Thesis (M.A.)--University of Colorado, 2005. / Includes bibliographical references (leaves 114-118). Also available online (PDF file) by a subscription to the set or by purchasing the individual file.

Thesis (M.A.)--University of Colorado, 2005. / Includes bibliographical references (leaves 114-118).

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Oxidase Alternativa (AOX) em plantas à codificada por uma pequena famÃlia multigÃnica de origem nuclear (DNA genÃmico). Essa famÃlia multigÃnica foi bem estudada em mono e dicotiledÃneas sendo subdividida em duas subfamÃlias: Aox1 e Aox2. Os genes Aox1 sÃo encontrados em mono e eudicotiledÃneas apresentando expressÃo mais relacionada a condiÃÃes de estresses enquanto que os genes Aox2 sÃo encontrados apenas em eudicotiledÃneas com expressÃo constitutiva. VÃrios genes Aox1 e apenas hum gene Aox2 sÃo encontrados na maioria das eudicotiledÃneas estudadas. Nesse trabalho, caracterizou-se a famÃlia multigÃnica da Aox no gÃnero Medicago (Medicago truncatula e Medicago sativa) pertencente à ordem Fabales. Em Medicago truncatula, a famÃlia multigÃnica da Aox foi carcterizada atravÃs de busca por bioinformÃtica em bancos de dados identificando-se 4 genes Aox (Aox1, Aox2a, Aox2b1 e Aox2b2) no genoma dessa espÃcie revelando pela primeira vez uma duplicaÃÃo do gene Aox2b. OligonucleotÃdeos especÃficos desenhados para cada um dos genes foram usados para amplificaÃÃo por PCR, clonagem e seqÃenciamento parcial dos referidos genes em Medicago sativa. A expressÃo gÃnica foi estudada, atravÃs de RT-PCR semiquantitativa, em sementes durante a germinaÃÃo (0, 24 e 48 horas de apÃs embebiÃÃo) e em raÃzes e folhas de Medicago sativa crescidas em meio hidropÃnico de Hoagland e submetidas a diferentes condiÃÃes de estresse (0, 6, 12 e 24 hs apÃs tratamento): controle, Ãcido salicÃlico (0,5 mM), PEG (100 g/L), H2O2 (10 mM) e cisteÃna (0,5 mM). Os resultados revelaram que todos os 4 genes Aox foram detectados em sementes de Medicago sativa, entretanto os genes Aox1, 2b1 e 2b2 apresentaram aumento da expressÃo durante a germinaÃÃo enquanto que o Aox2a mostrou-se constitutivo. Em folhas os genes Aox1, 2a e 2b1 foram detectados em todas as condiÃÃes testadas jà o Aox2b2 foi detectado mais intensamente apenas nas condiÃÃes de estresse. De maneira semelhante ao observado durante a germinaÃÃo os genes Aox1, 2b1 e 2b2 tiveram expressÃo aumentada em resposta a todas as condiÃÃes de estresse. O Aox2a mostrou-se constitutivo, mas foi intensamente expresso. Em raÃzes, todos os genes foram detectados e um perfil semelhante de induÃÃo dos genes Aox1, 2b1 e 2b2 tambÃm foi observado com exceÃÃo do tratamento com PEG onde apenas Aox1 foi induzido. O Aox2a tambÃm se mostrou constitutivo, mas foi fracamente expresso. Com a finalidade de se compreender melhor a co-expressÃo dos genes Aox1, 2b1 e 2b2 em Medicago sativa os promotores foram clonados e seqÃenciados revelando regiÃes idÃnticas entre eles indicando o envolvimento de elementos cis comuns na regulaÃÃo. Esses resultados corroboram com a co-expressÃo induzida por estresse de Aox1/Aox2b observada anteriormente em feijÃo. Contudo, em Medicago sativa, observamos expressÃo diferencial entre tecidos: no tecido radicular os genes Aox1, Aox2a e 2b1 podem sem induzidos por estresses e/ou mostram uma caracterÃstica de expressÃo. Por outro lado, em folhas o gene Aox2b2 diferenciou por apresentar apenas caracterÃstica de gene induzido em condiÃÃes de estresse. / In plants, Alternative Oxidase (AOX) is enconded by small milti gene family located in genomc DNA. This multi gene family was studied in mono and dicots plants being clusterd in two subfamilies: Aox1 and Aox2. Aox1 gene are found in all angiosperms tÃxon presenting gene expression related to stress situations while Aox2 genes are found in dicots plants only and presenting a houkeeping expression. Many