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Interactions Of Shiga-Like Toxin-2 (Stx-2) From Escherichia Coli O157:H7 And The Bcl-2 Family Of Proteins During Host Cell Programmed Cell Death, Lia K. Jeffrey

Doctoral Dissertations

Stx-2 is a major contributor to the pathogenesis of Escherichia coli 0157:H7. Prior reports suggest that Stx-2 increases necrosis and apoptosis of a variety of host cells including those of endothelial origin as well as immune cells such as neutrophils (156). However, the role Stx-2 plays in delayed apoptosis of neutrophils is not fully understood given that previous studies have shown conflicting results (118, 51). The process of apoptosis is mediated by the Bcl-2 protein family (2, 46, 226). The purpose of this research was to define the molecular mechanisms of Stx-2 and Bcl-2 protein family interactions. These studies examined …

Characterization Of The Expression Profile Of Polyamine Biosynthetic Genes (Spermidine Synthase) And Polyamine Metabolic Regulation In Arabidopsis, Lin Shao

Doctoral Dissertations

Polyamines are ubiquitously distributed cationic compounds, which play important roles in numerous cellular functions in plants. This study was aimed at elaborating the regulation of polyamine biosynthetic gene expression and polyamine metabolism. The organ/tissue specific expression patterns of two genes encoding the polyamine biosynthetic enzyme spermidine synthase ( AtSPDS1 and AtSPDS2) were studied in Arabidopsis at different developmental stages using promoter::reporter approach. The two homologues showed similar ubiquitous expression with subtle differences being observed in certain tissues (e.g. root, siliques, and embryos). Neither transgenic manipulation by over-expression of AtSPDSI alone nor its concomitant expression with genes encoding other biosynthetic enzymes …

Regulation Of The Catalytic And Allosteric Properties Of Photoreceptor Phosphodiesterase (Pde6) By The Glutamic Acid-Rich Protein-2 (Garp2), Wei Yao

Doctoral Dissertations

The photoreceptor phosphodiesterase (PDE6) must be precisely regulated to control the sensitivity, amplitude, and kinetics of the photoresponse during excitation, termination and adaptation to light stimulation in rod and cone photoreceptors. The central hypothesis of this thesis is that one PDE6 binding partner, the glutamic acid-rich protein (GARP2), may regulate PDE6 to reduce its "dark noise", enhance its sensitivity and conserve metabolic energy during rod photoresponse saturation.

The first aim of this research is to better understand the unique biochemical and biophysical characteristics of GARP2 in order to reveal its functional attributes for regulating PDE6 during phototransduction in rod photoreceptors. …

Calcium Homeostasis In Escherichia Coli, Muhammad Arif

Doctoral Dissertations

While the role of calcium ions as secondary chemical messengers has been well described in eukaryotic cells, little is known about calcium homeostasis in bacteria at the physiological and molecular level. No calcium influx gene has been identified so far. This dissertation focuses on calcium regulation of gene expression by (i) determining the effects of elevated and depleted calcium levels on global gene expression in wild-type cells (MG1655), (ii) employing transposon mutants (calcium-sensitive) and mutants that are defective in calcium transport, and (iii) performing quantitative analysis of a specific subset of 15 genes to elucidate their possible involvement in calcium …

A System For Automating The Interpretation Of Analytical Ultracentrifuge Data, Bradley W. Langhorst

Doctoral Dissertations

In order to accelerate the development of knowledge about protein associations, further improvements to acquisition, sharing, and analysis of Analytical Ultracentrifugation (AU) data must be made. XML data formats have been defined to allow complete exchange of the information associated with an AU experiment. Extensible formats to record solution identity, instrument setup parameters, acquired data, and analysis of that data have been developed. A normalized relational database to allow storage and searching of this data has also been created. A computer program called PANDaS (Protein Association Network Data Server) was built to interact with any software conforming to the data …

Studies Of Verticillium Wilt And Characterization Of Candidate Verticillium Wilt Resistance Genes In The Mint Species Mentha Longifolia (L) Huds, Kelly Jean Vining

Doctoral Dissertations

To investigate the genetic basis of verticillium wilt resistance in mint (Mentha L., Lamiaceae), wild-collected germplasm obtained from the United States Department of Agriculture was employed to develop breeding populations for wilt resistance screening and molecular genetic study, including cloning of candidate verticillium wilt resistance genes.

