A candidate gene approach was used to identify single nucleotide polymorphisms (SNPs) and their associations with body fat deposition and carcass merit traits in beef cattle. In total, 37 SNPs from 9 candidate genes have been genotyped on 463 hybrid, 206 Angus and 187 Charolais steers for association analyses with 10 different fat deposition and carcass merit traits. In single SNP analyses, 28 SNPs of 9 genes have been found significantly (P<0.05) associated with different traits in the cattle populations. Gene-specific linkage disequilibrium assessment of SNPs revealed the existence of haplotype blocks within 4 genes. Haplotype analyses have identified 31 haplotypes of 6 genes having significant associations (P<0.05) with different fat deposition and carcass merit traits in the cattle populations. These findings will provide insight into the genetic mechanism regulating body fat deposition in beef cattle and will assist the beef industry to improve beef quality through marker assisted selection. / Animal Science

The objective of this thesis was to construct radiation hybrid (RH) maps and estimate linkage disequilibrium (LD) using high density SNP markers on chromosomes 19 (BTA19) and 29 (BTA29) and use these as a tool to detect QTL in dairy and beef cattle. We have constructed RH maps of BTA19 and BTA29 consisting of 555 and 253 SNP markers respectively using a 12,000 rad whole genome RH panel. When aligned with the third draft of bovine genome sequence assembly, there was a significant internal rearrangement of the markers involving displacement, inversion and flips within the scaffolds with some scaffolds being misplaced in the genome assembly. Many of these mapped markers (370 and 186 SNP markers on BTA19 and 29 respectively) were further utilized to quantify the extent of LD using the square of the correlation coefficient (r2) and to study the pattern of selection signatures in beef (Angus) and dairy (Holstein) breeds of Bos taurus. Along the chromosomes, patterns of LD were variable in both breeds and a minimum of 30,000 informative and evenly spaced markers would be required for whole genome association studies in cattle. In addition, chromosomal regions showing evidence of selection for economically important traits in Angus and Holstein were identified. Furthermore, the dense SNP markers were used to perform chromosome-wide scan to detect QTL for different economically important traits in beef and dairy cattle. Two approaches, single marker LD regression and Bayesian Monte Carlo Markov Chain, were used to map QTL. QTL for 10 and 5 traits in dairy cattle and for 2 and 1 trait in beef cattle on BTA19 and 29 respectively were detected using both approaches of QTL mapping. The QTL detected in this study are a step towards the identification of positional candidate genes controlling these traits. In addition, we have detected several SNPs influencing economically important traits in both beef and dairy cattle. Some SNPs have been validated in an independent cattle population and has the potential of being utilized in the marker assisted selection of cattle. / Animal Science

Recent initiatives for biofuel production have increased research and development of sweet sorghum. Currently, the initial major limitation to integrating sweet sorghum into existing production systems is the lack of sweet sorghum hybrids adapted to industrial production systems. Hybrid development is now underway, and the application of genetic markers can be used to define the genetic basis of sugar yield and its components, as well as reduce the time required to deliver new sweet sorghum hybrids to market. The purpose of this research was to further characterize the genetic components that influence sweet sorghum productivity, agronomics, and composition. Specifically, a grain x sweet sorghum recombinant inbred line (RIL) population developed for quantitative trait locus (QTL) analysis related to sugar production was evaluated for 24 phenotypic traits including brix, percent moisture, and biomass yield across four environments. The 185 F4 RILs were derived from the parents 'BTx3197' and 'Rio', which are pithy stalk grain and juicy stalk sweet sorghums respectively. Following screening, two genetic maps were constructed with 372 and 381 single nucleotide polymorphisms (SNPs) evaluated using an Illumina GoldenGate assay. Analysis of the data in QTL Cartographer revealed a major and previously reported QTL for soluble solids on chromosome 3, but in contrast to previous studies, this QTL co-localized with other QTLs that have a negative influence on biomass and seed production. Therefore, selection for this QTL may not be advantageous. Because only a few QTLs for percent moisture were found, the results indicated that the pithy stalk phenotype does not have a major effect on percent moisture as measured in this study. Thus, breeding for high or low moisture content will be more challenging than previously expected. The absence of dominance effects indicated that brix must be high in both parents to produce high brix in the hybrid.

