimens collected and identified three other specimens of M. q. quadrifasciata, M. q. anthidioides and M. mandacaia. The data were subjected to cluster analyzes, principal component analysis, canonical variate analysis, Procrustes analysis of variance test and correlation matrices. Analyses of morphometric data are congruent and identified the M. q. quadrifascita as the identity of the species collected. The analysis of cross-validation and correlation matrices showed no significance, showing great similarity intrapopulation. Total DNA from each sample was subjected to analysis of PCR / RFLP (enzymes: Taq I, Vsp I and Mbo II), identifying a unique pattern of haplotypes for the bee population of the mouth, showing the high genetic similarity. All nests were located in palm trees that would be condemned by the low production due to damage caused by pests, besides the very low supply of other sites for nesting, which sets very high risk of population extinction. / No gênero Melipona, a abelha Melipona quadrifasciata é uma das espécies mais conhecida e está dividida em duas subespécies M. q. quadrifasciata e M. q. anthidioides. O presente trabalho teve como objetivo avaliar a identidade das abelhas mandaçaia do litoral norte do Estado de Sergipe por meio de técnicas moleculares e morfométrica, e verificar a possível barreira geográfica representada pelo Rio São Francisco. A pesquisa foi realizada em uma área total 30ha subdividida em três fragmentos nos municípios de Piaçabuçu AL, Brejo Grande SE e Ilha da Criminosa SE. Foram registradas 14 colônias de mandaçaia em Brejo Grande e coletados 10 espécimes de cada colônia. Nenhum ninho foi localizado na Ilha da Criminosa e em Piaçabuçu, o que reforça a hipótese de que o Rio São Francisco poderia está representando uma barreira geográfica para a dispersão das colônias. A distribuição espacial de ninhos em uma escala de 10ha, pode ser considerada como aleatória. Na morfometria foram utilizadas as asas dos espécimes coletados e de 3 outras amostras identificadas de M. q. quadrifasciata, M. q. anthidioides e M. mandacaia. Os dados foram submetidos a Análises de Agrupamento, Análise de Componentes Principais, Análise de Variáveis Canônicas, Análise de Variância de Procrustes e teste de correlação de matrizes. As análises dos dados morfométricos são congruentes e apontaram a M. q. quadrifascita como a identidade da espécie coletada. A análise de validação cruzada e a correlação de matrizes não apresentou significância, denotando grande similaridade intrapopulacional. O DNA total de cada amostra foi submetido a análise de PCR/RFLP (enzimas: Taq I, Vsp I e Mbo II), identificando um único padrão de haplótipos para a população de abelhas da foz, evidenciando a grande similaridade genética. É importante ressaltar que todos os ninhos estavam localizados em coqueiros que seriam condenados pela baixa produção ocasionada pelos danos decorrente de pragas, além da baixíssima oferta de outros locais para nidificação, o que configura altíssimo risco de extinção populacional.

A classificação de fungos microscópicos é limitada pela escassez de detalhes morfológicos e incertezas na caracterização bioquímica. Nesse trabalho desenvolvemos um método simples, baseado na amplificação de um segmento de cerca de 2.800 bares de bases seguida de digestão com DdeI e análise do padrão após eletroforese em agarose. Demonstramos que o método discrimina tão bem quanto a sequência de DNA do segmento após analisar mais de 400 amostras que foram classificadas em 33 espécies. Essa tecnologia facilita a busca por novos representantes da imensa diversidade existente, espécies que podem ser exploradas quanto ao seu valor potencial para a medicina e a indústria. Outras metodologias moleculares foram desenvolvidas para discriminar entre espécies próximas e na identificação de novas espécies. Em seguida, o trabalho esteve voltado para o desenvolvimento de um ensaio laboratorial que nos permitisse estudar a transição dimórfica em Candida albicans. Desenvolvemos dois novos ensaios, um biológico e outro sobre membrana artificial, para estudar a indução ou inibição da produção de hifas. A expressão de genes associados ao processo foi acompanhada de maneira quantitativa. Fracionando cromatograficamente o soro fetal bovino, obtivemos resultados indicativos de frações estimuladoras e inibidoras da transição morfológica. / Fungal identification is limited by few morphological details and uncertainty in the biochemical tests. In the present work we developed a simple procedure, based upon DNA amplification of a 2,800 base pairs region followed by the amplicon digestion with DdeI and electrophoretic analyzes in agarose gels. After analyzing more than 400 samples encompassing 33 species, we demonstrate that this method discriminates as well as DNA sequencing of the region. This technology simplifies the search for new representatives among the great diversity of fungi of medical and industrial interest. We devised also molecular methods to discriminate between closely related species and described a new putative species. Next our work was dedicated to develop an in vitro assay for the study of the dimorphic transition in Candida albicans. We devised two biological assays and one that used an artificial membrane to investigate the induction and inhibition of hyphal production. The expression of genes associated with the morphological transition was examined in a quantitative way. We fractionated bovine fetal serum by column chromatography and obtained results pointing to fractions that stimulate or inhibit hyphal production.

