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Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes. The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems. Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis. A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner. Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression. <b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling

<p>Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes.</p><p>The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems.</p><p>Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis.</p><p>A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner.</p><p>Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression.</p><p><b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling</p>

Today’s rapid development of powerful tools for geneexpression analysis provides unprecedented resources forelucidating complex molecular events. The objective of this workhas been to apply, combine andevaluate tools for analysis of differential gene expressionusing atherosclerosis as a model system. First, an optimisedsolid-phase protocol for representational difference analysis(RDA) was applied to twoin vitromodel systems. Initially, The RDA enrichmentprocedure was investigated by shotgun cloning and sequencing ofsuccessive difference products. In the subsequent steps,combinations of RDA and microarray analysis were used tocombine the selectivity and sensitivity of RDA with thehigh-throughput nature of microarrays. This was achieved byimmobilization of RDA clones onto microarrays dedicated forgene expression analysis in atherosclerosis as well ashybridisation of labelled RDA products onto global microarrayscontaining more than 32,000 human clones. Finally, RDA wasapplied for the investigation of the focal localisation ofatherosclerotic plaques in mice usingin vivotissue samples as starting material. A large number of differentially expressed clones wereisolated and confirmed by real time PCR. A very diverse rangeof gene fragments was identified in the RDA products especiallywhen they were screened with global microarrays. However, themicroarray data also seem to contain some noise which is ageneral problem using microarrays and should be compensated forby careful verification of the results. Quite a large number of candidate genes related to theatherosclerotic process were found by these studies. Inparticular several nuclear receptors with altered expression inresponse to oxidized LDL were identified and deserve furtherinvestigation. Extended functional annotation does not liewithin the scope of this thesis but raw data in the form ofnovel sequences and accession numbers of known sequences havebeen made publicly available in GenBank. Parts of the data arealso available for interactive exploration on-line through aninteractive software tool. The data generated thus constitute abase for new hypotheses to be tested in the field ofatherosclerosis. <b>Keywords:</b>representational difference analysis, geneexpression profiling, microarray analysis, atherosclerosis,foam cell formation

<p>Today’s rapid development of powerful tools for geneexpression analysis provides unprecedented resources forelucidating complex molecular events.</p><p>The objective of this workhas been to apply, combine andevaluate tools for analysis of differential gene expressionusing atherosclerosis as a model system. First, an optimisedsolid-phase protocol for representational difference analysis(RDA) was applied to two<i>in vitro</i>model systems. Initially, The RDA enrichmentprocedure was investigated by shotgun cloning and sequencing ofsuccessive difference products. In the subsequent steps,combinations of RDA and microarray analysis were used tocombine the selectivity and sensitivity of RDA with thehigh-throughput nature of microarrays. This was achieved byimmobilization of RDA clones onto microarrays dedicated forgene expression analysis in atherosclerosis as well ashybridisation of labelled RDA products onto global microarrayscontaining more than 32,000 human clones. Finally, RDA wasapplied for the investigation of the focal localisation ofatherosclerotic plaques in mice using<i>in vivo</i>tissue samples as starting material.</p><p>A large number of differentially expressed clones wereisolated and confirmed by real time PCR. A very diverse rangeof gene fragments was identified in the RDA products especiallywhen they were screened with global microarrays. However, themicroarray data also seem to contain some noise which is ageneral problem using microarrays and should be compensated forby careful verification of the results.</p><p>Quite a large number of candidate genes related to theatherosclerotic process were found by these studies. Inparticular several nuclear receptors with altered expression inresponse to oxidized LDL were identified and deserve furtherinvestigation. Extended functional annotation does not liewithin the scope of this thesis but raw data in the form ofnovel sequences and accession numbers of known sequences havebeen made publicly available in GenBank. Parts of the data arealso available for interactive exploration on-line through aninteractive software tool. The data generated thus constitute abase for new hypotheses to be tested in the field ofatherosclerosis.</p><p><b>Keywords:</b>representational difference analysis, geneexpression profiling, microarray analysis, atherosclerosis,foam cell formation</p>

Made available in DSpace on 2014-06-11T19:30:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-11-24Bitstream added on 2014-06-13T20:00:40Z : No. of bitstreams: 1 baeza_lc_dr_arafcf.pdf: 2250737 bytes, checksum: 666c7d374ebfa4ff8507fbfbe951a4b8 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / As dermatofitoses são processos infecciosos de pele, pêlo e unhas muito comuns no mundo inteiro. Trichophyton rubrum é o dermatófito mais freqüentemente isolado em lesões superficiais de pele e unha. Estudos envolvendo este patógeno são cada vez mais importantes devido ao aparecimento de linhagens resistentes aos medicamentos antifúngicos disponíveis no mercado e ao comportamento invasivo deste agente em pacientes com o sistema imune comprometido. Estes fatos e poucos estudos levam à necessidade de se ampliar o conhecimento sobre a biologia deste agente. Visando colaborar nesse sentido, o presente trabalho teve dois objetivos centrais: 1) Realizar a tipagem molecular de isolados clínicos de T. rubrum com e sem relação epidemiológica por RAPD (Amplificação Randômica de DNA Polimórfico) com duas seqüências randômicas diferentes (denominadas de 1 e 6), bem como a análise dos elementos repetitivos (TRS-1 e TRS-2) do espaço não transcrito (NTS) do DNA ribossomal (DNAr) e 2) Identificar transcritos envolvidos na interação deste patógeno com fonte humana de queratina, através do RDA (Análise de Diferença Representacional). A aplicação do RDA permitiu pela primeira vez a identificação de alguns transcritos expressos, provavelmente relacionados à patogênese deste microrganismo. / Dermatophytosis is common infection process which occurs in skin, hair and nails and Trichophyton rubrum is the most frequently isolated dermatophyte. Studies on this pathogen are becoming increasingly important because of frequent reports on resistant strains to antifungal drugs commercially available and the invasive behavior of these agents in immunocompromised patients. These facts, associated with few studies with this agent, indicate the need to expand the information about the fungal biology. As a contribution to this goal, the present study had two central objectives: 1) To compare different methodologies for molecular typing of clinical isolates of T. rubrum epidemiologically related and unrelated for RAPD (Randomly Amplified Polymorphic DNA) with two different random primers (denominated of 1 and 6); as well as the analysis of the repetitive elements (TRS-1 and TRS-2) of the space non-transcribed (NTS) of the ribosomal DNA (DNAr) and 2) To identify transcripts involved in the interaction of this pathogen with human keratin by RDA (Representational Difference Analysis). The application of the RDA allowed for the first time the identification of expressed transcripts during the microorganism proliferation that could be related to the T. rubrum virulence.

