published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Hematopoietic stem cells (HSCs) transplantations are widely used for the treatment of hematological and non-hematological disorders in clinic. Successful transplantation requires sufficient number and efficient homing of HSCs. Many studies have focused on developing an effective strategy to expand functional HSC population. Some regulatory molecules have been recently shown great promise for controlling the amplification of HSCs. In these dissertation studies, I first aim to identify gene(s) and their allelic variants contributing to strain-specific difference in HSC numbers between C57BL/6 (B6, low) and DBA/2 (D2, high) mice by using a classic forward genetic approach. Firstly, 3 quantitative trait loci (QTL) on chromosome (Chr) 3,5 and 18 were mapped by linkage analyses and confirmed in congenic mice. Secondly, Chr.3 QTL affected several HSC number-related biological processes. The D2 allele increased cycling and self-renewal whereas it decreased apoptotic rates of HSCs. Both actions conspired to increase HSC population size. Lastly, a small number of differentially-expressed genes was identified in Chr.3 congenic HSCs by a microarray-based candidate gene method, and the differential expression of one candidate, latexin, was found to relate to HSC number variations. Our studies report the strong evidence for the potential functions of latexin in HSC number regulation, and they are important for understanding molecular mechanisms of stem cell regulation and developing effective stem cell expansion strategies for clinical applications. In the second part of my studies, I studied homing and engraftment capabilities of HSCs. By using functional assays for progenitor and stem cells, I first reported the absolute homing efficiencies of murine young or old donor cells into young or old recipient mice. The results indicated that homing of primitive hematopoietic cells was not efficient and significantly decreased by aging of donors and recipients. The proliferation and differentiation states of HSCs were also impaired by homing itself, as well as by donors' and recipients age. Moreover, the hematopoietic reconstitution dynamics following transplantation were also affected by aging. Together, these findings will provide useful information for clinical applications especially when older individuals increasing serve as stem cell donors for elderly patients.

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting genetic regulation plays a role in HSC aging. In C57BL/6 (B6) mice, the HSC population steadily increases with age, whereas in DBA/2 (D2) mice, this population declines. Our lab has previously mapped a quantitative trait locus (QTL) to murine chromosome 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In these dissertation studies, I first aim to characterize the congenic mouse model which was generated by introgressing D2 alleles in the QTL onto a B6 background. Using a surrogate assay to mimic aging, I analyzed the cell cycle, apoptotic and self-renewal capabilities of congenic and B6 HSCs and show that D2 alleles in the QTL affect the apoptotic and selfrenewal capabilities of HSCs. In the second aim of these studies, I used oligonucleotide arrays to compare the differential expression of B6 and congenic cells using a population enriched for primitive stem and progenitor cells. Extensive analysis of the expression arrays pointed to two strong candidates, the genes encoding Retinoblastoma like protein 1 (p107) and Sorting nexin 5 (Snx5). B6 alleles were associated with increased p107 and Snx5 expression in old HSCs therefore both genes were hypothesized to be positive regulators of stem cell number in aged mice. Finally, in the third aim of these studies, I show that the individual overexpression of p107 and Snx5 in congeic HSCs increases day35 cobblestone area forming cell (CAFC) numbers, therefore confirming their roles as positive regulators of HSC number in vitro. These studies uncover novel roles for p107 and Snx5 in the regulation of HSC numbers and provide additional clues in the complex regulation of HSC aging.

TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).

