Dissertations / Theses on the topic 'Molecular genetics – Technique'

The classical approach to gene discovery has been to culture micro-organisms demonstrating a specific enzyme activity and then to recover the gene of interest through shotgun cloning. The realization that these standard microbiological methods provide limited access to the true microbial biodiversity and therefore the available microbial genetic diversity (collectively termed the Metagenome) has resulted in the development of environmental nucleic acid extraction technologies designed to access this wealth of genetic information, thereby avoiding the limitations of culture dependent genetic exploitation. In this work several gene discovery technologies was employed in an attempt to recover novel bacterial laccase genes (EC 1.10.3.2), a group of enzymes in which considerable biotechnological interest has been expressed. Metagenomic DNA extracted from two organic rich environmental samples was used as the source material for the construction of two genomic DNA libraries. The small insert plasmid based library derived from compost DNA consisted of approximately 106 clones at an average insert size of 2.7Kbp, equivalent to 2.6 Gbp of cloned environmental DNA. A Fosmid based large insert library derived from grape waste DNA consisted of approximately 44000 cfu at an average insert size of 25Kbp (1.1 Gbp cloned DNA). Both libraries were screened for laccase activity but failed to produce novel laccase genes. As an alternative approach, a multicopper oxidase specific PCR detection assay was developed using a laccase positive Streptomyces strain as a model organism. The newly designed primers were used to detect the presence of bacterial multicopper oxidases in environmental samples. This resulted in the identification of nine novel gene fragments showing identity ranging from 37 to 94% to published putative bacterial multicopper oxidase gene sequences. Three clones pMCO6, pMCO8 and pMCO9 were significantly smaller than those typically reported for bacterial laccases and were assigned to a recently described clade of Streptomyces bacterial multicopper oxidases.

Two PCR based techniques were employed to attempt the recovery of flanking regions for two of these genes (pMCO7 and pMCO8). The use of TAIL-PCR resulted in the recovery of 90% of the pMCO7 ORF. As an alternative approach the Vectorette&trade
system was employed to recover the 3&rsquo
downstream region of pMCO8. The complexity of the DNA sample proved to be a considerable technical challenge for the implementation of both these techniques. The feasibility of both these approaches were however demonstrated in principle. Finally, in an attempt to expedite the recovery of fulllength copies of these genes a subtractive hybridization magnetic bead capture technique was adapted and employed to recover a full &ndash
length putative multicopper oxidase gene from a Streptomyces strain in a proof of concept experiment. The StrepA06pMCO gene fragment was used as a &lsquo
driver&rsquo
against fragmented Streptomyces genomic DNA (&lsquo
tester&rsquo
) and resulted in the recovery of a 1215 bp open reading frame. Unexpectedly, this ORF showed only 80% identity to the StrepA06pMCO gene sequence at nucleotide level, and 48% amino acid identity to a putative mco gene derived from a Norcardioides sp JS614.

For organisms undergoing a developmental process it is ideal that specific genes are induced and repressed at the correct time and to the correct level in a coordinated manner. The process of meiosis and spore formation (collectively known as sporulation) in Saccharomyces cerevisiae provides a convenient system to elucidate transcriptional mechanisms of gene repression and the contribution such repression mechanisms offer to cells capable of undergoing a developmental process. This thesis focuses on transcriptional repression of sporulation-specific genes during both vegetative/mitotic conditions and sporulation. The fitness contribution of transcriptional repressors that regulate sporulationspecific genes during vegetative growth were investigated considering the similarities between meiosis and mitosis such as DNA replication, chromosome segregation and cytokinesis. Well-characterised sporulation genes of different functions were expressed in vegetative cells and ectopic expression of these genes was found not to be lethal. It was ascertained through strain competition studies that ectopic expression of the genes IME1, SMK1, SPR3 and DIT1 during mitotic growth did not affect cellular fitness. The expression of NDT80 in vegetative cells, however, caused a marked reduction in fitness and cells were also further compromised in the absence of the Sum1p repressor that regulates NDT80 transcription. The role of NDT80 as a transcriptional activator of middle sporulation genes, rather than the over-expression of NDT80 as a protein, caused the reduction of cell viability. Transcriptional regulation of the middle sporulation-specific gene SPR3 by the meiosis-specific Set3p repressor complex was investigated using synchronous sporulation cultures of the W303a/?? strain commonly used for sporulation studies. In a mutant W303a/?? ??set3/??set3 strain, lacking a key component of the Set3p repression complex, the transcription of SPR3 was uncharacteristically expressed at higher levels and derepressed during late sporulation. This SPR3 expression was consistent for both SPR3 transcript and SPR3::lacZ reporter protein studies. This preliminary work will enable future studies, using SPR3 promoter deletions fused to a lacZ reporter, aimed at determining the region of the SPR3 promoter that the Set3p complex may interact with to transcriptionally repress the gene during sporulation.

In this study different molecular techniques are contrasted (RAPD's, allozyme, sequencing mtDNA, sequencing ribosomal spacers) and appropriate analytical methods (allelic and infinite-sites approaches; inbreeding and coalescent models) used for estimating population genetic parameters in parasites. A range of population genetic questions at different scales were chosen to emphasise the importance of tailoring techniques and analytical methods to the particular question being investigated. The realisation that each question formulated has a particular scale means the appropriate technique and markers must be useful at that scale to attempt to answer the question. The useful scale of a technique depends several factors including the region of DNA examined, the density of sampling of the technique, and the mode of evolution of the markers. Each technique will produce a useful range of variability. Below the lower limit there is no variation, above the upper limit the variation is too high to produce useful comparisons. Parasites are of interest for many reasons, primarily because they can cause disease and thus impact on their host's population dynamics. They are often closely associated with their hosts and may undergo co-evolution, as well as causing an ongoing immunological "arms race" with their hosts. The parasitic mode of live is found throughout nearly all taxonomic groupings and thus classical models of population genetics based on sexual, diploid vertebrates do not fit well with the entire diversity of parasite groups. Genetic diversity within and among populations of Echinococcus granulosus was examined contrasting a RAPD dataset with an allozyme dataset. Two models of variation in Echinococcus have been proposed, those of Smyth and Rausch, and the expected genetic structure from each was compared to the observed genetic structure. The premise of Smyth’s model, predominant self-fertilisation, was supported, but the resultant pattern of genetic variation followed Rausch’s model. RAPD data, being dominant, present challenges to analysis. An approach to overcome this dominance problem and allow standard allelic frequency analysis is described using the selfing rate estimated from allozyme data. The RAPD data were also analysed using both band-sharing and nucleotide diversity approaches. A population genetic study of Ostertagia ostertagi in the USA was extended to two different scales: within an Australian state and between the USA and Australian continents. Three alternative explanations for the observed discrepancy between genetic structure and differentiation in an important biological trait, hypobiosis, were explored. A number of programs and analyses were compared including coalescent geneflow estimates. Variation among multiple copies of two spacer regions of rDNA was examined within individuals of Ostertagia ostertagi. Both the intergenic spacer and internal transcribed spacer 1 regions were found to include repeat regions, with different numbers of repeats creating length differences in clones from the same worm. Multi-copy genes present extra challenges in analysis to ensure that only homologous copies are being compared. Many studies fail to look for variation within populations or within individuals. The two major conclusions from these examples are that: 1). The study of variation necessarily involves an implicit scale, and markers must be chosen that are appropriate to the question being explored. 2). Using several methods of analysis of genetic data allows contrasts to be made, and if different methods produce similar results gives much more confidence in the conclusions drawn. Incongruence in results leads to new questions and reexamination of the assumptions of each analysis.

Evolution is the single cohesive logical framework in which all biological processes may exist simultaneously. Incremental changes in phenotype over imperceptibly large timescales have given rise to the enormous diversity of life we witness on earth both presently and through the natural record. The basic unit of evolution is mutation, and by perturbing biological processes, mutations may alter the fitness of an individual. However, the fitness effect of a mutation is difficult to infer from historical record, and complex to obtain experimentally in an efficient and accurate manner. We have recently developed a high throughput method to iteratively mutagenize regions of essential genes in yeast and subsequently analyze individual mutant fitness termed Exceedingly Methodical and Parallel Investigation of Randomized Individual Codons (EMPIRIC). Utilizing this technique as exemplified in Chapters II and III, it is possible to determine the fitness effects of all possible point mutations in parallel through growth competition followed by a high throughput sequencing readout. We have employed this technique to determine the distribution of fitness effects in a nine amino acid region of the Hsp90 gene of S. cerevisiae under elevated temperature, and found the bimodal distribution of fitness effects to be remarkably consistent with near-neutral theory. Comparing the measured fitness effects of mutants to the natural record, phylogenetic alignments appear to be a poor predictor of experimental fitness. In Chapter IV, to further interrogate the properties of this region, library competition under conditions of elevated temperature and salinity were performed to study the potential of protein adaptation. Strikingly, whereas both optimal and elevated temperatures produced no statistically significant beneficial mutations, under conditions of elevated salinity, adaptive mutations appear with fitness advantages up to 8% greater than wild type. Of particular interest, mutations conferring fitness benefits under conditions of elevated salinity almost always experience a fitness defect in other experimental conditions, indicating these mutations are environmentally specialized. Applying the experimental fitness measurements to long standing theoretical predictions of adaptation, our results are remarkably consistent with Fisher’s Geometric Model of protein evolution. Epistasis between mutations can have profound effects on evolutionary trajectories. Although the importance of epistasis has been realized since the early 1900s, the interdependence of mutations is difficult to study in vivo due to the stochastic and constant nature of background mutations. In Chapter V, utilizing the EMPIRIC methodology allows us to study the distribution of fitness effects in the context of mutant genetic backgrounds with minimal influence from unintended background mutations. By analyzing intragenic epistatic interactions, we uncovered a complex interplay between solvent shielded structural residues and solvent exposed hydrophobic surface in the amino acid 582-590 region of Hsp90. Additionally, negative epistasis appears to be negatively correlated with mutational promiscuity while additive interactions are positively correlated, indicating potential avenues for proteins to navigate fitness ‘valleys’. In summary, the work presented in this dissertation is focused on applying experimental context to the theory-rich field of evolutionary biology. The development and implementation of a novel methodology for the rapid and accurate assessment of organismal fitness has allowed us to address some of the most basic processes of evolution including adaptation and protein expression level. Through the work presented here and by investigators across the world, the application of experimental data to evolutionary theory has the potential to improve drug design and human health in general, as well as allow for predictive medicine in the coming era of personalized medicine.

