The studies in this thesis describe the application of genotyping and allele specific expression analysis to genetic studies. The role of the gene NPC1 in Triglyceride metabolism was explored in mouse models and in humans on the population level in study I. NPC1 was found to affect hepatic triglyceride metabolism, and to be relevant for controlling serum triglyceride levels in mice and potentially in humans. In study II the utility of the HapMap CEU samples was investigated for tagSNP selection in six European populations. The HapMap CEU was found to be representative for tagSNP selection in all populations while allele frequencies differed significantly in the sample from Kuusamo, Finland. In study III the power of Allele specific expression as a tool for the mapping of cis-regulatory variation was compared to standard eQTL analysis, ASE was found to be the more powerful type of analysis for a similar sample size. Finally ASE mapping was applied to regions reported to harbour long non-coding RNAs and associated SNPs were compared to published trait-associations. This revealed strong cis-regulatory SNPs of long non-coding RNAs with reported trait or disease associations.

Introdução: O sequenciamento completo do genoma humano foi finalizado em 2003, tornando-se responsável por uma revolução na prática médica, geralmente referenciada como a era da genômica e da medicina personalizada. O aumento na demanda por testes genéticos e a introdução de novos métodos de sequenciamento em larga escala tem gerado um inevitável crescimento no número de variantes raras mapeadas. Tal fato levou à expansão de áreas do conhecimento necessárias, tanto para uma adequada avaliação do real significado clínico desses achados, como para uma indicação mais criteriosa da investigação genética. Devido a isso, torna-se necessário otimizar os processos, desde a seleção dos casos sob suspeita até a coleta, armazenamento e análise dos dados clínico-laboratoriais e moleculares obtidos, visando a uma melhor acurácia no diagnóstico, tratamento e aconselhamento genético. Uma obtenção e interpretação adequada dos achados genético-moleculares, aliados a critérios clínicos de seleção adequados, resultaria no que hoje constitui a análise genética multifatorial ou integrada. Objetivo: O presente trabalho teve como objetivo implementar a análise genético-molecular integrada ao processo de investigação de pacientes com diagnóstico clínico de MODY (Maturity-Onset Diabetes of the Young). Materiais e métodos: A prevalência dos dois subtipos mais frequentes de MODY (MODY-GCK e MODY-HNF1A) foi investigada em uma grande coorte de famílias brasileiras. Todas as variantes identificadas como relacionadas ao fenótipo foram criteriosamente classificadas quanto a suas reais evidências de patogenicidade por meio da implementação das mais recentes diretrizes de obtenção e interpretação de achados genéticos. Resultados: Cento e nove probandos foram investigados, 45% (49/109) com suspeita clínica de MODYGCK e 55% (60/109) de MODY-HNF1A, além de 94 familiares, o que resultou na identificação de 25 variantes únicas candidatas no gene GCK em 30 probandos (61% - 30/49), além de 7 em 10 indivíduos com suspeita de MODYHNF1A (17% - 10/60). Dos 87 familiares sob risco rastreados, 43 eram portadores da mesma variante da família (37 - GCK e 6 - HNF1A). Um provável efeito fundador foi identificado com relação a uma deleção/inserção do tipo frameshift, presente em três probandos com MODY-GCK oriundos da mesma região do país. A implementação dos critérios da ACMG (The American College of Medical Genetics and Genomics) de avaliação de evidências de patogenicidade resultou na classificação de uma grande parcela das variantes como patogênica (36% - GCK / 86% - HNF1A) e provavelmente patogênica (44% - GCK / 14% - HNF1A), restando 16% com uma associação ainda incerta com o fenótipo investigado. Quatorze novas variantes foram identificadas (12 - GCK / 2 - HNF1A), ampliando, assim, o espectro de alterações associadas a MODY. Conclusões: Esta abordagem investigativa nos permitiu não apenas esclarecer a etiologia genética de inúmeros casos com diagnóstico clínico de MODY, como também determinar a classificação de patogenicidade das variantes de uma maneira mais detalhada, reforçando, principalmente, a provável relação com o fenótipo dentre aquelas ainda não descritas / Introduction: The full sequencing of the human genome was completed in 2003, becoming responsible for a revolution in medical practice, generally referred to as the era of genomics and personalized medicine. The increase in demand for genetic testing and the introduction of new methods of large-scale sequencing has raised an inevitable growth in the number of mapped rare variants. This fact led to the expansion of areas of knowledge required for a proper evaluation of the real clinical significance of these findings, as to a more solid indication of genetic research. Because of this, it is necessary to optimize the processes, from the selection of cases under suspicion until the collection, storage and analysis of the clinical laboratory and molecular data obtained, aiming a better accuracy in diagnosis, treatment and genetic counseling. A proper collection and interpretation of the molecular genetic findings, coupled with clinical criteria for appropriate selection would result in what today is the multifactorial or integrated genetic analysis. Objective: This current study aimed to implement the integrated molecular genetic analysis to the investigation process of patients with clinical diagnosis of MODY (Maturity Onset Diabetes of the Young). Materials and methods: The prevalence of the two most common MODY subtypes (MODYGCK and MODY-HNF1A) was investigated in a large cohort of Brazilian families. All variants identified as related to the phenotype were carefully classified to their actual evidence of pathogenicity by implementing the latest guidelines for obtainment and interpretation of genetic findings. Results: 109 probands were investigated, 45% (49/109) with clinical suspicion of MODY-GCK and 55% (60/109) of MODY-HNF1A, plus 94 family members under risk, which resulted in the identification of 25 unique variants candidates in the gene GCK in 30 probands (61% - 30/49), and also 7 in 10 individuals with suspected MODYxvi HNF1A (17% - 10/60). Of the 87 family members under risk, 43 were carriers of the same variant of the family (37 - GCK and 6 - HNF1A). A probable founding effect was identified, related to a frameshift deletion/insertion, present in three probands with MODY-GCK from the same region of the country. The implementation of the ACMG guidelines (The American College of Medical Genetics and Genomics) of evidence of pathogenicity assessment resulted in the classification of a large portion of the variants as pathogenic (36% - GCK / 86% - HNF1A) and likely pathogenic (44% - GCK / 14% - HNF1A), leaving 16% with a still uncertain association with the investigated phenotype. Fourteen new variants were identified (12 - GCK / 2 - HNF1A), broadening the spectrum of modifications associated to MODY. Conclusions: This investigative approach allowed us to not only clarify the genetic etiology of numerous cases with clinical diagnosis of MODY, as well as determine the pathogenicity classification of variants in a more detailed way, reinforcing the likely relationship with the phenotype among those not yet described

