<p>Sexual assault is a significant problem facing the South African society. In this context, efficient but also affordable genotyping systems are needed for positive identification of criminals in incidences of sexual violence. The aim of this study was therefore to develop non-commercial Y-STR genotyping systems suitable for sexual assault cases in South Africa. Y-chromosome STR loci constituting the minimal haplotype are still the most widely used loci in investigating sexual assault cases despite the fact that DYS391 and DYS392 have shown low levels of polymorphism in Xhosa populations in Cape Town. The minimal haplotype was, therefore, further investigated in the Cape Muslim population. The Cape Muslim population generally exhibited high GD values among all the South African populations. These values were higher than 0.5 for most loci, and ranged from 0.447 for DYS391 to 0.957 for DYS385. The highest number of alleles in most loci was also recorded in this population. The overall assessment of the minimal haplotype has shown that this system is still a useful in investigating sexual assault case in many South African subpopulations. Therefore the exercise of internal validation of the minimal haplotype system was successfully carried out in the laboratory. iii The properties of additional novel and widely used STRs were also investigated in this study. Loci were successfully sequenced and allele nomenclature was assigned to them according to the ISFG guidelines.</p>

The aim of the present study was to evaluate the power of discrimination and genetic (diversity) parameters in the Y chromosome extended haploytpe markers in populations of Tanzania for forensic and populations studies. Eleven Y chromosome extended haplotype markers were selected for this study, these includes Minimal haplotypes markers i.e. DYS19, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS389I/II and two additional markers DYS438 and DYS439. Six populations of Tanzania were investigated under this study. These populations were selected based on the language family categories / Niger Congo (Kuria and Sukuma), Nilo Saharan (Luo and Maasai) and Afro Asiatic (Iraqw and Alagwa).

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

The aim of the present study was to evaluate the power of discrimination and genetic(diversity) parameters in the Y chromosome extended haploytpe markers in populations of Tanzania for forensic and populations studies. Eleven Y chromosome extended haplotype markers were selected for this study, these includes Minimal haplotypes markers i.e. DYS19, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS389I/II and two additional markers DYS438 and DYS439. Six populations of Tanzania were investigated under this study. These populations were selected based on the language family categories; Niger Congo (Kuria and Sukuma), Nilo Saharan (Luo and Maasai) and Afro Asiatic (Iraqw and Alagwa).Buccal swabs were collected from unrelated males from Mwanza province (Sukuma),Mara (Kuria and Luo), Arusha (Maasai and Iraqw) and Dodoma province (Alagwa).Samples were typed using ABI 377 Genetic Analyser (Applied Biosystem) followed by analysis using softwares Gelprocessor, GeneScan 3.0.0 (Applied Biosystems) and Genotyper 3.7 (Applied Biosystems). The data obtained were analysed by GenePop 4.0,Arlequin 3.11 and Genetix v.4.05.2 software packages. Analyses such as AMOVA, Fst population pairwise comparison, Factorial component Analysis were used to obtain Allele frequency, haplotype frequency, gene diversities among various loci and levels of gene flow between populations.For the overall individuals, the highest Gene Diversity value was 0.8251 (DYS385) and the lowest was 0.2723 (DYS392). The overall Haplotype Diversity was 0.9984 and Discrimination capacity resulted 84.27%. A total of 225 distinct haplotypes were identified in 267 individuals, 28 were shared, the most frequent haplotype was present in 5 individuals. The levels of genetic diversity for the haplotypes per group as revealed by haplotype diversities confirmed that the most diverse group being Sukuma, Kuria,Iraqw, Maasai, Luo and Alagwa being the least diverse. The Discrimination capacity of these set of markers showed the highest value in Sukuma population (100%) subsequently followed by Iraqw, Luo, Maasai, Kuria and Alagwa (78.38%) being the lowest. Analysis of Molecular Variance showed a significant differentiation among populations, 93.96% of variance was found within population and 6.04% among population. Population pairwise results between all population pairs (except Sukuma and kuria and Alagwa and Luo) showed significant results (P < 0.05). Genetic heterogeneity that was found among Tanzanian populations could not be attributed to language barriers but was largely being contributed by a limited level of gene flow between these populations due to different ethnical, social, cultural and historical backgrounds between them. All Y chromosome extended haplotype loci used in this study (except DYS392 and DYS391 which showed the lowest level of polymorphism) were found to be likely useful for forensic application in Tanzania. Furthermore the extended haplotype markers used in this study may be useful in the establishment of the National DNA database following the enactment of the Human DNA Legislation in Tanzania (http://www.parliament.go.tz). / Magister Scientiae - MSc