A collection of fourteen Mentha longifolia accessions from Europe, Asia and South Africa was analyzed for morphological traits, oil composition, and verticillium wilt resistance. In addition, a preliminary molecular diversity assessment was conducted utilizing randomly amplified polymorphic DNA (RAPD) markers. The accessions were found to be diverse regarding all observed traits and the South …

Glycoprotein Hormone Receptors In Sea Lamprey (Petromyzon Marinus): Identification, Characterization And Implications For The Evolution Of The Vertebrate Hypothalamic-Pituitary-Gonadal/Thyroid Axes, Mihael Freamat

Doctoral Dissertations

The vertebrate hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) endocrine axes involve specific interaction between pituitary glycoprotein hormones GpH (lutropin LH, follitropin FSH, thyrotropin TSH) and glycoprotein hormone receptors GpH-R (LH-R, FSH-R and TSH-R respectively).

GpHs and GpH-Rs originate in a common ancestral ligand/receptor pair. Their duplications were followed by divergent evolution resulting in the emergence of a novel, pituitary/peripheral gland control level. It is estimated that this occurred more than 500 million years ago (mya), before or concomitant with the divergence of the jawed vertebrates (Gnathostomes) lineage from their jawless (Agnathan) ancestors. This coincides with the estimated time of divergence of …

The Development Of Tools To Allow Genetic And Genomic Analysis Of Frankia, Tania Rawnsley Spenlinhauer

Doctoral Dissertations

Frankia, a nitrogen-fixing actinomycete, forms a symbiotic association with a variety of woody dicotyledonous plants. The lack of standard genetic tools for this important bacterium has hindered studies on the molecular biology of this symbiosis. We are interested in developing tools for the genetic analysis of Frankia physiology and its interactions with its host plants. Our approach has focused on the development of a physical and genetic map of the Frankia chromosome by macrorestriction analysis. Agarose-embedded chromosomal DNA plugs were prepared from three Frankia isolates: Eul1c, EAN1pec and Ccl3. The genome sizes of the three strains were determined by pulse-field …

The Function Of Pab1 In Translation And In Puf3 Dependent Deadenylation, Darren J. Lee

Doctoral Dissertations

The involvement of the poly(A)-binding protein (PAB1) in deadenylation and translation is well known. How PAB1 inhibits deadenylation and promotes translation is not well understood. I have analyzed PAB1 variants, containing entire domain deletions and substitutions of yeast residues with human residues. Chapter I discusses and provides in vivo translation rates of strains containing PAB1 variants, defects in mRNA degradation proteins, and defects in translation components. In chapter II, I address the role of PAB1 in regulated deadenylation. For this analysis I studied the effect of PUF3, a member of the PUF family of proteins that bind specific 3' UTR …

Functional Analysis Of The Caf1 Protein, Takbum Ohn

Doctoral Dissertations

The CAF1 protein is a component of the CCR4-NOT deadenylase complex. While yeast CAF1 displays deadenylase activity, this activity is not required for its function in vivo, and CCR4 is the primary deadenylase in the complex. In order to identify CAF1-specific functional regions required for deadenylation in vivo, we targeted for mutagenesis six regions of CAF1 that are specifically conserved among CAF1 orthologs. Defects in three regions of the CAF1 protein (residues 173-175, residues 255-257 and residues 340-342, alleles caf1-1, caf1-3 and caf1-6, respectively) were found to dramatically reduce the rate of deadenylation in vivo and to result in typical …

