Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T18:47:19Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertacao-BrenoAlbuquerque.pdf: 1989114 bytes, checksum: 513b1650c4c44605e3bda0afef185321 (MD5) / Made available in DSpace on 2017-04-06T18:47:19Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertacao-BrenoAlbuquerque.pdf: 1989114 bytes, checksum: 513b1650c4c44605e3bda0afef185321 (MD5) Previous issue date: 2012 / O Papilomavírus bovino(BPV) é o agente etiológico da papilomatosebovina. Esta apresenta lesões que normalmente são benignas e tendem a regredir, porém podem progredir a uma neoplasia. Muitas metodologias utilizadas para detecção de BPV se mostram inespecíficas e apresentam reações cruzadas com outros organismos relacionados. No entanto, a reação quantitativa em tempo real emcadeia da polimerase (qPCR) é uma ferramenta de destaque na detecção, tipificação e quantificação de nucleotídeos e vem sendo utilizada na clínica para avaliar carga viral. O objetivo do trabalho foi desenvolver um novo protocolo de detecção, tipificação e quantificação de BPV através daqPCR. Foram desenhados cinco pares de primers, que possuem como alvo uma região conservada do genoma viral (gene L1) de diferentes BPVs. A seletividade dos primers foi testada in vitroe DNA extraído de células MDBK não infectadas foram utilizados como controle negativo. A técnica de qPCR permitiu detectar, tipificar e quantificar material viral dos BPVs 1, 2, 4, 5 e 6. O limiar relativo da detecção foi de 4fg de DNA,emtorno de 30-40 cópias de DNA/μL. Dos cinco pares de primers produzidos, quatro apresentaram mesmo perfil térmico durante a qPCR (qPCRBPV2, 4, 5 e 6), permitindo em um único procedimento detectar e tipificar os quatro tipos virais. A distinção das amostrasfoi realizada através da análise de meltingque permitiu tipificá-las. Através da metodologia desenvolvida foi observado que em lesões cutâneas de bovinos infectados com BPV a carga viral não se mostrou inferior a 1000 cópias/μL, enquanto que a técnica permite quantificar até um limiar de 40 copias de DNA/μl. Este trabalho possui relevância para validação de qPCR como diagnóstico da papilomatose bovina e particular importância quando aplicado em estudos da infecção pelo BPV e no monitoramento por veterinários da eficácia das futuras vacinas. / Bovine papillomavirus (BPVs) is the etiologic agent of bovine papilomatose which is characterized by hyper proliferative lesions. Papillomas in cattle are typically benignandoften regress, but occasionally lesions can persist and progress to malignant neoplasia.The majority of current techniques for identification of BPV is unspecific andpossessescross-reactivity with closely related organisms.The Real-time quantitative polymerase chain reaction assay (qPCR) has become an exceptional tool for detection and quantification of oligonucleotides and has been utilized increasingly on viral load evaluation.Aiming to develop a new protocol for fast detection, typification and quantification of BPV in qPCR, we designed five pairs of Oligonucleotides for BPV1, 2, 4, 5 and 6 focusing on L1 gene. The qPCR primers sets were testedin vitroandMadin-Darby Bovine Kidney Cells (MDBK)DNA was also used as negative control.The Real-time qPCR assay provided an accurate detection and quantification for the BPVs 1, 2, 4, 5 and 6. The relative detection limit for the assays was 4fg or 30 to 40genome equivalents. Four primers pairs (qPCRBPV2, 4, 5 and 6) had the same annealing temperature and their products showed differences on meltingpoints analyses. Through the meltingpoint analysis, samples can be identified and discriminated as a screening and then samples can be run for viral load. In our study we tested the viral load in bovine cutaneous skin warts and observed infections with 1000 copies/μl at least. However, this assay could reach levels of 40copies/μL. In conclusion, this methodology has an important impact on the validation of qPCR as a BPV diagnosis. Its relevance is proved when applied to BPV infection studies and the monitoring of the efficacy of future BPV vaccinesby veterinarians.