No description available.

<p dir="ltr">Humans encode approximately 100 deubiquitinating enzymes (DUBs) which are categorized into seven distinct subfamilies. Each family and representative has a unique expression, function and binding topology to ubiquitin. In addition to human DUBs, parasites, bacteria, and viruses contain DUBs with unique structures and functions. One subfamily of DUBs, the ubiquitin C-terminal hydrolases (UCH), has four structurally similar human members and two known members within the <i>Plasmodium falciparum</i> genome. Human UCHL1 and UCHL3 are genetically validated targets in oncology and <i>Plasmodium falciparum</i><i> </i>UCHL3 (PfUCHL3) is a prospective target for antimalarial drug development. Though these three UCH enzymes have potential as therapeutic targets, there is a significant lack of quality small molecule chemical probes to understand the underlying biology and function of the enzymes, pharmacologically validate the targets, and serve as leads for drug development in oncology and malaria.</p><p dir="ltr">The UCH enzymes are cysteine proteases, which our lab has leveraged to identify novel covalent small molecule inhibitors of each enzyme. The workflow for each hit identification and optimization campaign is similar. Covalent fragment screening of electrophilic small molecule libraries against the respective recombinant enzyme was performed to identify chemical space around each enzyme. Subsequent medicinal chemistry hit-to-lead optimization was undertaken to improve upon the moderately potent hit molecules to provide improved small molecule inhibitors for each enzyme. Inhibitor identification and optimization for UCHL1 is described in Chapter 2, revealing a novel scaffold and a cocrystal structure reveals a unique binding pose for UCHL1 inhibitors. These molecules were also characterized in breast cancer cells to validate UCHL1 as a therapeutic target in breast cancer. First-in-class covalent inhibitors of UCHL3 are described in Chapter 3. Medicinal chemistry optimization along with a cocrystal structure of the initial hit has revealed the molecular interactions of this novel inhibitory scaffold. PfUCHL3 inhibitor identification is described in Chapter 4. Characterization of these molecules against Plasmodium falciparum is described along with a comparison to a recently identified reversible PfUCHL3 inhibitor. Finally, conclusions and future directions toward the development of potent, drug-like inhibitors of each UCH enzyme is presented in Chapter 5.</p>

No description available.

