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No. of bitstreams: 1 silveira_cra_me_jabo.pdf: 1335776 bytes, checksum: 2c41b17b49dd121d78806d292d582d58 (MD5) Previous issue date: 2016-06-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do presente estudo foi avaliar o desempenho na produção embrionária in vivo e in vitro de doadoras da raça Holandesa não lactantes, verificar a ocorrência de polimorfismos no gene JY-1 e sua associação com a produção in vivo e in vitro de embriões. Primeiramente, foi verificada a similaridade na produção de embrião da mesma doadora entre as biotécnicas (produção in vivo e in vitro), sendo utilizadas 44 vacas e comparados os desempenhos nas produções de embrião. Na produção in vivo de embriões não foram observados efeitos de ano (P=0,13), estação dentro de ano (P=0,46) e touro dentro de estação e ano (P=0,20). Na produção in vitro, também não foram observados efeitos de ano (P=0,64) e estação dentro de ano (P=0,72), entretanto, o efeito do touro dentro da estação e ano foi significativo (P<0,01). Não há correlação entre as produções in vivo e in vitro dentro de doadora (r=- 0,04; P=0,56). Nenhuma probabilidade de semelhança entre as biotécnicas de produção de embriões, ou seja, se a mesma doadora tem o mesmo desempenho nas duas técnicas (acima ou abaixo da média), foi encontrada (P=0,12). Para verificar a ocorrência de polimorfismos no gene JY-1 e sua associação com a produção embrionária, dados retrospectivos de produções in vivo e in vitro de embriões foram obtidos de 144 vacas não lactantes da raça Holandesa. Para a identificação dos polimorfismos do gene JY-1, as regiões do éxon 2 e íntron 2 foram amplificadas e sequenciadas pelo método de PCR-sequenciamento. Foram detectados quatro SNPs [dois no éxon 2 (rs381676360; rs384600927) e dois no íntron 2 (rs378994837; rs211595914)]. O SNP rs381676360 provocou uma substituição não sinônima de aminoácido (leucina/isoleucina) e o rs384600927, uma substituição sinônima do aminoácido valina. Em relação ao equilíbrio de Hardy-Weinberg, uma das quatro frequências genotípicas não se encontrava em equilíbrio (rs211595914; P<0,01). Considerando o teste de desequilíbrio de ligação (DL), maiores valores de r2 (>0,33) foram observados entre os SNPs rs381676360, rs384600927 e rs378994837 (r2=0,767-0,973) e um SNP (rs211595914) apresentou baixos valores de r2 (r2=0,016-0,025). A associação dos SNPs com as características [número de embriões transferíveis produzidos in vivo (NET); taxa de embriões transferíveis in vivo; (%ET); número de embriões produzidos in vitro (NEV); taxa de clivagem (%CLIV) e taxa de embriões in vitro (%EV)] foi verificada. Nenhum dos SNPs isoladamente foi associado às características estudadas. Porém, quando todos os SNPs foram considerados em conjunto quanto à associação às características, valores significativos foram encontrados para o NET (P<0,01), NEV (P<0,01) e %EV (P<0,01). Desta forma, é possível concluir que o desempenho de produção de doadores de embriões depende da biotécnica utilizada. Ainda, que o gene JY-1 possui polimorfismos do tipo SNP em vacas não lactantes da raça Holandesa e que estes estão associados à produção embrionária. / The aim of this study was to evaluate the performance in in vivo and in vitro embryo production in non-lactating Holstein donors, verify the occurrence of JY-1 gene polymorphisms and their association with the in vivo and in vitro embryo production. First, the similarity in embryo production from the same donor between biotechnologies (in vivo and in vitro production) was verified, being used 44 cows and the embryo production performance was compared. In in vivo embryo production no effects of year (P=0.13), season within year (P=0.46) and sire within the season and year (P=0.20) were found. Regarding in vitro embryo production, no effects of year (P=0.64) and season within year (P=0.72) were found, however, the effect of sire within season and year was significant (P<0.01). There was no correlation concerning embryo production between techniques within donors (r=- 0.04, P=0.56). No probability of similarity between the embryo production techniques, i.e., if the same donor has the same performance in two techniques (above or below of the average), was found (P=0.12). To check the occurrence of JY-1 gene polymorphisms and their association with embryo production, retrospective data from in vivo and in vitro embryo production were obtained from 144 non-lactating Holstein cows. For the identification off JY-1 gene polymorphisms, the region of exon 2 and intron 2 was amplified and sequenced by PCR-sequencing method. Four SNPs were found [two in exon 2 (rs381676360; rs384600927) and two in intron 2 (rs378994837; rs211595914)]. SNP rs381676360 caused a non-synonymous amino acid substitution (leucine/isoleucine) and rs384600927 a synonymous substitution of the valine amino acid. Regarding the Hardy-Weinberg equilibrium, one of the four genotypic frequencies was not in equilibrium (rs211595914; P<0.01). For linkage disequilibrium test (LD) higher r2 values (>0.33) were observed between the SNPs rs381676360, rs384600927 and rs378994837 (r2=0.767 to 0.973) and one SNP (rs211595914) showed lower r2 values (r2=0.016 to 0.025). The association of SNPs with the characteristics [number of in vivo transferable embryos (NET); in vivo transferable embryo rate; (%ET); number of in vitro embryos (NEV); cleavage rate (% CLIV) and in vitro embryo rate (%EV)] was observed. None of the SNPs alone was associated with traits. However, when all SNPs were considered together for the association, significant values were found for NET (P<0.01), NEV (P<0.01) and %EV (P<0.01). Thus, it is possible to conclude that the donor embryo production performance depends on the implemented technique. Still, the JY-1 gene has SNP polymorphisms and they are associated with embryo production in non-lactating Holstein cows. / CNPq: 447172/2014-0 / FAPESP: 2014/13162-1