Aox1 genes e just one Aox2 are found in the most of dicots studied. However, in Fabales, as cowpea and soybean, is found a profile with two genes Aox2 (Aox2a , Aox2b) and one Aox1 gene. In this work was characterized a multi gene family of Aox genes in Medicago genus (Medicago truncatula e Medicago sativa) belonging to Fabales Order. Data mining in genBanks characterized four Aox genes (Aox1, Aox2a, Aox2b1 and Aox2b2) in Medicago truncatula genome revealing for first time a duplication of Aox2b genes. Specifics primers designed for each Aox gene and then were used to amplification by PCR, cloning and partial sequencing of these genes in Medicago sativa. Gene expression was carried out by semiquantitative RT-PCR in: germination seeds (0, 24, 48 hours of germination), roots and leaves from Medicago sativa grewth in Hoaglendâs medium and applied to disticts stress conditions (0, 6, 12 e 24 hs after treatment): control, salicylic acid (0,5mM), PEG (100g/L), H2O2 (10mM) e cisteÃne (0,5mM). The results revealed that all four Aox genes were detected in seed of Medicago sativa. However, Aox1, Aox2b1 and Aox2b2 genes presented high expression during germination while Aox2a showed constitutive expression. In leaves, Aox1, Aox2a and Aox2b1 were detected in all conditions tested, but Aox2b2 was observed more strong in stress situations only. Simirily observed in germination, Aox1, Aox2b1 and Aox2b2 increased transcripts levels in response to all stress conditions. Aox2a, one more time, had constitutive, but very strong expression. In roots, all genes were detected and a similar induction profile of Aox1, Aox2b1 and Aox2b2 were confirmed less to PEG treatment where just Aox1 was responsive. Aox2a had constitutive expression too, but it was weakly expressed. In order to understand the co-expresion of Aox1, Aox2b1 and Aox2b2 genes, the promoter regions were cloned and sequenced revealing close motifs among them suggesting th involviment of cis-elements in their regulation. Theses resuls corroborate with co-expression stress-induced of Aox1/Aox2b found in cowpea. However, in Medicago sativa, it was observed tissue-specific exression. Aox1, Aox2a and Aox2b1 with characteristics constitutive and induced in seeds germination and roots tissue while in leaves Aox2b2 differed by present stress-induced expression only.

De nos jours, l’obésité est un problème majeur de santé publique dont la prévalence mondiale ne cesse d’augmenter. Ainsi selon la National Health and Nutrition Examination Surveys (NHANES), le pourcentage d’adultes obèses aux Etats-Unis a presque doublé entre 1976-1980 et 1999-2002. De plus, pour la première fois dans l’histoire des Etats-Unis, la longévité de l’actuelle génération d’enfants est estimée à la baisse. Cette augmentation de l’obésité résulte à la fois de prédispositions génétiques et de facteurs environnementaux tels que l’accès à la nourriture, le tissu social, le régime alimentaire ou encore le manque d’activité physique. , Aussi, de récentes études ont suggéré que la flore microbienne intestinale jouait un rôle majeur dans la conversion énergétique des nutriments et son stockage. Bien que très peu d’expériences aient été réalisées, le rôle des Lactobacillus spp. a déjà été évoqué dans la prise de poids chez l’homme. Deux importantes études, l’une menée au Danemark et l’autre au sein même de notre laboratoire, ont montré que les Lactobacillus spp. étaient présents en quantité plus importante chez les sujets obèses par rapport aux contrôles et que leur taux corrélaient à la glycémie chez des patients diabétiques de type II (obèses). L’effet des Lactobacillus spp sur la prise de poids a été beaucoup plus étudié chez les animaux. En effet, l’agriculture représente l’un des domaines majeur d’utilisation des antibiotiques comme « growth promoters », notamment l’avoparcin qui empêche le développement