A leptospirose é uma zoonose disseminada mundialmente, causada por bactérias do gênero Leptospira. A melhor maneira de contornar o problema é por de medidas preventivas, já que a contenção da proliferação de roedores é inviável e não há vacina eficaz disponível. Estratégias de genômica funcional têm identificado um grande número de proteínas a serem estudadas. Esse fato aliado a dados da literatura, que identificaram proteínas envolvidas na patogenicidade e que são expressas somente em condição de virulência, levou à proposição da utilização da proteômica para canalizar os estudos. A metodologia envolveu obtenção de extratos protéicos de leptospiras retiradas de animais infectados, sua separação por gel bidimensional, e identificação dos spots por espectrometria de massas. O objetivo central foi a identificação de proteínas expressas em bactérias virulentas e ausentes nas não-virulentas. A identificação dessas proteínas pode facilitar a busca de proteínas envolvidas na virulência e infecção da leptospirose. Adicionalmente, é um avanço no esclarecimento da biologia e patogenicidade das leptospiras, bem como para o reconhecimento de candidatos potenciais para a composição de vacinas e/ou métodos diagnósticos mais eficientes. / Leptospirosis, one of the most spread zoonosis worldwide, is caused by bacteria of the genus Leptospira. Preventive measures are the best way to control the disease due to the difficulty to impair the proliferation of rodents and because no efficient vaccine is currently available. Functional genomics strategies have pointed a large number of proteins that could be important immunogens. This fact allied to published data reporting the identification of proteins involved in pathogenesis that are expressed only in virulent strains, led us to propose the use of proteomics as a tool to narrow down these studies. The methodology involved the preparation of protein extracts from tissuederived leptospires, separation by two-dimensional gel and identification of the spots by mass spectrometry. The central objective was to identify proteins expressed only in virulent bacteria. The identification of these proteins could help the search for proteins involved in virulence with a role during infection. Additionally, the data presented here represent a large step to clarify the biology and pathogenicity of leptospires, as well as the identification of potentially important vaccine candidates and/or proteins to compose more efficient diagnostic methods.

<p>The main objective of the reported study was to investigate how biomedical ontologies, logically structured representations of various aspects of the biomedical reality, can help researchers in analyzing experimental data. The dissertation reports two attempts to construct tools for the analysis of high-throughput experimental results using explicit domain knowledge representations. Furthermore, integrative efforts made by the community of Open Biomedical Ontologies (OBO), in which the author has participated, are reported, and a framework for consistently connecting the Gene Ontology (GO) with the Taxonomy of Species is proposed and discussed.</p>

<p>There are 42 members of the homeodomain-leucine zipper (HDZip) family of transcription factors in <i>Arabidopsis thaliana</i>. This thesis focuses on the functional analysis of one member of this family, ATHB16, and on the biochemical properties of HDZip proteins. </p><p>To assess the function of the <i>ATHB16</i> gene, the expression of <i>ATHB16</i> was altered in transgenic Arabidopsis plants by using sense and antisense RNA constructs under the control of the 35S promoter. The reciprocal phenotypic effects associated with elevated and reduced levels of <i>ATHB16</i> expression suggested that, in wild-type plants, ATHB16 acts as a mediator of blue and red light effects on the regulation of plant growth and the timing of the floral transition. </p><p>In wild-type Arabidopsis, expression of <i>ATHB16</i> is high in leaves, intermediate in adult roots and inflorescences, and low in stems and siliques. The expression of <i>ATHB16</i> in the root is markedly increased in response to exogenous abscisic acid (ABA) treatment, but is reduced in the ABA response mutants <i>abil </i>and <i>abi2</i>, suggesting that <i>ATHB16</i> may be involved in ABA signal transduction. This hypothesis was corroborated by observations of alterations in sensitivity to ABA inhibition of root growth in seedlings of a T-DNA insertion mutant of <i>ATHB16</i> and of transgenic plants with elevated <i>ATHB16</i> levels. </p><p>HDZip proteins bind DNA as dimers. DNA-binding studies showed that different HDZip proteins interact with very similar target sequences <i>in vitro</i> and that they selectively form heterodimers with each other. For example, it was demonstrated that ATHB16 can heterodimerize with ATHB6 and ATHB7 in yeast and with ATHB5 <i>in vitro</i>, suggesting that ATHB16 may interact with other HDZip proteins in Arabidopsis. This interaction may have functional significance, since it may provide a mechanism for the plant to integrate different input signals, like light of different spectral qualities and water availability in the regulation of its growth. </p>

