Orientador: Lilian Maria de Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faucldade de Ciencias Medicas / Made available in DSpace on 2018-08-12T08:43:08Z (GMT). No. of bitstreams: 1 Omoto_Ricardo_M.pdf: 806455 bytes, checksum: 919c6c6ffefecaf298fe52f6907eabfc (MD5) Previous issue date: 2008 / Resumo: Introdução: As bases moleculares responsáveis pelas variantes do gene GYPB ainda não estão estabelecidas para a população de Brasileiros descendentes de Africanos. O presente estudo foi realizado para analisar os mecanismos moleculares que originam o fenótipo S-s- e determinar a frequência do gene GYPB*S silencioso no fenótipo S-s+, em uma população de doadores de sangue descendentes de Africanos. Materiais e Métodos: Foram selecionadas 165 amostras de sangue de Brasileiros descendentes de Africanos (Nordeste do Brasil) fenotipados como S-s- (n=17) e S-s+ (n=148) por hemaglutinação. Com a finalidade de identificar as formas variantes do gene GYPB, realizamos a genotipagem dessas amostras pelas técnicas de PCR Alelo-específico (AS-PCR) e PCR-RFLP. Resultados: Em 13 das 17 amostras S-s- (76,5%) ambos os alelos GYPB*S e GYPB*s estavam deletados. Em 137 das 148 amostras fenotipadas como S-s+ (92,6%), o resultado da genotipagem pela técnica AS-PCR foi consistente com o fenótipo S-s+. Em 4 das amostras S-s- (23,5%) e em 11 das amostras S-s+ (7,4%) foi identificada a presença do alelo GYPB*S associado com o silenciamento do antígeno S. Das amostras de DNA de doadores com o fenótipo S-s- que demonstraram a presença do alelo GYPB*S, 2 apresentaram a variante GYP(P2), 1 a variante GYP(NY) e, em 1 amostra encontramos ambas as formas variantes de GYPB: GYP(P2) e GYP(NY) Em 11 doadores com o fenótipo S-s+ houve heterozigosidade para o alelo GYP(P2) (n=8) e heterozigosidade para o alelo GYP(NY) (n=3). Conclusão: Esse estudo relata pela primeira vez os mecanismos moleculares responsáveis pelo fenótipo S-s- em uma população de Brasileiros descendentes de Africanos e promove o conhecimento de uma nova informação sobre a frequência (7,4%) e as bases moleculares do gene GYPB*S silencioso nesta população. / Abstract: Background: The molecular background of variant forms of GYPB is not well studied in Brazilians of African descent. The present study was carried out to determine the molecular bases of the S-s- phenotype and the frequency of GYPB*S silent gene for the S-s+ phenotype in a blood donor population of African Brazilians. Methods: We selected 165 blood samples from African Brazilians (Northeastern Brazil) who phenotyped as S-s- (17) and S-s+ (148) by hemagglutination. AS-PCR and PCR-RFLP were used to identify the variant forms of GYPB. Results: In 13 of 17 S-s- samples (76.5%) both GYPB were deleted. In 137 of the 148 S-s+ samples (92.6%), the AS-PCR was consistent with the S-s+, phenotype. In 4 of the S-s- samples (23.5%) and 11 of the S-s+ samples (7.4%) showed the presence of the GYPB*S allele associated with silencing of the S antigen. In the 4 donors with the S-s- phenotype there was homozygosity (or hemizygosity) for the GYP(P2) allele (n=2), homozygosity (or hemizygosity) for the GYP(NY) allele (n=1) and heterozygosity for the GYP(P2) and GYP(NY) alleles (n=1). In the 11 donors with the S-s+ phenotype there was heterozygosity for GYP(P2) allele (n=8) and heterozygosity for GYP(NY) allele (n=3). Conclusion: This study reports for the first time the molecular mechanisms responsible for the S-s- phenotype in a population of African Brazilians and provides a new information about the frequency and molecular bases of GYPB*S silent gene (7.4%) in this population. / Mestrado / Clinica Medica / Mestre em Clinica Medica