Evolution is the single cohesive logical framework in which all biological processes may exist simultaneously. Incremental changes in phenotype over imperceptibly large timescales have given rise to the enormous diversity of life we witness on earth both presently and through the natural record. The basic unit of evolution is mutation, and by perturbing biological processes, mutations may alter the fitness of an individual. However, the fitness effect of a mutation is difficult to infer from historical record, and complex to obtain experimentally in an efficient and accurate manner. We have recently developed a high throughput method to iteratively mutagenize regions of essential genes in yeast and subsequently analyze individual mutant fitness termed Exceedingly Methodical and Parallel Investigation of Randomized Individual Codons (EMPIRIC). Utilizing this technique as exemplified in Chapters II and III, it is possible to determine the fitness effects of all possible point mutations in parallel through growth competition followed by a high throughput sequencing readout. We have employed this technique to determine the distribution of fitness effects in a nine amino acid region of the Hsp90 gene of S. cerevisiae under elevated temperature, and found the bimodal distribution of fitness effects to be remarkably consistent with near-neutral theory. Comparing the measured fitness effects of mutants to the natural record, phylogenetic alignments appear to be a poor predictor of experimental fitness. In Chapter IV, to further interrogate the properties of this region, library competition under conditions of elevated temperature and salinity were performed to study the potential of protein adaptation. Strikingly, whereas both optimal and elevated temperatures produced no statistically significant beneficial mutations, under conditions of elevated salinity, adaptive mutations appear with fitness advantages up to 8% greater than wild type. Of particular interest, mutations conferring fitness benefits under conditions of elevated salinity almost always experience a fitness defect in other experimental conditions, indicating these mutations are environmentally specialized. Applying the experimental fitness measurements to long standing theoretical predictions of adaptation, our results are remarkably consistent with Fisher’s Geometric Model of protein evolution. Epistasis between mutations can have profound effects on evolutionary trajectories. Although the importance of epistasis has been realized since the early 1900s, the interdependence of mutations is difficult to study in vivo due to the stochastic and constant nature of background mutations. In Chapter V, utilizing the EMPIRIC methodology allows us to study the distribution of fitness effects in the context of mutant genetic backgrounds with minimal influence from unintended background mutations. By analyzing intragenic epistatic interactions, we uncovered a complex interplay between solvent shielded structural residues and solvent exposed hydrophobic surface in the amino acid 582-590 region of Hsp90. Additionally, negative epistasis appears to be negatively correlated with mutational promiscuity while additive interactions are positively correlated, indicating potential avenues for proteins to navigate fitness ‘valleys’. In summary, the work presented in this dissertation is focused on applying experimental context to the theory-rich field of evolutionary biology. The development and implementation of a novel methodology for the rapid and accurate assessment of organismal fitness has allowed us to address some of the most basic processes of evolution including adaptation and protein expression level. Through the work presented here and by investigators across the world, the application of experimental data to evolutionary theory has the potential to improve drug design and human health in general, as well as allow for predictive medicine in the coming era of personalized medicine.

The hermaphroditic fish, Rivulus marmoratus, is the only vertebrate known to reproduce by internal self-fertilization; this process results in populations of homozygous clones. Most natural populations consist entirely of hermaphrodites, but phenotypically distinct, fertile males occur at frequencies up to 24% on some islands off the coast of Belize. The presence of large numbers of males in natural populations prompted this study to determine if males are involved in the mating system. The occurrence of cross-fertilization between males and hermaphrodites was determined by surveying progeny of field-caught hermaphrodites for non-segregation or segregation of DNA fingerprint markers as an indication of the homozygosity or heterozygosity of the parent.

DNA fingerprinting revealed no segregation of markers among the offspring in 12 of 12 Florida and Brazil laboratory lines and in 5 of 30 Belize Cay broods. These data indicate that the hermaphrodite parents were homozygous; thus, no detectable outcrossing has occurred in these populations. However, DNA fingerprinting revealed segregation of markers among the offspring in 25 of 30 Belize Cay broods. Twenty-four of these broods were from the island of Twin Cays. An average of 30% of the parental bands were segregating among the offspring; values ranged from 0.09 to 0.50. Offspring were, on average, 8% dissimilar to one another; values ranged from 2.08% to 15.09%. These data suggest that the 25 hermaphrodite parents were heterozygous; thus, males are involved in the mating system in some Belize Cay populations. These data are the first evidence of outcrossing in this species.

Master of Science

Due to the nature of the marine environment genetic studies allow insight into behaviour and natural history that is difficult or impossible to identify by direct field observation. Current as well as historical population demography and gene flow can be detected by using molecular techniques. Genetic studies on only a few commercially important marine species along the South African coast have been conducted, although many marine fish species utilize estuaries as nursery areas and little attention has been afforded to studying larval distribution and recruitment of these species from a molecular point of view. Many of these estuarine associated species, especially in the South African milieu, are important for recreational and subsistence use. Associated with southern African estuaries are 13 species of the family Sparidae of which Cape stumpnose Rhabdosargus holubi is the most abundant. Juveniles are mostly confined to estuaries while the adults are strictly marine. Rhabdosargus holubi are serial spawners but temporally separated spawning peaks have been recorded along the South African coastline. Within the first part of this dissertation, the general characteristics of marine fish populations and the marine environment along the South African coast are being discussed. The main aim of this study was to determine the population genetic structure from estimates of nuclear and mitochondrial genetic variation across the distributional range of Rhabdosargus holubi. Samples were collected from 13 geographic localities along the South African coastline from St Lucia in the northeast to Klein River in the southwest. Juveniles were sampled in estuaries and adults were collected in the marine intertidal zone. Mitochondrial DNA control region fragments of 368 bp in length were obtained from a total of 214 individuals from all sampling localities. A total of 36 alleles were identified from 34 polymorphic sites. Following an allele homogeneity test, samples from different localities were lumped to represent six distinct geographical regions. Mitochondrial DNA control region analyses of juveniles showed high haplotype diversity and low nucleotide diversity with no divergent maternal lineages. No pattern between haplotype genealogy and geographic locality was evident. Population genetic analyses using heterologous microsatellite amplification have been successfully completed for a number of studies, including numerous studies of variation within marine fish species. Microsatellite studies have proven to be more sensitive in detecting subtle population structure than mtDNA and/or protein polymorphisms in high gene flow species. A total of 113 microsatellite loci previously isolated from phylogenetically closely related marine fish species were tested for amplification. The success rate of heterologous microsatellite amplification was extremely low (0.02%), with only two polymorphic loci amplifying consistently for analysing 133 individuals sampled from six localities along the distributional range of R. holubi. Results from these two loci were insufficient to draw conclusions about the population genetic structure of R. holubi along the South African coast. Possible reasons for the low rate of amplification success and future research recommendations are discussed. The findings from this study suggest that R. holubi is not geographically restricted, has high gene flow among localities and likely exist as a single stock.
Dissertation (MSc (Genetics))--University of Pretoria, 2006.
Genetics
unrestricted

The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis. First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased. For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing.  Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites. To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.