Submitted by (edna.saturno@ufrpe.br) on 2017-04-20T16:36:59Z No. of bitstreams: 1 Itala Iara Medeiros de Araujo.pdf: 682787 bytes, checksum: fc0270c78b2defb1d6aa93cc96d0ae10 (MD5) / Made available in DSpace on 2017-04-20T16:36:59Z (GMT). No. of bitstreams: 1 Itala Iara Medeiros de Araujo.pdf: 682787 bytes, checksum: fc0270c78b2defb1d6aa93cc96d0ae10 (MD5) Previous issue date: 2013-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Candidate genes of the kappa-casein (CSN3) and beta-lactoglobulin (LGB) are involved in the composition, processing and quality of milk and are linked to production characteristics. This study aimed to evaluate the allele and genotype frequencies of kappa-casein gene (CSN3) and beta-lactoglobulin (LGB) and associate them with milk production of cattle participants Progeny Test Breed Girolando through analysis of variables milk yield in 305 days (P305) and predicted transmitting ability (PTA) for milk. For the study of polymorphisms were genotyped sires 138 and 729 cows (n = 867) for CSN3 and 737 and 131 bulls and cows (n = 868) to LGB. For the association analysis were evaluated 127 bulls and 536 cows (n = 663) and 127 for CSN3 bulls and 536 cows (n = 663) for LGB. The differentiation of allele A / B genes studied was obtained by PCR-RFLP. Allele frequencies, genotype and calculating the probability of Hardy-Weinberg equilibrium (HWE) were established through the program Popgen versão1.32 and tested by χ ² test at a significance level of 1%. The association study was performed by regression analysis using the GLM procedure of SAS 9.1. The allele and genotype frequencies for the gene CSN3 were, respectively, 0.7324 (AA), 0.2468 (AB) 0.0208 (BB) and 0.8558 (A) 0.1442 (B) and the LGB were, respectively, 0.2604 (AA), 0.4827 (AB) 0.2569 (BB) and 0.5017 (A) 0.4983 (B). The population is in HWE for both genes. No association was found between genes evaluated and analyzed variables. / Os genes candidatos da kappa-caseína (CSN3) e da beta-lactoglobulina (LGB) estão envolvidos na composição, processamento e qualidade do leite e estão ligados às características de produção. Objetivou-se avaliar as frequências alélicas e genotípicas do gene da kappa-caseína (CSN3) e da beta-lactoglobulina (LGB) e associá-las à produção de leite de bovinos participantes do Teste de Progênie da Raça Girolando, por meio de análise das variáveis de produção de leite em até 305 dias (P305) e de capacidade prevista de transmissão (PTA) de leite. Para o estudo do polimorfismo, foram genotipados 138 touros e 729 vacas (n = 867) para o CSN3 e 131 touros e 737 vacas (n = 868) para LGB. Para a análise de associação foram avaliados 127 touros e 536 vacas (n = 663) para CSN3 e 127 touros e 536 vacas (n = 663), para LGB. A diferenciação dos alelos A/B dos genes estudados foi obtida por meio da técnica de PCR-RFLP. As frequências alélicas, genotípicas e o cálculo da probabilidade de equilíbrio de Hardy-Weinberg (EHW) foram estabelecidos por meio do programa Popgen versão1.32 e testado pelo teste χ² ao nível de significância de 1%. O estudo de associação foi realizado por meio de análise de regressão utilizando o procedimento GLM do SAS 9.1. As frequências genotípicas e alélicas para o gene CSN3 foram, respectivamente, 0,7324 (AA); 0,2468 (AB); 0,0208 (BB) e 0,8558 (A); 0,1442 (B) e para o LGB foram, respectivamente, 0,2604 (AA); 0,4827 (AB); 0,2569 (BB) e 0,5017 (A); 0,4983 (B). A população encontra-se em EHW para ambos os genes. Não foi detectada associação entre os genes avaliados e as variáveis analisadas.