Orientador: Carmen Sílvia Bertuzzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T19:39:30Z (GMT). No. of bitstreams: 1 Marcelino_AlineRobertaBariani_M.pdf: 1077026 bytes, checksum: b7315e705aba4125ea2a4e2574a78b6b (MD5) Previous issue date: 2013 / Resumo: A Fibrose Cística (FC) é uma doença autossômica recessiva causada por mutações no gene CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) que acarretam em defeito ou na ausência da proteína por ele sintetizada. A CFTR, produto deste gene é uma proteína canal, localizada na membrana apical das células, responsável pela condução de íons cloreto. As mutações levam à ausência da proteína CFTR ou a alteração qualitativa/quantitativa da proteína, que acarreta no desequilíbrio osmótico entre os meios intra e extracelular. Como consequência há a ocorrência de muco viscoso e de difícil excreção nos pulmões e obstrução dos ductos pancreáticos, afetando desta forma o sistema respiratório e digestório. São conhecidas mais de 1.900 mutações no gene CFTR, sendo que mesmo em pacientes com mutações iguais como a F508del - com alta prevalência na população brasileira - há divergência entre os fenótipos observados. Dessa forma, o genótipo CFTR parece não ser determinante na modulação da gravidade clínica, uma vez que, indivíduos com mesmo genótipo CFTR apresentam manifestações clínicas diferentes. Outros genes, diferentes do CFTR foram associados à gravidade clínica dos pacientes, revelando que os produtos por eles expressos exercem algum tipo de ação modificadora do fenótipo da FC. Tais genes foram denominados modificadores e atuam em fatores secundários relacionados à evolução do quadro clínico, como a articulação do sistema imune. Os genes COX2 e IFRD1, com ação importante no sistema imune e no recrutamento de células de defesa foram identificados como modificadores da FC em estudos prévios realizados em uma população diferente da brasileira. No presente estudo, os polimorfismos -765G>C e 8473T>C no gene COX2 e 57460C>T no gene IFRD1, candidatos a modificadores, foram identificados nos pacientes e um estudo de associação genótipo-fenótipo foi conduzido a fim de verificar a ação moduladora de tais polimorfismos nos pacientes estudados. Nenhuma associação foi encontrada, exceto para o íleo meconial (p=0,028 - em pacientes com duas mutações identificadas no gene CFTR pertencentes à classe I, II e III) e para a polipose nasal (p=0,022 - em pacientes sem considerar o genótipo CFTR) para o polimorfismo 8473T>C no gene COX2 / Abstract: Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene mutations that lead to defective polypeptide or lack of the protein CFTR. The CFTR is a channel protein located in the apical cells membrane, responsible for chloride ions conductance. The mutations lead to an osmotic disequilibrium between intra and extracellular mediums, which causes viscous mucus production that is hard to be eliminated from lungs and pancreatic ducts, affecting, this way, respiratory and digestive systems. More than 1,900 CFTR different mutations are known, and even patients that carries identical mutations as F508del - the most common one in Brazilian population - shows a great discrepancy between the phenotypes that are observed. Thus, CFTR genotype seems not to be crucial in disease clinical course modulation, once different subjects carrying the same CFTR mutations reveal distinctive clinical manifestations. Genes besides CFTR were associated to CF patients clinical manifestation, revealing that the molecules they express have some kind of modifier activity in CF phenotype. Such genes were labeled as modifier genes and they act in secondary factors related to clinical course evolution as immune system response. The genes COX2 and IFRD1 have an important role in immune system and defense cell recruitment and they were identified as CF modifiers in previous studies that analyzed different population from the Brazilian one. In this current study, the polymorphisms -765G>C, 8473T>C and 57460C>T located in these genes were identified in our patients and association genotype-phenotype were carried out in order to verify the modulator activity of such variants in the studied