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No. of bitstreams: 2 Dissertação - Anniely Carvalho Rebouças Oliveira - 2016.pdf: 1878998 bytes, checksum: 1afecfd5948d2abb2a5374cbeb9ff005 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The acute respiratory infections (ARIs) are an important cause of morbidity and mortality worldwide. Viruses have an important role in the etiology of the respiratory infections. Children less than five years of age have about four to six ARIs per year and this is a common cause of hospitalization, mainly in developing countries. In Brazil, particularly in the West-Central region, studies that evaluated the circulation of respiratory viruses in the pediatric population are scarce. Thus, this study aimed to investigate the occurrence of respiratory viruses in the pediatric population, from Goiânia-Goiás. Between May/2014 and May/2015, samples of nasal swabs were collected from children from zero to six years of age presenting or not respiratory symptoms, attended at the Materno Infantil hospital in Goiânia. For the molecular screening, 16 viruses were investiged using three Multiplex Nested-PCR protocols. It two hundred and fifty one samples were collected, of wich 90 (35,9%) were positive for at least one respiratory agent, being rhinovirus (31%), respiratory syncytial virus (27,4%) and parainfluenza virus (13,3%) the most prevalent. Children under two years of age had greater overall positive, although not statistically significant. Similar detection rate was observed among the groups symptomatic (37%) and asymptomatic (34,5%). For the symptomatic group, children whose samples were collected during the dry period (p<0,05) and those with up to two years of age were most affected (p<0,05). In the asymptomatic group no statistical difference was observed. It was observed rate of co-detection of 6,4% (16/251), mostly in samples of symptomatic patients (13/16) (p<0,05). The evaluation of the temporal profile showed a higher detection rate during the dry season, less rainfall, relative humidity and cooler temperatures. The results reinforce the importance of respiratory viruses in children and contribute to further understanding of the epidemiological and temporal factors associated with different pathogens in our region. Thus, they open the way to new studies in the state of Goiás, also providing information to assist in the construction of control measures and more effective prevention of these infections. / As infecções do trato respiratório (ITRs) representam importante causa de morbimortalidade em todo o mundo, sendo os vírus importantes agentes etiológicos. Estima-se que grande parte das crianças menores de cinco anos de idade tenha a cada ano de quatro a seis ITRs, sendo esta causa comum de hospitalização, especialmente em países em desenvolvimento. No Brasil, em particular na Região Centro-Oeste, estudos que avaliem a circulação de vírus respiratórios em população pediátrica são escassos. Neste contexto, o presente estudo teve como objetivo investigar a ocorrência de diferentes vírus respiratórios em população pediátrica de Goiânia-Goiás, apresentando ou não quadro sintomático respiratório. Durante o período de maio/2014 a maio/2015, foram coletadas amostras de swab respiratório de crianças entre zero e seis anos de idade, apresentando quadro de infecção respiratória ou assintomáticas para a mesma, atendidas no Hospital Materno Infantil, em Goiânia. As amostras foram submetidas à triagem molecular para 16 vírus respiratórios por meio de três protocolos de multiplex nested-PCR. No total, 251 amostras foram coletadas, das quais 90 (35,9%) apresentaram positividade para pelo menos um agente respiratório, sendo rinovírus (31%), vírus sincicial respiratório (27,4%) e parainfluenza (13,3%) os mais frequentes. Crianças com até dois anos de idade apresentaram maior positividade global, contudo sem diferença estatística. Índice de detecção semelhante foi observado entre os grupos sintomático (37%) e assintomático (34,5%). Para as crianças do grupo sintomático, foram mais afetadas aquelas cujas amostras foram coletadas durante período de seca (p<0,05) e aquelas com até dois anos de idade (p<0,05). No grupo assintomático, nenhuma diferença estatística foi observada. Foi observado índice de co-detecção de 6,4% (16/251), sendo a maioria de indivíduos sintomáticos (13/16) (p<0,05). A avaliação do perfil temporal demonstrou maior índice de detecção nos meses de seca, com menor precipitação pluviométrica, umidade relativa do ar e temperaturas mais frias. Os resultados obtidos no presente estudo reforçam a importância dos vírus respiratórios na população pediátrica, auxiliando na compreensão dos fatores epidemiológicos e temporais associados aos diferentes patógenos em nossa região. Desta forma, abrem caminhos para novos estudos no estado de Goiás, fornecendo ainda informações que auxiliem na construção de medidas de controle e prevenção mais eficazes contra estas infecções.

