published_or_final_version / Biochemistry / Master / Master of Philosophy

Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP at the top of linkage group 1 of the M. truncatula genome. A NIP mapping population was established with the purpose of performing fine mapping in the region containing NIP. DNA from two M. truncatula ecotypes A17 and A20 can be distinguished through polymorphisms. Positional mapping of the NIP gene is based on the A17/A20 genetic map of M. truncatula. The NIP mapping population of 2277 plants was scored for their nodulation phenotype and genotyped with flanking molecular genetic markers 146o17 and 23c16d, which are located ~1.5 cM apart and on either side of NIP. This resulted in the identification of 170 recombinant plants, These plants' DNAs were tested further with different available genetic markers located in the region of interest, to narrow the genetic interval that contains the NIP gene. Segregation data from genotyping analysis of recombinant plants placed NIP in the region between 4L4 and 807 genetic markers.

Subsidiary mother cell (SMC) divisions during stomatal complex formation in Zea mays are asymmetric generating a small subsidiary cell (SC) and a larger epidermal cell. Mutants with a high number of abnormally shaped subsidiary cells include the brick1 (brk1) and discordia1 (dcd1) mutants. BRK1 is homologous to HSPC300, an ARP2/3 complex activator, and is involved in actin nucleation while DCD1 is a regulatory subunit of the PP2A phosphatase needed for microtubule generation (Frank and Smith, 2002; Wright et al. 2009). Possible causes of the abnormal SCs in brk1 mutants include a failure of the SMC nucleus to polarize in advance of mitosis, no actin patch, and transverse and/or no PPBs (Gallagher and Smith, 2000; Panteris et al 2006). The abnormal subsidiary mother cell division in dcd1 is due to correctly localized, but disorganized preprophase bands (PPBs; Wright et al. 2009). The observation that brk1 has defects in PPB formation and that the dcd1 phenotype is enhanced by the application of actin inhibitors led us to examine the dcd1; brk1 double mutant (Gallagher and Smith, 1999). We found that dcd1; brk1 double mutants demonstrate a higher percentage of aberrant SCs than the single mutants combined suggesting that these two mutations have a synergistic and additive effect on SC formation. Our observations and results are intriguing and the future step will be to quantitate the abnormal PPBs and phragmoplasts in the double and single mutants using immunolocalization of tubulin and actin as well as observations of live cells expressing tubulin-YFP.

To further facilitate mitochondrial DNA (mtDNA) sequence analysis for human identity testing, a better understanding of its mutation rate is needed. Prior to the middle 1990's the mutation rate applied to a forensic or evolutionary analysis was determined by phylogenetic means, This method involved calculating genetic distances as determined by amino acid or DNA sequence variability within or between species. The mutation rate as determined by this method ranged from 0.025-0.26 nucleotide substitutions/ site/ myr (million years). With the recent advent of mtDNA analysis as a tool in human identity testing an increased number of observations have recently come to light calling into question the mutation rate derived from the phylogenetic method. The mutation rate as observed from forensic analysis appears to be much higher than that calculated phylogenetically. This is an area that needs to be resolved in human identity testing. Mutations that occur within a maternal lineage can lead to a possible false exclusion of an individual as belonging to that lineage. A greater understanding of the actual rate of mutation within a given maternal lineage can assist in determining criteria for including or excluding individuals as belonging to that lineage. The method used to assess the mutation rate in this study was to compare mtDNA sequences derived from the HVI and HVII regions of the D-loop from several different maternal lineages. The sequence information was derived from five unrelated families consisting of thirty-five individuals. One intergenerational mutational event was found. This derives to approximately 1.9 nucleotide substitutions/ site/ myr. This mutation rate was very consistent with several other similar studies. This increased mutation rate needs to be considered by forensic testing laboratories performing mtDNA sequence analysis prior to formulating any conclusive results.