Colorectal cancer (CRC) is a common malignancy which develops due to old age and lifestyle factors, low percent of patients afflicted by a genetic disorders. Half of all colorectal cancer patients are diagnosed after metastasis. The high rate of the late detection, emphasizes on the requirement of convenient and inexpensive diagnostic methods for comprehensive screening programs. The aim of this study was to discover proto-oncogenes mutation and assessment of tumor suppressor genes expression. Formalin fixed paraffin embedded (FFPE) histologically verified colorectal cancer samples were used. APC, KRAS and BRAF mutations were investigated using polymerase chain reaction (PCR) fragments and direct sequencing. Gene expression assessment of MLH1, MGMT and CDKN2A were achieved via quantitative polymerase chain reaction (qPCR). In the present study we could detect a novel transversion heterozygous mutation in APC gene codon 1365 in three patients. BRAF codon 600 mutation were detected in one patient. KRAS codon 12 mutation was discovered in one sample and also a novel transition mutation in codon 15 was detected in 6 patients. In 80% of cases, MLH1 and MGMT expression were undetectable, in remaining 20%, MLH1 expression were reduced, but MGMT showed both reduced and increased expression compared to control. In 100% of patients CDKN2A expression was undetectable. The rate of mutations in predetermined hotspot codons and amount of uncommon mutations into APC, BRAF and KRAS in Nepalese patients indicates the requirement of further investigation in CRC patients from that part of the world. Also, the expression rate of MLH1, MGMT, CDKN2A and deficiency of an information source emphasizes the necessity of whole genome CRC expression profiling data to comparison and conclusion. / <p>-</p> / -

O vírus do Oeste do Nilo (VON) é um Flavivírus capaz de infectar muitas espécies de vertebrados, incluindo o homem. Embora reconhecida desde 1940, esta virose nunca havia sido descrita nas Américas, onde emergiu nos Estados Unidos ao final da década de 1990, com numerosos casos de meningoencefalite em humanos. Posteriormente, sua transmissão por transfusão de sangue e órgãos foi comprovada, levando à implantação de testes moleculares (NAT) para a triagem de doadores nos EUA e Canadá a partir de 2003. Nos anos seguintes, o VON foi sendo progressivamente detectado em países como México, Panamá e áreas do Caribe, sugerindo sua iminente introdução na América do Sul. De fato, evidências sorológicas foram reveladas em cavalos e aves na Colômbia, Venezuela, Argentina e muito recentemente no pantanal mato-grossense (em cavalos). A vigilância epidemiológica para este agente é de grande importância para a saúde pública, visto o potencial de morbimortalidade deste vírus para humanos. Sendo assim este trabalho tem o objetivo de investigar a presença do RNA do VON em amostras de doadores de sangue, pacientes com meningoencefalite ou febre de origem indeterminada e soros e amostras cerebrais de equinos. Foram analisadas 2.202 doações de sangue do Amazonas (HEMOAM), 3.144 do Mato Grosso do Sul (HEMOSUL); líquido cefalorraquidiano de 51 pacientes com suspeita de meningoencefalite viral (Hospital das Clínicas/FMUSP, São Paulo) e soro de 198 pacientes com síndrome febril aguda, negativos para Dengue e Malária (Fundação de Medicina Tropical de Manaus). Além disto, 293 amostras de soros de equinos da região do Pantanal e 63 biópsias de tecido cerebral de cavalos que foram a óbito por encefalite de etiologia desconhecida. Estas amostras foram submetidas ao teste automatizado cobas TaqScreen WNV (Roche) na plataforma cobas s201 em sistema de pool de 6 unidades (doações de sangue) ou individualmente (pacientes). Todas as amostras apresentaram amplificação satisfatória do controle da reação, porém nenhuma apresentou resultado positivo para a presença do RNA do VON. Embora já exista evidência da exposição de equinos no Brasil ao VON, não parece haver até o momento, disseminação importante deste agente entre humanos e equinos, uma vez que o RNA viral não foi detectado nem em doadores de sangue e nem em equinos, incluindo os de cidades próximas aos locais onde cavalos soropositivos foram encontrados (Corumbá MS). / The West Nile Virus (WNV) is a Flavivirus able to infect many species of vertebrates, including man. Recognized since 1940, this virus had never been described in the Americas, which emerged in the United States at the end of the 1990s, with numerous cases of meningoencephalitis in humans. Later, transmission