Diatoms associated with foraminifers of the genus Amphistegina were assessed using a combination of morphological and molecular techniques. These included: 1) microscopic identification of diatoms cultured from the host, 2) sequencing of portions of the small subunit of the ribosomal RNA gene (18S) and the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase [i.e., RubisCO] gene (rbcL) from DNA extracted directly from the Amphistegina hosts and also from diatoms cultured from these hosts, and 3) denaturing gradient gel electrophoresis (DGGE) profiles of rbcL and internal transcribed spacer 1 (ITS1) PCR amplicons from DNA extracted directly from hosts and from cultures. Consistent with previous culture studies, multiple species of pennate diatoms of the genera Nitzschia, Fragilaria (including Nanofrustulum), Amphora, and Navicula, were cultured from >900 host specimens collected from >20 sites in the western Atlantic and four sites in the Pacific. Diatoms of the genus Nitzschia grew in about half of all successful cultures. The genetic identities of selected cultures were consistent with those based on morphological taxonomy. Diatom sequences from DNA extracted directly from the cytoplasm of the Amphistegina hosts were species specific and distinct from sequences obtained from cultured diatoms and from sequences in GenBank of diatom taxa previously reported as endosymbionts. Multiple phylogenetic analyses revealed that the 18S and rbcL diatom sequences from specimens of A. gibbosa collected from the Atlantic sites and of Amphistegina spp. from Hawai’i were most similar to the 18S and rbcL sequences of an unnamed Fragilariaceae diatom in GenBank (Accession # JX413542.1 for 18S and JX413559.1 for rbcL) and other closely related diatoms in that family. Of diatom taxa previously reported as endosymbionts of larger foraminifers, Nanofrustulum shiloi was the most similar, but not identical, to the sequences from hosts collected from the Atlantic and Hawai’i. The 18S and rbcL diatom sequences from the Atlantic host species, A. gibbosa, were all nearly identical, but small intra-species differences (subclades) were observed from specimens collected from the deepest (75 m) site in the Florida Keys and also from the eastern-most site, Young Island near St. Vincent. The 18S and rbcL diatom sequences from the two host species from Hawai’i, A. lobifera and A. lessonii, were more variable but still within the family Fragilariaceae. The diatom sequences from A. radiata collected from two sites in Papua New Guinea (PNG) were most similar to diatoms of the family Plagiogrammaceae and therefore distinct from sequences obtained from other Amphistegina species in this study, as well as from all diatoms previously reported as endosymbionts. A small difference was observed between the diatom sequences from host specimens collected from a Pacific site as compared to a Bismarck Sea site. The ITS1 DGGE profiles of DNA extracted directly from A. gibbosa specimens at different depths, locations, and seasons in the western Atlantic were nearly identical. Differences were seen between rbcL DGGE profiles of DNA extracted directly from the different Amphistegina host species. The rbcL DGGE profiles directly from all hosts were clearly different from those extracted from diatoms cultured from the same host specimens, as well as from Nitzschia laevis, a commonly reported diatom endosymbiont in past culture-based studies. My findings are consistent with ultrastructural studies of endosymbionts of Amphistegina published in the early 1980s and congruent with recent molecular studies of endosymbionts in other diatom-bearing foraminifers, all of which indicate specificity. Nevertheless, the consistency with which several diatom taxa have been reported in culture studies from all oceans indicates the possibility of some relationship with Amphistegina spp., either as important food items, epiphytes, or minor opportunistic symbionts that can thrive in culture media.

The ability of a genetically-engineered Erwirzia carotovora subsp. carotovora (Ecc) strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or Pseudomonas fluorescens was tested on filters, within soil microcosms, and in planta. Ecc was engineered by chromosomal insertion of a disarmed endo-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and pBR322 were introduced separately into engineered Ecc clones. These strains served as donors in genetic transfer experiments. No transfer of the inserted kan marker or of pBR322 was observed under any experimental condition. In filter matings, RPI was transferred to wildtype Ecc at a frequency of 3.6 X 10^-2 transconjugants per donor (TPD) and to P. fluorescens at a frequency of 2.4 X 10^-5 TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10^-4 and 2.0 X 10^-4, while matings on potato slices yielded frequencies of 4.7 X 10^-2 and 2.3 X 10^-2. In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10^-3 and 8.4 X 10^-5 TPD.

Master of Science

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FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO
Introduction. The androgen insensitivity syndrome (AIS) is a rare disease (1:20,000 to 1:64.000)-linked X chromosome, which generates a disorder of sexual differentiation of the male fetus (XY) with a spectrum of phenotypes ranging from females complete (CAIS) to a male phenotype with discrete signs of androgen insensitivity. An increasing number of mutations have been cataloged and nearly 500 mutations have been related to CAIS and 1000 to the androgen receptor gene. The AR gene is located on Xq11-12, with eight exons, about 919 amino acids. Objective. To characterize the mutations in the AR gene families in the region of the "Bico do Papagaio" in the southwestern state of Maranhao. Methodology. We used molecular biology techniques such as DNA extraction, PCR, electrophoresis, purification of PCR products and sequencing. In addition, we analyzed the clinical and hormonal characteristics of 14 patients and their families. Results. In one family (with two twin affected), we found the mutation R753X and the third molecular diagnosis of CAIS in twins described in the World. In another family, with 12 patients, was identified a new mutation in exon 8, as described P893A, protein AR. Conclusion. This work enabled the application of molecular techniques for the accurate diagnosis of AIS, genetic counseling for relatives of affected patients, and contribute to the formation of more qualified human resources, aiming at the development of biotechnology in the state of Maranhão.
Introdução. A Síndrome de Insensibilidade Androgênica (AIS) é uma doença rara (1:20.000 a 1:64.000), de transmissão ligada ao cromossomo X, que gera um distúrbio da diferenciação sexual do feto masculino (XY) com um espectro de fenótipo que varia desde o feminino completo (CAIS) até um fenótipo masculino com discretos sinais de insensibilidade androgênica. Um número crescente de mutações tem sido catalogadas e quase 500 mutações já foram relacionadas à CAIS e cerca de 1000 ao gene do receptor androgênico. O gene AR localiza-se em Xq11-12, com 8 exons, com cerca de 919 aminoácidos. Objetivo. Caracterizar as mutações no gene AR em famílias da região do Bico do Papagaio , no sudoeste do Estado do Maranhão. Metodologia. Foram utilizadas técnicas de biologia molecular como extração de DNA, PCR, Eletroforese, Purificação de produtos de PCR e Sequenciamento automático. Além disso, foram analisados o quadro clínico e hormonal de 14 pacientes e de seus familiares. Resultados. Em uma das famílias (com duas gêmeas afetadas), foi encontrada a mutação R753X, sendo o terceiro diagnóstico molecular de CAIS em gêmeas descrito no Mundo. Em outra família, com 12 pacientes, foi a identificada uma mutação nova no exon 8, descrita como P893A, na proteína AR. Conclusão. Este trabalho possibilitou a aplicação de técnicas moleculares para o diagnóstico preciso de AIS, aconselhamento genético aos familiares das pacientes afetadas, além de contribuir para a formação de recursos humanos mais qualificados, visando o desenvolvimento da biotecnologia no Estado do Maranhão.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The tall coconut palm is about 70% of the coconut farm in Brazil. Nonetheless the information about the genetic variability existing in Brazilian populations and their genetic relationships are still incipient. Microsatellite markers or SSR (Simple Sequence Repeats) and morphological markers are the techniques most suitable for studies of genetic diversity. Thus, knowledge of the variability and genetic structure in the giant coconut palm, it is necessary to direct the activities of conservation and use of germplasm in breeding programs of this species. The objectives of this study were: 1) to analyze the distribution of the genetic variability of the original population of Tall-Brazil-Praia-Forte ( GBrPF - PO), located on the north coast of Bahia, and four coming accesses this population; 2) levels of diversity and genetic relationships between two accesses tall coconut palm collected in Brazil and introduced seven accessions of different geographical regions, conserved in Banco Internacional de Coco for Latin America and the Caribbean (ICG - LAC). The accessions were analyzed using 25 SSR primers specific morphological descriptors and 16 of the list of IPGRI, 1995. Accesses tall- Brazil-Praia-Forte (GBrPF) are conserved in physical bases in Ceará (GBrPF-CE), Pará (GBrPF-PA ) and ICG - LAC, the latter two physical bases in Sergipe: one in the experimental field of the Betume in the city of Neópolis (GBrPF-CEB) and the other in the experimental field Itaporanga in the municipality of Itaporanga d'Ajuda (GBrPF-CEI) . The other accesses greens: tall-do-Brazil-Merepe (GBrMe), collected in the coastal Northeast, tall-Malaysia (GML), tall-Vanuatu (GVT), tall-West African (GOA), tall-Polynesia (GPY), tall-Rennel (GRL), tall-Tonga (GTG) and tall-Rotuma (GRT) introduced in different geographic regions of the world, too are conserved in the ICG - LAC in the experimental field of the Betume. Three studies from this research project will be presented. In the first study, we found 18 polymorphic primers, 91 alelos, with a mean of 5.05 alleles/locus. Genotypic indices indicate greater genetic variability of access GBrPF-PA, GBrPF-CE and GBrPF-CEB, the analysis of gene structure identified an allele sharing and access of the population, suggesting that accesses listed represent the genetic structure of the original population. The grouping (UPGMA) showed the formation of 14 groups, with the GBrPF-CEB and GBrPF-PA showed greater similarity to the original population accesses. In the second study, for the study of genetic relationships among accessions of tall coconut palm, 19 primers were polymorphic, detecting 125 alleles, with an average of 6.57 alleles/locus. Genotypic indices indicate greater genetic variability among accessions of coconut - derived giant introduced the Pacific region. The analysis of gene structure led to the formation of five groups and accessions collected in Brazil showed genetic relationship with the African access and the emergence of ecotypes giant coconut palm in Brazil. Cluster analysis by the Nearest Neighbor method formed two main groups. In group I, the accessions were grouped into three subgroups: Ia (GTG, GRT and GPY), Ib (GRL and GVT) and Ic (GML). In group II, the accessions were separated into two subgroups: IIa (GOA) and IIb (GBrMe, GBrPF),indicating that the genetic relationships of the accessions are based on ecogeographic regions. In the third work, the study of genetic diversity through morphological markers using techniques of univariate and multivariate genetic variability was observed among genotypes. The results of principal component analysis, obtained from 16 morphological characters shows that three components were needed, that the variance explained by them reached a minimum of 80% and the selection of six characters with the highest contribution to the study of diversity. UPGMA was formed by five groups. Group I meets the GVT and GML access; group II with GPY, GTG and GBrPF; group III and IV each with one access, GRT and GOA, respectively, while group V with GBrMe and GRL. Groups showed an inconsistency with respect to the origins of the accessions, probably due to the quantitative nature of those characteristics that are controlled by many genes, being affected by environmental factors. Diversity and genetic structure evaluations demonstrate the variability and genetic relations in giant coconut palm. These results will guide decisions about the activities of conservation and use of coconut germplasm in the country
O coqueiro-gigante representa cerca de 70% da exploração do coqueiro no Brasil. Apesar disso, as informações sobre a variabilidade genética existente nas populações brasileiras e suas relações genéticas ainda são incipientes. Os marcadores microssatélites ou SSR (Simple Sequence Repeats), e os marcadores morfológicos, são as técnicas mais indicadas para os estudos de diversidade genética. Assim, o conhecimento da variabilidade e da estruturação genética em coqueiro-gigante, torna-se necessário para direcionar as atividades de conservação e utilização do germoplasma nos programas de melhoramento da espécie. Os objetivos do presente estudo foram: 1) analisar a distribuição da variabilidade genética da população original de gigante-do-Brasil-da-Praia-do-Forte (GBrPF-PO), localizada do litoral norte do Estado da Bahia, e de quatro acessos procedentes dessa população; 2) os níveis de diversidade e as relações genéticas entre dois acessos de coqueiro-gigante coletados no Brasil e sete acessos introduzidos de diferentes regiões geográficas do mundo, conservados no Banco Internacional de Coco para a América Latina e Caribe (ICG- LAC).Os acessos foram analisados por meio de 25 primers SSR específicos e 16 descritores morfoagronômicos da lista do IPGRI, 1995.Os acessos de gigante-do-Brasil-da-Praia-Forte (GBrPF) são conservados em bases físicas no Ceará (GBrPF-CE), Pará (GBrPF-PA) e no ICG-LAC, este último em duas bases físicas em Sergipe: uma no campo experimental do Betume, no município de Neopólis (GBrPF-CEB) e a outra no campo experimental de Itaporanga, no município de Itaporanga d’Ajuda (GBrPF-CEI). Os demais acessos de coqueiro: gigante-do-Brasil-de-Merepe (GBrMe), coletado no litoral do Nordeste do país, gigante-da-Malásia (GML), gigante-de-Vanuatu (GVT), gigante-do-Oeste-Africano (GOA), gigante-da-Polinésia (GPY), gigante-de-Rennel (GRL), gigante-de-Tonga (GTG) e gigante-de-Rotuma (GRT) introduzidos de diferentes regiões geográficas do mundo, também estão conservados no ICG-LAC no campo experimental do Betume. Três trabalhos oriundos deste projeto de pesquisa serão apresentados. No primeiro trabalho, constatou-se 18 primers polimórficos, 91alelos, com media de 5,05 alelos/loco. Os índices genotípicos indicam maior variabilidade genética dos acessos GBrPF-PA, GBrPF-CE e GBrPF-CEB, a análise da estrutura gênica identificou um compartilhamento de alelos da população e dos acessos, sugerindo que os acessos coletados, representam a estruturação genética da população original. O agrupamento (UPGMA), evidenciou a formação de 14 grupos, tendo os acessos GBrPF-CEB e GBrPF-PA mostrado maior similaridade com a população original. No segundo trabalho, para o estudo das relações genéticas entre acessos de coqueiro-gigante, 19 primers foram polimórficos, detectando 125 alelos, com média de 6,57 alelos/loco. Os índices genotípicos indicam uma maior variabilidade genética entre os acessos de coqueiros-gigantes introduzidos oriundos da região do Pacífico. A análise da estrutura gênica levou a formação de cinco grupos e os acessos coletados no Brasil apresentaram relação genética com o acesso Africano e o surgimento de ecótipos de coqueiro-gigante no Brasil. A análise de agrupamento pelo método do Vizinho mais Próximo formou dois grupos principais. No grupo I, os acessos foram agrupados em três subgrupos: Ia (GTG, GRT e GPY), Ib (GRL e GVT) e Ic (GML). No grupo II, os acessos foram separados em dois subgrupos : IIa (GOA) e IIb (GBrMe, GBrPF). Indicando que as relações genéticas dos acessos são fundamentadas nas regiões ecogeográficas. O terceiro trabalho,o estudo da diversidade genética, por meio de marcadores morfoagronômicos utilizando técnicas de análises uni e multivariadas, foi observada variabilidade genética entre os acessos. Os resultados da análise dos componentes principais, obtidos a partir de 16 caracteres morfoagronômicos mostra que foram necessários três componentes, para que a variância por eles explicada atingisse um mínimo de 80% e a seleção de seis caracteres de maior contribuição para o estudo da diversidade. Pelo método UPGMA formou-se cinco grupos. O grupo I reúne os acessos GVT e GML; o grupo II com o GPY, GTG e GBrPF; o grupo III e IV com apenas um acesso cada, GRT e o GOA, respectivamente e o grupo V com o GBrMe e GRL. Os grupos apresentaram uma incoerência com relação às origens dos acessos, provavelmente devido à natureza quantitativa das características avaliadas, que são controladas por muitos genes, sendo afetadas por fatores ambientais. As avaliações da diversidade e da estruturação genética evidenciam a variabilidade e as relações genéticas existentes em coqueiro-gigante. Esses resultados permitirão orientar as decisões sobre as atividades de conservação e uso do germoplasma do coqueiro no país
2017-01-04