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid advances have enormously expanded sequencing opportunities and applications, but also imposed heavy strains on steps prior to sequencing, as well as the subsequent handling and analysis of the massive amounts of sequence data that are generated, in order to exploit the full capacity of these novel platforms. The work presented in this thesis (based on six appended papers) has contributed to balancing the sequencing process by developing techniques to accelerate the rate-limiting steps prior to sequencing, facilitating sequence data analysis and applying the novel techniques to address biological questions.   Papers I and II describe techniques to eliminate expensive and time-consuming preparatory steps through automating library preparation procedures prior to sequencing. The automated procedures were benchmarked against standard manual procedures and were found to substantially increase throughput while maintaining high reproducibility. In Paper III, a novel algorithm for fast classification of sequences in complex datasets is described. The algorithm was first optimized and validated using a synthetic metagenome dataset and then shown to enable faster analysis of an experimental metagenome dataset than conventional long-read aligners, with similar accuracy. Paper IV, presents an investigation of the molecular effects on the p53 gene of exposing human skin to sunlight during the course of a summer holiday. There was evidence of previously accumulated persistent p53 mutations in 14% of all epidermal cells. Most of these mutations are likely to be passenger events, as the affected cell compartments showed no apparent growth advantage. An annual rate of 35,000 novel sun-induced persistent p53 mutations was estimated to occur in sun-exposed skin of a human individual.  Paper V, assesses the effect of using RNA obtained from whole cell extracts (total RNA) or cytoplasmic RNA on quantifying transcripts detected in subsequent analysis. Overall, more differentially detected genes were identified when using the cytoplasmic RNA. The major reason for this is related to the reduced complexity of cytoplasmic RNA, but also apparently due (at least partly) to the nuclear retention of transcripts with long, structured 5’- and 3’-untranslated regions or long protein coding sequences. The last paper, VI, describes whole-genome sequencing of a large, consanguineous family with a history of Leber hereditary optic neuropathy (LHON) on the maternal side. The analysis identified new candidate genes, which could be important in the aetiology of LHON. However, these candidates require further validation before any firm conclusions can be drawn regarding their contribution to the manifestation of LHON. / QC 20111115

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

>Magister Scientiae - MSc / Reference single nucleotide polymorphism (refSNP) identifiers are used to earmark SNPs in the human genome. These identifiers are often found in variant call format (VCF) files. RefSNPs can be useful to include as terms submitted to search engines when sourcing biomedical literature. In this thesis, the development of a bioinformatics software package is motivated, planned and implemented as a web application (http://sniphunter.sanbi.ac.za) with an application programming interface (API). The purpose is to allow scientists searching for relevant literature to query a database using refSNP identifiers and potential keywords assigned to scientific literature by the authors. Multiple queries can be simultaneously launched using either the web interface or the API. In addition, a VCF file parser was developed and packaged with the application to allow users to upload, extract and write information from VCF files to a file format that can be interpreted by the novel search engine created during this project. The parsing feature is seamlessly integrated with the web application's user interface, meaning there is no expectation on the user to learn a scripting language. This multi-faceted software system, called SNiPhunter, envisions saving researchers time during life sciences literature procurement, by suggesting articles based on the amount of times a reference SNP identifier has been mentioned in an article. This will allow the user to make a quantitative estimate as to the relevance of an article. A second novel feature is the inclusion of the email address of a correspondence author in the results returned to the user, which promotes communication between scientists. Moreover, links to external functional information are provided to allow researchers to examine annotations associated with their reference SNP identifier of interest. Standard information such as digital object identifiers and publishing dates, that are typically provided by other search engines, are also included in the results returned to the user. / National Research Foundation (NRF) /The South African Research Chairs Initiative (SARChI)