by transfusion of blood and organs was confirmed, leading to the deployment of molecular testing (NAT) for screening of donors in the U.S. and Canada since 2003. In the following years, WNV has been progressively detected in countries like Mexico, Panama and the Caribbean areas, suggesting their imminent introduction in South America In fact, serological evidence was revealed in horses and birds in Colombia, Venezuela and Argentina and most recently in Pantanal, Mato Grosso (horses). Epidemiological surveillance for this agent is of great importance to public health, given the potential morbidity and mortality of this virus to humans. Therefore this study aims to investigate the presence of WNV RNA in samples of blood donors, patients with meningoencephalitis or fever of unknown origin and serum and brain samples from horses. We analyzed 2202 blood donations from Amazon (HEMOAM), 3144 from Mato Grosso do Sul (HEMOSUL); cerebrospinal fluid of 51 patients with suspected viral encephalitis (Hospital das Clínicas / FMUSP, São Paulo) and serum samples from 198 patients with acute febrile syndrome, negative for Dengue and malaria (Foundation for Tropical Medicine in Manaus). In addition, more 293 serum samples from horses of the Pantanal and 63 biopsies of brain tissue from horses that died of encephalitis of unknown etiology. These samples were subjected to automated cobas TaqScreen WNV test (Roche) on the platform in cobas S201with a system of 6 units pool (blood donations) or individually (patients). All samples showed satisfactory control amplification, but none showed as positive for the presence of RNA VON. Although there is already evidence in horses in Brazil of exposure to WNV, there seems to be far that an important spread of this agent between humans and horses, since the viral RNA was not detected either in blood donors or in horses, including cities near the locations where seropositive horses were found (Corumbá - MS).

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No. of bitstreams: 1 Danielle Albuquerque Pires Rocha.pdf: 1640322 bytes, checksum: 517abde36a5e28961792c6d782a3d48b (MD5) Previous issue date: 2012-06-25 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / The lack of precision and speed in the laboratory diagnosis of some sexually transmitted infections (STI) are commom problems faced by professionals working in this area. Additionally, the lack of some of them in the public health system become more difficult to elucidate the diagnosis. Because of these difficulties, the STI are underdiagnosed, being treated indiscriminately, using only clinical diagnosis. The molecular diagnostic methods have arisen in recent years as an excellent alternative to fill some of the gaps left by traditional methods. The aim of this research was to study the epidemiology of some sexually transmitted pathogens at molecular level, using molecular biology techniques in women attended in public health system in the city of Coari, Amazonas, Brazil. Samples were collected from 361 women for cervical cytology (Pap Smear) and molecular examination. We carried out molecular diagnosis using the technique of Polymerase Chain Reaction (PCR) and real-time PCR for some sexually transmitted pathogens, which were: Human Papillomavirus, Herpes Simplex Virus 2, Human Cytomegalovirus, Chlamydia trachomatis, Neisseria gonorrhoeae and Thichomonas vaginalis. The results showed that 47.8% of these women were infected by any of the pathogens. There was a high prevalence of infection with Human Papilomavirus (29.1%), followed by T. vaginalis (12.7%), Human Cytomegalovirus (8.3%), C. trachomatis (6.4%), N. gonorrhoeae (1.4%) and Herpes Simplex Vírus 2 (0.6%). The prevalence of HPV type in the infected women was HPV 16 (58.1%), followed by HPV 58 (20.0%). The satisfactory slides for cytological diagnosis (n=321) showed that 7 women (2.1%) showed abnormal cytology (ASCUS, LSIL and HSIL), 5 of them being infected by HPV. There were no statistically significant associations between sexually transmitted infections and the variables related to socio-demographics, medical history and sexual behavior. / A falta de precisão e rapidez no diagnóstico laboratorial de algumas doenças sexualmente transmissíveis (DST) são problemas enfrentados pelos profissionais que atuam nesta área. Além dessas dificuldades inerentes aos testes, a falta de alguns deles na rede pública de saúde dificultam ainda mais a elucidação de diagnósticos. Por causa dessas dificuldades, as DST são subdiagnosticadas, sendo tratadas indiscriminadamente, valendo-se apenas do diagnóstico clínico. Os métodos moleculares de diagnóstico têm surgidos nos últimos anos como uma excelente alternativa para preencher algumas dessas lacunas deixadas pelos métodos tradicionais. Esta pesquisa teve como finalidade estudar a epidemiologia de alguns patógenos sexualmente transmissíveis a nível molecular, valendo-se para isso de técnicas de biologia molecular, em