Estudou-se Microsporum gypseum de diferentes fontes regiões geográficas do Brasil.Os fungos foram isolados do solo, de animais e obtidos em coleções de cultura. Em 692 amostras de solo e a taxa de recuperação foi de 19,2%. A atividade queratinolítica e elastinolítica foi avaliada quantitativamente em 138 amostras de M. gypseum. A biotipagem molecular foi realizado através da técnica de PCR-RFLP com a enzima MvaI. O seqüenciamento da região ITS1 do rDNA foi realizado em amostras representativas. M. gypseum de amostras clínicas e do solo mostraram diferenças quantitativas significantes na expressão das enzimas. A PCR-RFLP para a região ITS1 não mostrou diferença. Pelo seqüenciamento foram obtidas duas espécies sexuadas (A. gypseum e A. incurvatum). As atividades enzimáticas sugerem um importante papel na patogenicidade e uma provável adaptação desta espécie de fungo ao parasitismo animal. A análise dos resultados pode auxiliar a identificação e o conhecimento de mecanismos de virulência destes fungos. / Microsporum gypseum from different geographical sources and regions of Brazil were studied. The fungi were isolated from the soil, animals and obtained in collections of culture. In 692 samples of soil, the recovery rate was 19.2%. The keratinolytic and elastinolytic activities were quantitatively performed on 138 samples of M. gypseum. The molecular biotyping was carried out by the technique of PCR-RFLP with the enzyme MvaI. The sequencing of the region ITS1 rDNA was carried out on representative samples. Samples of M. gypseum from clinical isolates and from soil showed significant quantitative differences in the expression of enzymes. The PCR-RFLP for the ITS1 region showed no difference. For the sequencing was obtained two sexual species (A. gypseum and A. incurvatum). The enzyme activities suggest an important role in pathogenicity and a likely adjustment of this kind of parasitic fungus to feed. The results may help the identification and knowledge of mechanisms of virulence of these fungi.

Toxoplasma gondii, the causal agent of toxoplasmosis, is an important water and food borne protozoan parasite. T. gondii was previously shown to have a distinct clonal population structure composed of Type I, II and III lineages in North America and Europe. But more recent studies demonstrated high diversity in South America. In the present project we have conducted an intensive study of the population diversity of T. gondii and surveyed the extent of genetic variation among natural T. gondii isolates on a global scale in order to better understand the population dynamics and pathogenesis of this parasite. To this end, 948 T. gondii isolates have been collected from a broad range of animal hosts and different sites worldwide. Our initial multilocus PCR-RFLP genotyping analysis revealed high diversity (~140 distinct genotypes) with abundant unique genotypes in South America and a strong clonal population structure in North America, Europe, Asia and Africa. It also showed that the Type II is the most common lineage worldwide, followed by the type III strain. The Type I strain, though widely distributed, has been infrequently isolated. Several new clonal genotypes have been identified from South America. The newly identified 140 RFLP genotypes have been further analyzed by multilocus microsatellites and intron sequencing methods. The composite data set identified 11 different haplotypes, providing a framework for future study of molecular epidemiology and population genetics of T. gondii . Multilocus DNA sequencing of markers from each of the 14 chromosomes covering the entire genome has also been completed to help reveal more information about genome evolution and the origin of T. gondii . Taken together, this comprehensive epidemiological and population genetic study has revealed significant details on the diversity and extent of sexual recombination, which provides the basis for future studies to understand transmission patterns, population dynamics and origin of this successful apicomplexan parasite Toxoplasma gondii.