Magister Scientiae - MSc (Medical BioSciences) / Despite attempts to develop new drugs to reduce the worldwide mortality rate attributable to tuberculosis (TB), the illness remains a threat. Isoniazid (INH) has been used as a frontline drug for decades. However, several resistant strains of the organism - Mycobacterium tuberculosis (M. tuberculosis) - still emerge. The drug is mainly metabolised by a family of enzymes, arylamine N-acetyltransferases (NAT). The three human NAT genes - NAT1, NAT2 and the pseudogene, NATP - are found on chromosome 18p22. NAT1 and NAT2 are isoenzymes which differ at certain amino acid positions. Subsequently, the differences affect substrate specificity. NAT1 shows specificity to p-aminobenzoic acid (PABA) and paminosalicylate (PAS). Previously, computer algorithms were used to predict the efficacy of the enzyme with regard to the acetylation phenotype it confers. The two which were focused on, Sorting Intolerant From Tolerant (SIFT) program and Polymorphism phenotyping version 2 program (PolyPhen-2), showed conflicting results for the effect of SNPs on the acetylation rate and subsequent enzyme function. Further structural prediction methods were used to test the effect of V231G on the structure and consequent function of the native protein, NAT1.

Submitted by BÁRBARA ROCHA VENANCIO null (b.rvenancio@outlook.com) on 2017-06-01T12:26:01Z No. of bitstreams: 1 Dissertação_Bárbara Rocha Venancio_2017.pdf: 1455087 bytes, checksum: 52124957d6d5433b1fb34b032cc3d9c5 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-06-02T13:19:34Z (GMT) No. of bitstreams: 1 venancio_br_me_ilha.pdf: 1455087 bytes, checksum: 52124957d6d5433b1fb34b032cc3d9c5 (MD5) / Made available in DSpace on 2017-06-02T13:19:34Z (GMT). No. of bitstreams: 1 venancio_br_me_ilha.pdf: 1455087 bytes, checksum: 52124957d6d5433b1fb34b032cc3d9c5 (MD5) Previous issue date: 2017-04-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A presente dissertação de mestrado é composta por uma seção introdutória, seguida de uma revisão da literatura a qual antecede os três capítulos subsequentes. O primeiro capítulo aborda um conjunto de revisões de conhecimentos científicos contemporâneos sobre os efeitos da exploração madeireira em florestas tropicais e as práticas madeireiras utilizadas no Brasil, quais têm se demonstrado insuficientes para garantir a sustentabilidade tanto na produção genética quanto na produção madeireira. O segundo capítulo é um “primer note” descrevendo a identificação de 402 loci putativos (polimorfismos de nucleotídeo único – SNPs, inserções / deleções - INDELs) para Ipe (Handroanthus sp.), destinado à estudos de genética de populações, filogeografia e DNA fingerprinting. O último capítulo discute a viabilidade de DNA fingerprinting para espécies do gênero Handroanthus. Esse traz a análise da diversidade genética, diferenciação genética de populações de Handroanthus sp., bem como entre os países de origem das amostras, análises de auto atribuição de genótipos e testes de atribuição de madeira ao local de origem. / The present master dissertation is composed by an introductory section, followed by a review of literature, which prefaces the three subsequent chapters. The first chapter of this dissertation is a review assembly contemporary scientific knowledge about the effects of the forest logging in tropical rainforests and the actual logging practices used in Brazil, which seems insufficient to ensure sustainability in both genetic and timber production aspects. The second chapter is a primer note describing the identification of 402 putative loci (single nucleotide polymorphisms –SNPs; and insertion/deletions- INDELs) for Ipe (Handroanthus sp.), intended to help population genetics, phylogeography and DNA fingerprinting studies. The last chapter discuss the feasibility of DNA fingerprinting for Handroanthus species. It brings genetic diversity analysis, genetic differentiation of Handroanthus sp. sample-populations, as well as among countries, self-assignment and timber assignment tests analysis.