Magister Scientiae - MSc / Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community. / South Africa

<p>This thesis describes the application of multivariate methods in analyses of genomic DNA sequences, gene expression and protein synthesis, which represent each of the steps in the central dogma of biology. The recent finalisation of large sequencing projects has given us a definable core of genetic data and large-scale methods for the dynamic quantification of gene expression and protein synthesis. However, in order to gain meaningful knowledge from such data, appropriate data analysis methods must be applied.</p><p>The multivariate projection methods, principal component analysis (PCA) and partial least squares projection to latent structures (PLS), were used for clustering and multivariate calibration of data. By combining results from these and other statistical methods with interactive visualisation, valuable information was extracted and further interpreted.</p><p>We analysed genomic sequences by combining multivariate statistics with cytological observations and full genome annotations. All oligomers of di- (16), tri- (64), tetra- (256), penta- (1024) and hexa-mers (4096) of DNA were separately counted and normalised and their distributions in the chromosomes of three Drosophila genomes were studied by using PCA. Using this strategy sequence signatures responsible for the differentiation of chromosomal elements were identified and related to previously defined biological features. We also developed a tool, which has been made publicly available, to interactively analyse single nucleotide polymorphism data and to visualise annotations and linkage disequilibrium.</p><p>PLS was used to investigate the relationships between weather factors and gene expression in field-grown aspen leaves. By interpreting PLS models it was possible to predict if genes were mainly environmentally or developmentally regulated. Based on a PCA model calculated from seasonal gene expression profiles, different phases of the growing season were identified as different clusters. In addition, a publicly available dataset with gene expression values for 7070 genes was analysed by PLS to classify tumour types. All samples in a training set and an external test set were correctly classified. For the interpretation of these results a method was applied to obtain a cut-off value for deciding which genes could be of interest for further studies.</p><p>Potential biomarkers for the efficacy of radiation treatment of brain tumours were identified by combining quantification of protein profiles by SELDI-MS-TOF with multivariate analysis using PCA and PLS. We were also able to differentiate brain tumours from normal brain tissue based on protein profiles, and observed that radiation treatment slows down the development of tumours at a molecular level.</p><p>By applying a multivariate approach for the analysis of biological data information was extracted that would be impossible or very difficult to acquire with traditional methods. The next step in a systems biology approach will be to perform a combined analysis in order to elucidate how the different levels of information are linked together to form a regulatory network.</p>

<p>DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. </p><p>In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. </p><p>To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken.</p><p>The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis.</p><p>The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.</p>

<p>Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We developed a novel multiplexed method for SNP genotyping based on four-color fluorophore tag-microarray minisequencing. This method allows simultaneous genotyping of 80 samples and up to 200 SNPs in any allele combination. In study I we set up the method for a panel of SNPs from genes in the type I interferon system, and applied it in study III. In study II we used the technique to genotype SNPs from the coding region of the mitochondrial genome. A panel of 150 SNPs was genotyped in 265 individuals representing nine different populations. We demonstrated that the multiplexed SNP genotyping method for mitochondrial DNA increases the power of forensic identification in combination with sequencing of the hypervariable region of mitochondrial DNA. </p><p>In study III we performed a genetic association study of SNPs in genes related to the type I Interferon system in Systemic Lupus Erythematosus (SLE). SLE is a chronic autoimmune inflammatory disease with a complex etiology. The SNPs were genotyped in DNA samples from Swedish, Finnish, and Icelandic patients with SLE, unaffected family members, and unrelated controls. The analysis identified SNPs in two genes, the tyrosine kinase 2 (TYK2) and interferon regulatory factor 5 (IRF5) genes that are highly associated with SLE with p-values <10^-7 for joint linkage and association. </p><p>Study IV describes the analysis of the TYK2 and IRF5 SNPs in a large Rheumatoid Arthritis (RA) sample cohort. We found that SNPs in the IRF5 gene were significantly associated with RA with a p-value = 0.00008. In contrast, we did not detect an association with SNPs in the TYK2 gene. These findings demonstrate that SLE and RA may have a common genetic background in the case of IRF5, while the TYK2 variants appear to be unique for SLE. </p>