Aging is a biological process that has many detrimental effects due to the accumulation of oxidative damage to key biomolecules due to the action of free radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each form has been shown to reduce a specific enantiomer of bound and free oxidized methionine. Effects of Msr have yet to be studied in the major developmental stages of Drosophila melanogaster despite the enzymes elevated expression during these stages. A developmental timeline was determined for MsrA mutant, MsrB mutant, and double null mutants against a wild type control. Results show that the Msr double mutant is delayed approximately 20 hours in the early/mid third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon is due to an issue gaining mass. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2013. / FAU Electronic Theses and Dissertations Collection

The research efforts refer to tracking homologus loci in the chromosomes of a pair of a species. The purpose is to infer the extent of maximum syntenic correlation when an exhaustive set of orthologs of the species are searched. Relevant bioinformatic analyses use comparative mapping of conserved synteny via Oxford grid. In medical diagnostic efforts, deducing such synteny correlation can help screening chromosomal aberration in genetic disorder pathology. Objectively, the present study addresses: (i) Cytogenetic framework of syntenic correlation and, (ii) applying information-theoretics to determine entropy-dictated synteny across an exhaustive set of orthologs of the test pairs of species. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Cytogenetics is a study on the genetic considerations associated with structural and functional aspects of the cells with reference to chromosomal inclusions. Chromosomes are structures within the cells containing body's information in the form of strings of DNA. When atypical version or structural abnormality in one or more chromosomes prevails, it is defined as chromosomal aberrations (CA) depicting certain genetic pathogeny (known as genetic disorders). The present study assumes the presence of normal and abnormal chromosomal sets in varying proportions in the cytogenetic complex ; and, stochastical mixture theory is invoked to ascertain the information redundancy as a function of fractional abnormal chromosome population. This bioinformatic measure of redundancy is indicated as a track-parameter towards the progression of genetic disorder, for example, the growth of cancer. Lastly, using the results obtained, conclusions are enumerated, inferences are outlined and directions for future studies are considered. / by Jagadeshwari Karri. / Thesis (M.S.C.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