Submitted by Gislaine Aparecida Querino (gislainequerino@hotmail.com) on 2018-09-28T01:32:53Z No. of bitstreams: 1 Querino,GA.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-09-28T12:55:41Z (GMT) No. of bitstreams: 1 querino_ga_dr_bot.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5) / Made available in DSpace on 2018-09-28T12:55:41Z (GMT). No. of bitstreams: 1 querino_ga_dr_bot.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5) Previous issue date: 2018-07-25 / A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae que pode se manifestar sob diferentes formas clínicas a depender da resposta imune celular do hospedeiro. Vários marcadores genéticos têm sido definidos e, devido à participação dos alelos HLA na resposta imune, estes têm sido amplamente estudados na hanseníase. O HLA-DP constitui uma das moléculas clássicas de classe II, com poucos estudos em hanseníase. O objetivo deste trabalho foi conduzir um estudo de associação genética de base populacional em hanseníase para os genes HLA-DPA1 e HLA-DPB1 com duas amostras caso-controle: a primeira de Rondonópolis-MT, uma região hiperendêmica para hanseníase, e a segunda do Estado de São Paulo, uma região com índices epidemiológicos controlados. Foram realizados estudos com 9 tag SNPs na região dos genes HLA-DPA1 e HLA-DPB1 na população de Rondonópolis–MT (411 casos e 357 controles). O SNP rs9277341 que apresentou dados de associação com significância estatística após a correção de Bonferroni foi então testado na população de São Paulo (570 casos e 380 controles). Para avaliar o efeito funcional deste marcador foi determinada estudoa produção das citocinas IL-10 e TNF após estímulo com antígeno sonicado de M. leprae em 21 controles saudáveis. Na população de Rondonópolis-MT foi realizada a tipificação dos alelos HLA-DPA1* e HLA-DPB1* através da técnica de PCR-SSO empregando-se o kit Labtype-SSO (One Lambda, CA, USA). Os resultados de genotipagem mostraram associação com hanseníase per se para o marcador rs9377341 do gene HLA-DPA1 e dois marcadores do gene HLA-DPB1 (rs1431402 e rs9277469). Associações de proteção para a forma multibacilar da hanseníase foram encontradas para os marcadores rs9277341 e rs2301220 do gene HLA-DPA1 e para o marcador rs31350221 do gene HLA-DPB1. O SNP rs9277341, com dados significativos após a correção de Bonferroni e testado na população do Estado de São Paulo, não apresentou associação com hanseníase. A avaliação funcional mostrou que os carreadores do alelo G do rs9277341 apresentaram níveis maiores de IL-10 e TNF. Na tipificação dos alelos HLA, o alelo HLA-DPB1*03 foi associado à hanseníase per se e o genótipo HLA-DPB1*02/03 foi associado à hanseníase per se e à proteção para a forma MB. Esses dados sugerem a participação dos genes HLA-DPA1 e HLA-DPB1 nos desfechos da hanseníase. / Leprosy, a chronic infectious disease caused by Mycobacterium leprae, displays different clinical manifestations depending on the immune response of the host. Several genetic markers have been defined and due to the involvement of the HLA alleles in the immune response they were extensively studied in leprosy. However, HLA-DP is a classical class II molecule with few studies on leprosy. The objective of this study was to conduct a population-based genetic association study in leprosy for the HLA-DPA1 and HLA-DPB1 genes with two case-control samples: the first from Rondonópolis - Mato Grosso (MT), a hyperendemic region in leprosy and the second from the state of São Paulo, a region with controlled epidemiological indices. Studies were carried out with 9 Tag Single Nucleotide Polymorphism (SNP) in HLA-DPA and HLA-DPB genes in the population from Rondonópolis – MT (411 cases and 357 controls). The SNP rs9277341 who presented association data with statist significance after the Bonferroni correction was tested in the São Paulo population (570 cases and 380 controls). To evaluate the functional effect of this marker was carried a study of the production of IL-10 and TNF cytokines after stimulation with the sonicated antigen of M. leprae in 21 healthy controls. In the Rondonópolis populations was performed the HLA-DPA1* and HLA-DPB1* allele typing by Sequence-Specific Oligonucleotide – Polymerase Chain Reaction (SSO- PCR) using the Labtype-SSO kit (One Lambda, CA, USA). The genotyping results showed association with leprosy per se for the SNP rs9277341 in the HLA-DPA1 gene and two markers in the HLA-DPB1 gene (rs1431402 and rs9277469).The protections associations for the multibacilar form the leprosy was found for the markers rs9277341 and rs2301220 in the HLA-DPA1 gene and the marker rs 31350221 in the HLA-DPB1 gene. The rs9277341 with significatif datas after the Bonferroni correction and tested in the São Paulo state showed no association with leprosy. The functional study showed significantly increased IL-10 and TNF levels in G carriers. HLA typing analysis the HLA-DPB1*03 allele was associated with leprosy per se and the HLA-DPB1*02/03 genotype was associated with leprosy per se and the protection with MB form. These data suggest an association of HLA-DPA1 and HLA-DPB1 genes with leprosy.