A survey of gastro-intestinal parasites was conducted on faecal samples collected from 379 feral cats and 851 native fauna from 16 locations throughout Western Australia. The prevalence of each parasite species detected varied depending upon the sampling location. Common helminth parasites detected in feral cats included Ancylostoma spp. (29.8%), Oncicola pomatostomi (25.6%), Spirometra erinaceieuropaei (14%), Taenia taeniaeformis (4.7%), Physaloptera praeputialis (3.7%) and Toxocara cati (2.6%). The most common protozoan parasites detected in feral cats were Isospora rivolta (16.9%) and I. felis (4.5%). The native mammals were predominately infected with unidentified nematodes of the order Strongylida (59.1%), with members of the orders Rhabditida, Spirurida and Oxyurida also common. Oxyuroid nematodes were most common in the rodents (47.9%) and western grey kangaroos (27.8%). Several species of Eimeria were detected in the marsupials whilst unidentified species of Entamoeba and coccidia were common in most of the native fauna. Primers anchored in the first and second internal transcribed spacers (ITS1 and ITS2) of the ribosomal DNA (rDNA) were used to develop a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique to differentiate the species of Ancylostoma detected in feral cats. Amplification of the ITS+ region (ITS1, ITS2 and 5.8S gene) followed by digestion with the endonuclease RsaI produced characteristic patterns for A. tubaeforme, A. ceylanicum and A. caninum, which were detected in 26.6%, 4.7% and 0% of feral cats respectively. Giardia was detected in a cat, dingo, quenda and two native rodents. Sequence analysis at the small subunit rDNA gene (SSU-rDNA) identified the cat and dingo as harbouring G.duodenalis infections belonging to the genetic assemblages A and D respectively. Subsequent analysis of the SSU-rDNA and elongation factor 1 alpha (ef1á) identified a novel species of Giardia occurring in the quenda. Attempts to genetically characterise the Giardia in the two native rodents were unsuccessful. Serological detection of Toxoplasma gondii was compared to a one tube hemi-nested PCR protocol to evaluate its sensitivity. PCR was comparable to serology in detecting T. gondii infections, although PCR was a much more definitive and robust technique than serology for large numbers of samples. Amplification of T. gondii DNA detected infections in 4.9% of feral cats and 6.5% of native mammals. The distribution of T. gondii does not appear to be restricted by environmental factors, which implies that vertical transmission is important for the persistence of T. gondii infections in Western Australia. These results demonstrate that cats carry a wide range of parasitic organisms, many of which may influence the survival and reproduction of native mammals. As such, the large-scale conservation and reintroduction of native fauna in Western Australia must not disregard the potential influence parasites can have on these populations.

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Listeria monocytogenes is a foodborne pathogen that has been isolated from a variety of food sources. It is the cause of the food-borne disease, listeriosis that shows symptoms such as meningitis, encephalitis and abortion. Different strains of L. monocytogenes exist and not all are thought to be pathogenic to humans. The aim of this study was to evaluate and compare conventional methods, culturing on selective (Oxford agar) and chromogenic (RAPID’L.mono agar) media, as well as speciesspecific and multiplex polymerase chain reaction (PCR) methods for the detection and identification of 94 L. monocytogenes isolates from various areas in an avocado processing facility, as well as the final product. To achieve a better understanding of the genetic diversity of the confirmed L. monocytogenes strains isolated from the avocado facility, two subtyping techniques, PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE), were employed. All of the isolates were identified as Listeria species on both Oxford and RAPID’L.mono agar. On the RAPID’L.mono agar, 76 of the 94 isolates produced colonies typical of L. monocytogenes, with the remaining 18 showing colonies typical of L. innocua (n=13) and L. ivanovii (n=5). The species-specific