Chez B. subtilis, le régulateur Mta régule l'expression de deux gènes codant pour des pompes d'efflux de drogues, bmr et blt. Ce régulateur joue un rôle physiologique beaucoup plus large que la régulation des gènes impliqués dans la résistance aux drogues puisqu'il régule l'expression d'au moins 18 autres gènes. Dans un mutant mta la quantité de protéine CpgA est très diminuée. Cette protéine est une GTPase impliquée dans l'assemblage du ribosome et dans la formation de la paroi. Cette diminution est supprimée par une mutation secondaire qui restaure un niveau intracellulaire de CpgA supérieur à celui de la souche sauvage. Cette mutation confère également un avantage de croissance, sur la souche sauvage et sur le mutant mta, au double mutant résultant. L'augmentation de la quantité de CpgA est corrélée avec l'augmentation du niveau d'expression du gène cpgA ainsi que de celui des gènes def et prpC avec lesquels cpgA est en opéron. Le gène def code pour une déformylase, une protéine impliquée dans la traduction, et prpC pour une Ser/Thr phosphatase capable de déphosphoryler CpgA. Dans le mutant supprimé, la surexpression de l'opéron def-cpgA est accompagnée d'un phénotype drastique de cellules enchaînées et immobiles en phase exponentielle de croissance (phénotype Mhp). Ces cellules contiennent le facteur alternatif de transcription SigD nécessaire à l'expression des gènes dont les produits sont impliqués dans la mobilité cellulaire et dans le clivage des septa lors de la division cellulaire, mais SigD est inactif dans ces cellules (SigD OFF). / In B. subtilis, the regulator Mta regulates the expression of two genes encoding drug efflux pumps, bmr and blt. This regulator has a physiological role far wider than the regulation of genes involved in resistance to drugs since it regulates the expression of at least 18 other genes. In a mta mutant, the amount of the CpgA protein is markedly reduced. This protein is a GTPase involved in ribosome assembly and in cell wall expansion. This decrease is suppressed by a secondary mutation that restores a higher level of intracellular CpgA than that of the wild type strain. This mutation also confers a growth advantage to the resulting double mutant over the wild type and the mta mutant strains. The increased amount of CpgA is correlated with increased level of cpgA gene expression and of the prpC and def genes with which cpgA is in operon. The def gene encodes a deformylase, a protein involved in translation, and prpC for a Ser / Thr phosphatase able to dephosphorylate CpgA. In the double mutant, overexpression of the def-cpgA operon is accompanied by a drastic phenotype of chained non-motile cells in the exponential phase of growth (Mhp phenotype). These cells contain SigD, an alternative sigma factor, necessary for the expression of genes whose products are involved in cell motility and in the cleavage of septa during cell division, but SigD is inactive in these cells (SigD OFF). In exponential growth phase, a wild type strain of B. subtilis shows a heterogeneous population consisting of cells SigD ON, motile and isolated, and cells SigD OFF, chained and non-motile. In the mta strain, the transition from one state to another is reversible as in the wild type strain.

Experimentally naive rats exhibit varying degrees of novelty exploration. Some rats display high rates of locomotor reactivity to novelty (high responders; HR), and others display low rates (low responders; LR). The novelty-seeking phenotype (LRHR) is introduced as a model of stress responsiveness. In this thesis I examined effects of chronic variable physical and social stress or control handling on the levels of various neurotrophins in the hippocampus, and changes in mossy fibre terminal fields in LRHR rats. A positive correlation is seen between histone deacetylase 2 and brain-derived neurotrophic factor (BDNF) levels both of which are oppositely regulated in LRHR CA3 fields in response to chronic social stress. Increase in BDNF levels in CA3 field accompanied increase in supra-pyramidal mossy fibre terminal field size (SP-MF) in HRs, and decrease in BDNF levels accompanied decrease in SP-MF volume in LRs. Epigenetic regulation of neurotrophic support underlying these structural changes is discussed. / by Ozge Oztan. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Sponges are an important source of bioactive marine natural products, or secondary metabolites. The common Caribbean reef sponge, Axinella corrugata, produces an antitumor and antibacterial chemical, stevensine. This study determined whether environmental stressors, such as elevated temperature and exposure to Amphibalanus amphitrite larvae, affect the production of stevensine by A.corrugata and if the stressors caused A.corrugata to exhibit differential gene expression. Temperature stress resulted in no significant change in the production of stevensine; only two genes were significantly differentially expressed, including hsp70. Larval stressed resulted in increased production of stevensine and significant differential gene expression (more than seventy genes). This study suggests that A.corrugata may be resilient to elevations in temperature and that one of stevensine's roles in nature is as an antifoulant. / by Jennifer Grima. / Thesis (M.S.)--Florida Atlantic University, 2013 / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Chan Kai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Title --- p.i / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Abstract (English) --- p.vii / Abstract (Chinese) --- p.ix / Table of contents --- p.xi / List of Figures --- p.xvi / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Human Hemoglobin --- p.1 / Chapter 1.2 --- Hemoglobinopathies --- p.4 / Chapter 1.3 --- Hereditary Persistence of Fetal Hemoglobin (HPFH) and β - Thalassemias --- p.6 / Chapter 1.4 --- Globin Genes Switching --- p.7 / Chapter 1.5 --- Pharmaceutical Agents for γ-Globin Gene Reactivation --- p.9 / Chapter 1.6 --- Discovery of LC978: A Novel Fetal Hemoglobin Inducing Agent --- p.10 / Chapter 1.7 --- Aim of Study --- p.11 / Chapter Chapter 2: --- Specific Induction of Gamma Globin Gene Transcription in K562 Leukemia Cell Line by Lead Compound LC978 --- p.12 / Chapter 2.1 --- K562 Cell Line as a Model for Gamma Globin Gene Induction Studies --- p.12 / Chapter 2.2 --- LC978-Induced Fetal Hemoglobin Expression in K562 Cell Line --- p.12 / Chapter 2.3 --- Materials --- p.14 / Chapter 2.3.1 --- "Chemicals, Kits and Reagents" --- p.14 / Chapter 2.3.2 --- Buffers and Solutions --- p.15 / Chapter 2.3.3 --- Cell Line --- p.16 / Chapter 2.3.4 --- Instruments and Equipments --- p.16 / Chapter 2.3.5 --- Enzymes --- p.16 / Chapter 2.3.6 --- Nucleic Acids --- p.17 / Chapter 2.3.7 --- Oligo Primers --- p.17 / Chapter 2.4 --- Methods --- p.17 / Chapter 2.4.1 --- In vitro Bioassay for Total Hemoglobin Production --- p.17 / Chapter (a) --- Preparation of Treatment Cell Culture Plates --- p.17 / Chapter (b) --- Treatment of K562 Cells by LC978 --- p.18 / Chapter (c) --- Signal Development --- p.18 / Chapter 2.4.2 --- Detection of Fetal Hemoglobin Production by HbF Sandwich ELISA --- p.18 / Chapter (a) --- Treatment of K562 Cells by LC978 --- p.18 / Chapter (b) --- Preparation of Capture Antibody-Coated and BSA-Blocked ELISA Plate --- p.19 / Chapter (c) --- Preparation of K562 Cell Lysates --- p.19 / Chapter (d) --- Antigen Capture and Signal Development --- p.19 / Chapter 2.4.3 --- Detection of Gamma Globin mRNA Level by Real-time RT-PCR --- p.20 / Chapter (a) --- Treatment of K562 Cells by LC978 --- p.20 / Chapter (b) --- Preparation of K562 Cell Lysate in Guanidium Thiocyanate (GT) Solution --- p.20 / Chapter (c) --- Isolation of Total RNA from LC978-treated K562 Cells --- p.21 / Chapter (d) --- cDNA Synthesis from mRNA by Reverse Transcriptase (RT) --- p.22 / Chapter (e) --- Real-Time Quantitative Polymerase Chain Reaction (PCR) --- p.23 / Chapter 2.5 --- Results --- p.24 / Chapter (a) --- In vitro Bioassay for Total Hemoglobin Production --- p.24 / Chapter (b) --- Fetal Hemoglobin Sandwich ELISA --- p.24 / Chapter (c) --- LC978-Induced Gamma Globin mRNA Accumulation --- p.25 / Chapter 2.6 --- Discussion --- p.31 / Chapter Chapter 3: --- Construction of Promoter-Reporter Plasmid Constructs --- p.33 / Chapter 3.1 --- The Human Gamma Globin