Mitochondria are a major source of reactive oxygen species and are particularly vulnerable to oxidative stress. Mitochondrial dysfunction, methionine oxidation, and oxidative stress are thought to play a role in both the aging process and several neurodegenerative diseases. Two major classes of methionine sulfoxide reductases, designated MsrA and MsrB are enzymes that function to repair the enatiomers of methionine sulfoxide, met-(o)-S and met-(o)- R, respectively. This study focuses on the effect of Msr deficiencies on mitochondrial function by utilizing mutant alleles of MsrA and MsrB. The data show that loss of only one form of Msr in the mitochondria does not completely impair the function of the mitochondria. However, loss of both Msr proteins within the mitochondria leads to an increased ROS production and a diminished energy output of the mitochondria. These results support the hypothesis that Msr plays a key role in proper mitochondrial function. / by Jennifer Verriotto. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

by Yeung Sze Mang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 123-133). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / 槪論 --- p.iii / ABBREVIATIONS --- p.iv / AMINO ACIDS SHORTHAND --- p.vi / TABLE OF CONTENTS --- p.vii-x / Chapter CHAPTER 1 --- LITERATURE REVIEW / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Structural Analysis of SL --- p.1 / Chapter 1.3 --- Location of SL-producing cells and Expression of SL --- p.5 / Chapter 1.4 --- Possible Functions of SL --- p.9 / Chapter 1.4.1 --- Adaptation to various backgrounds and Intensities of Illuminations --- p.9 / Chapter 1.4.2 --- Control of Reproduction and Maturation --- p.10 / Chapter 1.4.3 --- Responses to Stress --- p.12 / Chapter 1.4.4 --- Regulation of P034- and Ca2+ Metabolism --- p.12 / Chapter 1.4.5 --- Acid - Base Balance --- p.14 / Chapter 1.4.6 --- Regulation of Energy Metabolism --- p.15 / Chapter 1.4.7 --- Regulation of Fat Metabolism --- p.15 / Chapter 1.5 --- Regulation of SL Gene Expression --- p.19 / Chapter 1.5.1 --- Pit-1 Related Gene Regulation --- p.19 / Chapter 1.5.2 --- Regulation of Hormone Secretion --- p.21 / Chapter 1.5.2.1 --- Hypothalamic Factors --- p.21 / Chapter 1.5.2.2 --- Steroids --- p.23 / Chapter 1.6 --- Aims of Thesis --- p.23 / Chapter 1.6.1 --- Identification of SLII from Goldfish (Carassius auratus) --- p.23 / Chapter 1.6.2 --- Aims --- p.27 / Chapter CHAPTER 2 --- PCR ANALYSIS OF GFSLII GENE AND ITS EXPRESSION IN GOLDFISH TISSUE / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and Methods --- p.31 / Chapter 2.2.1 --- Materials --- p.31 / Chapter 2.2.2 --- Methods --- p.33 / Chapter 2.2.2.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.33 / Chapter 2.2.2.1.1 --- PCR Cloning of Goldfish SLII Gene --- p.33 / Chapter 2.2.2.1.2 --- Restriction Enzyme Digestion of the PCR Clones --- p.33 / Chapter 2.2.2.1.3 --- Subcloning of the Digested Fragments --- p.33 / Chapter 2.2.2.1.4 --- DNA Sequencing of the Subcloned Fragments --- p.34 / Chapter 2.2.2.2 --- Tissue Distribution Studies Using RNA Assay --- p.35 / Chapter 2.2.2.2.1 --- Tissue Preparation --- p.35 / Chapter 2.2.2.2.2 --- Total RNA Extraction --- p.35 / Chapter 2.2.2.2.3 --- Electrophoresis of RNA in Formadehyde Agarose Gel --- p.36 / Chapter 2.2.2.2.4 --- First Strand cDNA Synthesis --- p.37 / Chapter 2.2.2.2.5 --- Goldfish SLII Specific PCR --- p.37 / Chapter 2.2.2.2.6 --- PCR to Test DNA Contamination --- p.38 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.39 / Chapter 2.3.2 --- Tissue Distribution Studies Using RNA Assay --- p.40 / Chapter 2.4 --- Discussion --- p.45 / Chapter 2.4.