Analysis Of The Role Of Poly(A) -Binding Protein (Pab1) In The Mrna Degradation Process In Yeast, Gang Yao

Doctoral Dissertations

The mRNA deadenylation process influences multiple aspects of protein synthesis and is known to be the major factor controlling mRNA decay rates. My data demonstrates that yeast PAB1 plays both positive and negative roles in controlling deadenylation, and I have identified particular regions of PAB1 involved in controlling different aspects of the mRNA degradative process. I have found that yeast PAB1 does not play a simple, obstructionist role in regulating CCR4 deadenylation. Instead, PAB1-PAB1 protein interactions, as mediated by the PAB1 proline-rich region (P domain) and the RRM1 domain, are required for the CCR4 deadenylase activity. The P and RRM1 …

A Molecular And Morphological Investigation Of The Red Seaweed Genus Porphyra (Bangiales, Rhodophyta) In The Northwest Atlantic, Troy Lee Bray

Doctoral Dissertations

To evaluate the possible occurrence of cryptic Porphyra taxa in the Northwest Atlantic, extensive field collections were made during winter and spring periods when relatively few previous collections had been made. Approximately 100 different sites extending from Chance Harbor, New Brunswick, Canada to Rye, New York, USA, were sampled during multiple years (2001-2005). Historical specimens from several herbaria (NHA, FH, BM, WTU, MICH, US) were also examined for possible cryptic taxa. A combination of morphological and molecular tools was used to screen both recent and historical collections.

Sequences from the plastid-encoded, ribulose-1, 5-bisphosphate carboxylase-oxygenase large subunit (rbcL) gene and rbcL- …

The Taxonomic And Systematic Relationships Of Several Salt Marsh Fucus Taxa (Heterokontophyta, Phaeophyceae) Within The Gulf Of Maine And Ireland Examined Using Microsatellite Markers, Aaron L. Wallace

Doctoral Dissertations

The present thesis utilizes microsatellite markers to examine genetic affinities between several salt marsh Fucus ecads in order to ascertain their relationships with attached parental species. Chapter I provides an introduction to the genus Fucus and discusses morphological plasticity, systematic difficulties, and studies of hybridization between different Fucus species.

Chapter II describes my development of microsatellite markers for Fucus. Four polymorphic microsatellite loci were used to determine the origin of a dwarf muscoides-like Fucus from the Brave Boat Harbor (ME) salt marsh. Similar forms were originally described in Europe, and appear to be derived from F. vesiculosus L. However, my …

Function, Expression And Evolution Of The *Src Family Kinases In Caenorhabditis Elegans, Chery Angel Whipple

Doctoral Dissertations

The vertebrate proto-oncogene, Src, is the prototype of a family of membrane-associated protein tyrosine kinases involved in cell signaling pathways that control cell growth, development, and differentiation. Activation of Src by mutation or overexpression has been implicated in a range of cancers, particularly breast and colon cancer. Inappropriate activation of Src has pronounced oncogenic effects on cell morphology, adhesion, and motility. Despite its implications in cancer, the normal biological role of Src is not well understood.

I used the nematode C. elegans to investigate the expression, function, and evolution of two src genes (src-1 and src-2) utilizing a combination of …

Analysis Of Signal Output By The Ethylene Receptor Etr1 From Arabidopsis, Xiang Qu

Doctoral Dissertations

Ethylene is one of the most important plant hormones and regulates many processes during plant growth and development. In Arabidopsis, the ethylene receptor family consists of five members: ETR1 and ERS1 have a functional histidine kinase domain and form subfamily 1; members of subfamily 2, including ETR2, ERS2, and EIN4, possess a highly diverged histidine kinase domain predicted not to be functional. To analyze signal output by the ethylene receptor ETR1 from Arabidopsis, mutant-based approaches were taken. Initially, the role of the proposed signal output region of ETR1 in ethylene signaling was examined. For this purpose, the ability of mutant …