Introdução: A cardiomiopatia hipertrófica (CH) é uma doença cardíaca estrutural primária, caracterizada por hipertrofia do ventrículo esquerdo, sem dilatação, geralmente assimétrica e predominantemente septal. Na população geral a prevalência estimada da CH é de 0,2% (1:500), correspondendo a 0,5% de todas as cardiopatias. Atualmente estão descritas mais de 1400 mutações associadas à CH em 20 genes relacionados com os miofilamentos do sarcômero, o disco-Z e o transporte de cálcio, sendo que os três mais associados são os genes MYH7, MYBPC3 e TNNT2, responsáveis por 50% do casos com diagnóstico molecular positivo no Brasil. Dessa forma, o advento de novas tecnologias de sequenciamento de DNA de alta performance promete revolucionar o diagnóstico molecular, tornando mais rápida e barata a identificação de alterações genéticas, impactando positivamente na custo-efetividade do manejo diagnóstico e terapêutico de pacientes e famílias com o diagnóstico de CH. Materiais e Métodos: Noventa e uma amostras de uma casuística de pacientes não relacionados, portadores de CH com diagnóstico molecular prévio para os 3 genes mais associados (19 positivas e 72 negativas) foram utilizadas juntamente com uma amostra referência do HapMap (NA12878) na validação de um pipeline proposto para a identificação de alterações genéticas em um painel com 74 genes associados à cardiomiopatias hereditárias, utilizando a plataforma Ion Torrent PGM. A etapa de chamada de variantes foi testada em dois limiares diferentes de cobertura de sequenciamento (30x e 10x) e três limiares de frequência de alelo variante (35%, 25% e 20%). A amostra NA12878 foi utilizada na aferição de valores de reprodutibilidade intra e inter-ensaio. As amostras da casuística de CH com diagnóstico molecular prévio negativo foram utilizadas na análise de ganho diagnóstico. Eram consideradas alterações potencialmente patogênicas aquelas que apresentassem associação prévia com CH ou classificação deletéria em dois de três algoritmos de predição de impacto funcional (PROVEAN, SIFT, PolyPhen2) e MAF<0,01, se disponível. Resultados: A plataforma de sequenciamento utilizada apresentou desempenho aceitável, gerando em média 165,9 ±13,1 Mb, com um valor médio de 146,9 ± 11,54 Mb acima de PhredQ>=20, por amostra. O valor médio de cobertura de sequenciamento por amostra foi de 250 ± 23,94x, com 95,2% das regiões alvo cobertas pelo menos 10x. A sensibilidade máxima observada para SNVs foi de 96,7% enquanto que para InDels foi 28,5%. Os valores de reprodutibilidade inter e intra-ensaio de 89,5% e 87,3%, respectivamente. Das 72 amostras negativas, 35 puderam ser reclassificadas como positivas, sendo que os dois genes com mais ocorrências de alterações genéticas foram FLNC e TRIM63, ambos já relacionados com CH. Vinte e duas amostras foram reclassificadas como inconclusivas e 15 permaneceram negativas. O ganho diagnóstico foi de 21,5%. Conclusões: A plataforma Ion Torrent PGM apresenta potencial no sequenciamento de genes relacionados à cardiomiopatias hereditárias e o pipeline validado mostrou valores analíticos praticáveis em uma rotina diagnóstica. A utilização do painel genético ampliado se mostrou viável na detecção de alterações genéticas, propiciando uma boa margem de ganho diagnóstico em comparação com o sequenciamento apenas dos três genes mais associados à CH
Introduction: Hypertrophic cardiomyopathy (HCM) is a primary cardiac disease, mainly characterized by unexplained left ventricle hypertrophy, in the absence of dilatation, usually asymmetric and predominantly septal. The estimated prevalence is 1:500 individuals in the general population, corresponding for 0.5% of all cardiac diseases. Up to now, more than 1400 mutations are associated with HCM in 20 genes related with sarcomeric myofibrils, Z-disc and calcium homeostasis, wherein the 3 most associated genes are MYH7, MYBPC3 and TNNT2, accounting for 50% of cases with positive molecular diagnostics in Brazil. Thus, the advent of new high throughput DNA sequencing technologies promise to revolutionize the use of molecular diagnostics, turning the identification of genetic mutations in a fast and cheap practice, increasing the cost-effectiveness of diagnostic and treatment of patients and families with HCM. Materials and Methods: Ninety one samples from an HCM casuistic of unrelated individuals with previous molecular diagnostics for the three most HCM-associated genes (19 positives and 72 negatives) were processed along with a reference HapMap sample (NA12878) in the validation process of a pipeline proposed for the detection of genetic alterations in a genetic panel composed of 74 genes associated with inherited cardiomyopathies, using Ion Torrent PGM platform. The variant call step was tested for two cutoffs of sequencing coverage (30x and 10x) and three cutoffs of variant allele frequency (35%, 25% and 20%). The sample NA12878 was used in the assessment of intra and inter-assay reproducibility. Negative samples from the HCM casuistic were used in the assessment of diagnostic yield. Variants were considered potentially pathogenic if previously described as associated with HCM or if presenting a deleterious score in at least two of three impact prediction algorithms tested (PROVEAN, SIFT and PolyPhen-2) and MAF < 0.01, if available. Results: The chosen next-generation sequencing platform presented an acceptable performance, with a mean throughput of 165,9 ±13,1 Mb, with a mean value of 146,9 ± 11,54 Mb above PhredQ >= 20. Mean sequencing coverage was 250 ± 23,94x, wherein 95.2% of target bases were covered at least 10x. Maximum achieved sensitivity for SNVs was 96.7% while for InDels was 28.5%. Both values of inter and intra-assay reproducibility were 89.5% and 87.3%, respectively. Of all 72 negative samples, 35 were reclassified as positive with the two most frequently mutated genes being FLNC and TRIM63, both already associated with HCM. Twenty two samples were reclassified as inconclusive and 15 remained negatives. Diagnostic yield was 21.5%. Conclusions: Ion Torrent PGM platform presented a feasible potential for the sequencing of inherited cardiomyopathies-associated genes and the designed pipeline presented reliable analytical values for diagnostic use. The expanded panel proved to be a good strategy for the detection of genetic alteration providing a good value of diagnostic yield in comparison with the sequencing of the three most HCM-associated genes alone