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.45 / Chapter 2.4.2 --- Tissue Distribution Studies Using RNA Assay --- p.46 / Chapter CHAPTER 3 --- ANALYSIS OF GOLDFISH SLII GENE / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Materials and Methods --- p.49 / Chapter 3.2.1 --- Materials --- p.49 / Chapter 3.2.2 --- Methods --- p.54 / Chapter 3.2.2.1 --- Screening of Goldfish Genomic Library --- p.54 / Chapter 3.2.2.1.1 --- Preparation of the Plating Host --- p.54 / Chapter 3.2.2.1.2 --- Preparation of the Probe --- p.54 / Chapter 3.2.2.1.3 --- Primary Screening of Goldfish Genomic Library --- p.55 / Chapter 3.2.2.1.4 --- Isolation of the Positive Clones --- p.56 / Chapter 3.2.2.1.5 --- Phage Titering --- p.56 / Chapter 3.2.2.1.6 --- Purification of the Positive Clones --- p.57 / Chapter 3.2.2.1.7 --- Phage DNA Preparation --- p.57 / Chapter 3.2.2.1.8 --- Find out the Target Gene Size of the Positive Clones --- p.58 / Chapter 3.2.2.1.9 --- Cloning of the PCR Fragments into pUC18 Vector --- p.59 / Chapter 3.2.2.1.10 --- Checking the Cloned Insert Size --- p.60 / Chapter 3.2.2.1.11 --- Restriction Enzyme Digestion to Release the Inserts --- p.61 / Chapter 3.2.2.1.12 --- Mini prep of the Positive Clones for Further Investigations --- p.61 / Chapter 3.2.2.1.13 --- DNA Sequencing of the Positive Clones --- p.61 / Chapter 3.2.2.1.14 --- Restriction Enzyme Mapping of the Positive Clones --- p.62 / Chapter 3.2.2.1.15 --- Subcloning of Clone 2A and5A / Chapter 3.2.2.1.16 --- Determination of the Promoter Region of Clone 2A Using Universal Genome Walker Kit --- p.63 / Chapter 3.2.2.2 --- Southern Blot Analysis of Goldfish and Catfish Genomic DNA --- p.66 / Chapter 3.2.2.2.1 --- Genomic DNA Preparation from Goldfish and Catfish Tissues --- p.66 / Chapter 3.2.2.2.2 --- Restriction Enzyme Digestion of the Genomic DNA --- p.67 / Chapter 3.2.2.2.3 --- Alkaline Transfer of the Digested Genomic DNA --- p.67 / Chapter 3.2.2.2.4 --- Hybridization of the Digested Genomic DNA --- p.67 / Chapter 3.3 --- Results --- p.69 / Chapter 3.3.1 --- Screening of the Goldfish Genomic Library --- p.69 / Chapter 3.3.2 --- Mapping the Target Genes --- p.69 / Chapter 3.3.3 --- DNA Sequencing of the 2 Positive Clones --- p.69 / Chapter 3.3.4 --- Southern Blot Analysis of Goldfish and Catfish Genomic DNA --- p.81 / Chapter 3.4 --- Discussion --- p.83 / Chapter CHAPTER 4 --- EXPRESSION OF RECOMBINANT GOLDFISH SOMATOLACTIN IN ESCHERICHIA COLI (E. COLI) / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Materials and Methods --- p.89 / Chapter 4.2.1 --- Materials --- p.89 / Chapter 4.2.2 --- Methods --- p.96 / Chapter 4.2.2.1 --- Transformation of the Recombinant Protein Carrying Plasmid into E. coli. (BL21) --- p.96 / Chapter 4.2.2.2 --- Small Scale Expression of Recombinant Goldfish SLII Protein --- p.96 / Chapter 4.2.2.3 --- Large Scale Expression of Recombinant Goldfish SLII Protein --- p.97 / Chapter 4.2.2.4 --- Preparation of the Recombinant Protein for Purification --- p.99 / Chapter 4.2.2.5 --- Protein Purification Using Novagen His-Bind Resin Kit --- p.99 / Chapter 4.2.2.6 --- Production of Polyclonal Antibody in Rabbits --- p.100 / Chapter 4.2.2.7 --- Enzyme Linked Immunosorbant Assay (ELISA) --- p.101 / Chapter 4.2.2.8 --- Western Blot Analysis of the Recombinant Hormones --- p.103 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- Expression of the Recombinant Goldfish SLII --- p.105 / Chapter 4.3.2 --- Purification of the Recombinant Goldfish SLII --- p.105 / Chapter 4.3.3 --- ELISA Analysis --- p.105 / Chapter 4.3.4 --- Western Blot Analysis --- p.110 / Chapter 4.4 --- Discussion --- p.113 / Chapter 4.4.1 --- Expression of the Recombinant Goldfish SLII --- p.113 / Chapter 4.4.2 --- Purification of the Recombinant Goldfish SLII --- p.114 / Chapter 4.4.3 --- Analysis of the Recombinant Goldfish SLII --- p.114 / Chapter CHAPTER 5 --- GENERAL DISCUSSION AND CONCLUSIONS --- p.116 / REFERENCES --- p.123