mulheres atendidas na Atenção Primária à Saúde do Município de Coari, Amazonas (AM). Foram colhidas amostras cervicais de 361 mulheres para exame citológico (Papanicolaou) e exame molecular. Foi realizado o diagnóstico molecular através da técnica de Reação em Cadeia da Polimerase (PCR) e PCR em tempo real para alguns patógenos sexualmente transmissíveis, que foram: Papilomavírus Humano, Herpes Vírus Simples 2, Citomegalovírus Humano, Chlamydia trachomatis, Neisseria gonorrhoeae e Trichomonas vaginalis. Os resultados obtidos mostraram que 47,8% dessas mulheres estavam infectadas por algum dos patógenos estudados. Constatou-se alta prevalência de infecção por Papilomavírus Humano (29,1%), seguida pelo T. vaginalis (12,7%), Citomegalovírus Humano (8,3%), C. trachomatis (6,4%), N. gonorrhoeae (1,4%) e Herpes vírus Simples 2 (0,6%). O tipo prevalente de HPV nas mulheres infectadas foi o HPV 16 (58,1%), seguido pelo HPV 58 (20,0%). As lâminas citológicas satisfatórias para diagnóstico (n=321) mostraram que 7 mulheres (2,1%) exibiram alterações citológicas (ASCUS, LSIL e HSIL), estando 5 delas contaminadas pelo HPV. Não foram encontradas associações estatisticamente significativas entre as infecções por patógenos sexualmente transmissíveis e variáveis relacionadas à condições sócio-demográficas, história clínica e comportamento sexual.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Queijo de Coalho à um dos produtos lÃticos mais consumidos no Nordeste do Brasil. No estado do CearÃ, sua produÃÃo à considerada tradicional e apresenta bastante significÃncia econÃmica e social. O queijo de Coalho à tradicionalmente feito com leite cru, o que representa um risco em potencial para a saÃde do consumidor, devido à possibilidade de veiculaÃÃo de microrganismos patogÃnicos. O processo de pasteurizaÃÃo do leite, alÃm de destruir os microrganismos patogÃnicos, reduz tambÃm as bactÃrias Ãcido lÃticas (BAL) que sÃo os microrganismos responsÃveis pelas propriedades sensoriais dos alimentos fermentados. AlÃm do mais, a substituiÃÃo das BAL endÃgenas por fermento lÃtico comercial tem levado a perdas nas propriedades sensoriais do queijo. Este trabalho teve por objetivo identificar, pela tÃcnica da ReaÃÃo em Cadeia da Polimerase (PCR), BAL dos gÃneros Lactococcus e Lactobacillus, previamente identificadas bioquimicamente, isoladas de leite, massa de queijo e queijos de Coalho artesanais produzidos no estado do CearÃ. Os 44 isolados selecionados apresentavam propriedades tecnolÃgicas de interesse para fabricaÃÃo de queijo de Coalho como: capacidade de acidificar o leite, baixa atividade proteolÃtica, capacidade de produÃÃo de aroma e capacidade de tolerÃncia ao NaCl na concentraÃÃo de 3%. As PCR identificaram 20,4% dos isolados como Lactococcus lactis subsp. lactis e 27,3% como Lactobacillus paracasei. Nenhum isolado de Lactobacillus plantarum teve sua identificaÃÃo confirmada pela tÃcnica molecular. Ao todo, 47,7% dos isolados foram confirmados pela PCR, assegurando que estes microrganismos podem ser usados na fabricaÃÃo de queijo de Coalho a partir de leite pasteurizado, mantendo as caracterÃsticas sensoriais tÃpicas deste produto. Este estudo mostra a importÃncia da implantaÃÃo de tÃcnicas moleculares aumentando a qualidade e a eficiÃncia na identificaÃÃo de microrganismos. / Coalho Cheese is one of the dairy products more consumed in Northeast of Brazil. In Cearà state, its fabrication is traditional and has social and economic importance. Traditionally, it is made with raw milk, which represents potential risk for consumers health due to the possibility of foodborne pathogens transmission. The milk pasteurization process eliminates pathogenic microorganisms, but also reduces the lactic acid bacterias (LAB) â the microorganism responsible for sensorial characteristics of fermented foods. Moreover, the substitution of indigenous LAB by commercial lactic ferment has led to losses in the sensorial properties of the cheese. This work aimed to identify, by Polymerase Chain Reaction (PCR), LAB of the genus Lactococcus and Lactobacillus, previously biochemical identified, isolated from milk, curd and traditional Coalho cheeses produced in the state of CearÃ. The forty-four strains selected presented interesting technological properties for the Coalho cheese manufacture: ability to acidify the milk, low proteolytic activity, ability to produce flavor and ability to tolerate 3% NaCl. The PCR identified 20,4% of the isolates as Lactococcus lactis subsp. lactis and 27,3% as Lactobacillus paracasei. No one Lactobacillus plantarum was identified by the molecular technique employed. A total of 47.7% of the isolates were confirmed by PCR, assuring that these microorganisms can be used for Coalho cheese manufacture made from pasteurized milk, preserving the typical characteristics of this product. This work shows the relevance of molecular approaches, increasing the quality and efficiency in the microorganisms identification.