de nombreuses bactéries dont des Gram positives comme les Actinobacteria et les Firmicutes à l’exception toutefois des Lactobacillus spp. Les probiotiques sont aussi utilisés comme « growth promoters » et il a été montré que l’addition de 106 Lactobacillus spp. dans les régimes alimentaires d’animaux permettait de modifier leur flore microbienne intestinale et d’entrainer un gain de poids important. A partir de toutes ces constatations, nous avons émis l’hypothèse que les bactéries pouvaient jouer un rôle dans l’obésité via une modification de la composition de la flore intestinale. Il nous est également apparu essentiel d’évaluer l’effet de l’addition de concentration bactérienne importante dans l’alimentation quotidienne. L’objectif de notre étude est ainsi d’évaluer le rôle des Lactobacillus spp. en tant que facteur de croissance et d’évaluer leur impact sur la prise de poids. / The prevalence of obesity is a major world health problem that is rapidly increasing. In the USA, according to the National Health and Nutrition Examination Surveys (NHANES), the percentage of obese adults nearly doubled between NHANES 1976–1980 and NHANES 1999– 2002. Moreover, because of the increase in prevalence of American childhood obesity, the current children generation is predicted to be the first in United States to see a decrease in longevity. Obesity results from a mixture of genetic background and environmental factors including food availability, social networks, diet, and physical activity. Recent evidence suggests that gut microbiota plays a major role in energy intake, conversion and storage. Although very few reports have investigated gut microbiota and its association with obesity, several evidences support a role of Lactobacillus spp. in weight gain within human. Lactobacillus spp. were found at higher levels among obese subjects in two major studies, one conducted by us and one made in Denmark. Moreover, the rate of Lactobacillus spp. was correlated with blood glucose in subjects with type II diabetes (obese). In agriculture avoparcin which is one of the most used antibiotics to cause weight gain in animals, is effective on most bacteria including Gram-positive Actinobacteria and Firmicutes, with the notable exception on some Lactobacillus spp.. In addition, probiotics are also used as growth promoters and the addition of even 106 Lactobacillus spp in the diet of farm animals results in gut microbiota changes and weight gain. As a result, we hypothesized that bacteria could play a role in the obesity pandemic notably with the ingestion of probiotics that modified the gut microbiota structure. In addition, we stressed the necessity for further investigations to assess the effects of routinely adding high amounts of bacteria in food. The objective of our study was to evaluate the putative role of Lactobacillus sp. in growth promotion.

No description available.

Briefly, some findings in my research are as follows: (1) The genetic variants of the CA repeats in IGF1 promoter 1 can affect the activity of promoter 1, and the CA repeat showed a suppressive effect on the activity of the promoter 1 of IGF1 gene. EMSA results have shown that the CA repeats could bind to certain nuclear protein. (2) The SNPs T/C (rs5742612) and T/A (rs2288377) can also affect the activity of the promoter 1 in IGF1 gene, and the activity of C-A haplotype is significantly higher than that of T-T haplotype. EMSA results have shown that the SNP T/A (rs2288377) could bind to certain nuclear protein. (3) I developed the new dual reporter assay method to investigate the transactivation interaction between the SNP T/G (rs2071430) and C/A (rs17000900) in the MxA promorer. This new method can not only improve the detection limit for small difference between haplotypes, but also calculate the model of transactivation effect between these two SNPs. The