Li, Lei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 85-91). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Phytochromes are the photoreceptors mainly responsible for the detection of red and far-red (FR) light and the following responses. HFR1 is a basic helix-loop-helix type putative transcription factor involved in Phytochrome A signaling pathway. First we look at the early phenotype of mutant seedlings lacking a functional HFR1 gene and we show that auxin is involved in the increased hypocotyl phenotype of these seedlings. Northern blots and RT-PCRs showed that ATHB-2, a gene involved in shade avoidance is regulated by HFR1 under FR light. Microarray experiments were performed to find the genes that are early targets of regulation by HFR1.

The purpose of this thesis for the degree of Master of Science was to use molecular and immunological techniques to study cellular responses to dengue virus infection. In the initial study, Differential Display was used to compare mRNA expression in dengue-infected K562 cells and mock-infected cells. Cloning and sequencing were then used to identify cellular genes that were potentially up-regulated in response to Dengue virus infection. These genes included bleomycin hydrolase and a dystrophin homologue. The goal of the later part of this research was to construct a recombinant vaccinia virus expressing the dengue 1 Hawaii NS3 protein. Cytotoxic T-lymphocyte assays and protein gel electrophoresis showed that the NS3 protein was being expressed. This construct was then used to study the cytotoxic T-cell response of a dengue 1 vaccine recipient. The results of this study showed that this individual has dengue 1 NS3 specific T-cells and also that this vaccinia virus can be used for subsequent T-cell studies.

"Host resolution of viral infection is dependent upon components of the innate and acquired immune system. The mammalian protein Apobec3 plays an important role as part of the immune system’s innate defenses through its modification of reverse transcribed viral DNA. Recently, Apobec3 was found to directly inhibit HIV-1 and HBV replication through deaminating newly transcribed deoxycytidine residues to deoxyuridine. The ability of mouse and simian Apobec3 variants to inhibit human retroviruses and vice versa highlights the utility of analyzing cross-species homologues. To better understand this editing enzyme, differentially pathogen-susceptible inbred mice were used as an experimental model. The purpose of this project is to examine the effects of murine Apobec3 (muA3) alternative splicing on its DNA-editing characteristics. Three distinct Apobec3 isoforms were isolated from pathogen-susceptible BALB/cByJ (“C”) inbred mice, and two Apobec3 isoforms came from pathogen-resistant C57BL/6ByJ (“Y”) mice. The five muA3 isoforms were cloned, sequenced, and expressed from a constitutive promoter in a haploid Saccharomyces cerevisia strain. MuA3 DNA-editing activity was measured via the CAN1 forward mutation assay. The five isoforms studied in this project were discovered to be strain-specific. One isoform from each mouse strain mutated the yeast CAN1 locus significantly. Additionally, both muA3 isoform mRNAs derived from the pathogen-resistant Y mice were found to persist at a higher level (2.7 -12.4 fold) than any of the C mouse isoforms. This suggests that the absence of exon 5 or some other signal in the Y mice may influence transcript stability. Evidence also suggests that the murine Apobec3 start codon is actually 33bp upstream of its reference start, with implications for previous research performed using muA3. Sequencing analysis of genomic DNA revealed the presence of a 4bp insertion in a region of BALB/cByJ muA3 which may have disrupted an intronic splicing enhancer signal. Furthermore, a novel BALB/cByJ Apobec3 isoform was characterized. This is the first report of strain-specific processing with regard to muA3."

MicroRNAs play a key role in post-transcriptional gene regulation. They regulate target gene expression with mRNA degradation or translation repression. Each miRNA is estimated to regulate dozens of genes in human, and dysregulation of miRNA leads to various diseases, such as cancer, heart disease and depression. Therefore, it is critical to understand the mechanism of miRNA biogenesis and targeting. This work integrated gene and miRNA expression profile from various cancer projects to screen for potential miRNA biogenesis regulators and activity modulators. In this analysis, we identified several genes that regulate miRNA pathway and found their association with tumor progression and clinical outcome.

