Les selenoproteïnes constitueixen una família diversa de proteïnes, caracteritzada per la presència del Seleni (Se), en forma de l'amino àcid atípic, la selenocisteïna (Sec). La selenocisteïna, coneguda com l'amino àcid 21, és similar a la cisteïna (Cys) amb un àtom de seleni en lloc de sofre (S). Les selenoproteïnes són els responsables majoritaris dels efectes biològics del seleni i s'ha observat que poden estar implicades en la infertilitat masculina, el càncer, algunes malalties coronàries,l'activació de virus latents i l'envelliment. La selenocisteïna es codifica pel codó UGA, normalment codó de parada (STOP). Per a la recodificació correcta del UGA són necessaris diversos factors. A la part 3' de la regió no traduïda (UTR) dels transcrits dels gens de selenoproteïnes en organismes eucariotes s'hi troba una estructura de "stem-loop" anomenada SECIS. La proteïna SBP2 interactua amb el SECIS, així com amb el ribosoma, i forma un complex amb el factor d'elongació EFsec i el tRNA de la selenocisteïna, el tRNASec. Donat que el codó TGA normalment significa fi de la traducció, les formes tradicionals de cerca de gens no el reconeixen com a codó codificant. Per aquesta raó ha estat necessari desenvolupar una metodologia específica per a la predicció de gens de selenoproteïnes. En els últims anys, hem contribuït a la descripció del selenoproteoma eucariota amb el descobriment de noves famílies (Castellano et al., 2005), amb l'elaboració de nous mètodes (Taskov et al., 2005; Chapple et al., 2009) i l'anotació de diferents genomes (Jaillon et al., 2004; Drosophila 12 genomes Consortium, 2007; Bovine Genome Sequencing and Analysis Consortium, 2009). Finalment, hem identificat el primer animal que no té selenoproteïnes (Drosophila 12 genomes Consortium, 2007; Chapple and Guigó, 2008), un descobriment soprenent donat que, fins el moment, es creia que les selenoproteïnes eren essencials per la vida animal. / Selenoproteins are a diverse family of proteins containing the trace element Selenium (Se)in the form of the non-canonical amino acid selenocysteine (Sec). Selenocysteine, the 21st amino acid, is similar to cysteine (Cys)but with Se replacing Sulphur. In many cases the homologous gene of a known selenoprotein is present with cysteine in the place of Sec in a different genome. Selenoproteins are believed to be the effectors of the biological functions of Selenium and have been implicated in male infertility, cancer and heart diseases, viral expression and ageing. Selenocysteine is coded by the opal STOP codon (TGA). A number of factors combine to achieve the co-translational recoding of TGA to Sec. The 3' Untranslated regions (UTRs) of eukaryotic selenoprotein transcripts contain a stem-loop structure called a Sec Insertion Sequence (SECIS) element. This is recognised by the Secis Binding Protein 2 (SBP2), which binds to both the SECIS element and the ribosome. SBP2, in turn, recruits the Sec-specific Elongation Factor EFsec, and the selenocysteine transfer RNA, tRNASec. The dual meaning of the TGA codon means that selenoprotein genes are often mispredicted by the standard annotation pipelines. The correct prediction of these genes, therefore, requires the development of specific methods. In the past few years we have contributed significally to the description of the eukaryotic selenoproteome2 with the discovery of novel families (Castellano et al., 2005), the elaboration of novel methods (Taskov et al., 2005; Chapple et al., 2009) and the annotation of different genomes (Jaillon et al., 2004; Drosophila 12 genomes Consortium, 2007; Bovine Genome Sequencing and Analysis Consortium, 2009). Finally, and perhaps most importantly, we have identified the first animal to lack selenoprotein genes (Drosophila 12 genomes Consortium, 2007; Chapple and Guigó, 2008). This last finding is particularly surprising because it had previously been believed that selenoproteins were essential for animal life.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 215-225). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves : 325-355). Also available on the Internet.