Magister Scientiae - MSc (Biotechnology) / The objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population. The first step was to design the multiplex system. This was done by using inhouse SNPs. A total of seven multiplexes were designed and optimised, each consisting of two, three or four different SNPs respectively. A total of 143 saliva and buccal samples were collected from male Johannesburg Coloureds. DNA was extracted from the saliva samples using an optimised organic method. DNA was extracted from the buccal samples using an optimised salting out method. DNA was successfully extracted from 77 of the male samples. A total of 69 samples were screened using Multiplex 1; of the 69 samples 56 samples were successfully screened to infer the paternal lineage of the samples. The results show that the most frequent haplogroup of the Johannesburg male samples was haplogroup CF (39%). The second most frequent haplogroup was haplogroup DE (38%). Under further analysis of haplogroup DE it was seen that 37% of those samples were derived for the haplogroup E1b1b.

Au cours de la dernière décennie, les réseaux (les graphes) se sont révélés être un outil efficace pour modéliser des systèmes complexes. La problématique de détection de communautés est une tâche centrale dans l’analyse des réseaux complexes. La majeur partie des travaux dans ce domaine s’intéresse à la structure topologique des réseaux. Cependant, dans plusieurs cas réels, les réseaux complexes ont un ensemble d’attributs associés aux nœuds et/ou aux liens. Ces réseaux sont dites : réseaux attribués. Mes activités de recherche sont basées principalement sur la détection des communautés dans les réseaux attribués. Pour aborder ce problème, on s’est intéressé dans un premier temps aux attributs relatifs aux liens, qui sont un cas particulier des réseaux multiplexes. Un multiplex est un modèle de graphe multi-relationnel. Il est souvent représenté par un graphe multi-couches. Chaque couche contient le même ensemble de nœuds mais encode une relation différente. Dans mes travaux de recherche, nous proposons une étude comparative des différentes approches de détection de communautés dans les réseaux multiplexes. Cette étude est faite sur des réseaux réels. Nous proposons une nouvelle approche centrée "graine" pour la détection de communautés dans les graphes multiplexes qui a nécessité la redéfinition des métriques de bases des réseaux complexes au cas multiplex. Puis, nous proposons une approche de clustering dans les réseaux attribués qui prend en considération à la fois les attributs sur les nœuds et sur les liens. La validation de mes approches a été faite avec des indices internes et externes, mais aussi par une validation guidée par un système de recommandation que nous avons proposé et dont la détection de communautés est sa tâche principale. Les résultats obtenus sur ces approches permettent d’améliorer la qualité des communautés détectées en prenant en compte les informations sur les attributs du réseaux. De plus, nous offrons des outils d’analyse des réseaux attribués sous le langage de programmation R. / In complex networks analysis field, much effort has been focused on identifying graphs communities of related nodes with dense internal connections and few external connections. In addition to node connectivity information that are mostly composed by different types of links, most real-world networks contains also node and/or edge associated attributes which can be very relevant during the learning process to find out the groups of nodes i.e. communities. In this case, two types of information are available : graph data to represent the relationship between objects and attributes information to characterize the objects i.e nodes. Classic community detection and data clustering techniques handle either one of the two types but not both. Consequently, the resultant clustering may not only miss important information but also lead to inaccurate findings. Therefore, various methods have been developed to uncover communities in networks by combining structural and attribute information such that nodes in a community are not only densely connected, but also share similar attribute values. Such graph-shape data is often referred to as attributed graph.This thesis focuses on developing algorithms and models for attributed graphs. Specifically, I focus in the first part on the different types of edges which represent different types of relations between vertices. I proposed a new clustering algorithms and I also present a redefinition of principal metrics that deals with this type of networks.Then, I tackle the problem of clustering using the node attribute information by describing a new original community detection algorithm that uncover communities in node attributed networks which use structural and attribute information simultaneously. At last, I proposed a collaborative filtering model in which I applied the proposed clustering algorithms.