Orientador: Ana Carla Pereira Latini / Resumo: A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae que pode se manifestar sob diferentes formas clínicas a depender da resposta imune celular do hospedeiro. Vários marcadores genéticos têm sido definidos e, devido à participação dos alelos HLA na resposta imune, estes têm sido amplamente estudados na hanseníase. O HLA-DP constitui uma das moléculas clássicas de classe II, com poucos estudos em hanseníase. O objetivo deste trabalho foi conduzir um estudo de associação genética de base populacional em hanseníase para os genes HLA-DPA1 e HLA-DPB1 com duas amostras caso-controle: a primeira de Rondonópolis-MT, uma região hiperendêmica para hanseníase, e a segunda do Estado de São Paulo, uma região com índices epidemiológicos controlados. Foram realizados estudos com 9 tag SNPs na região dos genes HLA-DPA1 e HLA-DPB1 na população de Rondonópolis–MT (411 casos e 357 controles). O SNP rs9277341 que apresentou dados de associação com significância estatística após a correção de Bonferroni foi então testado na população de São Paulo (570 casos e 380 controles). Para avaliar o efeito funcional deste marcador foi determinada estudoa produção das citocinas IL-10 e TNF após estímulo com antígeno sonicado de M. leprae em 21 controles saudáveis. Na população de Rondonópolis-MT foi realizada a tipificação dos alelos HLA-DPA1* e HLA-DPB1* através da técnica de PCR-SSO empregando-se o kit Labtype-SSO (One Lambda, CA, USA). Os resultados de genotipagem mostraram asso... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, displays different clinical manifestations depending on the immune response of the host. Several genetic markers have been defined and due to the involvement of the HLA alleles in the immune response they were extensively studied in leprosy. However, HLA-DP is a classical class II molecule with few studies on leprosy. The objective of this study was to conduct a population-based genetic association study in leprosy for the HLA-DPA1 and HLA-DPB1 genes with two case-control samples: the first from Rondonópolis - Mato Grosso (MT), a hyperendemic region in leprosy and the second from the state of São Paulo, a region with controlled epidemiological indices. Studies were carried out with 9 Tag Single Nucleotide Polymorphism (SNP) in HLA-DPA and HLA-DPB genes in the population from Rondonópolis – MT (411 cases and 357 controls). The SNP rs9277341 who presented association data with statist significance after the Bonferroni correction was tested in the São Paulo population (570 cases and 380 controls). To evaluate the functional effect of this marker was carried a study of the production of IL-10 and TNF cytokines after stimulation with the sonicated antigen of M. leprae in 21 healthy controls. In the Rondonópolis populations was performed the HLA-DPA1* and HLA-DPB1* allele typing by Sequence-Specific Oligonucleotide – Polymerase Chain Reaction (SSO- PCR) using the Labtype-SSO kit (One Lambda, CA, USA). The genotyp... (Complete abstract click electronic access below) / Doutor