Les relations structure/fonction des hemoglobines humaines a fonction modifiee, exprimees a l'etat heterozygote sont etudiees. Des proprietes de 2 nouveaux mutants a affinite augmentee pour l'oxygene sont decrits. L'analyse tridimensionnelle met en evidence que la substitution a pour consequence un deplacement de la region helicale ef avec une modification d'accessibilite du dpg dans la carte centrale

Un nouveau facteur de transcription, appartenant à la famille des protéines à domaine bHLH, a récemment été isolé dans notre laboratoire. Initialement appelé « clone bc8 » puis HRT1, ce facteur présentait des similitudes avec les protéines Hairy and Enhancer of split qui interviennent notamment dans le phénomène d’inhibition latérale lors de la formation du tissu neural. Des études d’hybridation in situ réalisées chez l'embryon de xénope ont suggéré un rôle important de XHRT1, la protéine HRT1 de xénope, dans le développement neural. Nous avons recherché les partenaires protéiques de XHRT1 par la technique du double-hybride afin de mieux comprendre son mécanisme d’action moléculaire dans la neurogenèse.

Tout d’abord nous avons construit les outils appropriés pour l’élaboration du travail, à savoir, les clones de levures exprimant les appâts spécifiques des domaines de la protéine étudiée et la création d’une banque d’ADNc du xénope au stade de la neurulation. Ensuite, trois criblages ont été réalisés. Dans le premier cas, nous avons recherché les partenaires des domaines bHLH et Orange (bHLH-O). Le domaine bHLH est en effet responsable de la dimérisation de ce type de protéine. Le domaine Orange qui suit le domaine bHLH, pourrait participer dans le choix du partenaire d’hétérodimérisation. Nous avons isolé deux facteurs de type bHLH-Orange apparentés à HRT1, XHairy1 et XHairy2b et confirmé leur interaction avec XHRT1. Les domaines impliqués dans ces interactions sont les bHLH-O pour les trois facteurs. Ce même criblage nous a permis d’isoler un nouvel ADNc qui code une protéine sans domaine apparent connu actuellement. Nous avons montré que cette protéine reconnaissait spécifiquement le domaine Orange de HRT1 mais pas celui des autres facteurs de type bHLH-O. Elle a été baptisée BOIP pour Bc8 Orange Interacting Protein. Le rôle physiologique de cette interaction n’a pu être démontré. Nous avons établi que la protéine BOIP pouvait aussi s’homodimériser. Nous avons aussi déterminé son profil d’expression chez le xénope et la souris. Son transcrit est hautement présent dans les testicules adultes. La protéine pourrait donc jouer un rôle important dans la spermatogenèse. Les deux autres criblages, utilisant les domaines situés dans la partie C-terminale de XHRT1, ont apporté des nouveaux partenaires potentiels, mais ces interactions n’ont pu être confirmées dans un système indépendant.

Enfin, en étudiant plus en détail les interactions entre XHRT1 et XHairy1 ou XHairy2b, nous avons mis à jour une possible fonction de spécificité dans le choix du partenaire dans la région C-terminale de HRT1. La formation de ces dimères pourrait jouer un rôle dans la formation du tube neural mais également dans d’autres différenciations tissulaires.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