Mechanistic understanding of gene regulation is crucial for rational engineering of new genetic systems through synthetic biology. Genetic engineering efforts in new organisms are often hampered by a lack of knowledge about how regulatory components function in new host contexts. This dissertation focuses on efforts to overcome these challenges through the development of generalizable experimental methods for studying the behavior of DNA regulatory sequences in diverse species at large-scale. Chapter 2 describes experimental approaches for quantitatively assessing the functions of thousands of diverse natural regulatory sequences through a combination of metagenomic mining, high-throughput DNA synthesis and deep sequencing. By employing these methods in three distinct bacterial species, we revealed striking functional differences in gene regulatory capacity. We identified regulatory sequences with activity levels with activity levels spanning several orders of magnitude, which will aid in efforts to engineer diverse bacterial species. We also demonstrate functional species-selective gene circuits with programmable host behaviors that may be useful for microbial community engineering. In Chapter 3 we provide evidence for the evolution of altered stringency in σ70-mediated transcriptional activation based on patterns of initiation and activity from promoters of diverse compositions. We show that the contrast in GC content between a regulatory element and the host genome dictates both the likelihood and the magnitude of expression. We also discuss the potential implications of this proposed mechanism on horizontal gene transfer. The next two chapters focus on efforts aimed at extending the high-throughput methods described in earlier chapters to new organisms. Chapter 4 presents an in vitro approach for multiplexed gene expression profiling. Through the development and use of cell-free expression systems made from diverse bacteria, it was possible to rapidly acquire thousands of transcriptional measurements in small volume reactions, enabling functional comparisons of regulatory sequence function across multiple species. In Chapter 5 we characterize the restriction-modification system repertoires of several commensal bacterial species. We also describe ongoing efforts to develop methods for bypassing these systems in order to increase transformation efficiencies in species that are difficult or impossible to transform using current approaches.

Finally, the isolation and cloning of black sea bream SL receptor using PCR cloning and protein pull down assay were also attempted. Based on the PCR cloning results, the phylogenetic analysis of nonsalmonids fish GHR1 and SLR protein sequence, the GHR1 data of tissue distribution and effects of environmental salinity and fasting in tilapia, along with the results of far western blot, black sea bream GHR1 is probably a receptor for SL, however there is also a SL specific receptor in black sea bream. / In hormone treated primary cell culture of nonspawning black sea bream pituitary, 10-8 M E2 significantly increases SL mRNA level but 10-10 M, 10-9 M, 10-8 M of E2 inhibit GH mRNA level in female black seabream; 10-8 M E2 also inhibits SL and GH mRNA expression in bisexual black sea bream; 10-8 M MT inhibits SL mRNA expression in male black sea bream but any concentration of MT detected shows no significant effect on GH mRNA level. / Key words. somatolactin (SL), monthly changes, SL promoter, pit-1 and SL receptor / Somatolactin, SL, is a novel member of GH family of pituitary hormone only found in fish. It is considered to be a member of the GH gene family after gene duplication. Two types of SL, SL alpha and SL beta were identified, and SL 13 seems only in fresh water fish, such as goldfish, catfish, rainbow trout, eel and zebrafish. Black sea bream is a marine fish, and there is only SL alpha found from sequencing of over 100 SL cDNA clones. / The cDNAs encoding for transcription factor pit-1 variants were cloned and the transactivation of these Pit-1 isoforms on SL gene promoter were studied. Three variants of Pit-1 are first identified in fish. Pit-1b and Pit-1c can enhance SL promoter activity in Hepa-T1 cells respectively to about 2 fold and 12 fold, but pit-1a failed to activate the SL gene it in the same cells. All the three pit-1s of black sea bream couldn't reverse the inhibition of SL promoter in GH3 cells. The data suggest that N terminal 60 amino acid residues are critical in transactiation on SL promoter and SL promoter activity is possibly limited to fish SL secreting cells. / The SL gene promoter was obtained for gene regulation studies aiming to search for possible regulatory elements controlling the transcription of SL gene in black seabream. SL gene promoter is active in HepaT1 cells, but is inhibited in GH3 cells. Seven putative pit-1 response elements were confirmed with EMSA and super shift assay. / To study the physiological function of SL in black seabream, we initiated a study of monthly expressions of SL mRNA and gonadal somatic index (GSI) to determine whether SL is related to reproduction in black seabream, with GH mRNA levels were also detected for comparison. The results imply that function of SL is possibly related to early development of testis, while GH probably plays some roles in testis and ovary maturation. / by Tian, Jing. / "October 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4574. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 156-170). / 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.

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