Orientadores: Sophie Françoise Mauricette Derchain, Luis Otavio Sarian / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T00:16:06Z (GMT). No. of bitstreams: 1 Moraes_DenisedaRochaPittaLimade_M.pdf: 959384 bytes, checksum: dcd7eaa366994d0d3d606ecc04a19503 (MD5) Previous issue date: 2008 / Resumo: Introdução: Este estudo faz parte de uma linha de pesquisa cuja finalidade é avaliar testes de detecção de papilomavírus humano (HPV) envolvidos na carcinogênese e rastreamento de câncer cervical. Até recentemente, a captura híbrida 2 (HC2) para detecção de um pool de HPV de alto risco oncogênico foi o método clínico mais estudado por este grupo. Entretanto, frente à evidente diferença no risco de evolução das lesões cervicais, a genotipagem do HPV através de análise do ácido nucléico passou a ser relevante. Objetivo: Avaliar a distribuição de infecções simples e múltiplas de diferentes tipos de HPV em mulheres com neoplasia intra-epitelial cervical de alto grau (NIC2 e NIC3). Sujeitos e Métodos: Foram avaliados os genótipos específicos de HPV da amostra cervical de 106 mulheres utilizando Roche Linear Array® human papillomavirus (LA-HPV) genotyping assay (Roche Diagnostics,USA). O material foi coletado antes da conização cervical. Foram comparadas as proporções de NIC2 e NIC3 em grupos de mulheres infectadas com tipos de HPV dos grupos filogenéticos Alfa7 (A7) e Alfa9 (A9). Três situações foram consideradas: mulheres com 1) infeccão simples; 2) infecção múltipla; 3) infecção simples e múltipla. Foram comparadas as proporções de diferentes combinações de tipos de HPV em grupos de mulheres com NIC2 e NIC3. Resultados: Pelo menos um tipo de HPV foi detectado em 99% das amostras. Infecções múltiplas foram detectadas em 68 (64,7%) das amostras. Os genótipos de alto risco mais freqüentemente detectados em infecção simples ou múltipla foram HPV16 (57,1%), HPV58 (24,7%), HPV33 (15,2%), HPV52 (13,3%), HPV31 (10,4%) e HPV51 (7,6%) e HPV18 (6,6%). A probabilidade de mulheres com NIC3 serem infectadas com HPV da espécie A9 foi maior. Os HPV 16 e ou 18, associados ou não com outros tipos virais foram mais frequentemente encontrados nas mulheres com NIC3 do que naquelas com NIC2. Conclusão: A severidade da NIC esteve associada com a presença de tipos de HPV incluídos na classificação filogenética A9 e por infecções que incluem HPV16 e 18 combinados ou não com outros genótipos de HPV / Abstract: Objective: To evaluate the distribution of single and multiple infections of different human papillomavirus (HPV) types in women with high grade cervical intraepithelial neoplasia (CIN2 and CIN3) and to assess the relation of the various combinations of virus with the severity of CIN. Subject and methods: Cervical samples from 106 women treated due to CIN2 (18) or CIN3 (88) were examined for specific HPV genotypes using Roche Linear Array® Human Papillomavirus (LA-HPV)(Roche Diagnostics, USA). The material was collected immediately before cervical conization. The proportion of CIN2 and CIN3 in groups of women infected with varying HPV phylogenetic groups Alpha7 (A7) and Alpha9 (A9) was compared. Three situations were considered: women with 1) single infection; 2) multiple infections; 3) the whole sample. The proportions of CIN2 and CIN3 in groups of women with different combinations of HPV types were compared. Results: At least one HPV type was detected in 99% of the whole series. Multiple infections were detected in 68 (64.7%) samples. The most frequent high-risk genotypes detected (single/multiple) were HPV16 (57.1%), HPV58 (24.7%), HPV33 (15.2%), HPV52 (13.3%), HPV31 (10.4%), HPV51 (7.6%) and HPV18 (6.6%). Women with CIN3 were more infected with HPV of species A9. HPV16 and/or HPV18, associated or not with other viral types, were more frequently found in specimens of women with CIN3 than in those of women with CIN2. Conclusions: the severity of high-grade CIN may be associated by the presence of HPV types included in the A9 phylogenetic classification and by infections including HPV16 and 18 combined or not with other HPV genotypes / Mestrado / Ciencias Biomedicas / Mestre em Tocoginecologia