Este trabalho demonstrou a construção de minibibliotecas de \"Expressed Sequence Tags\" (EST) com o uso de RT-PCR de baixa estringencia e \"primer\" consenso-degenerado. Através do estudo de parâmetros críticos como concentração de sais, temperatura e velocidade de ciclagem, composição dos \"primers\" e qualidade do RNA mensageiro, foi possível padronizar um protocolo. Tal protocolo permitiu um aumento do numero de seqüências por minibiblioteca em relação a protocolos similares existentes na literatura (Dias neto et al., 1997 e 2000) sem perda de características desejáveis como amplificação preferencial do centro dos genes e nomalização das mensagens. As seqüências produzidas levaram a um significativo aumento das seqüências de EST de S. mansoni disponíveis publicamente e permitiu a detecção da existência de novos fragmentos de genes expressos na fase adulta do parasita. Neste trabalho também clonamos uma apirase de S. mansoni cuja a seqüência não havia sido descrita anteriormente. O gene possui cerca de 2,7 mil pares de bases e codifica para uma proteína de 544 aminoácidos. Esta foi expressa em sistema heterologo e utilizada para obtenção de anticorpos policlonais contra esta proteína. Utilizando estes anticorpos foi possível detectar a proteína no tegumento do parasita. / This work demonstrates the construction of EST minilibraries employing low stringency RT-PCR and consensus-degenerate primers. Through the study of critical parameters such as salt concentration, cycling temperature and ramp speed, composition of primers and quality of messenger RNA a standard protocol was obtained. Such protocol allowed an increase in the number of sequences per minilibrary in relation to similar protocols in the literature (Dias Neto et al., 1997 and 2000) without no loss in desirable characteristics such as preferential amplification of the central portion of messages and normalization of messages. The sequences produced allowed a significant increase in the publicly available EST sequences of S. mansoni and permitted discovery of new gene fragments expressed in adult stage of the life cycle of the parasite. In this work we also cloned an apyrase of S. mansoni whose sequence had never been described previously. The gene has about 2.7 kilobases and codes for a protein of 544 aminoacids. This protein was expressed in a heterologous system and was used for production of a polyclonal antibody. This antibody was used for detection of this protein in the tegument of the parasite.

Blastocladiella emersonii é um fungo aquático da classe dos quitridiomicetos, notável pelas mudanças morfogenéticas que ocorrem durante o seu ciclo de vida. Neste trabalho isolamos 8.495 seqüências expressas (ESTs) deste fungo, que representam 3.226 seqüências únicas putativas. Destas seqüências, 37% foram classificadas segundo o processo biológico onde estariam envolvidas, de acordo com o sistema de anotação do Gene Ontology (GO). Analisamos os perfis de expressão in silico das ESTs usando estatística Bayesiana e os resultados obtidos foram validados por Northern blot para sete perfis de expressão selecionados. Pudemos encontrar boa correlação entre vários padrões de expressão e determinados processos biológicos. Foram selecionadas algumas seqüências potencialmente envolvidas com a esporulação do fungo para melhor caracterização. Analisamos a expressão de dois genes codificando centrinas (BeCenl e BeCen2) pertencentes a subfamílias distintas. Centrinas são proteínas ligantes de cálcio envolvidas em diferentes processos como o direcionamento do aparelho flagelar e a duplicação dos centros organizadores de microtúbulos (MTOCs). Observamos que os níveis da proteína BeCenl, que não havia sido descrita em fungos, apresentam um máximo aos 150 min da esporulação, defasado do pico de expressão do seu mRNA que ocorre aos 90 min deste estágio. A proteína BeCen2 está presente em níveis constantes durante todo a ciclo de vida do fungo, embora o seu mRNA apresente um pico de expressão aos 120 min da esporulação. Experimentos de imunofluorescência localizaram a proteína BeCenl no citoplasma e no corpo basal do zoósporo. Estes dados sugerem que BeCenl atue na re-orientação e movimento dos corpos basais e BeCen2 na duplicação dos MTOCs. Investigamos também a expressão de dois genes codificando proteína-quinases dependentes de ciclina putativas (BeCdkl e BeCdk2). Apenas uma Cdk (Cdkl) foi descrita em fungos como diretamente envolvida no controle do ciclo celular. Ambos os genes apresentam expressão diferencial, com níveis máximos de mRNA para os dois casos aos 90 min da esporulação. Por outro lado, a proteína BeCdkl está presente durante todo o ciclo de vida do fungo e foi localizada no núcleo e no capacete nuclear dos zoósporos. Um transportador putativo de hexose (Bemst) foi também analisado, com base na regulação por glicose de genes envolvidos com o ciclo celular observada em eucariotos. Verificou-se que os níveis do mRNA de Bemst diminuem drasticamente durante a esporulação, mas glicose ou outras hexoses não afetaram a expressão de Bemst. / Blastocladiella emersonii is an aquatic fungus that belongs to the class of chytridiomycetes, notable for the morphogenetic processes which occur during its life cycle. In this work we have isolated 8,495 expressed sequence tags (ESTs) from this fungus, representing 3,226 putative unigenes. From these unigenes, 37% were classified into a biological process, as a result of Gene Ontology (GO) annotation. Furthermore, we analyzed the expression profile in silico of each transcript using Bayesian Statistics and seven of these profiles were validated by Northern blot analysis. In addition, we found a good correlation between several of these expression patterns and certain biological processes. Some ESTs potentially involved in the sporulation of the fungus were selected to be further characterized. We analyzed the expression of two genes encoding two centrins (BeCenl and BeCen2) of distinct subfamilies. Centrins are calcium-binding proteins involved in different processes such as basal body orientation and duplication of the microtubuleorganizing centers (MTOCs). The amount of BeCenl, a centrin ortholog not previously described in fungi, presents a maximum at 150 min of sporulation, whereas the peak of its mRNA occurs at 90 min of this stage. Protein BeCen2 presents constant levels during the entire life cycle of the fungus, even though its mRNA shows a peak of expression at 120 min of sporulation. In addition, immunofluorescent studies localized BeCenl in the cytoplasm and the basal body of zoospores. These results suggest that BeCenl plays a role in re-orientation and movement of basal bodies and BeCen2 in MTOCs duplication. We also investigated the expression of two genes encoding putative cyclin-dependent protein kinases (BeCdkl and BeCdk2). Only one type of Cdk (Cdkl) directly involved in cell cycle control has been described in other fungi. Both genes were found to be differentially expressed, with maximum mRNA levels being detected in either case at 90 min of sporulation. In contrast, BeCdk1 is present throughout the life cycle of the fungus and was immunolocalized in the nuc1eus and the nuclear cap of zoospores. A putative hexose transporter (Bemst) was also investigated, taking into account the regulation by glucose of cell cycle controlled genes in eukaryotes. We found that Bemst mRNA levels decrease drastically during sporulation, but glucose and other hexoses had no effect on Bemst expression.

No / Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.

No / Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

Tse, Sui Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 95-104). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Motivation --- p.1 / Chapter 1.1.1 --- Basic Concepts and Definitions --- p.2 / Chapter 1.2 --- Evolutionary Algorithms --- p.5 / Chapter 1.2.1 --- Chromosome Encoding --- p.6 / Chapter 1.2.2 --- Selection --- p.7 / Chapter 1.2.3 --- Crossover --- p.9 / Chapter 1.2.4 --- Mutation --- p.10 / Chapter 1.2.5 --- Elitism --- p.11 / Chapter 1.2.6 --- Niching --- p.11 / Chapter 1.2.7 --- Population Manipulation --- p.13 / Chapter 1.2.8 --- Building Blocks --- p.13 / Chapter 1.2.9 --- Termination Conditions --- p.14 / Chapter 1.2.10 --- Co-evolution --- p.14 / Chapter 1.3 --- Local Search --- p.15 / Chapter 1.4 --- Memetic Algorithms --- p.16 / Chapter 1.5 --- Objective --- p.17 / Chapter 1.6 --- Summary --- p.17 / Chapter 2 --- Background --- p.18 / Chapter 2.1 --- Multiple Drugs Tumor Chemotherapy --- p.18 / Chapter 2.2 --- Bioinformatics --- p.22 / Chapter 2.2.1 --- Basics of Bioinformatics --- p.24 / Chapter 2.2.2 --- Applications on Biomedical Systems --- p.26 / Chapter 3 --- A New Drug Administration Dynamic Model --- p.29 / Chapter 3.1 --- Three Drugs Mathematical Model --- p.31 / Chapter 3.1.1 --- Rate of Change of Different Subpopulations --- p.32 / Chapter 3.1.2 --- Rate of Change of Different Drug Concen- trations --- p.35 / Chapter 3.1.3 --- Toxicity Effects --- p.35 / Chapter 3.1.4 --- Summary --- p.36 / Chapter 4 --- Memetic Algorithm - Iterative Dynamic Program- ming (MA-IDP) --- p.38 / Chapter 4.1 --- Problem Formulation: Optimal Control Problem (OCP) for Mutlidrug Optimization --- p.38 / Chapter 4.2 --- Proposed Memetic Optimization Algorithm --- p.40 / Chapter 4.2.1 --- Iterative Dynamic Programming (IDP) . . --- p.40 / Chapter 4.2.2 --- Adaptive Elitist-population-based Genetic Algorithm (AEGA) --- p.44 / Chapter 4.2.3 --- Memetic Algorithm 一 Iterative Dynamic Programming (MA-IDP) --- p.50 / Chapter 4.3 --- Summary --- p.56 / Chapter 5 --- MA-IDP: Experiments and Results --- p.57 / Chapter 5.1 --- Experiment Settings --- p.57 / Chapter 5.2 --- Optimization Results --- p.61 / Chapter 5.3 --- Extension to Other Mutlidrug Scheduling Model . --- p.62 / Chapter 5.4 --- Summary --- p.65 / Chapter 6 --- DNA Sequencing by Hybridization (SBH) --- p.66 / Chapter 6.1 --- Problem Formulation: Reconstructing a DNA Sequence from Hybridization Data --- p.70 / Chapter 6.2 --- Proposed Memetic Optimization Algorithm --- p.71 / Chapter 6.2.1 --- Chromosome Encoding --- p.71 / Chapter 6.2.2 --- Fitness Function --- p.73 / Chapter 6.2.3 --- Crossover --- p.74 / Chapter 6.2.4 --- Hill Climbing Local Search for Sequencing by Hybridization --- p.76 / Chapter 6.2.5 --- Elitism and Diversity --- p.79 / Chapter 6.2.6 --- Outline of Algorithm: MA-HC-SBH --- p.81 / Chapter 6.3 --- Summary --- p.82 / Chapter 7 --- DNA Sequencing by Hybridization (SBH): Experiments and Results --- p.83 / Chapter 7.1 --- Experiment Settings --- p.83 / Chapter 7.2 --- Experiment Results --- p.85 / Chapter 7.3 --- Summary --- p.89 / Chapter 8 --- Conclusion --- p.90 / Chapter 8.1 --- Multiple Drugs Cancer Chemotherapy Schedule Optimization --- p.90 / Chapter 8.2 --- Use of the MA-IDP --- p.91 / Chapter 8.3 --- DNA Sequencing by Hybridization (SBH) --- p.92 / Chapter 8.4 --- Use of the MA-HC-SBH --- p.92 / Chapter 8.5 --- Future Work --- p.93 / Chapter 8.6 --- Item Learned --- p.93 / Chapter 8.7 --- Papers Published --- p.94 / Bibliography --- p.95

Background Coagulase-negative staphylococci (CoNS) and in particular Staphylococcus epidermidis have emerged as major pathogens primarily causing nosocomial infections in patients with indwelling medical devices. These infections are often caused by multidrug-resistant strains of S. epidermidis (MDRSE). Other clinical entities due to CoNS are lower urinary tract infections (UTI) in women and native valve endocarditis. The purpose of this work was to investigate the frequency of antibiotic resistance and the molecular epidemiology of both hospital and community-associated isolates of S. epidermidis in order to examine if certain clones are related to MDRSE infections. Furthermore, we aimed to explore if specific clones of S. saprophyticus are associated with UTI in women. Methods A total of 359 hospital-associated methicillin-resistant isolates of CoNS obtained from 11 hospitals in northern Europe and 223 community-associated staphylococcal isolates were examined. Furthermore, 126 isolates of S. saprophyticus isolated from women with uncomplicated UTI from five different locations in northern Europe were analyzed. Pulsed-field gel electrophoresis (PFGE) was used for genotyping. Additionally, some of the S. epidermidis isolates were analyzed with multilocus sequence typing (MLST). Antibiotic susceptibility was determined for all isolates by the disc diffusion test. Results 293 of the 359 (82%) hospital-associated and 124 of the 223 (56%) community-associated isolates belonged to the species S. epidermidis. Among the hospital-associated S. epidermidis isolates, two dominating PFGE types (type A and B) were distinguished, comprising 78 (27%) and 51 (17%) isolates, respectively. Type A, which was detected in a Norwegian and eight Swedish hospitals, corresponded with a novel sequence type (ST215). Type B was discovered in a German, a Danish and seven Swedish hospitals and corresponded with ST2. In contrast, community-associated isolates of S. epidermidis were genetically extremely diverse with no predominating genotype, and showed a low rate of antibiotic resistance; only two (1.6%) methicillin-resistant strains were detected. Among 126 analyzed isolates of S. saprophyticus, 47 different PFGE profiles were identified. Several clusters of genetically highly related isolates were detected among isolates obtained from different locations and periods of time. Conclusion We have demonstrated the occurrence, persistence and potential dissemination of two multidrug-resistant S. epidermidis (MDRSE) genotypes, including a novel sequence type (ST215), within hospitals in northern Europe. Community-associated isolates of S. epidermidis showed a low rate of methicillin-resistance and were genetically heterogeneous. These results indicate that MDRSE by large are confined to the hospital setting in our region. Moreover, although the S. saprophyticus population was quite heterogeneous, indistinguishable isolates of S. saprophyticus causing lower UTI in women were identified in different countries 11 years apart, indicating the persistence and geographical spread of some clones of S. saprophyticus.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Running title: Seasonal affective disorder and melanopsin. Typescript (photocopy).