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54 matches in the database. These are records: 1 - 30.

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1. Adya, Neeraj. Mechanism of human T cell leukemia virus type-I gene (HTLV-I) regulation as mediated by regulatory protein, Tax.

Degree: PhD, Molecular Biology and Microbiology, 1994, Case Western Reserve University

► Human T-lymphotropic retrovirus type I (HTLV-I) is the causative agent of adult… (more)

▼ Human T-lymphotropic retrovirus type I (HTLV-I) is the causative agent of adult T-cell leukemia/lymphoma (ATL) and tropical spastic paraparesis (TSP) or HTLV-I associated myelopathy (HAM). The 3′ end of the HTLV-I genome encodes a transactivator, Tax, that stimulates transcription from the viral LTR. In addition to its essential role in viral replication, Tax activates transcription of various cellular genes involved in mediating cell growth. The ability of Tax to alter viral and cellular gene expression appears to be causally related to ATL and TSP/HAM. Our research efforts have focused on elucidating the molecular mechanism of Tax function. We previously demonstrated that CREB, a cellular DNA binding protein of the bZip family activates transcription from the HTLV-I LTR synergistically with Tax. Three cellular proteins that bind specifically to the cAMP response element (CRE) in the HTLV-I 21 bp repeats were subsequently identified. These three binding proteins are composed of homodimers and a heterodimer formed by CREB and a highly related protein, ATF-1. Tax was shown to interact specifically with the CREB subunit in CREB homodimer and CREB:ATF-1 heterodimer. Sequence comparison and domain switching between CREB and ATF-1 indicate that Tax interacts specifically with the s p282AAR residues near the conserved DNA binding domain of CREB. The Tax:CREB complex exhibits increased DNA recognition specificity and assembles preferentially on CRE motif flanked by 5′ G rich and 3′ C rich sequences present in the viral 21 bp repeats. The tethering of Tax to the viral enhancer via CREB is crucial for Tax transactivation. Characterization of Tax mutants further indicates that the structural integrity in the NH2-terminus of Tax is crucial for its interaction with CREB and support the notion that a transactivation domain resides in the COOH-terminal region of Tax. These results suggest a model whereby the recruitment of Tax via CREB to the HTLV-I enhancer facilitates the interaction between the transactivation domain of Tax and the basal transcriptional machinery leading to increased HTLV-I gene expression. Advisors/Committee Members: Giam, Chou-Zen.

Subjects: Biology, Molecular

Keywords: Mechanism human T cell leukemia virus type I gene regulation mediated regulatory protein Tax

2. Altier, Craig. Salmonella typhimurium interaction with intestinal epithelial cells: Identification of a novel invasion locus.

Degree: PhD, Molecular Biology and Microbiology, 1996, Case Western Reserve University

► Salmonella typhimurium is a gram-negative bacterial pathogen of world-wide significance, causing disease… (more)

▼ Salmonella typhimurium is a gram-negative bacterial pathogen of world-wide significance, causing disease in both humans and animals. An early and required step in the pathogenesis of this organism is its penetration of intestinal epithelium as a way to reach deeper tissues. The penetration of epithelial cells requires the expression of numerous bacterial genes. We have attempted to identify bacterial determinants of this process by selecting for genes that are specifically expressed upon bacterial contact with cultured epithelial cells. This selection relies on transcriptional fusions to the phage P1 recombinase, cre, to induce a chromosomal rearrangement that is both permanent and detectable, and so can identify transiently-expressed genes. Our initial characterization of seven putative promoters found in this way has shown four to be novel loci. Of the remaining three, one is derived from the oligopeptide permease C (oppC) gene; another maps near btuB, required for vitamin B12 uptake; and the third carries at least two unrelated DNA fragments. We have analyzed mutants of the opp operon and shown them not to be required for the penetration of cultured epithelial cells. We have also tried to identify negative regulators of all of the seven clones without success. We created chromosomal deletion mutants at three of the cloned loci and found one that was unable to invade Caco-2 or HEp-2 cultured epithelial cells. The resulting locus, derived from the clone carrying unrelated DNA fragments, has been termed nil, for novel invasion locus nil maps to 61 minutes on the Salmonella chromosome, linked to relA. Mutants at this locus have no detectable phenotype other than loss of epithelial cell invasion. Sequence analysis places nil within an open reading frame capable of encoding a 42 amino acid hydrophilic peptide without homology to known proteins. nil also appears to function in the established Salmonella invasion pathway, since a double mutant of nil and invA, a characterized invasion gene, is not substantially less invasive than a strain with either single mutation Advisors/Committee Members: Maurer, Russell.

Subjects: Biology, Molecular

Keywords: Salmonella typhimurium; Intestinal epithelial cells

3. Antczak, Michael Richard. Growth factor- and oncogene-induced transformation in chicken embryo fibroblasts and normal diploid human fibroblasts.