Gene Promoter --- p.33 / Chapter 3.2 --- SEAP as a Reporter Gene for Promoter Deletion Study --- p.34 / Chapter 3.3 --- Materials --- p.35 / Chapter 3.3.1 --- "Chemicals, Kits and Reagents" --- p.35 / Chapter 3.3.2 --- Buffers and Solutions --- p.35 / Chapter 3.3.3 --- Bacterial Strain --- p.36 / Chapter 3.3.4 --- Cell Line --- p.36 / Chapter 3.3.5 --- Enzymes --- p.37 / Chapter 3.3.6 --- Nucleic Acids --- p.37 / Chapter 3.3.7 --- Oligo Primers --- p.37 / Chapter 3.4 --- Methods --- p.38 / Chapter 3.4.1 --- Molecular Cloning of A-Gamma Globin Gene Promoter and 3' Enhancer into pBlueScript II SK (-) --- p.38 / Chapter (a) --- Design and Synthesis of Oligo Primers --- p.38 / Chapter (b) --- Isolation of Genomic DNA from K562 Cells --- p.39 / Chapter (c) --- PCR Amplification of Gamma Globin Gene Promoter and 3' Enhancer --- p.40 / Chapter (d) --- Ligation of PCR Fragments into EcoR V-cut pBlueScript II SK (-) --- p.41 / Chapter (e) --- Preparation of E coli DH5a Competent Cells --- p.43 / Chapter (f) --- Heat-Shock Transformation of E. coli DH5a Competent Cells --- p.44 / Chapter (g) --- PCR Screening and Plasmid Purification of Putative pBlu2SKM-γAP and pBlu2SKM-γAE --- p.44 / Chapter (h) --- Isolation of Putative pBlu2SKM-γAP and pBlu2SKM-γAE Plasmid DNA --- p.45 / Chapter (j) --- Nucleotide Sequencing of Putative pBlu2SKM-yAP and pBlu2SKM-γAE --- p.47 / Chapter (j) --- Graphical Summary of Section 3.6.1 Sub-cloning Procedures --- p.49 / Chapter 3.4.2 --- Molecular Cloning of A-Gamma Globin Gene Promoter and 3' Enhancer into pSEAP2-Enhancer --- p.51 / Chapter (a) --- Sub-cloning of Promoter Fragment into pSEAP2-Enhancer --- p.51 / Chapter (b) --- Sub-cloning of 3' Enhancer Fragment into p 1224γAP-SEAP2 --- p.52 / Chapter (c) --- Graphical Summary of Section 3.6.2 Sub-cloning Procedures --- p.54 / Chapter 3.4.3 --- Construction of p 1224γAP-SEAP2-γAE Promoter Deletions Constructs --- p.56 / Chapter (a) --- Restriction Digestion at 5' End of A-Gamma Promoter Deletions --- p.56 / Chapter (b) --- Restriction Digestion at 3' Ends of A-Gamma Promoter Deletions --- p.56 / Chapter (c) --- Blunting 5'-overhangs and Self-Ligation of Linearized Plasmid Constructs --- p.57 / Chapter (d) --- Graphical Summary of Section 3.6.3 5，Deletions Procedures --- p.58 / Chapter 3.5 --- Results --- p.59 / Chapter (a) --- Nucleotide Sequence Confirmed by Cycle Sequencing --- p.60 / Chapter (b) --- "Resulting Plasmid Constructs p 1224γAP-SEAP2-yAE, p754yAP-SEAP2-yAE and p205yAP-SEAP2-γAE" --- p.64 / Chapter 3.6 --- Discussion --- p.67 / Chapter Chapter 4: --- Mapping of LC978-Responsive Elements on Human A-Gamma Globin Gene Promoter --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- pSV-β-Galactosidase as a Transfection Normalization Standard --- p.69 / Chapter 4.3 --- pSV-β-Galactosidase as a Transfection Normalization Standard --- p.70 / Chapter 4.4 --- Materials --- p.72 / Chapter 4.4.1 --- "Chemicals, Kits and Reagents" --- p.72 / Chapter 4.4.2 --- Buffers and Solutions --- p.73 / Chapter 4.4.3 --- Cell Line --- p.74 / Chapter 4.4.4 --- Nucleic Acids --- p.74 / Chapter 4.4.5 --- Instruments and Equipments --- p.74 / Chapter 4.5 --- Methods --- p.74 / Chapter 4.5.1 --- Determination of Optimal Dose of Transfection Reagent for --- p.74 / Chapter (a) --- Sterilization of Plasmid DNA for Transfection --- p.74 / Chapter (b) --- Transient Transfection of K562 Cells by pEGFP-N 1 --- p.75 / Chapter (c) --- Examination of EGFP Expression Level --- p.76 / Chapter 4.5.2 --- β-Galactosidase as Normalization Standard for K562 Transfections --- p.76 / Chapter (a) --- Transient Transfection of K562 Cells by pSV-β-Gal --- p.76 / Chapter (b) --- Determination of β-Galactosidase Expression Level --- p.76 / Chapter 4.5.3 --- Mapping of LC978-Responsive Elements on Human Gamma Globin Gene Promoter --- p.77 / Chapter (a) --- Co-Transfection of K562 Cells by p1224/754/205γAP-SEAP2 -γAE and pSV-β-Gal --- p.77 / Chapter (b) --- Treatment of Co-Transfected K562 Cells by LC978 --- p.77 / Chapter (c) --- Determination of β-Galactosidase Expression Level --- p.78 / Chapter (d) --- Determination of Secreted Alkaline Phosphatase (SEAP) Expression