by Chiu Wing Lok Abe Kurtz. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 218-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Tsang Po Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 135-152). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract 摘要 --- p.iv / List of Figures --- p.vi / List of Tables --- p.viii / List of Abbreviations --- p.ix / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The Role of Glucose in Biological System --- p.1 / Chapter 1.2 --- Glucose Transporter Families --- p.1 / Chapter 1.2.1 --- Na+-Dependent Glucose Transporters --- p.2 / Chapter 1.2.2 --- Facilitative Glucose Transporters --- p.3 / Chapter 1.3 --- Glucose Transporter Type1 --- p.7 / Chapter 1.3.1 --- Primary Structure of the Glutl Protein --- p.7 / Chapter 1.3.2 --- Secondary Structure --- p.8 / Chapter 1.3.3 --- Tertiary Structure --- p.8 / Chapter 1.3.4 --- Kinetics Properties --- p.11 / Chapter 1.3.5 --- Tissue Distribution --- p.12 / Chapter 1.3.6 --- Multifunctional Property --- p.13 / Chapter 1.3.7 --- Characterization of GLUT1 Gene --- p.13 / Chapter 1.3.8 --- Regulation of GLUT1 Expression --- p.14 / Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.16 / Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome (GIutlDS) --- p.19 / Chapter 1.5.1 --- Backgronnd of GIutlDS --- p.19 / Chapter 1.5.2 --- Clinical Features of GIutlDS --- p.23 / Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24 / Chapter 1.5.4 --- Diagnosis --- p.26 / Chapter 1.5.5 --- Manage nent --- p.27 / Chapter 1.5.5.1 --- Ketogenic Diet --- p.27 / Chapter 1.6 --- Hypothesis and Objectives --- p.29 / Chapter Chapter 2: --- Biochemical and Molecular Analysis of GLUT1 in a Suspected GlutlDS Case --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patient --- p.32 / Chapter 2.1.2 --- Blood Samples --- p.32 / Chapter 2.1.3 --- Reagents and Buffers for Reverse Transcription --- p.32 / Chapter 2.1.4 --- Reagents and Buffers for TA Cloning --- p.34 / Chapter 2.1.5 --- Reagents for Genomic DNA Extraction --- p.34 / Chapter 2.1.6 --- Reagents and Buffers for Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.35 / Chapter 2.1.8 --- Reagents for Zero-trans 3-OMG Influx in Erythrocytes --- p.37 / Chapter 2.1.9 --- Reagents for Zero-trans 3-OMG Efflux from Erythrocytes --- p.38 / Chapter 2.1.10 --- Reagents for Erythrocytes Membrane Extraction and Detection --- p.39 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- GLUT1 Gene Analysis --- p.44 / Chapter 2.2.2 --- Zero-trans 3-OMG Influx into Erythrocytes --- p.51 / Chapter 2.2.3 --- Zero-trans 3-OMG Efflux from Erythrocytes --- p.52 / Chapter 2.2.4 --- Glutl Protein Expression --- p.54 / Chapter 2.2.5 --- Statistics --- p.57 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Molecular Analysis of the GLUT1 Gene of a Suspected GlutlDS Patient --- p.58 / Chapter 2.3.2 --- Functional Analysis of the GlutlDS Patient's Glutl Protein --- p.61 / Chapter 2.3.3 --- Glutl Protein Expression in the GlutlDS Patient --- p.64 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3: --- Pathogenicity Studies of GLUT1 Mutations --- p.71 / Chapter 3.1 --- Materials --- p.72 / Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.72 / Chapter 3.1.2 --- Cell Lire --- p.73 / Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.73 / Chapter 3.1.4 --- Cell Culture Wares --- p.75 / Chapter 3.1.5 --- Reagents for Transfection --- p.75 / Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.76 / Chapter 3.1.7 --- Consumables for Confocal Microscopy --- p.77 / Chapter 3.1.8 --- Reagents and Buffers for Flow Cytometry --- p.77 / Chapter 3.1.9 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.77 / Chapter 3.2 --- Methods --- p.79 / Chapter 3.2.1 --- Cell Culture Methodology --- p.79 / Chapter 3.2.2 --- Construction of GLUT1 Mutants --- p.80 / Chapter 3.2.3 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.84 / Chapter 3.2.4 --- Protein Expression Study --- p.85 / Chapter 3.2.5 --- 2-DOG Influx Assay in CHO-K1 Cells --- p.87 / Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.89 / Chapter 3.2.7 --- Statistics --- p.90 / Chapter 3.3 --- Results --- p.91 / Chapter 3.3.1 --- Molecular Analysis of 1034-1035Insl2 Mutation --- p.91 / Chapter 3.3.2 --- Expression of the Wild Type and Mutant GFP-Glutl Fusion Proteins --- p.92 / Chapter 3.3.3 --- Functional Analysis of the 1034-1035Insl2 Mutant --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter Chapter 4: --- GLUT1 Promoter Study --- p.100 / Chapter 4.1 --- Materials --- p.101 / Chapter 4.1.1 --- Construction of GLUT1 Promoter Vectors --- p.101 / Chapter 4.1.2 --- Cell Lines --- p.102 / Chapter 4.1.3 --- Cell Culture Media and Other Reagents --- p.103 / Chapter 4.1.4 --- Dual Luciferase Reporter Assay System --- p.103 / Chapter 4.2 --- Methods --- p.105 / Chapter 4.2.1 --- Bioinformatics --- p.105 / Chapter 4.2.2 --- Cell Culture --- p.105 / Chapter 4.2.3 --- Construetion of GLUT1 Promoter Vectors --- p.105 / Chapter 4.2.4 --- 5'-Deletion Analysis of GLUT1 Promoter --- p.108 / Chapter 4.2.5 --- Determination of the Activities of GLUT1 Promoter Fragments --- p.110 / Chapter 4.2.6 --- Statistics --- p.113 / Chapter 4.3 --- Results --- p.114 / Chapter 4.3.1 --- Determination of the Promoter Activity of the 5'-deletion Fragments --- p.114 / Chapter 4.3.2 --- Prediction of Transcription Factors in the 5'-deletion Fragments --- p.119 / Chapter 4.4 --- Discussion --- p.121 / Chapter Chapter 5: --- General Conclusion and Future Perspectives --- p.133 / References --- p.135

This dissertation reviews the development and implementation of integrative, systems biology methods designed to parse driver mutations from high- throughput array data derived from human patients. The analysis of vast amounts of genomic and genetic data in the context of complex human genetic diseases such as Glioblastoma is a daunting task. Mutations exist by the hundreds, if not thousands, and only an unknown handful will contribute to the disease in a significant way. The goal of this project was to develop novel computational methods to identify candidate mutations from these data that drive the molecular differentiation of glioblastoma into the mesenchymal subtype, the most aggressive, poorest-prognosis tumors associated with glioblastoma.