<p>Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA.</p><p>In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products.</p><p>Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees.</p><p>In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.</p>

Glucocorticoids (GCs) have been widely used as a therapeutic agent for diverse inflammatory ocular diseases. However, a high percentage of patients undergoing this treatment develop high intraocular pressure (IOP), which if left unsupervised may lead to glaucoma. It is believed that the IOP elevation in response to GC treatment has a genetic determinant. In order to test this hypothesis, we analyzed in 52 patients the presence of single nucleotide polymorphisms (SNPs) in the glucocorticoid receptor gene (GR), the principal mediator of GCs uptake by the cells. We studied six GR SNPs previously reported to be associated with sensitivity and resistance to GCs: GluArg22/23GluLys (codon 22-23), Asn363Ser (codon 363), IVS2+646C>G (intron 2/BclI), IVS3-46G>C (intron 3), IVS4-16G>T (intron 4), Asn766Asn (Codon 766). Nevertheless, the results of this preliminary study did not show any specific correlation between SNPs in the GR gene and IOP elevation. Therefore, we proceeded to perform a whole genome SNP screen with the DNA samples of these patients to search for possible target genes responsible for the elevated IOP after GC treatment. As a result, we identified forty-eight SNPs in thirty-three genes that correlate with the high IOP response. The gene showing the strongest association is a poorly known G-protein coupled receptor. In addition, four SNPs hit a single transporter gene. Other candidate genes identified are a translation elongation factor, an F-box protein, an oxysterol binding protein, and a solute carrier family gene. These results support our hypothesis that IOP elevation following GC treatment is a genetically determined response. GCs are a common treatment for innumerable medical conditions; we believe that a genetic association between GC treatment and its physiological response may be important for improving treatment management and drug development for retinal diseases as well as for other medical ailments. However, further studies need to be performed to analyze in depth the association between the candidate genes identified in this study and the steroid response.

<p>Many Single Nucleotide Polymorphisms (SNPs) found in coding or regulatory regions within the human genome lead to phenotypic differences that make prediction of physical appearance, based on genetic analysis, potentially useful in forensic investigations. Complex traits such as pigmentation can be predicted from the genome sequence, provided that genes with strong effects on the trait exist and are known. Phenotypic traits may also be associated with variations in gene expression due to the presence of SNPs in promoter regions. In this project, the identification of genes associated with these physical traits of potential forensic relevance have been collated from the literature using a text mining platform and hand curation. The SNPs associated with these genes have been acquired from public SNP repositories such as the International HapMap project, dbSNP and Ensembl. Characterization of different population groups based on the SNPs has been performed and the results and data stored in a MySQL database. This database contains SNP genotyping data with respect to physical phenotypic differences of forensic interest. The potential forensicrelevance of the SNP information contained in this database has been verified through in silico SNP analysis aimed at establishing possible relationships between SNP occurrence and phenotype. The software used for this analysis is MATCH&trade / .</p>

DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken. The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis. The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We developed a novel multiplexed method for SNP genotyping based on four-color fluorophore tag-microarray minisequencing. This method allows simultaneous genotyping of 80 samples and up to 200 SNPs in any allele combination. In study I we set up the method for a panel of SNPs from genes in the type I interferon system, and applied it in study III. In study II we used the technique to genotype SNPs from the coding region of the mitochondrial genome. A panel of 150 SNPs was genotyped in 265 individuals representing nine different populations. We demonstrated that the multiplexed SNP genotyping method for mitochondrial DNA increases the power of forensic identification in combination with sequencing of the hypervariable region of mitochondrial DNA. In study III we performed a genetic association study of SNPs in genes related to the type I Interferon system in Systemic Lupus Erythematosus (SLE). SLE is a chronic autoimmune inflammatory disease with a complex etiology. The SNPs were genotyped in DNA samples from Swedish, Finnish, and Icelandic patients with SLE, unaffected family members, and unrelated controls. The analysis identified SNPs in two genes, the tyrosine kinase 2 (TYK2) and interferon regulatory factor 5 (IRF5) genes that are highly associated with SLE with p-values &lt;10-7 for joint linkage and association. Study IV describes the analysis of the TYK2 and IRF5 SNPs in a large Rheumatoid Arthritis (RA) sample cohort. We found that SNPs in the IRF5 gene were significantly associated with RA with a p-value = 0.00008. In contrast, we did not detect an association with SNPs in the TYK2 gene. These findings demonstrate that SLE and RA may have a common genetic background in the case of IRF5, while the TYK2 variants appear to be unique for SLE.