results were better than those of traditional method, and it gave a clear-cut demonstration of the effect of interaction between these two SNPs on the activity of MxA promoter. / In addition, in the IGF1 study, the core promoter region of promoter 2 was identified through 5' deletion mutagenesis methods. Moreover, a cell-type specific mechanism of bidirectional activation of promoter was found. / Recently, more and more studies focus on gene function with the completion of the Human Genome Project. It is well known that polymorphism of human genome sequence is a common phenomenon in the human population. Specially, a lot of genetic polymorphisms, including single nucleotide polymorphisms (SNPs) and microsatellites, have been reported in the regulatory region of many genes. However, the effects of most of these genetic polymorphisms on gene expression are still unknown. The polymorphisms in the promoter can play an important role in the gene regulation. For example, some SNPs located in the transcription factor binding site (TFBS) can affect gene transcription. So, it is very necessary to directly study the effect of genetic variants on promoter transactivation activities. In this study, we studied the effect of genetic polymorphisms on gene expression through reporter gene assay, electrophoretic mobility shift assay (EMSA), and so on. And the candidate genes include insulin-like growth factor 1 (IGF1) and myxovirus resistence 1 (MxA). Some SNPs and microsatellites have been reported in the promoters of these genes. In our previous researches, we focused on the study of the association between these polymorphisms and some diseases, and it was found that a few SNPs significantly associated with relevant diseases. Based on the previous results, in my project, I developed new functional assays and also improved existing methods to analyse the functional effect of these genetic variants of promoters on transactivation activities. / by Huang, Wei. / "March 2008." / Adviser: Nelson Leung Sang Tang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 139-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Lau Suk Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 128-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Table of content --- p.ii / List of Figures --- p.x / List of Tables --- p.xiv / Abbreviation --- p.xv / Abstract --- p.xviii / 論文摘要 --- p.xx / Chapter 1 --- Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Tetraodon nigroviridis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Genomic Sequencing Project --- p.3 / Chapter 1.1.3 --- Tetraodon nigroviridis as Study Model --- p.4 / Chapter 1.1.3.1 --- Genomic Comparison --- p.4 / Chapter 1.1.3.2 --- Gene Order and Structural Studies --- p.5 / Chapter 1.1.3.3 --- Genomic Evolution --- p.6 / Chapter 1.2 --- Transcriptional Regulation and Transcription Factors Binding Sites Prediction --- p.7 / Chapter 1.2.1 --- Transcriptional Regulation --- p.7 / Chapter 1.2.1.1 --- Chromatin Remodeling --- p.7 / Chapter 1.2.1.2 --- Locus Control Regions (LCR) and Boundary Elements --- p.8 / Chapter 1.2.1.3 --- Promoter Structure --- p.9 / Chapter 1.2.1.4 --- Transcriptional Machinery Assembly --- p.10 / Chapter 1.2.2 --- Transcription Factors and Their Binding Sites --- p.11 / Chapter 1.2.3 --- Transcription Factor Binding Site Prediction --- p.12 / Chapter 1.3 --- Acetylcholinesterase --- p.15 / Chapter 1.3.1 --- Background --- p.15 / Chapter 1.3.2 --- Regulation ofAChE --- p.17 / Chapter 1.3.2.1 --- Transcriptional Level --- p.17 / Chapter 1.3.2.2 --- Post-transcriptional Level --- p.19 / Chapter 1.3.2.3 --- Post-translational Level --- p.20 / Chapter 1.3.2.3.1 --- Oligomerization --- p.20 / Chapter 1.3.2.3.2 --- Glycosylation --- p.21 / Chapter 1.3.2.3.3 --- Phosphroylation --- p.22 / Chapter 1.3.3 --- Functions of AChE --- p.23 / Chapter 1.3.3.1 --- Hydrolysis Acetylcholine --- p.23 / Chapter 1.3.3.2 --- Embryonic Development --- p.23 / Chapter 1.3.3.3 --- Haemotopotesis and Thrombopsiesis --- p.24 / Chapter 1.3.3.4 --- Neuritogensis --- p.24 / Chapter 1.3.3.5 --- Amyloid Fibre Assembly --- p.24 / Chapter 1.3.3.6 --- Apoptosis --- p.25 / Chapter 1.3.4 --- AChE and Alzheimer's disease --- p.25 / Chapter 1.3.4.1 --- Treatment for AD Patients --- p.27 / Chapter 1.4 --- Inducible Cell Expression Systems --- p.28 / Chapter 1.5 --- Objectives --- p.32 / Chapter 2 --- Chapter 2 Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Primer Design --- p.34 / Chapter 2.2.2 --- Cell Culture --- p.34 / Chapter 2.2.3 --- Transformation --- p.35 / Chapter 2.2.4 --- Plasmids Preparation --- p.35 / Chapter 2.2.5 --- Plasmids Screening --- p.36 / Chapter 2.2.6 --- RNA Extraction --- p.36 / Chapter 2.2.7 --- Reverse Transcriptase Polymerase Chain Reaction and Construction tnAChE/pCR4 vector --- p.37 / Chapter 2.2.8 --- Genomic Analysis --- p.37 / Chapter 2.2.9 --- Protein Sequence Analysis --- p.38 / Chapter 2.2.10 --- Genomic DNA Extraction --- p.39 / Chapter 2.2.11 --- Construction of Reporter Vectors ptnAChE_565/pGL3 and ptnAChK1143/pGL3 --- p.39 / Chapter 2.2.12 --- Luciferase Assay --- p.40 / Chapter 2.2.13 --- Transcription Factors and Promoter Prediction --- p.40 / Chapter 2.2.14 --- Protein Assay --- p.41 / Chapter 2.2.15 --- AChE Activity Determined by Ellman's Method --- p.41 / Chapter 2.2.16 --- Histochemistry --- p.42 / Chapter 2.2.17 --- Protein Extraction from Tissues --- p.42 / Chapter 2.2.18 --- Construction of Bacterial Expression Vector His-MBP-tnAChEAC/pHISMAL --- p.43 / Chapter 2.2.19 --- Protein Expression in Bacterial Expression System --- p.43 / Chapter 2.2.20 --- Purification and Thrombin Cleavage of His-MBP- tnAChEAC --- p.44 / Chapter 2.2.21 --- SDS Electrophoresis --- p.44 / Chapter 2.2.22 --- Western Blotting --- p.45 / Chapter 2.2.23 --- Construction of Tet-Off Expression Vector --- p.45 / Chapter 2.2.24 --- Transient Expression of tnAChEAC --- p.46 / Chapter 2.2.25 --- Establishment of Stable Tet-Off CHO Cell Lines Overexpressing tnAChEAC --- p.47 / Chapter 2.2.26 --- MTT Assay --- p.47 / Chapter 2.2.27 --- Partial Purification of tnAChEΔC --- p.48 / Chapter 3 --- Chapter 3 Sequence Analysis of AChE Gene of Tetraodon nigroviridis --- p.49 / Chapter 3.1 --- Results --- p.49 / Chapter 3.1.1 --- Cloning of tnAChE from Tetraodon nigroviridis Brain --- p.49 / Chapter 3.1.2 --- "Comparative genomic analysis of tnAChE with Human, Rat, Mouse, Takifugu rubripes, ZebrafishAChE" --- p.49 / Chapter 3.1.3 --- Primary Sequence Analysis --- p.52 / Chapter 3.1.4 --- Promoter and Transcriptional Factors Predictedin tnAChE Promoter Region --- p.60 / Chapter 3.1.4.1 --- Promoter Region Analysis In Silico --- p.60 / Chapter 3.1.4.2 --- Promoter Activity Analysis --- p.76 / Chapter 3.2 --- Discussion --- p.78 / Chapter 4 --- Characterization of tnAChE in Prokaryotic and Eukaryotic Tet-Off Inducible Expression System --- p.91 / Chapter 4.1 --- Results --- p.91 / Chapter 4.1.1 --- AChE Expresses in Tetraodon nigroviridis --- p.91 / Chapter 4.1.2 --- Expression of recombinant tnAChE in Bacterial Expression System --- p.94 / Chapter 4.1.2.1 --- Construction of His-MBP-tnAChEΔC/pHISMAL Construct --- p.94 / Chapter 4.1.2.2 --- His-MBP-tnAChEAC Expression in E. coli Strains BL21 (DE) and C41 --- p.94 / Chapter 4.1.3 --- Expression of tnAChEAC in Mammalian Expression System --- p.99 / Chapter 4.1.3.1 --- Construction of tnAChEAC/pTRE2hgyo Mammalian Expression Vector --- p.99 / Chapter 4.1.3.2 --- Transient Expression of tnAChEAC --- p.99 / Chapter 4.1.3.3 --- Establishment of Tet-Off CHO Cells Stably Expressing the Inducible tnAChEAC --- p.101 / Chapter 4.1.3.4 --- Characterization of Tet-Off tnAChEAC Stably Transfected Cell Clones --- p.103 / Chapter 4.1.3.5 --- Effect of