Orientador: Angélica Zaninelli Schreiber / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T21:59:44Z (GMT). No. of bitstreams: 1 ReichertLima_Franqueline_M.pdf: 2034183 bytes, checksum: 203b846210e23f42a824280eb3cadda2 (MD5) Previous issue date: 2014 / Resumo: O gênero Cryptococcus engloba duas espécies consideradas patogênicas: C.neoformans e C.gattii. Apesar dos avanços na área médica, a criptococose permanece uma das infecções fúngicas sistêmicas mais importantes no Brasil. Anfotericina B (AMB) associada a flucitosina (5FC) é a terapia de indução indicada porém, no Brasil, 5FC não está disponível e o tratamento segue somente com AMB ou em associação com fluconazol (FCL). Este trabalho avaliou a ocorrência de espécies e os genótipos de isolados clínicos de pacientes atendidos no Hospital de Clínicas da UNICAMP em um período de 5 anos; isolados ambientais coletados na cidade de Campinas-SP e o perfil de suscetibilidade aos antifúngicos sozinhos contra isolados clínicos e ambientais e o efeito de combinações de antifúngicos frente a isolados clínicos de Cryptococcus spp.. A identificação de espécies e genótipos foi realizada por testes bioquímicos, Restriction Fragment Length Polymorphism do gene URA5 (URA5-RFLP) e sequenciamento da região Internal Transcribed Spacer (ITS) do DNA ribossomal. Testes de suscetibilidade para AMB, 5FC, FCL, voriconazol (VRC), itraconazol (ITC) e terbinafina (TRB) isolados foram realizados conforme CLSI M27-A3 (2008). Os testes com antifúngicos combinados (AMB+5FC; AMB+FCL; AMB+TRB; FCL+TRB), foram realizados pelo método do "tabuleiro de xadrez" para determinação do Coeficiente de Inibição Fracional (CIF) para avaliar o tipo de interação entre as substâncias (sinergismo, indiferença ou antagonismo). Dentre 75 isolados clínicos reativados, foram identificados 66 C.neoformans e 9 C.gattii. Todos C.gattii pertenceram ao genótipo VGII enquanto que 62 isolados de C.neoformans ao genótipo VNI e apenas 4 ao genótipo VNII. Foram obtidos 92 isolados ambientais de Cryptococcus, pertencentes às espécies C.neoformans (genótipo VNI), C.laurentii, C.albidus, C.flavescens e Cryptococcus spp.. Os valores dos intervalos de CIM para C.neoformans clínicos foram AMB: ? 0,125-1 µg/mL; 5FC: ? 0,125-2 µg/mL; FCL: 0,25-8 µg/mL; VRC: ? 0,015-0,125 µg/mL; ITC: 0,03-0,25 µg/mL e TRB: 0,125-2 µg/mL. Intervalos de CIM para C.gattii variaram de 0,25-1 µg/mL para AMB; 0,5-4 µg/mL para 5FC; 2-16 µg/mL para FCL; 0,06-0,25µg/mL para VRC; 0,06-0,5 µg/mL para ITC e 0,5-4 µg/mL para TRB. Foram observados elevados valores de CIM para os antifúngicos 5FC e FCL frente aos isolados ambientais de C.albidus e C.laurentii. O genótipo VNI de C.neoformans clínico mostrou 75,80% de interação sinérgica para AMB+5FC; 79,03% para AMB+FCL; 77,42% para AMB+TRB e 95,16% para FCL+TRB. O genótipo VNII apresentou 100% de sinergismo em todas as combinações. C.gattii (VGII) apresentou 88,9% de sinergismo nas combinações AMB+5FC e AMB+FCL; 100% para AMB+TRB e FCL+TRB. Não foi observado efeito antagônico nas combinações de antifúngicos. Foi observado bom desempenho nas combinações realizadas, especialmente naquelas envolvendo a TRB para ambas as espécies C.neoformans e C.gattii. O