Level --- p.78 / Chapter (e) --- Determination of Fetal Hemoglobin Expression Level --- p.79 / Chapter 4.5.4 --- Mapping of Hydroxyurea-Responsive Elements on Human Gammm Globin Gene Promoter --- p.80 / Chapter (a) --- Determination of Optimal Biological Dose (OBD) of Hydroxyurea --- p.80 / Chapter (b) --- Co-Transfection of K562 Cells and Subsequent Treatment by Hydroxyurea --- p.80 / Chapter (c) --- "Assay for β-Galactosidase (β-Gal), Secreted Alkaline Phosphatase (SEAP) and Fetal Hemoglobin (HbF) Expression Level" --- p.81 / Chapter 4.5.5 --- Sodium Butyrate-Induced SEAP Expression --- p.81 / Chapter (a) --- Determination of Optimal Biological Dose (OB(d) of Sodium Butyrate --- p.81 / Chapter (b) --- Co-Transfection of K562 Cells and Treatment by Sodium Butyrate --- p.82 / Chapter (c) --- "Assay for p-Galactosidase (β-Gal), Secreted Alkaline Phosphatase (SEAP) and Fetal Hemoglobin (HbF) Expression Level" --- p.83 / Chapter 4.5.6 --- Data Analysis --- p.83 / Chapter (a) --- "Data Processing, Normalization and Graphing" --- p.83 / Chapter (b) --- Statistical Analysis --- p.83 / Chapter 4.6 --- Results --- p.84 / Chapter 4.6.1 --- Optimal Dose of Transfection Reagent for K562 --- p.84 / Chapter 4.6.2 --- β-Galactosidase as Normalization Standard for K562 Transfections --- p.84 / Chapter 4.6.3 --- LC978 Induction on Co-Transfected K562 Cells --- p.84 / Chapter 4.6.4 --- Hydroxyurea Induction on Co-Transfected K562 Cells --- p.85 / Chapter 4.6.5 --- Sodium Butyrate Induction on Co-Transfected K562 Cells --- p.86 / Chapter 4.7 --- Discussion --- p.98 / Chapter 4.7.1 --- Theme Question to be Answered --- p.98 / Chapter 4.7.2 --- Optimal Dose of DMRIE-C Transfection Reagent on K562 Cell Line --- p.98 / Chapter 4.7.3 --- pSV-β-gal as an Internal Normalization Control --- p.99 / Chapter 4.7.4 --- Responsive Element Mapping --- p.99 / Chapter (a) --- LC978-Induced Response --- p.100 / Chapter (b) --- Hydroxyurea-Induced Response --- p.100 / Chapter (c) --- Sodium Butyrate-Induced Response --- p.101 / Chapter 4.7.5 --- LCR-Dependent Gamma Globin Gene Reactivation --- p.101 / Chapter 4.7.6 --- Induction of Gamma Globin by Histone Deacetylase Inhibitor --- p.104 / Chapter 4.7.7 --- Basal SEAP Expression Levels of the Promoter-Reporter Constructs --- p.105 / Chapter 4.7.8 --- Summary --- p.105 / Chapter Chapter 5: --- General Discussion --- p.106 / References Cited --- p.109

"The objective of this thesis project was to begin identifying which regulatory transcription factors are involved in the up-regulation of the gene promoter for the Ą6 subunit of the gamma-alpha-butyric acid (GABAA-Ą6) receptor in cerebellar granule cell neurons (GCNs). Although a 150 base pair sequence proximal to the GABAA-Ą6 gene promoter had been characterized previously using electrophoretic mobility shift assays (EMSAs), the specific transcription factor(s) needed to express the GABAA-Ą6 gene had not been examined. This project utilized EMSAs to investigate this 150 base pair sequence further. It was found that when this sequence proximal to the gene promoter was divided into two overlapping halves, both shortened sequences were able to compete for binding with nuclear extracts. The full-length sequence was further divided into six sub-regions, and double-stranded competitors were generated from synthetic oligonucleotides. The only oligonucleotide to compete was the one that corresponded to the region of overlap between the left and right halves. This overlap region contains consensus sites for OCT-1, STAT, and the regulatory transcription factor NF-1. An NF-1 consensus sequence was able to compete DNA-protein complexes. Supershift assays showed that a xenopus NF-1 antibody, previously shown to compete in gel shift assays, caused a mobility shift of the DNA-probe complex. Analysis of extracts from granule cell neurons, cultured from 0 to 6 days in vitro (DIV) indicated NF-1 to be present all time points. Northern analyses were performed using probes for NF-1A, NF-1B, NF-1C and NF-1X. NF-1A transcripts were observed from 0 to 6 DIV, while NF-1B and NF-1X transcripts were present at 2 and 4 DIV. NF-1C RNA was barely detectable at any time point."