INTRODUÇÃO: A hiperplasia adrenal macronodular independente de ACTH (AIMAH) é uma doença rara, caracterizada pela presença de macronódulos funcionantes nas adrenais e por uma produção aumentada, autônoma e sustentada de cortisol. Constitui uma causa incomum de síndrome de Cushing (SC). A forma esporádica da doença parece ser a mais frequente, no entanto, se desconhece a real prevalência de sua forma familial. Apesar de ser uma entidade clínica conhecida há quase 50 anos, o processo fisiopatológico que culminaria com a AIMAH, as alterações genéticas predisponentes e aspectos clínicos, laboratoriais e radiológicos relevantes da doença ainda não foram elucidados de forma clara. O diagnóstico recente de uma grande família portadora da doença viabilizou a realização do presente trabalho. OBJETIVOS: 1) Caracterizar a evolução da AIMAH em sua forma familial, correlacionando as manifestações clínicas, os dados laboratoriais e os achados radiológicos; 2) investigar a possível associação entre a AIMAH e a ocorrência de meningiomas intracranianos; 3) avaliar a atividade metabólica das adrenais hiperplasiadas na AIMAH; 4) definir o padrão de herança genética da doença na família estudada; e 5) mapear regiões cromossômicas e loci potencialmente relacionados à etiologia genética da AIMAH familial. MÉTODOS: 96 membros da família estudada foram inicialmente submetidos a uma avaliação clínica e laboratorial pormenorizada. Em seguida, foram realizados exames de tomografia computadorizada para a caracterização radiológica das adrenais. Exames de ressonância magnética e de tomografia por emissão de pósitrons com fluordesoxiglicose marcada, acoplada à tomografia computadorizada (18F-FDGPET/CT) foram realizados em pacientes com as formas familial e esporádica da doença para, respectivamente, investigar a presença de meningiomas intracranianos e caracterizar a atividade metabólica das adrenais hiperplasiadas. Foram também realizados testes in vivo para a pesquisa de receptores hormonais aberrantes nos pacientes com a forma familial da doença. Em uma outra etapa do estudo, diferentes técnicas de biologia molecular foram empregadas para a investigação da etiologia genética da AIMAH familial. Desta forma, realizou-se: o sequenciamento do gene do receptor do ACTH (MC2R), um estudo de ligação genética utilizando microssatélites específicos, um estudo de ligação genética em escala genômica utilizando polimorfismos de nucleotídeo único (SNPs) e o sequenciamento de genes suspeitos. RESULTADOS: A avaliação dos indivíduos pertencentes à genealogia permitiu o diagnóstico de 15 casos da doença (7 mulheres e 8 homens) em três gerações consecutivas. A AIMAH era transmitida para as gerações subsequentes tanto pelo sexo masculino como feminino e acometia cerca de metade dos irmãos em alguns segmentos da família. A idade média ao diagnóstico da doença foi de 52,8 +-11,3 anos (32 a 74 anos) e cerca de 86% (12/14) desses pacientes apresentavam SC subclínica. As dosagens do cortisol salivar à meia-noite e do cortisol em urina de 24 horas demonstraram baixa sensibilidade (21% e 14%, respectivamente) para o diagnóstico da doença em sua forma familial. O valor do ACTH plasmático encontrava-se baixo ( < 10 pg/mL) em 46% (5/11) dos pacientes doentes. Em cerca de 62% (8/13) dos casos, foi demonstrada uma redução do valor sérico do sulfato de desidroepiandrosterona (SDHEA). Por regressão logística simples, foi observado que a probabilidade (odds ratio) de um indivíduo apresentar a doença na família era maior diante da presença de pletora, após o diagnóstico de diabetes ou pré-diabetes ou diante do relato de ganho ponderal progressivo. O espessamento de ambas as adrenais associado à presença de nódulos bilaterais foi o achado radiológico mais frequente na forma familial da doença. No entanto, em um terço dos pacientes (5/15) foram encontradas alterações radiológicas em somente uma das adrenais. Durante os testes in vivo para pesquisa de receptores hormonais aberrantes, foram observadas, com frequência, respostas distintas entre os indivíduos doentes pertencentes à família. Nos pacientes submetidos ao exame de ressonância magnética, foram demonstradas imagens típicas de meningiomas intracranianos em um terço (5/15) dos casos. No exame 18F-FDG-PET/CT, foi observado um aumento da atividade metabólica das adrenais hiperplasiadas, tanto nos pacientes com SC manifesta como naqueles com a forma subclínica da doença. O estudo molecular permitiu delimitar nos cromossomos 16 e 11 algumas regiões genômicas potencialmente relacionadas à etiologia genética da AIMAH familial. O sequenciamento de alguns genes suspeitos (GPR56, GPR97 e GPR114), localizados nessas regiões, não demonstrou a presença de mutações. CONCLUSÕES: Na genealogia estudada, o padrão de transmissão da AIMAH foi autossômico dominante, e a SC subclínica foi a forma mais frequente de manifestação da doença. O teste de supressão com 1 mg de dexametasona via oral à meia-noite demonstrou ser o exame laboratorial de escolha para a avaliação inicial dos pacientes suspeitos de apresentarem AIMAH familial, em função, sobretudo, da baixa sensibilidade do cortisol salivar à meia-noite e do cortisol urinário para o diagnóstico da doença. Valores normais do ACTH plasmático foram um achado laboratorial frequente na AIMAH familial e valores baixos do SDHEA sérico demonstraram ser um indício relativamente precoce da SC subclínica associada à doença. Diferentes padrões radiológicos foram demonstrados nas tomografias das adrenais dos pacientes com AIMAH familial, não sendo infrequente a presença de assimetria entre as duas glândulas. Os resultados dos testes in vivo para a pesquisa de receptores hormonais aberrantes foram mais condizentes com a hipótese de que a expressão desses receptores seria um epifenômeno do processo fisiopatológico, resultante da proliferação e desdiferenciação celular. Uma alta prevalência de meningiomas intracranianos foi observada nos pacientes com AIMAH, tanto na forma familial da doença como na forma esporádica. Demonstrou-se também, pela primeira vez, que as adrenais na AIMAH podem exibir uma captação aumentada de 18F-FDG no exame de PET/CT, de forma semelhante às metástases e aos carcinomas da glândula. Por fim, foram delimitadas no cromossomo 16 (16p12.1, 16p11.2, 16q12.1, 16q13 e 16q21) e no cromossomo 11 (11q23.1) as principais regiões do genoma suspeitas de estarem ligadas à etiologia genética da AIMAH familial (genoma de referência: NCBI36/hg18)
INTRODUCTION: ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a rare disease characterized by functioning adrenal macronodules and increased, autonomous and sustained cortisol production. This condition is an uncommon cause of Cushing\'s syndrome (CS). While the sporadic form of the disease appears to be the most frequent, the true prevalence of its familial form is unknown. Despite being a known clinical entity for almost 50 years, the pathophysiological process that leads to AIMAH, the predisposing genetic alterations and important clinical, laboratory and radiological aspects of the disease have not been fully clarified. The recent identification of a large group of relatives with familial AIMAH allowed the accomplishment of the present study. OBJECTIVES: The following were the aims of this study: 1) characterize the development of familial AIMAH through correlations between clinical manifestations, laboratory data and radiological findings; 2) investigate the possible association between AIMAH and the occurrence of intracranial meningioma; 3) characterize the metabolic activity of the adrenal glands in this disease; 4) define the inheritance pattern of the disease in the family studied; and 5) map chromosomal regions and loci potentially related to the genetic etiology of familial AIMAH. METHODS: 96 members of the family studied were initially subjected to a detailed clinical and laboratory evaluation. Computed tomography (CT) scans were performed for the radiological characterization of the adrenal glands. Magnetic resonance imaging scans and 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) scans were performed on patients with both forms of the disease (familial and sporadic) to investigate the presence of intracranial meningioma and characterize the metabolic activity of the adrenal glands, respectively. In vivo studies for aberrant hormone receptors were also conducted on those patients with familial AIMAH. In another phase of the study, different molecular biology techniques were employed to investigate the genetic etiology of familial AIMAH. For such, sequencing of the ACTH receptor gene (MC2R), a linkage study using specific microsatellite markers, a single nucleotide polymorphism (SNP)-based genome-wide linkage study and the sequencing of suspect genes were performed. RESULTS: The evaluation of the family revealed the diagnosis of 15 cases of the disease (7 women and 8 men) in three consecutive generations. AIMAH was transmitted to subsequent generations by both genders and half of the siblings were affected in some segments of the family. Mean age at diagnosis was 52.8 +-11.3 years (range: 32 to 74 years) and about 86% (12/14) of the patients exhibited subclinical CS. Both midnight salivary cortisol and 24-hour urinary cortisol demonstrated low sensitivity (21% and 14%, respectively) for the diagnosis of familial AIMAH. Plasma ACTH levels were low ( < 10 pg/ml) in 46% (5/11) of patients with the disease. In about 62% (8/13) of cases, serum dehydroepiandrosterone sulphate (DHEAS) levels were below the normal range. Simple logistic regression models revealed that the probability (odds ratio) of an individual having the disease in the family was greater in the presence of plethora, progressive weight gain or after the diagnosis of diabetes or prediabetes. Adrenal thickening associated with the presence of bilateral nodules was the most common radiological finding in familial AIMAH. However, radiological abnormalities were found in only one of the adrenal glands in one third of the patients (5/15). Throughout the in vivo studies for aberrant hormone receptors, distinct responses were frequently observed among the individuals with familial AIMAH. One third (5/15) of the patients who underwent magnetic resonance imaging scans had typical images of intracranial meningiomas. The 18F-FDG-PET/CT scan revealed increased metabolic activity of the hyperplastic adrenals in patients with both overt and subclinical CS. The molecular studies delimited genomic regions on chromosomes 16 and 11 potentially related to the genetic cause of familial AIMAH. Some suspected genes (GPR56, GPR97 and GPR114), located in these genomic regions, were sequenced, but no mutations were found. CONCLUSIONS: In the extended family studied, AIMAH followed an autosomal dominant pattern of inheritance and subclinical CS was the most common presentation of the disease. The 1 mg overnight dexamethasone suppression test proved to be the screening test of choice for the initial evaluation of patients suspected to have familial AIMAH, due mainly to the low sensitivity of midnight salivary cortisol and 24-hour urinary cortisol as screening tests. A normal level of plasma ACTH was a common laboratory finding in familial AIMAH. Low serum levels of DHEAS proved to be a relatively early finding associated with the subclinical CS determined by the disease. Adrenal CT scans revealed different radiological patterns among patients with familial AIMAH, with a fairly frequent rate of asymmetry between glands. The distinct responses observed throughout the in vivo studies for aberrant hormone receptors, among family members, favor the hypothesis that these receptors may be an epiphenomenon resulting from cell proliferation and dedifferentiation. An increased prevalence of intracranial meningioma was demonstrated in both the familial and sporadic forms of AIMAH. For the first time, it was shown that AIMAH may exhibit increased 18FFDG uptake on the PET/CT scan, similarly to adrenal carcinoma and metastasis. The main genomic regions potentially associated with familial AIMAH were delimited on chromosome 16 (16p12.1, 16p11.2, 16q12.1, 16q13 and 16q21) and chromosome 11 (11q23.1) (reference genome: NCBI36/hg18)