genótipo VNI foi o predominante entre os genótipos que afetam os pacientes com criptococose na região de Campinas. Em infecções de difícil tratamento ou que não respondem aos antifúngicos convencionais, a combinação de diferentes antifúngicos como AMB+TRB ou FCL+TRB podem vir a ser uma alternativa em países onde 5FC não está disponível, como no Brasil. Mais estudos são necessários para avaliar o papel dos genótipos na sensibilidade aos antifúngicos, assim como estudos de combinações de antifúngicos in vitro e in vivo para que novas estratégias possam ser empregadas no tratamento da criptococose / Abstract: Cryptococcus genus comprises two species considered pathogenic: C.neoformans and C.gattii. Despite advances in the medical field, cryptococcosis remains one of the most important systemic fungal infections in Brazil. Amphotericin B (AMB) associated with flucytosine (5FC) induction therapy is indicated but, in Brazil, 5FC is not available and treatment follows only with AMB or in combination with fluconazole (FCL). This study evaluated the prevalence of species and molecular subtypes of clinical isolates from patients treated at the Hospital of UNICAMP in a period of 5 years; environmental isolates collected in Campinas ¿ SP and in vitro antifungal susceptibility profile of antifungal agents alone against clinical and enviromental isolates of Cryptococcus spp. and the effect of combinations of antifungal agents against clinical isolates of Cryptococcus spp.. The species identification and subtyping was performed by biochemical tests, Restriction Fragment Length Polymorphism of URA5 gene (RFLP - URA5) and sequencing of the Internal Transcribed Spacer region (ITS) of ribosomal DNA. Susceptibility tests for AMB, 5FC, FCL, voriconazole (VRC), itraconazole (ITC) and terbinafine (TRB) isolated were performed according to CLSI M27-A3 (2008). Combined antifungals tests (AMB +5FC; AMB+FCL; AMB +TRB, FCL+TRB), were performed by the "Checkerboard" method to determine the Fractional Inhibitory Coefficient index (FIC) to assess the type of interaction between substances (synergism, indifference or antagonism). Among the 75 viable clinical isolates, 66 were identified as C.neoformans and 9 as C.gattii. All C.gattii belonged to subtype VGII while 62 isolates belonged C.neoformans VNI genotype and only 4 VNII. 92 environmental isolates of Cryptococcus were obtained. The species C.neoformans (subtype VNI) C.laurentii, C.albidus, C.flavescens and Cryptococcus spp.were identified. The MIC range values of clinical C.neoformans for AMB were: ? 0.125 -1 µg/mL; 5FC: ? 0.125 to 2 µg/mL; FCL: 0.25-8 µg/mL; VRC: ? 0.015 to 0.125 µg/mL; ITC: 0.03 to 0.25 µg/mL and TRB: 0.125 to 2 µg/mL. MIC for C.gattii ranged from 0.25-1 µg/mL for AMB; 0.5-4 µg/mL for 5FC; 2-16 µg/mL for FCL; 0.06 to 0.25 µg/mL for VRC; 0.06 to 0.5 µg/mL for ITC and 0.5-4 µg/mL for TRB. High MIC values were observed for FCL and 5FC against environmental isolates of C.albidus and C.laurentii. The VNI C.neoformans genotype showed 75.80 % of synergistic interaction for AMB+5FC; 79.03 % for AMB + FCL; 77.42 % for AMB + TRB and 95.16 % for FCL+TRB. The VNII genotype showed 100% synergism in all combinations. C.gattii (VGII) showed 88.9 % of synergism in combinations AMB+5 FC and AMB+FCL; 100 % for AMB+TRB and TRB+FCL. No antagonistic effect was observed in all evaluated antifungal combinations. Good performance was observed in all combinations performed, especially those involving TRB for both C.neoformans and C.gattii species. The VNI was the predominant genotype among genotypes affecting patients with cryptococcosis in Campinas region. In difficult to treat infections or unresponsive to conventional antifungal agents, the combination of different antifungals so as AMB+FCL or TRB+TRB may become an alternative in countries where 5FC is not available, as in Brazil. More studies are needed to evaluate the role of genotypes in sensitivity to antifungal agents, as well as antifungal agents combination studies in vitro and in vivo so that new strategies can be employed in the treatment of cryptococcosis / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Cartas al Editor