<p>Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community.</p>

This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected. / <p>QC 20130821</p>

Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, a minute chromosome that leads to the creation of the fusion gene BCR/ABL and the transcription of the fusion protein BCR/ABL in transformed cells. The constitutively active tyrosine kinase BCR/ABL confers enhanced proliferation and survival on leukemic cells. CML has in only a few decades gone from being a disease with very bad prognosis to being a disease that can be effectively treated with oral tyrosine kinase inhibitors (TKIs). TKIs are drugs inhibiting BCR/ABL as well as other tyrosine kinases. In this thesis, the focus has been on the immune system of CML patients, on immune escape mechanisms present in untreated patients and on how these are affected by TKI therapy. We have found that newly diagnosed, untreated CML patients exert different kinds of immune escape mechanisms. Patients belonging to the Sokal high-risk group had higher levels of myeloid-derived suppressor cells (MDSCs) as well as high levels of the programmed death receptor 1 (PD-1)-expressing cytotoxic T cells compared to control subjects. Moreover, CML patients had higher levels of myeloid cells expressing the ligand for PD-1, PD-L1. CML patients as well as patients with B cell malignacies had high levels of soluble CD25 in blood plasma. In B cell malignacies, sCD25 was found to be released from T regulatory cells (Tregs). Treatment with the TKIs imatinib or dasatinib decreased the levels of MDSCs in peripheral blood. Tregs on the other hand increased during TKI therapy. The immunostimulatory molecule CD40 as well as NK cells increased during therapy, indicating an immunostimulatory effect of TKIs. When evaluating immune responses, multiplex techniques for quantification of proteins such as cytokines and chemokines are becoming increasingly popular. With these techniques a lot of information can be gained from a small sample volume and complex networks can be more easily studied than when using for example the singleplex ELISA. When comparing different multiplex platforms we found that the absolute protein concentration measured by one platform rarely correlated with the absolute concentration measured by another platform. However, relative quantification was better correlated.

Nous présentons dans ce document notre projet de recherche dans le cadre de la maitrise en informatique de l'université du Québec à Montréal. Dans le cadre de cette maîtrise nous avons conçu un langage de modélisation de réseaux sociaux permettant de modéliser plusieurs réseaux sociaux simultanément. Nous voulions que ce langage soit proche du langage naturel afin d'être accessible aux néophytes en informatique. Nous avons également réalisé une plate-forme permettant l'exécution de ce langage. La conception de ce langage est motivée par le fait que généralement au sein d'un ensemble social donné il existe plusieurs types d'interactions sociales simultanément. Par exemple si nous considérons le groupe social constitué par l'ensemble des employés d'une entreprise, il existe souvent des relations de collaboration mais également des relations d'amitié et/ou des relations de conseil entre les employés de l'entreprise. L'étude des réseaux sociaux doit souvent prendre en compte tous les types de relations présentes dans un ensemble social afin de bien comprendre l'évolution de cet ensemble social. Dans le but de mettre en pratique le langage que nous avons conçu nous avons étudié la corrélation entre plusieurs réseaux sociaux. Nous avons également comparé notre plate-forme avec les outils d'analyse et de simulation de réseaux disponibles sur le marché actuellement. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : réseaux sociaux, analyse de réseaux, simulation, réseaux multiplexes

The in situ proximity ligation assay (in situ PLA) is an immunoassay that enables directvisualisation of single protein targets or protein interactions in cell or tissue samples. This project revolves around designing and introducing several novel multiplexable components tobe used in conjunction with Olink Bioscience's Duolink product line. In this report, a novel in silico approach to DNA oligomer interaction design is presented. Using this in silico method, a multiplexed system of DNA oligomers has been designed andevaluated using in situ PLA and fluorescence microscopy.