casuistic. There was not found association, except for meconium ileus (p=0,028 - in patients with two CFTR mutations from class I, II and III) and for nasal polyposis (p=0,022 - in patients whose CFTR genotype was not considered) to 8473T>C polymorphism in COX2 gene / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Background: The clinical and pathological presentation of COPD is heterogeneous. ‘Chronic bronchitis’ is a phenotype of COPD, which is a clinical diagnosis of a productive cough of ≥ 3 months for ≥ 2 consecutive years. Chronic bronchitis is associated with worse lung function, frequent exacerbations, recurrent hospitalisations and premature death in patients with COPD. Chronic bronchitis sufferers can be further subphenotyped into those who produce sputum during exacerbation or during winter months only and those who are ‘persistent sputum producers,’ who experience mucous hypersecretion throughout the year. An improved understanding of persistent sputum producers is the object of this thesis. Aims: 1) To compare the clinical characteristics and airway inflammatory biomarker profile of COPD persistent sputum producers to that of COPD sputum non-producers 2) To investigate the short term repeatability of sputum parameters in COPD persistent sputum producers 3) To study the expression and relationship of mucins, hypoxia inducible factor (HIF-1α) and carbonic anhydrase IX (CAIX) in COPD persistent sputum producers. Methods: 1) Lung physiology, health status, sputum inflammatory biomarkers and sputum culture results were compared between COPD persistent sputum producers and sputum non-producers 2) Repeatability of spontaneous and induced sputum parameters at 8 weeks was assessed in COPD persistent sputum producers 3) Immunohistochemistry was performed on bronchial biopsies of COPD persistent sputum producers and control groups (COPD sputum non-producers, smokers with normal lung function and lifelong healthy non-smokers with normal lung function) to study the expression of MUC5AC, MUC5B, HIF-1α and CAIX 4) The association between HIF-1α and MUC5B expression was investigated in vitro. Results and Conclusions: The findings suggest that 1) COPD persistent sputum producers have clinically more severe disease, increased airway inflammation, increased impact on health status, increased rate of bacterial colonization and higher number of exacerbations compared to COPD sputum non-producers 2) Induced sputum is repeatable over short term in COPD persistent sputum producers 3) Expression of MUC5B, HIF-1α and CAIX is increased in COPD persistent sputum producers compared to COPD sputum non-producers, smokers with normal lung function and healthy non-smokers 4) HIF-1α can potentially cause increased MUC5B expression. This work reveals potential targets for the development of novel therapies to limit mucous hypersecretion in COPD.

Campylobacter species are the primary cause of bacterial food-borne diarrhoea worldwide. Comparative genomic analyses of Campylobacter strains reveal genome plasticity providing insight into the evolution of virulence traits. The goal of this study was to identify genes important for infectivity and for naturally occurring variability in phenotypic traits in C. jejuni and C. coli strains. Transcriptome and phenotype analyses were conducted to determine if genetic and phenotypic characteristics could be attributed to the source of the strains. Isolates from water sources had higher biofilm formation than animal strains. Clinical strains had decreased sensitivity to hydrogen peroxide as well as increased adherence and invasion when compared to animal strains. A number of genetic differences were observed; however, without further analysis it is difficult to determine which of these impact virulence in Campylobacter. Ultimately, this project will lead to the identification of markers associated with strains of Campylobacter causing illness.