Genetic Diversity And Structure Of Calanoid Copepods: Molecular Evolutionary Patterns In Coastal Estuaries (Acartia Tonsa) And The Open Ocean (Calanus Spp), Robert Sean Hill

Doctoral Dissertations

Calanoid copepods are an important part of marine and estuarine ecosystems. However, it has been difficult to study their life histories, population structure, and evolution because they share a conserved morphology that complicates species identification. A primary focus of this study was the genetic and physiological variation of the calanoid copepod Acartia tonsa from four estuaries along the East Coast of the USA (Great Bay, NH, Buzzards Bay, MA, Narragansett Bay, RI, and Beaufort Inlet, NC). Based on DNA sequence variation for the mitochondrial cytochrome oxidase I (mtCOI) gene, significant population structure was observed between all pairs of estuarine populations …

Transcriptional Elongation Defects Enhance Upstream Poly (A) Site Utilization And Spt5 Affects Mrna Degradation Through Its Physical And Functional Interaction With Ccr4 -Not Complex, Yajun Cui

Doctoral Dissertations

While a number of proteins are involved in elongational processes, the mechanism of action of most of these factors remains unclear primarily because of the lack of suitable in vivo model systems. We have identified in yeast several genes, each of which contain internal poly (A) sites, whose full-length mRNA formation is reduced by mutations in RNA polymerase II subunit RPB2, elongation factor SPT5, or TFIIS. RPB2 and SPT5 defects also promoted the utilization of upstream poly (A) sites for genes that contain multiple 3 ' poly (A) signaling sequences, supporting a role for elongation in differential poly (A) site …

Nuclear And Cytoplasmic Genetic Variation In Picea: Dna Markers For Evaluating Past Migration, Introgression And Evolutionary History, Joselle Germano-Presby

Doctoral Dissertations

Mitochondrial and chloroplast haplotypes were identified in range-wide populations of white (Picea glauca), black (P. mariana ) and red spruce (P. rubens). The chloroplast genome exhibited more intraspecific variation than the mitochondrial genome. Red spruce displayed the most total chloroplast genetic diversity (H T = 0.52). Neighbor-joining analysis arranged the chloroplast haplotypes into three monophyletic groups that were nearly 100% species-specific. These results strongly refute a previously proposed progenitor/derivative relationship of black/red spruce. Red and black spruce were estimated to have diverged from their common ancestor ∼0.6--3.5 million years ago. Mitochondrial diversity detected in black spruce was attributed to interspecific …

Thermoionization And Dissociation Of Fullerenes And Endohedral Fullerenes, Rongping Deng

Doctoral Dissertations

Electron emission and unimolecular dissociation of energetically excited fullerenes and endohedral fullerenes are studied with mass spectrometry. Three experimental approaches have been developed for these studies. These are UV laser excitation time-of-flight mass spectrometry (TOF-MS), laser desorption and ionization mass spectrometry (LDI-MS), and ion-molecule collision mass spectrometry (IMC-MS).

Our first experimental effort is to investigate delayed electron emission from multiphoton excited C60 molecules. A lower limit of the probability of electron emission from multiphoton excited C60 molecules is determined to be about 2.6%. This result indicates that electron emission is not merely parasitic to dissociation.

The second experimental effort is …

Calcium -Dependent Protein Kinases Are Myristoylated And Associated With Different Membranes In Arabidopsis Thaliana, Sheen Xun Lu

Doctoral Dissertations

In plants, calcium-dependent protein kinases (CDPKs) are the predominant calcium-stimulated kinases and are known to be involved in many cellular processes. CDPK enzymatic activity previously has been detected in many locations in plant cells, including the membrane fraction. However, little is known about the subcellular locations of individual CDPKs or the mechanisms involved in targeting them to those locations. Arabidopsis contains 34 genes that are predicted to encode CDPKs and 28 of the predicted CDPK proteins have potential myristoylation motifs at their amino termini. Myristate is a 14-carbon saturated fatty acid that is attached co-translationally to the amino-terminal glycine of …