Orientador: Maria José Soares Mendes Giannini / Banca: Clarice Queico Fujimura Leite / Banca: Celia Maria de Almeida Soares / Banca: Paulo Inácio da Costa / Banca: Mário Hiroyuki Hirata / Resumo: As dermatofitoses são processos infecciosos de pele, pêlo e unhas muito comuns no mundo inteiro. Trichophyton rubrum é o dermatófito mais freqüentemente isolado em lesões superficiais de pele e unha. Estudos envolvendo este patógeno são cada vez mais importantes devido ao aparecimento de linhagens resistentes aos medicamentos antifúngicos disponíveis no mercado e ao comportamento invasivo deste agente em pacientes com o sistema imune comprometido. Estes fatos e poucos estudos levam à necessidade de se ampliar o conhecimento sobre a biologia deste agente. Visando colaborar nesse sentido, o presente trabalho teve dois objetivos centrais: 1) Realizar a tipagem molecular de isolados clínicos de T. rubrum com e sem relação epidemiológica por RAPD (Amplificação Randômica de DNA Polimórfico) com duas seqüências randômicas diferentes (denominadas de 1 e 6), bem como a análise dos elementos repetitivos (TRS-1 e TRS-2) do espaço não transcrito (NTS) do DNA ribossomal (DNAr) e 2) Identificar transcritos envolvidos na interação deste patógeno com fonte humana de queratina, através do RDA (Análise de Diferença Representacional). A aplicação do RDA permitiu pela primeira vez a identificação de alguns transcritos expressos, provavelmente relacionados à patogênese deste microrganismo. / Abstract: Dermatophytosis is common infection process which occurs in skin, hair and nails and Trichophyton rubrum is the most frequently isolated dermatophyte. Studies on this pathogen are becoming increasingly important because of frequent reports on resistant strains to antifungal drugs commercially available and the invasive behavior of these agents in immunocompromised patients. These facts, associated with few studies with this agent, indicate the need to expand the information about the fungal biology. As a contribution to this goal, the present study had two central objectives: 1) To compare different methodologies for molecular typing of clinical isolates of T. rubrum epidemiologically related and unrelated for RAPD (Randomly Amplified Polymorphic DNA) with two different random primers (denominated of 1 and 6); as well as the analysis of the repetitive elements (TRS-1 and TRS-2) of the space non-transcribed (NTS) of the ribosomal DNA (DNAr) and 2) To identify transcripts involved in the interaction of this pathogen with human keratin by RDA (Representational Difference Analysis). The application of the RDA allowed for the first time the identification of expressed transcripts during the microorganism proliferation that could be related to the T. rubrum virulence. / Doutor

Newts have the incredible ability to regenerate many different organs and tissues as adults, including the limbs. Limb regeneration occurs via the dedifferentiation of stump tissue and the formation of a blastema, which provides the majority of cells for the regenerate. Despite all that we have learned about dedifferentiation and blastema formation, the cellular and molecular mechanisms underlying these processes are still poorly understood. We used representational difference analysis (RDA) to identify genes involved in the early dedifferentiation process in newt forelimb regeneration. Our analysis identified approximately 410 unique genes that were differentially regulated during this process. Microarray analysis was used to determine the expression profile of these genes throughout limb and tail regeneration. We used quantitative PCR (qPCR) to validate the expression of a subset of these genes [β-catenin, wntless, dapper, thymosin-β 4 (Tβ4), and thymosin-β 10/15 (Tβ10/15)] in regenerating limb and tail tissue, as well as in differentiating newt myoblasts. We also verified the expression of these genes in the regenerating newt limb using immunohistochemistry (IHC) and in situ hybridization (ISH). Finally, we performed a functional analysis on β-catenin, wntless, dapper, and Tβ4 by overexpressing these genes in mouse myoblasts to examine their effects on differentiation and potential roles in dedifferentiation. Quantitative PCR verified the expression of β-catenin, wntless, dapper, and Tβ4 during limb regeneration and IHC/ISH localized the β-catenin and Tβ4 proteins to the blastema during regeneration. Tβ10/15 was shown by qPCR to be expressed in the tail during regeneration. Overexpression of newt β-catenin, wntless, dapper, and Tβ4 in mouse myoblasts showed that each of these genes has an inhibitory effect on the differentiation of myoblasts into myotubes and, therefore, may play a role in promoting or maintaining the dedifferentiated state. Our work has identified a large number of genes with potential roles in regulating the dedifferentiation process during newt forelimb regeneration. We have also laid a framework from which much more work can be done by drawing on the genes we have identified and the microarray data, which indicate ideal follow-up candidates. Our analysis of specific genes has also increased our understanding of the molecular events occurring during the dedifferentiation process in the regenerating newt limb.