PCR successfully amplified a 730 base pairs region of the hly gene of 80 of the 94 isolates. For the same 80 isolates the multiplex PCR successfully amplified 800, 517 and 238 base pair (bp) fragments of the inlA, inlC and inlJ genes, respectively. The remaining 14 isolates included the 13 isolates identified as L. innocua, as well as an isolate identified as L. monocytogenes on RAPID’L.mono. The results obtained on the Oxford agar showed a 100 % positive correlation when compared to the PCR results in identifying Listeria species, while the RAPID’L.mono had a 4 % false negative result in identifying L. monocytogenes compared to the PCR results. Sixty-four of the confirmed L. monocytogenes isolates were subtyped using PCRRFLP and PFGE. For the PCR-RFLP analysis, a 733 bp fragment of the inlA gene was successfully amplified for all of the isolates, followed by digestion with the restriction enzymes, AluI and Tsp509I. AluI produced three different banding patterns and Tsp509I produced two different banding patterns. Subtyping of the isolates using PFGE was carried out by macrorestriction of the genomic DNA with ApaI and AscI. The restriction fragments were resolved by PFGE and the fingerprints were classified into four clusters. In the combined analyses, cluster I contained forty-eight isolates (n=48), cluster II 1 isolate (n=1), cluster III fifteen isolates (n=15) and cluster IV 1 isolate (n=1). The PCR-RFLP results had a 98 % correlation with the PFGE results. The results of this study indicated inconsistencies between the results obtained by conventional and molecular detection methods for the identification of L. monocytogenes. Species-specific and multiplex PCR, however, proved useful to accurately detect and identify L. monocytogenes in a shorter period of time and could replace the use of conventional agar during identification. Both PCR-RFLP and PFGE proved useful in the subtyping of L. monocytogenes isolates with the PCR-RFLP being less expensive and results obtainable in a shorter period of time. / AFRIKAANSE OPSOMMING: Listeria monocytogenes is ‘n patogeen afkomstig van voedsel wat uit ‘n verskeidenheid voedselbronne geisoleer kan word. Dit is die oorsaak van die voedsel afkomstigde siekte, listeriosis met simptome soos harsingvliesontsteking, ensefalitis en aborsie. ‘n Verskeidenheid L. monocytogenes stamme bestaan, maar nie almal word as patogenies beskou nie. Die doel van hierdie studie was om konvensionele metodes, naamlik mikrobiologiese kweking op selektiewe (Oxford agar) en chromatografiese (RAPID’L.mono agar) media, sowel as spesies-spesifieke en multipleks polimerase ketting reaksie (PKR) metodes te evalueer en vergelyk vir die deteksie en identifikasie van 94 L. monocytogenes isolate geisoleer vanuit verskeie areas in ‘n avokado prosesseringsfasiliteit sowel as die finale produk. Om ‘n beter begrip van die genetiese diversiteit van die isolate wat as L. monocytogenes bevestig is te verkry, is twee subtiperingstegnieke, PKR-restriksiefragmentlengte polimorfisme (PKR-RFLP) en pulsveld jel-elektroforese (PVJE) toegepas. Beide Oxford en RAPID’L.mono agar het al die isolate as Listeria spesies geidentifiseer. Op die RAPID’L.mono agar het 76 van die 94 isolate kolonies tipies van L. monocytogenes gevorm, 13 kolonies was tipies van L. innocua (n=13) en vyf kolonies tipies van L. ivanovii (n=5). Die spesies-spesifieke PKR het ‘n 730 basis paar (bp) streek van die hly geen suksesvol geamplifiseer vir 80 van die 94 isolate. Die multipleks PKR het 800, 517 en 238 bp fragmente van die inlA, inlC and inlJ gene onderskeidelik, vir dieselfde 80 isolate suksesvol geamplifiseer. Die oorblywende 14 isolate het die 13 isolate wat as L. innocua geïdentifiseer is en die een isolaat wat as L. monocytogenes op RAPID’L.mono geïdentifiseer is ingesluit. Resultate verkry met die Oxford agar het 100 % ooreengestem met die PKR resultate vir die identifikasie van Listeria spesies. Die RAPID’L.mono het ‘n 4 % vals negatiewe resultaat gelewer in vergelyking met die PKR resultate. Vier-en-sestig van die bevestigde L. monocytogenes isolate is gesubtipeer deur PKR-RFLP en PVJE. Tydens die PKR-RFLP analise is ‘n 733 bp fragment van die inlA geen suksesvol geamplifiseer, gevolg deur vertering met die restriksie-ensieme, AluI