This dissertation includes molecular and phytochemical investigations of the harmful, bloom-forming alga, Prymnesium parvum, including analysis of known polyketide metabolites as a function of salinity and growth. Initially, the development of molecular and phytochemical tools was necessary for the detection and quantification of P. parvum and its associated toxins. Suites of oligonucleotides and molecular beacons were designed for conventional and quantitative multiplex PCR to amplify four species- and gene-specific products simultaneously that were used for the detection and quantitation of P. parvum. This built-in redundancy provided increased confidence in reactions with the positive confirmation of four discrete products. Techniques were also developed for the chemical enrichment of toxins produced by P. parvum. Until now, isolation of “prymnesins” has never been reproduced. Polyketide prymnesins possess unique spectral properties that were used to generate an LC-MS fingerprint that comprised 13 ion species. Preliminary investigations using chemifluorimetric methods were also capable of detecting prymnesins in the pico- and nano-molar range. Environmental samples were tested as an independent assessment of these methods. Lastly, the roles of polyketide prymnesins were analyzed with respect to total hemolytic activity (HA) as a function of culture age and salinity. Variation in HA of supernatants was statistically significant relative to both variables (p << 0.05). Salinity was inversely related to HA wherein cultures growing in 5-25 psu were 150-200% more hemolytic. Total HA was inversely related to culture age during the first three weeks, but positively related to it during the next three weeks. Interestingly, no hemolysis was detected in fractions containing prymnesins from culture supernatants and the majority of hemolysins remained in the aqueous phase. Prymnesins extracted from cells varied significantly over the 6-week observation period (p << 0.05); HA was positively correlated during the first half and inversely related during the last half of the study. Salinity was directly related to HA from cell extracts, but these effects were not significantly different until the last three weeks. These investigations suggest that polyketide prymnesins are present at much lower quantities than previously believed, and they may not be the key compounds associated with hemolysis due to P. parvum. / text

This dissertation develops technology for microfluidic point-of-care immunoassay devices. This research (2004–2007) improved microfluidic immunoassay performance by reducing reagent consumption, decreasing analysis time, increasing sensitivity, and integrating processes using a lab-on-a-chip. Estimates show that typical hospital laboratories can save $1.0 million per year by using microfluidic chips. Our first objective was to enhance mixing in a microfluidic channel, which had been one of the main barriers to using these devices. Another goal of our studies was to simplify immunoassays by eliminating surfactants. Manufacturers of latex immunoassays add surfactants to prevent non-specific aggregation of microspheres. However, these same surfactants can cause false positives (and negatives) during diagnostic testing. This work, published in Appendix A (© 2006 Elsevier) shows that highly carboxylated polystyrene (HCPS) microspheres can replace surfactants and induce rapid mixing via diffusion in microfluidic devices. Our second objective was to develop a microfluidic device using fiber optics to detect static light scattering (SLS) of microspheres in Appendix B (© 2007 Elsevier). Fiber optics were used to deliver light emitting diode (LED) or laser light. A miniature spectrometer was used to measure 45° forward light scattering collected by optical fiber. Latex microspheres coated with PR3 proteins were used to test for the vasculitis marker, anti-PR3. No false negatives or positives were observed. A limit of detection (LOD) of 50 ng mL⁻¹ was demonstrated. This optical detection system works without fluorescence or chemiluminescence markers. It is cost effective, small, and re-usable with simple rinsing. The final objective in this dissertation, published in Appendix C (© 2007 Elsevier), developed a multiplex immunoassay. A lab-on-a-chip was used to detect multiple antibodies using microsphere light scattering and quantum dot (QD) emission. We conjugated QDs onto microspheres and named this configuration “nano-on-micro” or “NOM”. Upon radiation with UV light, strong light scattering is observed. Since QDs also provide fluorescent emission, we are able to use increased light scattering for detecting antigen-antibody reactions, and decreased QD emission to identify which antibody is present.