The genus Enterococcus comprises of a group of commensal organisms of the human gut which has been associated with cases of endocarditis and urinary tract infections. In the present study, 12 Enterococcus isolates were obtained from clinical specimens and characterized using genotyping techniques that have become an integral part of clinical research. There were three different genotyping methods used to identify the enterococci to species level and to determine the level of genetic diversity among the selected strains. These techniques were, randomly amplified polymorphic DNA-PCR (RAPD-PCR), 16S rDNA ribotyping analysis and pulse field gel electrophoresis (PFGE) respectively. The minimum inhibitory concentration (MIC) to penicillin and vancomycin were also determined using a disc diffusion assay and a microtitre plate dilution assay. All twelve strains were found to be vancomycin resistant enterococci (VRE) at a MIC value greater than 100μg/ml. Penicillin growth inhibition based on MIC values were categorized into three groups, susceptible (< 0.25 μg/ml), intermediate (≤ 3μg/ml) and resistant (≥ 4μg/ml) respectively. RAPD-PCR was performed using four random primers. Primers yielding the highest discriminative power were used for phylogenetic analysis. The phylogenetic analysis indicated that all 12 strains yielded clonal dissemination, therefore a low genetic diversity between them. The 16S rDNA of all strains were used to identify the enterococci at species level. The rDNA were sequenced and analysed using the NCBI BLAST algorithm and found to belong to three species of Enterococcus. These were E.faecalis, E.faecium and E.durans. PFGE analysis was performed by restriction of all 12 strain’s genomic DNA with the restriction enzyme SmaI. The PFGE patterns were divided into two groups with low genetic diversity. Compared with the RAPD PCR patterns PFGE gives a higher discriminatory power as a higher dissimilarity between the strains was observed. Similar penicillin MICs for each of the strains in the three categories are grouped together in the phylogenetic trees for both PFGE and RAPD-PCR. RAPDPCR is a sensitive, faster, specific and cost effective technique, PFGE analysis has given a higher discriminatory power, higher reproducibility of the results and the polymorphism seen in the patterns suggest that PFGE has a potential of being an essential tool in clinical diagnostics.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Wong Ka Lok.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 121-129).
Abstracts in English and Chinese.
Acknowledgments --- p.i
Abstract --- p.ii
Table A --- p.v
Table B --- p.vi
Table of Contents --- p.vii
Abbreviations --- p.xi
Chapter Chapter 1 --- Molecular authentication of endangered crocodiles and snakes
Chapter 1.1 --- Introduction --- p.1
Chapter 1.2 --- Traditional method of snake and crocodile identification
Chapter 1.2.1 --- Morphology --- p.7
Chapter 1.2.2 --- Chemical Analysis --- p.9
Chapter 1.3 --- Molecular Technology in Authentication
Chapter 1.3.1 --- Polymerase Chain Reactions (PCRs) --- p.11
Chapter 1.3.2 --- Random-primed amplification reaction --- p.12
Chapter 1.3.3 --- Sequence Characterized Amplified Region (SCAR) --- p.13
Chapter 1.3.4 --- PCR-RFLP --- p.13
Chapter 1.3.5 --- DNA sequencing --- p.14
Chapter 1.4 --- Objectives and strategies of the study --- p.15
Chapter Chapter 2 --- Materials and General Methods
Chapter 2.1 --- Reagents and Buffers
Chapter 2.1.1 --- Buffers for Total DNA Extraction --- p.17
Chapter 2.1.2 --- Reagents for Agarose Gel Electrophoresis --- p.17
Chapter 2.1.3 --- Reagents for Plasmid DNA Preparation --- p.18
Chapter 2.1.4 --- Medium for Bacterial Culture --- p.18
Chapter 2.1.5 --- Reagents for Preparation of Competent Cells --- p.19
Chapter 2.2 --- DNA Isolation
Chapter 2.2.1 --- Extraction of DNA from meats --- p.20
Chapter 2.2.2 --- Extraction of DNA from blood --- p.20
Chapter 2.3 --- Phenol/Chloroform Extraction --- p.21
Chapter 2.4 --- Ethanol Precipitation --- p.22
Chapter 2.5 --- DNA Concentration/Purity Estimation --- p.22
Chapter 2.6 --- Mitochondrial DNA amplification --- p.23
Chapter 2.7 --- Random-Primed Polymerase Chain Reactions --- p.24
Chapter 2.8 --- SCAR for Snake samples --- p.24
Chapter 2.9 --- SCAR for Crocodile samples --- p.25
Chapter 2.10 --- Restriction fragment length polymorphism analysis --- p.25
Chapter 2.11 --- Agarose Gel Electrophoresis of DNA --- p.26
Chapter 2.12 --- Purification of PCR product --- p.26
Chapter 2.13 --- Preparation of Escherichia coli Competent Cells --- p.27
Chapter 2.14 --- Ligation and transformation of E. coli --- p.27
Chapter 2.15 --- Plasmid preparation --- p.28
Chapter 2.16 --- Screening of Plasmid DNA by Restriction Digestion --- p.29
Chapter Chapter 3 --- DNA sequencing of snakes & construction of snake database
Chapter 3.1 --- Introduction --- p.30
Chapter 3.2 --- Materials and methods
Chapter 3.2.1 --- Snake samples --- p.32
Chapter 3.2.2 --- "DNA Extraction, mitochondrial gene amplification and DNA sequencing" --- p.33
Chapter 3.2.3 --- Construction of database --- p.33
Chapter 3.3 --- Results
Chapter 3.3.1 --- Cytochrome b gene amplification and sequencing --- p.34
Chapter 3.3.2 --- Gene amplification and sequencing of 16S rRNA --- p.42
Chapter 3.3.3 --- Cytochrome b sequence database --- p.50
Chapter 3.3.4 --- 16S rRNA sequence database --- p.53
Chapter 3.4 --- Discussion
Chapter 3.4.1 --- Cytochrome b and 16S rRNA genes of snake species --- p.55
Chapter 3.4.2 --- Cytochrome b and 16S rRNA databases --- p.55
Chapter Chapter 4 --- Application of PCR-RFLP and SCAR in snake species identification
Chapter 4.1 --- Introduction --- p.57
Chapter 4.2 --- Material and Methods
Chapter 4.2.1 --- DNA extraction and PCR-RFLP --- p.58
Chapter 4.2.2 --- RAPD and SCAR --- p.58
Chapter 4.3 --- Results
Chapter 4.3.1 --- PCR-RFLP of cytochrome b genes of snakes --- p.59
Chapter 4.3.2 --- PCR-RFLP of 16S rDNA --- p.61
Chapter 4.3.3 --- RAPD & SCAR analysis --- p.67
Chapter 4.4 --- Discussion --- p.72
Chapter Chapter 5 --- "Application of DNA sequencing, PCR-RFLP and SCAR to identify crocodile species"
Chapter 5.1 --- Introduction --- p.74
Chapter 5.2 --- Materials and methods
Chapter 5.2.1 --- "Crocodile, human and four animal samples" --- p.75
Chapter 5.2.2 --- "DNA Extraction, mitochondrial gene amplification and DNA sequencing" --- p.75
Chapter 5.2.3 --- PCR-RFLP and SCAR --- p.76
Chapter 5.3 --- Results
Chapter 5.3.1 --- Isolation of crocodiles DNA --- p.77
Chapter 5.3.2 --- Isolation of DNA from Human and four animal species --- p.78
Chapter 5.3.3 --- Cytochrome b gene amplification and sequencing --- p.78
Chapter 5.3.4 --- 16S rRNA gene amplification and sequencing --- p.84
Chapter 5.3.5 --- PCR-RFLP of cytochrome b --- p.89
Chapter 5.3.6 --- PCR-RFLP of 16S rRNA --- p.91
Chapter 5.3.7 --- SCAR primers for four crocodile species --- p.93
Chapter 5.4 --- Discussion --- p.97
Chapter Chapter 6 --- A case report - authentication of animal samples using DNA sequencing
Chapter 6.1 --- Introduction --- p.99
Chapter 6.2 --- Material and methods
Chapter 6.2.1 --- Materials --- p.101
Chapter 6.2.2 --- DNA Extraction and sequencing --- p.101
Chapter 6.3 --- Result and discussion
Chapter 6.3.1 --- Cytochrome b gene sequencing --- p.102
Chapter 6.3.2 --- Sequence homology among samples and meats obtained from the market --- p.111
Chapter 6.3.3 --- Identity of samples B & D --- p.113
Chapter Chapter 7 --- General Discussion
Chapter 7.1 --- Advantages and weakness of DNA technology --- p.116
Chapter 7.2 --- Choosing appropriate molecular markers --- p.118
Chapter 7.3 --- Further suggested work --- p.119
Chapter 7.4 --- Conclusion --- p.119
References --- p.121
Appendix --- p.130

Indiana University-Purdue University Indianapolis (IUPUI)
The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.

Indiana University-Purdue University Indianapolis (IUPUI)
The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

Student Number : 9704202D - MSc dissertation - School of Clinical Microbiology and Infectious Diseases - Faculty of Science
Meningococcal disease is an important cause of morbidity and mortality worldwide, particularly in children and young adults. Epidemics caused by Neisseria meningitidis continue to plague many countries on a global scale, none more so than countries of the African ‘meningitis belt’, where attack rates can reach up to 1000/100,000 population. It has been well recognized that most epidemic and endemic cases of meningococcal disease are caused by a limited number of genetically defined clonal groups. The objective of this molecular epidemiological study was to genotypically characterize strains of N. meningitidis collected in South Africa from July 1999 to July 2002. Characterization of meningococcal strains belonging to serogroup A, B, C, W135 and Y, by PFGE and MLST allowed us to determine the genetic population structure of N. meningitidis in South Africa, and thus identify the predominant clonal groups responsible for the majority of meningococcal disease in the country over this period. The results from the genotypic characterization revealed that the greatest majority of meningococcal disease in South Africa was caused by a strains belonging to only a few “hyperinvasive lineages”, most notably strains of the ST-44 complex (lineage III), ST-32 complex (ET-5 complex), ST-11 complex (ET-37 complex), and the ST-1 complex (subgroup I/II) which have all been responsible for major epidemics worldwide. These findings have direct implications on public health decision, particularly with regards to the development of effective intervention and control strategies, and emphasize the need for continuous long-term monitoring of the circulation of these strains in the population.

Title.: Utilisation of molecular cytogenetic techniques in reproductive genetics Chromosomal abnormalities constitute one of the most important causes of birth defects, fertilization failure and/or human infertility. Approximately, 40-50% of human conceptuses are chromosomally abnormal, 6% of the abortions during the first trimester of gestation are directly linked to chromosomal abnormality, while at term 0.6% of livebirths present with such features. Most of these abnormalities originate from the gametogenesis and arise through disturbed meiotic processes. Each gamete is a final and original product of the meiosis carrying a unique chromosomal set. Therefore, cytogenetic examination of individual gametes represents an important scientific challenge for our undrstandidng of the formation, incidence and etiology of aforementioned chromosomal abnormalities. Nonetheless, it is very technically demanding to perform efficient chromosomal investigation on gametes, hence single cells. My Ph.D. thesis is focused on the development of new techniques for the detection of chromosomal abnormalities in gametes and embryos. We developed a PNA-based technique as an alternative to conventional FISH and PRINS-based methods for fast, efficient and robust in situ detection of chromosomal abnormalities in human...