Infectious diseases that spread from person-to-person and continent-to-continent are a cause for concern for any health entity. One such disease is malaria, a mosquito-borne infection instigated by the protozoan parasite, Plasmodium falciparum. Hundreds of millions of people are affected annually and it is responsible for nearly 1 million deaths. It is the most fatal species causing malaria and proliferates in human red blood cells with a life cycle occurring every 48 hours. At this time, the parasite’s late stage form or schizont bursts from the erythrocyte releasing immune-inducing particles and infective forms (merozoites) into the bloodstream. The merozoites go on to infect other red blood cells as human immunity leads to fever. Fever is a hallmark symptom of malaria and effectively inhibits the growth of late stage parasites. Plasmodium still manages to complete its life cycle as early stages or rings are not affected by febrile temperatures. It is this facet of parasite biology that prompts our research into identifying genetic factors associated with fever. The parasite’s response under elevated body temperature may offer further insight into its adaptive mechanism. A heat shock assay was developed in order to simulate fever in vitro. Mutant parasite cultures were subjected to 41°C for 8 hours and returned to normal body temperature or 37°C for the remainder of the life cycle. The piggyBac mutagenesis system allows for the evaluation of phenotypes associated with a particular genotype as the transposon inserts randomly into the gene. This often leads to changes in function that may cause delays in invasion or attenuation of growth. Determining the genes responsible for these phenotypes would be a great advantage to the field of drug discovery. Collaborative efforts to develop vaccines and new antimalarial drugs are underway as resistance to current methods of treatment is on the rise. Such circumstances require new technologies for detecting novel drug targets or pathways in the parasite that can be significantly affected by these therapeutics. QISeq is a next generation sequencing tool that identifies genes with a particular phenotype that may alter intraerythrocytic development of P. falciparum. This technique was utilized in our study to confirm the heat shock phenotype with a high-throughput approach. The genomic DNA of pooled parasite cultures was sequenced to reveal those mutants sensitive and/or resistant to febrile temperature exposure. Through bioinformatics analyses, functional associations between genes can be made that lead to biological pathways of interest for therapeutic research.

Autism spectrum disorders (ASD) are a group of related neurodevelopmental diseases displaying significant genetic and phenotypic heterogeneity. Despite recent progress in ASD genetics, the nature of phenotypic heterogeneity across probands is not well understood. Notably, likely gene-disrupting (LGD) de novo mutations affecting the same gene often result in substantially different ASD phenotypes. We find that truncating mutations in a gene can result in a range of relatively mild decreases (15-30%) in gene expression due to nonsense-mediated decay (NMD), and show that more severe autism phenotypes are associated with greater decreases in expression. We also find that each gene with recurrent ASD mutations can be described by a parameter, phenotype dosage sensitivity (PDS), which characterizes the relationship between changes in a gene’s dosage and changes in a given phenotype. Using simple linear models, we show that changes in gene dosage account for a substantial fraction of phenotypic variability in ASD. We further observe that LGD mutations affecting the same exon frequently lead to strikingly similar phenotypes in unrelated ASD probands. These patterns are observed for two independent proband cohorts and multiple important ASD-associated phenotypes. The observed phenotypic similarities are likely mediated by similar changes in gene dosage and similar perturbations to the relative expression of splicing isoforms. We also identify patterns of developmental and cell type-specific expression that additionally contribute to the variability of several autism phenotypes.

In biomedical research, ontologies are widely used to represent knowledge as well as to annotate datasets. Many of the existing ontologies cover a single type of phenomena, such as a process, cell type, gene, pathological entity or anatomical structure. Consequently, there is a requirement to use multiple ontologies to fully characterize the observations in the datasets. Although this allows precise annotation of different aspects of a given dataset, it limits our ability to use the ontologies in data analysis, as the ontologies are usually disconnected and their combinations cannot be exploited. Motivated by this, here we present novel ontology design methods for combining pathology and anatomy concepts. To this end, we use a dataset of mouse models which has been characterized through two ontologies: one of them is the mouse pathology ontology (MPATH) covering pathological lesions while the other is the mouse anatomy ontology (MA) covering the anatomical site of the lesions. We propose four novel ontology design patterns for combining these ontologies, and use these patterns to generate four ontologies in a data-driven way. To evaluate the generated ontologies, we utilize these in ontology-based data analysis, including ontology enrichment analysis and computation of semantic similarity. We demonstrate that there are significant differences between the four ontologies in different analysis approaches. In addition, when using semantic similarity to confirm the hypothesis that genetically identical mice should develop more similar diseases, the generated combined ontologies lead to significantly better analysis results compared to using each ontology individually. Our results reveal that using ontology design patterns to combine different facets characterizing a dataset can improve established analysis methods.