Characterization Of Prokaryotic Diversity In A Chlorinated Solvent-Contaminated Bedrock Aquifer Using Denaturing Gradient Gel Electrophoresis, Ribosomal Dna Sequencing, And Real Time Pcr, Walid Naser

Doctoral Dissertations

A culture-independent molecular phylogenetic approach was implemented to investigate spatial and temporal variations in microbial diversity and abundance in a chlorinated solvent-contaminated bedrock aquifer. Core and groundwater samples were collected from several boreholes and included the following samples: (1) groundwater that was associated with the core, (2) open fractures and (3) partially mineralized sealed fractures (PMSF) exposed by striking the core with a surface-sterilized geology hammer. DNA representing the attached and unattached microbial communities was extracted from these samples, and the 16S rRNA gene was amplified with primers specific for Bacteria, Archaea, sulfate reducers, Geobacteraceae family, Dehalorespirers (Dehalococcoides sp. and …

Regulation Of Ethylene Receptor Expression In Arabidopsis, Xue-Chu Zhao

Doctoral Dissertations

The ethylene receptor family of Arabidopsis consists of five members, ETR1, ETR2, ERS1, ERS2, and EIN4. Regulation of ethylene receptor expression in Arabidopsis was studied using three different approaches. First, the effect of ethylene-pathway mutations upon expression of ETR1 was examined. Ethylene-insensitive mutations in ETR1 resulted in a post-transcriptional increase in levels of the mutant receptor. Treatment of seedlings with silver, which leads to ethylene insensitivity, also resulted in an increase in levels of ETR1. Mutations in other components of the ethylene pathway had little effect upon expression of ETR1. Second, microarray analysis was performed using reciprocal mutants to examine …

Transgenic Manipulation Of Polyamine Metabolism In Poplar, Pratiksha Bhatnagar

Doctoral Dissertations

Polyamines are low molecular weight polycations found in all living organisms. They are involved in plant stress response and development. The study was aimed at analyzing the effects of altered polyamine metabolism on the polyamine and related pathways in transformed poplar cells overexpressing a mouse odc cDNA under the control of 2X 35S CaMV promoter. The transgenic cells (line 2E) showed elevated levels of mouse ornithine decarboxylase enzyme activity, several fold higher amounts of putrescine, a small increase in spermidine, and a small reduction in spermine as compared with non-transgenic (NT) cells. The conversion of labeled ornithine into putrescine was …

The Enzymatic Function Of The Ccr4 Protein, Junji Chen

Doctoral Dissertations

The CCR4-NOT complex from Saccharomyces cerevisiae, which exists in two forms in vivo: 1.9 and 1.0 x 10 6 MDa in size, is a general transcriptional regulatory complex. It regulates mRNA transcription in initiation, elongation and degradation.

The 1.0 MDa complex was purified to near homogeneity, and mass spectrometric analysis was used to identify all the components of the complex. The 1.0 MDa complex contains CCR4, CAF1, NOT1--5, and two new proteins, CAF40 and CAF130. CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes. Immunoprecipitation and gel filtration experiments confirm that …

Identification Of Suppressors Of Spt10 That Affect Adh2 Expression In The Yeast Saccharomyces Cerevisiae, Bradley David Anderson

Doctoral Dissertations

Suppression of the effects of an spt10 mutation on ADH2 expression is a phenotype shared by a small number of genes involved in the regulation of gene expression. All six known suppressors of spt10 are either components of the CCR4-NOT Complex (CCR4, CAF1, NOT4), or have been shown to interact with the complex ( DBF2, SRB9, SRB10). This indicated that spt10 suppression may be characteristic of CCR4-like gene regulation. In this work, I conducted a screen for additional suppressors of spt10 at ADH2. The screen identified no less than ten complementation groups, including ADH2 and two known suppressors of spt10, …

The Relationship Between Fruit Color Traits And Anthocyanin Genes In Diploid Fragaria, Chaoyang Deng