Indiana University-Purdue University Indianapolis (IUPUI)
Forensic DNA profiling is widely used as an identification tool for associating an individual with evidence of a crime. Analysis of a DNA sample involves observation of data in the form of an electropherogram, and subsequently annotating a DNA “profile” from an individual or from the evidence. The profile obtained from the evidence can be compared to reference profiles deposited in a national DNA database, which may include the potential contributor. Following a match, a random match probability is calculated to determine how common that genotype is in the population. This is the probability of obtaining that same DNA profile by sampling from a pool of unrelated individuals. Each state has adopted various laws requiring suspects and/or offenders to submit a DNA sample for the national database (such as California’s law that all who are arrested must provide a DNA sample). These profiles can then be associated with past unsolved crimes, and remain in the database to be searched in the event of future crimes. In the case of database samples, a physical sample of the offender’s DNA must be kept on file in the laboratory indefinitely so that in the event of a database hit, the sample is able to be retested. Current methods are to collect a buccal swab or blood sample, and store the DNA extracts under strict preservation conditions, i.e. cold storage, typically -20° C. With continually increasing number of samples submitted, a burden is placed on crime labs to store these DNA extracts. A solution was required to help control the costs of properly storing the samples. FTA™ paper was created to fulfill the need for inexpensive, low maintenance, long term storage of biological samples, which makes it ideal for use with convicted offender DNA samples. FTA™ paper is a commercially produced, chemically treated paper that allows DNA to be stored at room temperature for years with no costly storage facilities or conditions. Once a sample is required for DNA testing, a small disc is removed and is to be used directly in a PCR reaction. A high quality profile is important for comparing suspect profiles to unknown or database profiles. A single difference between a suspect and evidentiary sample can lead to exclusion. Unfortunately, the DNA profile results yielded from the direct addition have been unfavorable. Thus, most crime laboratories will extract the DNA from the disc, leading to additional time and cost to analyze a reference sample. Many of the profiles from the direct addition of an FTA™ disc result in poor quality profiles, likely due to an increase in PCR inhibitors and high concentrations of DNA. Currently, standardized protocols regarding the recommended locations for removal of a sample disc from a bloodspot on an FTA™ card does not exist. This study aims to validate the optimal location by comparing DNA profiles obtained from discs removed from the center, halfway, and edge locations of a bloodspot from 50 anonymous donors. Optimal punch location was first scored on the number of failed, partial or discordant profiles. Then, profile quality was determined based on peak characteristics of the resulting DNA profiles. The results for all three disc locations were 5.3% failed amplifications, 4.2% partial amplifications, and one case of a discordant profile. Profile quality for the majority of the samples showed a high incidence of stutter and the absence of non-template adenylation. Of the three disc locations, the edge of the blood stain was ideal, due to a presumably lower concentration of DNA and likely more dilute amount of the PCR inhibitor heme. Therefore, based on the results of this study, there is a greater probability of success using a sample from the edge of a blood stain spotted in FTA™ paper than any other location of the FTA™ card.

Brettanomyces/Dekkera is a wine contaminating yeast responsible for serious economic losses due to the production of volatile phenols and the consequent negative impact on wine quality. Despite its importance to the wine industry it has remained poorly understood at the genetic level for a long period of time. In recent years, a genomic survey sequencing was implemented on Dekkera bruxellensis by some research groups providing resources for further investigation on this species. This thesis aims to make a review on how the acquired genetic knowledge has been applied for the development of molecular techniques for the detection, identification and enumeration of Brettanomyces/Dekkera in wine. Furthermore it is evaluated whether the accessibility to the whole genome sequence of D. bruxellensis allows a better understanding of the adaptation and survival mechanisms of this yeast in wine and the genetic bases of some relevant phenotypic traits. It is extremely important to find rapid and effective ways to detect Brettanomyces/Dekkera in wine. Conventional methods are based on culturing the yeast on selective media, but these are laborious and time consuming, delaying corrective actions on the wine processing. Various studies based on molecular methods have been developed as alternatives to the traditional ones. They offer faster results and increased sensibility and specificity. However some limitations can be pointed out such as high detection limits and the quantification of non viable cells as is the case of QPCR. Beyond this, high cost equipment and skilled technicians are needed to execute the methods and for interpretation of the results. Various genes and proteins were identified and related with the capacity of Brettanomyces/Dekkera to survive in the wine environment. These genes are responsible for glucose transport, carbon metabolism, anaerobic growth, fermenting characteristics, nitrate assimilation and osmotic stress response. Proteins involved in cell wall budding, adhesion and pseudohyphal growth were also identified and seem to be related with the capacity of cells to survive on the surface of grapes and barrels. The analysis of the genome sequence has also revealed one ORF that encodes a protein related to sulphite tolerance of Brettanonomyces/Dekkera. However, further studies on the expression and regulation of these genes are still necessary. The production of volatile phenols is the major spoilage activity of Brettanonomyces/Dekkera. This requires the activity of two enzymes, the phenolic acid decarboxylase (PAD) and the vinylphenol reductase (VPR). The sequencing of Brettanomyces/Dekkera genome revealed the presence of the DbPAD gene. Two paralogous genes that encode PAD were identified and they differ in the gene expression and type of metabolism, which may explain the differences found between different isolates of Brettanomyces/Dekkera. The VPR protein has been isolated and purified but the gene sequence has yet to be found. Although the sequencing of the genome of Brettanomyces/Dekkera has brought new opportunities to better understand the behaviour of this yeast in wine, the molecular mechanisms are still poorly understood and further investigation is necessary.
Brettanomyces/Dekkera é uma levedura contaminante do vinho que tem originado importantes perdas económicas devido à produção de fenóis voláteis e ao consequente impacto negativo na qualidade do vinho. Apesar da sua importância para a indústria do vinho, durante um longo período de tempo foi pouco compreendida ao nível genético. Nos últimos anos, a sequenciação genómica de Dekkera bruxellensis tem sido implementada por alguns grupos de pesquisa, o que fornece recursos para uma investigação mais aprofundada sobre esta espécie. Esta tese tem como objetivo fazer uma avaliação sobre de que forma o conhecimento genético adquirido tem sido aplicado para o desenvolvimento de técnicas moleculares para a deteção, identificação e quantificação de Brettanomyces/Dekkera em vinho. Além disso, é avaliado se o acesso a toda a sequência genómica de D. bruxellensis permite uma melhor compreensão dos mecanismos de adaptação e sobrevivência desta levedura em vinho e as bases genéticas de algumas características fenotípicas. É extremamente importante encontrar formas rápidas e eficazes para detetar Brettanomyces/Dekkera em vinho. Os métodos convencionais são baseados no cultivo da levedura em meios seletivos, o que os tornam laboriosos e morosos, atrasando possíveis ações corretivas durante o processamento do vinho. Vários estudos baseados em métodos moleculares têm sido desenvolvidos como alternativa aos tradicionais. Estes oferecem resultados mais rápidos e um aumento de sensibilidade e de especificidade. No entanto, os métodos moleculares apresentam algumas limitações como limites de deteção elevados e a quantificação de células não viáveis como é no caso do QPCR. Para além destas limitações são necessários equipamentos de elevado custo e técnicos especializados para executar os métodos e interpretar dos resultados. Vários genes e proteínas foram identificados e relacionados com a capacidade de Brettanomyces / Dekkera sobreviver em ambiente vínico. Esses genes são responsáveis pelo transporte da glucose, metabolismo do carbono, crescimento anaeróbio, características fermentativas, assimilação do nitrato e resposta ao stress osmótico. Foram também identificadas proteínas envolvidas na gemulação, na adesão e no crescimento de pseudo-hifas que parecem estar relacionadas com a capacidade de sobrevivência na superfície das uvas e na superfície dos barris. A análise da sequência do genoma de Brettanomyces/Dekkera revelou a presença de um ORF que codifica a proteína que confere tolerância ao sulfito, contudo são necessários mais estudos de expressão e regulação destes genes. A produção de fenóis voláteis é a principal causa de deterioração dos vinhos por Brettanonomyces / Dekkera. A produção destes compostos requer a atividade de duas enzimas, a hidroxinamato descarboxilase (PAD) e a vinifenol reductase (VPR). A sequenciação do genoma de Brettanomyces/Dekkera revelou a presença do gene DbPAD. Foram ainda identificados dois genes parálogos que codificam o gene PAD, estes diferem na expressão génica e no tipo de metabolismo, o que pode explicar as diferenças encontradas nos diferentes isolados de Brettanomyces/Dekkera. A proteína da enzima VPR foi isolada e purificada, contudo sequência do gene ainda não foi encontrada. Embora a sequenciação do genoma de Brettanomyces/Dekkera tenha trazido novas oportunidades para compreender melhor o comportamento desta levedura em vinho, os mecanismos moleculares ainda estão mal compreendidos sendo necessária mais investigação.