and Tsp509I. AluI het drie verskillende bandpatrone opgelewer en Tsp509I twee verskillende bandpatrone. Subtipering deur PVJE is uitgevoer deur makro-restriksie van die genomiese DNA met ApaI en AscI. Die restriksie fragmente is geskei deur PVJE en die vingerafdrukke is in vier groepe geklassifiseer. Groep I het 48 isolate (n=48), groep II 1 isolaat (n=1), groep III 15 isolate (n=15) en groep IV 1 isolaat (n=1) gehad tydens die gekombineerde analise. Die PKR-RFLP resultate het 98 % ooreengestem met die van die PVJE. Die resultate van hierdie studie het teenstrydighede tussen die resultate van konvensionele en molekulêre deteksie metodes opgelewer vir die identifikasie van L. monocytogenes. Die spesies-spesifieke en multipleks PKR het egter beide goed te pas gekom vir die akkurate deteksie en identifikasie van L. monocytogenes en kan heel moontlik die gebruik van konvensionele agar tydens identifikasie vervang. Beide PKRRFLP en PVJE was nuttig vir die subtipering van L. monocytogenes isolate. PKR-RFLP is egter ‘n goedkoper tegniek en die resultate is in ‘n korter tydsperiode beskikbaar.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-06-12T18:57:15Z No. of bitstreams: 2 tarcisio_tomas_cabral_sousa.pdf: 1757108 bytes, checksum: 835c7914569f767d1b2d01ecdb785268 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-06-14T19:16:53Z (GMT) No. of bitstreams: 2 tarcisio_tomas_cabral_sousa.pdf: 1757108 bytes, checksum: 835c7914569f767d1b2d01ecdb785268 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-14T19:16:53Z (GMT). No. of bitstreams: 2 tarcisio_tomas_cabral_sousa.pdf: 1757108 bytes, checksum: 835c7914569f767d1b2d01ecdb785268 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / APERAM BioEnergia / VERACEL / GERDAU / A produ??o de eucalipto vem sofrendo grandes preju?zos com o ataque de uma nova praga, o Thaumastocoris peregrinus, seu nome popular ? percevejo bronzeado devido seu h?bito alimentar, succivoro. Essa alimenta??o causa danos diretos ?s ?rvores, deixando as folhas com aspecto bronzeado, diminuindo a fotoss?ntese, chegando a secar e cair, o que pode culminar com a morte da planta. Poucos s?o os estudos relacionados a essa praga, n?o existindo um controle eficaz. Com o intuito de conhecer sobre a morfologia do sistema digest?rio, bem como a intera??o deste com micro-organismos e visando fornecer informa??es ao controle dessa praga, foram realizadas a histologia do sistema digest?rio e sequenciamento de fragmentos de DNA obtidos de micro-organismos internos ao inseto. A histologia permitiu observar que o percevejo n?o tem os cecos g?stricos, parte do intestino m?dio que armazena micro-organismos que auxiliam na digest?o dos alimentos e que ? possuidor de um par de gl?ndulas salivares que s?o compostas de dois l?bulos cada. As an?lises moleculares possibilitaram verificar que, possivelmente, existem micro-organismos presentes no sistema digest?rio vivendo em associa??o com o mesmo. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The Eucalyptus production has suffered great losses from the attack of a new pest, the Thaumastocoris peregrinus, popularly known as bronze bug to cause silvering on eucalyptus leaves. This symptom occurs because of your eating habits, because it sucks the sap of its host. This feeding causes direct damage to trees, because its leaves stay with tanned look, reducing photosynthesis, reaching dry and fall off, which can lead to the death of the plant. There are few studies relating to this pest, with no effective control. With intuited to know about the digestive morphology and the interaction of this with micro-organisms, targeting subsidies to the control of this pest were performed histology of the digestive system and sequencing of DNA fragments obtained from internal micro-organisms to the insect. The histology allowed to observe that the bronze bug does not have the gastric cecum of the midgut that stores micro-organism to assist in digestion of food, and their digestive system is divided into three parts, foregut, middle and posterior, which is possessor of a pair of salivary glands that are composed of two lobes each. Molecular analysis enabled us to verify that, possibly, there are micro-organisms present in the digestive system living in association with the same.