Ce travail de thèse intitulé «Evaluation de marqueurs génétiques du complexe Mycobacterium tuberculosis combinée à l'utilisation d'outils bioinformatiques: apport en épidémiologie et phylogénie de la tuberculose» a consisté en la sélection et l'évaluation de marqueurs minisatellites dans le cadre d'études épidémiologiques et phylogénétiques du bacille tuberculeux, Une première partie a ainsi porté sur l'épidémie de tuberculose en Guadeloupe, Martinique, et Guyane française ainsi que dans un pays continental à faible incidence de tuberculose, la Suède, Les résultats montrent les disparités existant entre les populations de patients tuberculeux des 3 départements français d'Amérique (DFA) ainsi que les similitudes génotypiques existant entre les bacilles tuberculeux. Par ailleurs le rôle majeur joué par les cas d'importation dans l'épidémie de tuberculose a été montré aussi bien dans les DFA qu'en Suède. Nous nous sommes ensuite intéressés à une utilisation optimale des minisatellites pour une meilleure discrimination de la famille génotypique émergeante « Beijing» circulant au Japon (Osaka, Kobe et Okinawa), ainsi qu'en Russie. Nous avons constaté le faible pouvoir discriminant du format classique 12-locus MIRU (Mycobacterial lnterspersed Repetitive Units) et proposons plusieurs nouvelles stratégies de typage basées plusieurs marqueurs minisatellites incluant certains locus hypervariables. Enfin, dans un troisième volet, nous avons étudié la diversité génétique des bacilles tuberculeux circulant actuellement dans la Caraïbe. laquelle semble refléter le passé historique très particulier de cette zone géographique au croisement d'une multitude de peuplements. / This thesis entitled "Evaluation of genetic markers of Mycobacterium tuberculosis combined with the use of bioinformatics tools: input for epidemiology and phylogeny of tuberculosis" deals with the selection and evaluation of minisatellite markers in the context of epidemiological and phylogenetic studies of the tubercle bacillus M. tuberculosis. The first part of the present work has focused on the epidemic of tuberculosis in Guadeloupe, Martinique and French Guiana as well as in a low incidence continental country, i.e Sweden. The results show differences between populations of TB patients in the three French Departments of America and similarities between the genotypes of the circulating tubercle bacilli Moreover, results highlight the important role played by imported cases for TB epidemic the three French departments of America and Sweden. ln a second part, minisatellite markers have been evaluated for a better discrimination of strains of the emerging genotype "Beijing" circulating in Japan (Osaka, Kobe and Okinawa), and Russia. A low discriminatory power of classical format 12-locus MIRU (Mycobacterial lnterspersed Repetitive Units) was observed and new combinations of minisatellites have also been proposed including hypervariable locus. Finally, the genetic diversity of tubercle bacilli circulating in the Caribbean was investigated which currently seem to reflect historical past of this very special region at the intersection of a multitude of cultures and peoples.