No description available.

No description available.

The burden of liver diseases is increasing worldwide, accounting for two million deaths annually. In the past decade, tremendous progress has been made in the basic and translational research of liver tissue engineering, which seeks to build physiologically relevant liver models to better understand liver diseases, accelerate drug development, and advance regenerative medicine. Liver microtissues are small, three-dimensional (3D) hepatocyte cultures that recapitulate liver physiology and have been used in many biomedical applications. However, sourcing of high-quality human hepatocytes for microtissue fabrication poses a significant challenge. Since the inception of induced pluripotent stem cell (iPSC) technology, iPSC-derived hepatocyte-like cells (HLCs) have demonstrated significant improvement over other hepatocyte cell sources in many studies. Despite their promising potential, HLCs face certain challenges: they resemble fetal hepatocytes rather than adult hepatocytes; they undergo dedifferentiation quickly after reaching maturity; they are produced on a small scale; and they exhibit large donor-to-donor and batch-to-batch variability. This doctoral thesis focuses on engineering patient-specific liver microtissues with prolonged phenotypic maintenance and disease modeling potential. Chapter 1 provides a review of recent advances, challenges, and future directions in liver microtissue research. 3D microtissues can be generated by scaffold-free assembly or scaffold-assisted methods using macroencapsulation, droplet microfluidics, and bioprinting. Optimization of the hepatic microenvironment entails incorporating the appropriate cell composition for enhanced cell-cell interactions and niche-specific signals, and creating scaffolds with desired chemical, mechanical and physical properties. Perfusion-based culture systems such as bioreactors and microfluidic systems are used to achieve efficient exchange of nutrients and soluble factors in the microtissues. Chapter 2 describes our efforts in optimizing methods of generating human HLCs from the peripheral blood of selected donors. Peripheral blood mononuclear cells (PBMCs) were first reprogrammed to iPSCs using Sendai viruses carrying the four Yamanaka factors. We developed an optimized protocol for hepatocyte differentiation from iPSCs, and obtained HLCs that exhibited hepatocyte-specific phenotypes and functions that were comparable to other reports. We then demonstrated the one-step generation of homogeneous, microencapsulated liver microtissues in Chapter 3. Droplet microfluidics was used to produce double emulsion droplets that served as individual microenvironments where HLCs were encapsulated in methylated collagen and alginate. The cells self-assembled in <16 hours through dynamic interactions with methylated collagen, and individual spheroids were encapsulated in polymerized alginate gel to prevent cell fusion and attachment. HLC spheroids remained viable and functional for >24 days, whereas 2D HLCs underwent dedifferentiation within 7 days of reaching maturity. The spheroids showed further maturation compared to the 2D HLCs at peak maturity. Co-culture of HLCs with human endothelial cells was also investigated in the 3D system, but no improvement was observed over monoculture spheroids with our current methods. To our knowledge, this is the first study to utilize droplet microfluidics to generate homogeneous, compartmentalized droplets that serve as optimized 3D microenvironments for HLC aggregation and maturation. It demonstrated the potential of using high-throughput droplet microfluidics to produce and encapsulate mature, functional human HLCs for long-term applications. In Chapter 4, we developed a TM6SF2 knockout and overexpression model in iPSCs to investigate its molecular function and potential role in nonalcoholic fatty liver disease (NAFLD). Transmembrane 6 superfamily member 2 (TM6SF2) is a protein of unknown function, and analysis from our model suggested that TM6SF2 dysregulation has a biphasic response. Our data showed that both knockout and overexpression can result in the upregulation of cholesterol biosynthesis and a defect in the proper processing of lipid droplets. Additionally, high expression of the TM6SF2 rs58542926 variant has an increased risk for cholesterol upregulation, compared to the major allele. Future works will focus on generating liver microtissues from the TM6SF2 knockout and transgene-expressing cells using droplet microfluidics, and validating our hypotheses with established biochemical and functional assays.