Data de aprova??o ausente. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-06-08T22:19:37Z No. of bitstreams: 2 luana_martins_santos.pdf: 1170564 bytes, checksum: 215e11cc17c654f8760cc6085cca3464 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-06-22T15:18:34Z (GMT) No. of bitstreams: 2 luana_martins_santos.pdf: 1170564 bytes, checksum: 215e11cc17c654f8760cc6085cca3464 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-22T15:18:34Z (GMT). No. of bitstreams: 2 luana_martins_santos.pdf: 1170564 bytes, checksum: 215e11cc17c654f8760cc6085cca3464 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A produ??o de muda ? uma etapa primordial e decisiva para a implanta??o de uma floresta. No entanto, devido a diversos fatores de ordem t?cnica, as perdas no setor de produ??o s?o bastante relevantes. Dentre estes fatores est? o descarte do substrato, que quando feito de forma err?nea favorece a prolifera??o de micro-organismos fitopatog?nicos em viveiro. Isso pode desencadear em perdas significativas e ainda se tornar um passivo ambiental. Raramente o substrato ? reutilizado nos viveiros comerciais, uma vez que presume-se que as caracter?sticas f?sica, qu?mica e biol?gicas formam perdidas durante a produ??o das mudas. Contudo, h? poucos estudos sobre micro-organismos em substratos. A import?ncia deste estudo fundamenta-se na escassez de pesquisa sobre essa problem?tica que afeta o setor de produ??o de mudas, bem como buscar alternativas sustent?veis que pressup?e a demanda de uso desse insumo. A aplica??o de t?cnicas moleculares vem sendo empregadas para detectar e identificar os micro-organismos em diversos ambientes. Neste contexto, o objetivo deste estudo foi detectar a diversidade microbiol?gica em amostras de substratos descartados pela t?cnica de PCR-RFLP. Foram coletados dez amostras de substratos em diferentes viveiros no estado de Minas Gerais em diferentes fases de produ??o e estado de tecnifica??o. O DNA gen?mico total foi extra?do e amplificado pela t?cnica de PCR com oligonucleot?deos espec?ficos para fungos, bact?ria e archaea. Os produtos amplificados foram submetidos ? clivagem com enzimas de restri??o HaeIII, BamHI, TaqI e HindIII, para detec??o de poss?veis polimorfismos entre as amostras por meio da t?cnica de PCR-RFLP. Foi poss?vel verificar diferen?as entre as amostras de substratos, tanto em rela??o ao tamanho da regi?o do DNA amplificada, bem como em rela??o ? presen?a de s?tios de restri??o. Com base no ?ndice de similaridade, detectou-se uma maior varia??o da diversidade dentro da amostra em diferentes fases do que nas amostras entre os diferentes viveiros. Portanto, estes resultados podem ser relevantes para o conhecimento da diversidade microbiol?gica em substrato. Por?m mais estudos se fazem necess?rios para maior compreens?o destes micro-organismos e sua fun??o no substrato. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The production of changes is a crucial and decisive step for the implementation of a forest. However, due to several technical factors, losses in the production sector are quite relevant. Among these factors is the disposal of substrate, which when done wrongly favors the proliferation of phytopathogenic microorganisms in nursery. This can trigger significant losses and still become a environmental liabilities. Rarely the substrate is reused in commercial nurseries, since it is assumed that the physical, chemical and biological characteristics were lost during the production of seedlings. However, there are few studies on microorganisms on substrates. The importance of this study is based on the scarcity of research on this issue that affects the production of seedlings, as well as seek sustainable alternatives that assumes the use of this raw material demand. The application of molecular techniques have been employed to detect and identify microorganisms in various environments. In this context, the objective of this study was to detect microbiological diversity in substrate samples dropped by PCR-RFLP technique. Ten samples were collected of substrates in different nurseries in the State of Minas Gerais in different stages of production and State of modern farms. Total genomic DNA was extracted and amplified by the PCR technique with specific oligonucleotides to fungi, bacteria and archaea. The amplified products were submitted to cleavage with HaeIII restriction enzymes, BamHI, HindIII, and TaqI for detection of possible polymorphisms between the samples by PCR-RFLP technique. It was possible to check differences between samples of substrates, both in relation to the size of the amplified DNA region as well as in relation to the presence of restriction sites. Based on the index of similarity, if a greater variation of diversity within the sample at different stages than in samples between different nurseries. Therefore, these results may be relevant to the knowledge of microbiological diversity in substrate. However more studies are needed to better understanding of these microorganisms and their role in the substrate.