>Magister Scientiae - MSc / Insertion-deletion polymorphisms (indels) have been underutilized in forensic identification of individuals in comparison with single nucleotide polymorphisms (SNPs) and short tandem repeat (STRs) systems. The use of indels for the purpose of human identification is more advantageous than previously used methods as it combines desirable characteristics of both the SNPs and STRs i.e. low costs and simplistic typing methods as well as indels having small amplicons size, making them suitable for genotyping highly degraded DNA. Currently there is only one commercial kit available for the forensic community, the Investigator® DIPlex kit (Qiagen), which cover a total of 30 indel loci distributed over 19 autosomal chromosomes. The objective of this study was to evaluate the Qiagen Investigator® DIPplex kit for forensic application in South Africa. The kit‘s performance was evaluated by comparing different extraction methods; sensitivity, robustness and reproducibility were evaluated and forensic parameters (match probability, power of discrimination, polymorphism information content, power of exclusion and typical paternity index) were estimated based on population data generated from five South African populations (Afrikaner, Mixed Ancestry, Indian-Asian, Xhosa and Zulu). Population comparisons were performed using Fst-analysis, factorial component analysis as well as phylogenetic tree construction. DNA was extracted from buccal swabs and whole blood collected from a total of 512 individuals from the five South African population groups and genotyped using the Qiagen Investigator® DIPplex kit. Sanger DNA sequencing and sequence alignments confirmed the presence of a null allele at locus HLD97 which was present in high frequency in the Xhosa and Zulu populations. This observation was made in 14 individuals belonging to the Xhosa and Zulu populations. Null allele frequencies in all five South African populations were also estimated. Null alleles were estimated for all loci using analytical methods i.e. Charkraborty null allele estimator, Brookfield null allele estimators 1 and 2 and ML-NullFreq software program. The kit performed well in the laboratory, not requiring any additional reagents or instrumentation and successfully generating profiles with input DNA amounts as low as 0.2 ng/μL. Although well suited for forensic application, the Qiagen Investigator® DIPplex kit showed some drawbacks with regards to application on South African populations. The presence of a null allele at the HLD97 locus as well as indication of population substructure affects allele frequency estimates for the South African populations. Correction for population substructure as present within the South African populations should be considered using FST analysis and it is recommended that the HLD97 locus should be excluded from any kinship analysis performed on South African populations.

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Orientadores: Fernando Lopes Gonçales Junior, Neiva Sellan Lopes Gonçales / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T22:44:35Z (GMT). No. of bitstreams: 1 Tonetto_PriscilaAparecida_M.pdf: 2027435 bytes, checksum: ec4453a66ae541917395dc0f14142ed8 (MD5) Previous issue date: 2006 / Resumo: O vírus da hepatite B (VHB) pode ser classificado em oito principais genótipos (A-H), e essa classificação tem uma distribuição geográfica determinada. Os genótipos do VHB podem influenciar na progressão de doença. O objetivo foi determinar os genótipos e os subtipos do VHB e correlacioná-los com as variáveis clínicas epidemiológicas, laboratoriais e histológicas. Foram determinados os genótipos de 139 amostras de soro de pacientes infectados pelo VHB, coletadas em Campinas, no estado de São Paulo, Brasil. O método para genotipagem utilizado foi o seqüenciamento parcial do gene S do VHB. Os primers utilizados foram desenhados a partir de seqüências do gene S, com genótipo determinado, depositadas no GenBank. Todas as seqüências obtidas foram comparadas com as seqüências depositadas no GenBank para determinação dos genótipos. O genótipo A (55%) do VHB foi o mais predominante na população, seguido pelos genótipos C (3%), D (38%) e F (4%). Entre os pacientes infectados pelos genótipos A e D, observou-se uma provável descendência africana de 18% (14/76) e 11% (6/53), respectivamente. Entre os quatro pacientes infectados pelo genótipo C, dois possuíam descendência