Over Expressed tnAChEAC on cell viability --- p.103 / Chapter 4.1.3.6 --- Partial Purification of tnAChEAC from Stably Transfected Cells --- p.107 / Chapter 4.1.3.7 --- tnAChE and tnAChEAC in Different pH Values --- p.112 / Chapter 4.1.3.8 --- Kinetic Study of tnAChEAC --- p.112 / Chapter 4.1.3.9 --- Inhibition of AChE Activity of Partial Purified tnAChEAC by Huperzine --- p.112 / Chapter 4.2 --- Discussion --- p.116 / Chapter 4.2.1 --- Bacterial Expression System --- p.116 / Chapter 4.2.2 --- Expression of tnAChEΔC in Mammalian System --- p.119 / Chapter 5 --- General Discussion --- p.124 / Chapter 5.1 --- Summaries --- p.124 / Chapter 5.2 --- Further works --- p.126 / Chapter 6 --- References --- p.128 / Appendix 1 internet software and database used in this project --- p.151 / Appendix 2 tnAChE mRNA sequence --- p.152 / Appendix 3 ptnAChE-1143 sequence --- p.154 / Appendix 4 Six open reading frame translation of ptnAChE-1143 --- p.156

by Kwong-Kwok Wong. / Thesis (Ph.D)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 171-183. / TITLE PAGE --- p.I / ABSTRACT --- p.II / STATEMENT --- p.V / ACKNOWLEGEMENTS --- p.VI / ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XVI / Chapter Chapter 1. --- Introduction and Literature Review --- p.1 / Chapter I . --- Introduction --- p.1 / Chapter A. --- General introduction --- p.1 / Chapter B. --- Purpose of study --- p.5 / Chapter II. --- Literature review --- p.6 / Chapter A. --- Global control of aerobic-anaerobic shift --- p.6 / Chapter B. --- Identified anaerobiosis-inducible genes --- p.8 / Chapter C. --- Genetics of anaerobic regulation --- p.15 / Chapter i. --- Redox control --- p.15 / Chapter ii. --- DNA conformation --- p.15 / Chapter iii. --- fnr (oxrA) regulatory gene --- p.16 / Chapter iv. --- narL gene --- p.18 / Chapter v. --- Other regulatory genes --- p.19 / Chapter vi. --- Proposed FNR and NarL recognition sequences --- p.20 / Chapter D. --- future prospect --- p.23 / Chapter Chapter 2. --- Isolation of Anaerobiosis-inducible Promoters --- p.25 / Chapter I . --- Introduction --- p.25 / Chapter A. --- Properties of promoter-probe plasmid pKK232.8 --- p.26 / Chapter B. --- Properties of promoter-probe plasmid pFZYl --- p.28 / Chapter II. --- Materials and methods --- p.30 / Chapter A. --- Bacterial strains and plasmids --- p.30 / Chapter B. --- Media --- p.30 / Chapter C. --- Solutions --- p.31 / Chapter D. --- Small scale prepartaion of plasmid DNA --- p.32 / Chapter E. --- Large scale preparation of plasmid DNA --- p.33 / Chapter F. --- Digestion of DNA with restriction endonucleases --- p.35 / Chapter G. --- Analysis of DNA samples with agarose gel electrophoresis --- p.36 / Chapter H. --- Dephosphorylation of DNA fragments --- p.37 / Chapter I. --- Partial digestion of Chromosomal DNA with restriction enzyme Sau3A. --- p.37 / Chapter J. --- Ligation of DNA --- p.38 / Chapter K. --- Preparation of competent cells --- p.38 / Chapter L. --- Transformation --- p.40 / Chapter M. --- Chloramphenicol resistance levels test for promoter clones with plasmid pKK232.8 --- p.41 / Chapter N. --- Preparation of crude cell extract for chloramphenicol acetyltransferase (CAT) assays --- p.41 / Chapter O. --- CAT assay --- p.42 / Chapter P. --- Protein assay --- p.42 / Chapter Q. --- β-galactosidase assay --- p.43 / Chapter III . --- Results --- p.45 / Chapter A. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pKK232.8 --- p.45 / Chapter B. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pFZYl --- p.54 / Chapter IV. --- Summary and Discussion --- p.70 / Chapter A. --- Cloning with promoter-probe plasmid pKK232.8 --- p.70 / Chapter B. --- Cloning with promoter-probe plasmid pFZYl --- p.71 / Chapter C. --- Number of anaerobiosis inducible promoters --- p.73 / Chapter Chapter 3. --- Subcloning and Sequencing --- p.74 / Chapter I . --- Introduction --- p.74 / Chapter II. --- Materials and methods --- p.74 / Chapter A. --- Bacterial strains and bacteriophages --- p.74 / Chapter B. --- Preparation of M13mp RF plasmid --- p.75 / Chapter C. --- DNA sequencing by the chain termination method --- p.75 / Chapter D. --- Polymerase chain reaction (PCR) for the amplification of DNA fragments cloned in plasmid pFZYl --- p.79 / Chapter E. --- Using Exonuclease III to construct unidirectional deletions to generate nested clones --- p.80 / Chapter F. --- Direct gel electrophoresis --- p.81 / Chapter G. --- C-testiscreening for the orientation of insert in M13 phage --- p.81 / Chapter III . --- Results --- p.82 / Chapter A. --- Subcloning and sequencing of pFE29 and pFE117 --- p.82 / Chapter B. --- Subcloning and sequencing of pHSKl --- p.90 / Chapter C. --- Subcloning and sequencing of pHSK8 --- p.100 / Chapter D. --- "Subclonig and sequencing of pFSl, pFS22 and pFS3 4" --- p.109 / Chapter IV. --- Summary and Discussion --- p.113 / Chapter A. --- Trimming down size of DNA fragments to smaller fragments which still contained anaerobiosis-inducible promoters --- p.113 / Chapter B. --- Nucleotide sequencing --- p.113 / Chapter C. --- Sucloning and sequencing strategy --- p.115 / Chapter Chapter 4. --- Expression of Anaerobiosis-inducible Promoters --- p.120 / Chapter I . --- Introduction --- p.120 / Chapter II. --- Materials and methods --- p.122 / Chapter A. --- Bacterial strains and phages --- p.122 / Chapter B. --- Media --- p.125 / Chapter C. --- Transformation in Salmonella typhimurium --- p.125 / Chapter D. --- Genetic techniques --- p.126 / Chapter III. --- Results --- p.129 / Chapter A. --- Expression of Escherichia coli qlpT promoter --- p.129 / Chapter B. --- "Expression of Salmonella typhimurium anaerobiosis-inducible promoters cloned in pHSK8, pFS22 and pFS34" --- p.134 / Chapter IV. --- Summary and Discussion --- p.137 / Chapter A. --- A pair of divergent promoters were both regulated by anaerobiosis and glucose. --- p.137 / Chapter B. --- fnr(oxrA) dependent and independent promoters --- p.137 / Chapter C. --- Effect of nitrate on anaerobiosis expression. --- p.138 / Chapter Chapter 5. --- Analysis of Anaerobiosis-inducible Promoter-containing DNA sequences and Final Discussion --- p.141 / Chapter I. --- Analysis of anaerobiosis-inducible promoter-containing DNA sequences --- p.141 / Chapter A. --- "Search for initiation codon, conserved ""-10"" and ""-35"" regions" --- p.141 / Chapter B. --- Search for FNR binding sites and NarL binding sites. --- p.151 / Chapter C. --- Homology search among the promoter sequences of all anaerobiosis-inducible genes. --- p.156 / Chapter II. --- Final Discussions. --- p.164 / Chapter A. --- Summary of the properties of the sequenced and characterized promoters cloned in this study --- p.164 / Chapter B. --- Further studies. --- p.167 / REFERENCES --- p.168

Orientador: José Paes de Almeida Nogueira Pinto / Banca: Luciano dos Santos Bersot / Banca: Vera Lúcia Mores Rall / Resumo: No presente estudo foram avaliadas carcaças de frangos convencionais (originárias de aves que recebem promotores de crescimento) e alternativos (que não recebem promotores de crescimento), com o objetivo de: a) avaliar o efeito da não utilização de tais drogas sobre a prevalência de Salmonella spp. em frangos alternativos, comparando-a com a dos convencionais; b) determinar e comparar o perfil de resistência a antimicrobianos das cepas de Salmonella spp. isoladas de ambos os tipos de frangos; c) avaliar o efeito das operações de abate sobre a contaminação das carcaças, tanto em frangos convencionais quanto nos alternativos. Foram avaliadas 50 carcaças convencionais e 48 alternativas, as quais apresentaram prevalências de Salmonella spp. de 58% e 56,3%, respectivamente, ao longo da linha de abate. Na entrada dos frangos no abatedouro, não houve diferença