Doctoral Dissertations

A candidate gene approach was used to determine the likely molecular identity of the c locus (yellow fruit color) in Fragaria vesca, a diploid (2n = 2x = 14) strawberry. Using PCR with degenerate primer pairs, intron-containing segments of structural genes coding for the enzymes: chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanindin synthase (ANS), and one Del-like regulatory gene (RAN) in the anthocyanin biosynthetic pathway were amplified, cloned and sequenced. Intron length polymorphisms for each of these genes were detected among three diploid varieties: F. vesca Alpine variety 'Yellow Wonder' (YW) (Europe); DNIC, an …

Regulation Of Mammalian Photoreceptor Phosphodiesterase (Pde) By Its Noncatalytic Gaf Domains And Its Two Small Subunits, Gamma And Delta, Hongmei Mou

Doctoral Dissertations

cGMP phosphodiesterase (PDE) is the central effector of visual transduction in retinal rod photoreceptors. The overall goal of this work was to investigate the possible regulatory mechanisms of PDE in mammalian rod photoreceptors by its noncatalytic cGMP binding sites on the PDE catalytic dimer (Palphabeta), and by the interactions between the inhibitory Pgamma subunit and Palphabeta in their phosphorylated and non-phosphorylated states.

First, the binding of cGMP to the noncatalytic sites on membrane-associated PDE (PDE-M) and soluble PDE (PDE-S) was characterized. We found that PDE-M holoenzyme contains 2 non-identical high-affinity cGMP binding sites; one of the cGMP binding sites is …

Characterization Of The Interactions Within The Ccr4 -Not Complex And The Interactions Between Adr1 And Tfiid Components, Yongli Bai

Doctoral Dissertations

The mechanisms of transcriptional regulation are well conserved from yeast to human cells. Most genes are regulated at the transcriptional level, particularly, at the transcription initiation step. In Saccharomyces cerevisiae at least one transcriptional activator, ADR1, and one transcription complex, CCR4-NOT, participate in the activation of the ADH2 gene. The core 1 x 106 dalton CCR4-NOT complex consists of CCR4, CAF1, the five NOT proteins (NOT1-NOT5), and three other uncharacterized proteins. Deletion of NOT3, NOT4, or NOT5 did not affect the association of CCR4 with CAF1 in the gel filtration analysis. In contrast, NOT2 inactivation caused partial destabilization of the …

Isolation And Characterization Of A Caenorhabditis Elegans Src Loss-Of-Function Allele Using Reverse Genetics, Jennifer Dignan Hogan

Doctoral Dissertations

The vertebrate proto-oncogene Src is a protein-tyrosine kinase that has been implicated as a component of receptor-mediated signal transduction pathways important for cell growth and differentiation. Consistent with this notion, overexpression or activation of Src by mutation induces neoplastic transformation in cell culture, leads to tumorigenesis in laboratory animals, and has been observed in a number of human tumors. Despite years of intensive investigation, neither its role in oncogenesis nor its normal, biological role is understood.

To diminish the issue of redundancy that has complicated analysis of Src function in vertebrates and Drosophila, I have chosen to study Src function …

The Role Of Cytokines In The Pathogenesis Of Experimental Legionella Pneumophila Infections, Corinna Mary Krinos

Doctoral Dissertations

Legionnaires' disease is an acute lobar pneumonia caused, primarily by the facultative intracellular pathogen Legionella pneumophila. This organism when inhaled by humans descends into the lower respiratory tract and parasitizes alveolar macrophages. L. pneumophila adhered to U-937 cells, A549 cells and peritoneal macrophages from A/J mice in an opsonin-independent fashion. Following attachment, the organism penetrated the cell membrane, replicated within these cells eventually inducing lysis. To better define the adhesion of L. pneumophila to host cells, an E. coli clone (LP 116), expressing the 25 kDa major outer membrane protein (MOMP) of L. pneumophila was used in binding studies. This …