asiática e dois eram caucasianos. Todos os pacientes infectados pelo genótipo F eram caucasianos sem ascendência indígena relatada. Aproximadamente 30% dos pacientes eram HBeAg positivo e 70% eram HBeAg negativo. A carga viral do DNA-VHB foi aproximadamente cinco vezes mais alta entre os HBeAg positivo quando comparada aos HBeAg negativo. Os genótipos A e D são os mais prevalentes entre os pacientes, aparentemente em virtude da imigração européia em nossa região / Abstract: Hepatitis B virus (HBV) can be classified into eight major genotypes (A-H) that have mainly a geographic distribution. The HBV genotype may influence disease progression. Our objective was to determine the genotypes and the subtypes of HBV and to correlate them with the with variables clinical epidemiologies, laboratories and histological. Hepatitis B virus genotypes were determined in 139 plasma samples collected in Campinas, in the state of São Paulo, Brazil from HBV-infected patients. A method for genotyping hepatitis B virus by partial HBsAg gene sequencing with primers common to all known genotypes was developed. The results of sequencing corresponded to those found in HBV isolates obtained from GenBank, including all of the known HBV genotypes. HBV genotype A was predominant in our sample, appearing in 76 patients (55%), while genotypes C, D and F was found in 4 (3%), 53 (38%) and 6 (4%) of the patients, respectively. Among the patients infected by genotypes A and D, were observed a probably African descendents of the 18.3% (14/76) and 11.3% (6/53), respectively. Among the genotype C infected patients, 2 (50%) were of Asian descendents and 2 were Caucasians. The genotype F infected patients were all Caucasians without told indigenous origin. About 30% of the patients were HBeAg positive and 70% were HBeAg negative. The viral load of HBV-DNA was about 5 times higher among HBeAg positive than in HBeAg-negative patients. Genotypes A and D were the most prevalent among our HBV-infected patients, apparently a consequence of the types of immigration to our region / Mestrado / Ciencias Basicas / Mestre em Ciências Médicas

Over half the human genome consists of repetitive sequences. One major class is the tandem repeats (TRs), which are defined by their location in the genome, repeat unit, and copy number. TRs loci that exhibit variant copy numbers are called Variable Number Tandem Repeats (VNTRs). High VNTR mutation rates of approximately 0.0001 per generation make them suitable for forensic studies, and of interest for potential roles in gene regulation and disease. TRs are generally divided into three classes: 1) microsatellites or short tandem repeats (STRs) with patterns <7 bp; 2) minisatellites with patterns of seven to hundreds of base pairs; and 3) macrosatellites with patterns of >100 bp. To date, mini- and macrosatellites have been poorly characterized, mainly due to a lack of computational tools. In this thesis, I utilize a tool, VNTRseek, to identify human minisatellite VNTRs using short-read sequencing data from nearly 2,800 individuals and developed a new computational tool, MaSUD, to identify human macrosatellite VNTRs using data from 2,504 individuals. MaSUD is the first high-throughput tool to genotype macrosatellites using short reads. I identified over 35,000 minisatellite VNTRs and over 4,000 macrosatellite VNTRs, most previously unknown. A small subset in each VNTR class was validated experimentally and in silico. The detected VNTRs were further studied for their effects on gene expression, ability to distinguish human populations, and functional enrichment. Unlike STRs, mini- and macrosatellite VNTRs are enriched in regions with functional importance, e.g., introns, promoters, and transcription factor binding sites. A study of VNTRs across 26 populations shows that minisatellite VNTR genotypes can be used to predict super-populations with >90% accuracy. In addition, genotypes for 195 minisatellite VNTRs and 22 macrosatellite VNTRs were shown to be associated with differential expression in nearby genes (eQTLs). Finally, I developed a computational tool, mlZ, to infer undetected VNTR alleles and to detect false positive predictions. mlZ is applicable to other tools that use read support for predicting short variants. Overall, these studies provide the most comprehensive analysis of mini- and macrosatellites in human populations and will facilitate the application of VNTRs for clinical purposes.