A espécie Escherichia coli constitui um grupo de bactérias tipicamente não patogênicas e que fazem parte do trato intestinal de humanos e animais. As amostras são sorotipadas com base em seus antígenos de superfície O (somático), H (flagelar) e K (capsular). O antígeno flagelar correspondente ao filamento é formado pela polimerização da flagelina, codificada pelo gene fliC. O presente trabalho empregou a técnica de PCR-RFLP para analisar os padrões de antígenos flagelares de 112 amostras de EPEC e STEC. Quatorze amostras não amplificaram o gene fliC, 17 tiveram seu antígeno flagelar determinado apenas por PCR-RFLP e 75 amostras tiveram seus antígenos flagelares confirmados por esta técnica. Três antígenos H com padrões irregulares foram clonados e sequenciados. Após o sequenciamento, inserções e remoções de nucleotídeos foram encontradas. Até o momento, poucos estudos utilizam um número abrangente de amostras de STEC e EPEC provenientes de diferentes animais para a determinação do antígeno H empregando a técnica de PCR-RFLP do gene fliC. De acordo com os resultados encontrados neste estudo, podemos concluir que a técnica de PCR-RFLP do gene fliC é mais rápida, menos trabalhosa e mais eficiente que a metodologia de sorotipagem clássica. / The Escherichia coli species consists of a group of typically non-pathogenic bacteria present in the intestinal tract of humans and animals. Strains are serotyped according to their O (somatic), H (flagellar) and K (capsular) surface antigens, in order to distinguish these microorganisms from the non-pathogenic members of the intestinal microbiota. The flagellar antigen corresponding to the filament is formed by the polymerization of the flagellin, codified by the fliC gene. This study employed the PCR-RFLP technique to analyze flagellar antigen patterns from 112 EPEC and STEC strains. Fourteen strains have not amplified the fliC gene, 17 had their flagellar antigen determined only by the PCR-RFLP and 75 strains had their flagellar antigen confirmed by this technique. Three H antigens with irregular patterns were cloned and sequenced. After sequencing, insertions and deletions of nucleotides were discovered. So far, few studies used a significant number of STEC and EPEC strains originated from different animals to determine H antigens employing the PCR-RFLP technique of the fliC gene. According to the findings of this study, we assumed that PCR-RFLP of the fliC gene is faster, less laborious and more efficient than classic serotyping methodology.

Magister Scientiae - MSc / Sexual assault is a significant problem facing the South African society. In this context, efficient but also affordable genotyping systems are needed for positive identification of criminals in incidences of sexual violence. The aim of this study was therefore to develop non-commercial Y-STR genotyping systems suitable for sexual assault cases in South Africa. Y-chromosome STR loci constituting the minimal haplotype are still the most widely used loci in investigating sexual assault cases despite the fact that DYS391 and DYS392 have shown low levels of polymorphism in Xhosa populations in Cape Town. The minimal haplotype was, therefore, further investigated in the Cape Muslim population. The Cape Muslim population generally exhibited high GD values among all the South African populations. These values were higher than 0.5 for most loci, and ranged from 0.447 for DYS391 to 0.957 for DYS385. The highest number of alleles in most loci was also recorded in this population. The overall assessment of the minimal haplotype has shown that this system is still a useful in investigating sexual assault case in many South African subpopulations. Therefore the exercise of internal validation of the minimal haplotype system was successfully carried out in the laboratory. The properties of additional novel and widely used STRs were also investigated in this study. Loci were successfully sequenced and allele nomenclature was assigned to them according to the ISFG guidelines. / South Africa