There are 42 members of the homeodomain-leucine zipper (HDZip) family of transcription factors in Arabidopsis thaliana. This thesis focuses on the functional analysis of one member of this family, ATHB16, and on the biochemical properties of HDZip proteins. To assess the function of the ATHB16 gene, the expression of ATHB16 was altered in transgenic Arabidopsis plants by using sense and antisense RNA constructs under the control of the 35S promoter. The reciprocal phenotypic effects associated with elevated and reduced levels of ATHB16 expression suggested that, in wild-type plants, ATHB16 acts as a mediator of blue and red light effects on the regulation of plant growth and the timing of the floral transition. In wild-type Arabidopsis, expression of ATHB16 is high in leaves, intermediate in adult roots and inflorescences, and low in stems and siliques. The expression of ATHB16 in the root is markedly increased in response to exogenous abscisic acid (ABA) treatment, but is reduced in the ABA response mutants abil and abi2, suggesting that ATHB16 may be involved in ABA signal transduction. This hypothesis was corroborated by observations of alterations in sensitivity to ABA inhibition of root growth in seedlings of a T-DNA insertion mutant of ATHB16 and of transgenic plants with elevated ATHB16 levels. HDZip proteins bind DNA as dimers. DNA-binding studies showed that different HDZip proteins interact with very similar target sequences in vitro and that they selectively form heterodimers with each other. For example, it was demonstrated that ATHB16 can heterodimerize with ATHB6 and ATHB7 in yeast and with ATHB5 in vitro, suggesting that ATHB16 may interact with other HDZip proteins in Arabidopsis. This interaction may have functional significance, since it may provide a mechanism for the plant to integrate different input signals, like light of different spectral qualities and water availability in the regulation of its growth.

The main objective of the reported study was to investigate how biomedical ontologies, logically structured representations of various aspects of the biomedical reality, can help researchers in analyzing experimental data. The dissertation reports two attempts to construct tools for the analysis of high-throughput experimental results using explicit domain knowledge representations. Furthermore, integrative efforts made by the community of Open Biomedical Ontologies (OBO), in which the author has participated, are reported, and a framework for consistently connecting the Gene Ontology (GO) with the Taxonomy of Species is proposed and discussed.

A majority of methods for identifying sequences in the human genome involve target sequence amplification through PCR. This work presents novel methods for amplifying circularized DNA and presents solutions for some major limitations of PCR. We have developed a novel method to amplify circularized DNA molecules based on a serial rolling-circle replication reaction, called circle to circle amplification (C2CA). Amplified DNA circles can be detected in array-based analyses or in real-time using molecular beacons. The amplification mechanism allows higher precision in quantification than in exponential amplification methods like PCR, and more products can be generated than in PCR. A major limitation of PCR is that amplification artifacts arise when large numbers of specific primer pairs are simultaneously added to a reaction. We have developed a solution to this problem that enables multiplex PCR amplification of specific target sequences without producing amplification artifacts. The procedure is based on oligonucleotide constructs, called selectors. The selectors identify defined target nucleic acid sequences, and they act as ligation templates to direct circularization of these targets. The selectors contain a general primer-pair motif that allows the circularized targets to be amplified in multiplex using a universal PCR primer pair. We also developed a computer program, PieceMaker, that finds an optimal design of selector probes for a given selector application. We demonstrate the method by performing a 96-plex PCR of specific DNA sequences with high success-rate and reproducibility.

Over the last few years, genome-wide association studies (GWAS) have been used to identify numerous obesity associated SNPs in the human genome. By using linkage studies, candidate obesity genes have been identified. When SNPs in the first intron of FTO were found to be associated to BMI, it became the first gene to be linked to common obesity. In order to look for causative explanations behind the associated SNPs, a re-sequencing of FTO had been performed on the SOLiD sequencing platform. In-house candidate gene, SLCX, was also sequenced in order to evaluate a potential obesity association. The purpose of this project was to analyse the sequences and also to evaluate the quality of the SOLiD sequencing. A part of the project consisted in performing PCRs and selecting genomic regions for future sequencing projects. I developed and implemented a sequence analysis strategy to identify obesity associated SNPs. I found 39 obesity-linked SNPs in FTO, a majority of which were located in introns 1 and 8. I also identified 3 associated intronic SNPs in SLCX. I found that the SOLiD sequencing coverage varies between non-repetitive and repetitive genomic regions, and that it is highest near amplicon ends. Interestingly, coverage varies significantly between different amplicons even after repetitive sequences have been removed, which indicates that it is affected by features inherent to the sequence. Still, the observed allele frequencies for known SNPs were highly correlated with the SNP frequencies documented in HapMap. In conclusion, I verify that SNPs in FTO are associated with obesity and also identify a previously unassociated gene, SLCX, as a potential obesity gene. Re-sequencing of genomic regions on the SOLiD platform was proven to be successful for SNP identification, although the difference in sequencing coverage might be problematic.

<p>The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.</p>

The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.

Our understanding of tree growth and development has increased substantially in the last few decades and is expected to increase much more as we fully exploit newly developed genomic tools. A major milestone in tree genomics was the sequencing of the entire genome of Populus trichocarpa, and the realization that we understand the function of very few of the 45,000 predicted genes in this genome. To advance our knowledge of gene function in Populus, we have created the largest population of mutant poplars to date which will enable us to link altered phenotypes with genes that are responsible. This thesis describes this mutant population, provides preliminary results on the complexity of mutants identified and examines one distinct mutant called shriveled leaf. These results clearly demonstrate the power of this population for gene function analysis and reveal that this population will be a valuable genomic resource for tree biotechnology for many years to come. / Thesis (Master, Biology) -- Queen's University, 2008-01-31 16:09:03.458