significativa (p > 0,05) nas prevalências entre os dois grupos testados (38% nos convencionais e 47,9% nos alternativos), sendo que ao final do processamento os convencionais apresentaram 47,9% de positividade contra 14,6% dos alternativos, diferença essa significativa (p < 0,05). S. Enteritidis foi o sorotipo predominante entre as cepas identificadas. Quanto ao perfil de resistência, nossos resultados mostraram que, quando observada, ela foi maior nas cepas oriundas dos frangos convencionais. Conclui-se que: a) a não utilização dos promotores de crescimento não levou a um aumento da prevalência de Salmonella spp. nas aves alternativas; b) as cepas oriundas de frangos alternativos revelaram uma tendência a um menor nível de resistência aos antimicrobianos testados quando comparadas às dos convencionais; c) as operações de abate, no caso das aves alternativas, foram responsáveis por uma diminuição na contaminação inicialmente observada no tocante à Salmonella spp, sendo que no caso das convencionais, pelo seu aumento. / Abstract: Conventional chicken (receiving growth promoters) and alternative chickens (not receiving growth promoters) carcasses had been valuated in the present study, with the following objectives: a) to estimate the effect of no utilization of such drugs on the prevalence of Salmonella spp. in alternative chicken as compared to that of the conventional ones; b) to determine and to compare the resistance profile to antimicrobial agents of Salmonella spp. strains isolated from both types of chickens; c) to valuate the effect of the slaughter operations on the carcasses contamination in conventional chickens and in alternative ones. Fifty conventional and 48 alternative carcasses had been valuated, with Salmonella spp. prevalences of 58% and 56.3%, respectively, along the slaughter line. At the entry of the chickens in the slaughter-house there was no significant difference (p > 0.05) in the prevalences between the two tested groups (38% in the conventional and 47,9% in the alternative), at the end of the processing the conventional presenting 47,9% of positiveness compared to 14,6% of the alternative, this difference being significant (p < 0,05). S. Enteritidis was the predominant serotype among the identified strains. In relation to the resistance profile, our results showed that when it had been observed, it was larger in the strains originated from the conventional chicken. It had been concluded that: a) the removal of the growth promoters did not produce an increase in the Salmonella spp. prevalence in the alternative chicken; b) the strains originated from alternative chicken showed a trend toward a smaller resistance level to the tested antimicrobial agents compared to those from the conventional ones: c) the slaughter operations in the case of the alternative chickens were responsible for a decrease in the contamination initially observed regarding Salmonella spp. and for its increase in the case of the conventional. / Mestre

Existing methods to systematically characterize sequence-intrinsic activity of promoters are limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than a billion DNA fragments in parallel for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a barcode and decoded by paired-end sequencing. This library is transfected into cells and transcribed barcodes are quantified in the RNA by high-throughput sequencing. By computationally analyzing the resulting data using generalized linear models, we succeed in delineating subregions within promoters that are relevant for their activity on a genomic scale, and making accurate predictions of expression levels that can be used to inform minimal promoter reporter construct design. We also show how our approach can be extended to analyze the differential impact of single-nucleotide polymorphisms (SNPs) on gene expression.