<p>Most human traits and common diseases have a complex genetic makeup involving more than one gene. The work presented in this thesis investigates standing body height and the common disease type 2 diabetes mellitus (T2DM). In study I we analyzed two single nucleotide polymorphisms (SNPs) in the TCF7L2 gene that had been shown to be associated with T2DM. Analysis was performed in the ULSAM population cohort of ~1500 males. We were able to replicate the association to type 2 diabetes and in addition to that we made a novel find, showing association between the risk alleles and increased proinsulin levels. In study II we analyzed four genes identified to be associated with T2DM in a genome-wide association study. We analyzed SNPs in these genes in the ULSAM population cohort and found an association between SNPs in the HHEX gene and insulin responses and insulin levels. </p><p>The aim of studies III-V was to identify genes affecting normal variation in standing body height. Using a candidate gene approach in study III, 17 genes were screened in the ULSAM population cohort using SNPs. A suggestive association of the ESR1 gene with height was found and confirmed as significant in males from the PIVUS population cohort. In study IV, as a part of the GenomEUtwin project, we performed genetic fine mapping of a linked locus for body height on the X-chromosome. By analyzing 1377 SNPs in 780 Finnish twins, we mapped a region spanning 65kb of this locus with linkage to body height in males. This region contains the GPC3 and PHF6 genes that have known connections to syndromes were standing body height is affected. In study V significant linkage and association to standing body height in males was found for the COL1A11 gene, using population cohorts from Finland and Iceland. </p>

Most human traits and common diseases have a complex genetic makeup involving more than one gene. The work presented in this thesis investigates standing body height and the common disease type 2 diabetes mellitus (T2DM). In study I we analyzed two single nucleotide polymorphisms (SNPs) in the TCF7L2 gene that had been shown to be associated with T2DM. Analysis was performed in the ULSAM population cohort of ~1500 males. We were able to replicate the association to type 2 diabetes and in addition to that we made a novel find, showing association between the risk alleles and increased proinsulin levels. In study II we analyzed four genes identified to be associated with T2DM in a genome-wide association study. We analyzed SNPs in these genes in the ULSAM population cohort and found an association between SNPs in the HHEX gene and insulin responses and insulin levels. The aim of studies III-V was to identify genes affecting normal variation in standing body height. Using a candidate gene approach in study III, 17 genes were screened in the ULSAM population cohort using SNPs. A suggestive association of the ESR1 gene with height was found and confirmed as significant in males from the PIVUS population cohort. In study IV, as a part of the GenomEUtwin project, we performed genetic fine mapping of a linked locus for body height on the X-chromosome. By analyzing 1377 SNPs in 780 Finnish twins, we mapped a region spanning 65kb of this locus with linkage to body height in males. This region contains the GPC3 and PHF6 genes that have known connections to syndromes were standing body height is affected. In study V significant linkage and association to standing body height in males was found for the COL1A11 gene, using population cohorts from Finland and Iceland.

This thesis describes three readout formats for molecular analyses. A common feature in all works is probing techniques that upon specific target recognition ideally results in equimolar amounts of DNA circles. These are then specifically amplified and detected using any of the techniques presented herein. The first paper presents a method that enables homogeneous digital detection and enumeration of biomolecules, represented as fluorescence-labelled DNA macromolecules. This method offers precise measurements to be performed with a wide linear dynamic range. As an application, two closely related bacterial species were selectively detected. The second paper further investigates and optimizes the properties of the technique presented in paper one. The third paper demonstrates a platform that enables simultaneous quantitative analysis of large numbers of biomolecules. The array format and decoding scheme together propose a digital strategy for decoding of biomolecules. The array and the decoding procedure were characterized and evaluated for gene copy-number measurements. The fourth paper examines a new strategy for non-optical measurements of biomolecules. Characteristics of this technique are investigated, and compared to its optical equivalent, fluorescence polarization.

The chimpanzee is humankind’s closest living relative and the two species diverged ~6 million years ago. Comparative studies of the human and chimpanzee genomes and transcriptomes are of great interest to understand the molecular mechanisms of speciation and the development of species-specific traits. The aim of this thesis is to characterize differences between the two species with regard to their genome sequences and the resulting transcript profiles. The first two papers focus on indel divergence and in particular, indels causing premature termination codons (PTCs) in 8% of the chimpanzee genes. The density of PTC genes is correlated with both the distance to the telomere and the indel divergence. Many PTC genes have several associated transcripts and since not all are affected by the PTC we propose that PTCs may affect the pattern of expressed isoforms. In the third paper, we investigate the transcriptome divergence in cerebellum, heart and liver, using high-density exon arrays. The results show that gene expression differs more between tissues than between species. Approximately 15% of the genes are differentially expressed between species, and half of the genes show different splicing patterns. We identify 28 cassette exons which are only included in one of the species, often in a tissue-specific manner. In the fourth paper, we use massive parallel sequencing to study the chimpanzee transcriptome in frontal cortex and liver. We estimate gene expression and search for novel transcribed regions (TRs). The majority of TRs are located close to genes and possibly extend the annotations. A subset of TRs are not found in the human genome. The brain transcriptome differs substantially from that of the liver and we identify a subset of genes enriched with TRs in frontal cortex. In conclusion, this thesis provides evidence of extensive genomic and transcriptomic variability between human and chimpanzee. The findings provide a basis for further studies of the underlying differences affecting phenotypic divergence between human and chimpanzee.

Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity. Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections. To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays. The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.