Degree: PhD, Molecular Biology and Microbiology, 1993, Case Western Reserve University

► Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro… (more)

▼ Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of a family of v-src, c-src and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to this oncogene's fibroblast transforming abilities. The tumorigenic potentials detected by direct oncogene transfection faithfully reproduced the findings of similar studies using infectious, oncogenic retroviruses. Our studies establish the utility of CEF transformation by direct DNA transfection. Deregulated expression of the TGFα and EGF-R genes has been implicated in the development of a range of mammalian malignancies, most notably those of human origin. While introduction of the TGFα/EGF genes into immortalized rodent cell lines has frequently resulted in cellular transformation, attempts to generate similar results following simple, exogeneous TGFα/EGF treatment have proven largely unsuccessful. The potential role(s) played by these growth factors in the transformation of normal cells, derived from primary cultures has, heretofore, not been closely examined. In chapter 3 it is reported that both normal chicken embryo fibroblasts (CEF) and normal, diploid, human foreskin fibroblasts (HFF) can be efficiently transformed, in an apparent single-step fashion, following exogeneous TGFα/EGF treatment. CEF cells infected with a retrovirus carrying the TGFα gene generated unusually large, aggressively growing soft agar colonies. The ligand-induced transformation of CEF and HFF is affected by culture age. Cultures consistently responded less efficiently as they underwent increasing numbers of population doublings, yet there was no detectable, concomitant, diminution in the number of receptors per cell. This is the first report of efficient transformation of normal cells derived from primary cultures by TGFα and EGF, without the need for other complementing growth factors or oncogenes. Advisors/Committee Members: Kung, Hsing-Jien.

Keywords: Growth factor oncogene chicken embryo fibroblasts diploid human fibroblasts

4. Bendezu, Felipe Oseas. CELL SHAPE DETERMINATION IN ESCHERICHIA COLI.

Degree: PhD, Molecular Biology and Microbiology, 2008, Case Western Reserve University

► The load-bearing exoskeleton, known as the murein sacculus, is believed to impart… (more)

▼ The load-bearing exoskeleton, known as the murein sacculus, is believed to impart the shape of bacterial cells. The chemical composition and enzymes responsible for building this bag-like structure are fairly well understood. What is lacking, however, is a detailed understanding of how the bacterial cell coordinates the spatio-temporal synthesis of the sacculus to achieve a complex shape. It is now clear that many genera of bacteria use the cytoskeletal actin protein MreB to accomplish this. However, the precise mechanism by which MreB actin contributes to complex cell shape determination is not known. A popular model states that MreB spiral-like cyto-filaments serve as a track to direct the placement of new murein material. In the well-studied bacterium Escherichia coli, MreB is one of five known proteins needed for rod-shape growth, with the others being MreC, MreD, PBP2 and RodA. How loss of one or more of these proteins affects cell physiology has been a confusing issue. In attempts to bring clarity into this field of research, I created a defined and comprehensive set of shape mutants and analyzed their phenotypes. These studies showed that all five are only conditionally essential. Cells lacking one or more of these proteins were able to survive when grown under conditions of slow mass increase or when the essential division protein FtsZ is over-expressed. Intriguingly, when grown under conditions of fast mass increase, mutant cells grow into giant spheres that are unable to regulate important cellular processes and eventually die. These spheres accumulate excess membrane in their interior that compete with the cell membrane for essential division proteins and I propose that this contributes significantly to the lethal division defect under non-permissive conditions. I used the fact that extra FtsZ can rescue life to shape-defective mutants as the basis for a genetic screen to identify additional shape mutants. This identified an uncharacterized membrane protein we named RodZ. Cells lacking RodZ display an impressive set of media and temperature-influenced shape defects. Also, using the first fully function fluorescent fusion protein to MreB, I was able to show that RodZ is essential for proper MreB assembly. Advisors/Committee Members: de Boer, Piet.

Subjects: Cellular biology; Genetics; Microbiology; Molecular biology

Keywords: bacterial cell shape; MreB; bacterial actin

5. Boerkoel, Cornelius Franciscus III. Modulation of CSV 5' and 3' LTR transcription in c-myc activation.

Degree: PhD, Molecular Biology and Microbiology, 1991, Case Western Reserve University

► Chicken syncytial virus (CSV) activates c-myc in chicken B-cell lymphomas by integrating… (more)

▼ Chicken syncytial virus (CSV) activates c-myc in chicken B-cell lymphomas by integrating within the c-myc locus and promoting c-myc transcription from the 3′ LTR. Such myc-associated proviruses are frequently partially deleted, and, in contrast to wild-type proviruses which initiate >98% of viral transcription in the 5′ LTR, these proviruses initiate >98% of viral transcription in the 3′ LTR. Structural and transcriptional analysis of one of these myc-associated CSV proviruses shows that ∼80% of the viral genome is deleted and that 5′ LTR transcription is inhibited by deletion of a positive regulatory element in the leader sequence and possibly by a silencer within the envelope gene. The 5′ LTR is intact, however, and outside of the proviral context, it is transcriptionally competent. Within the proviral context, the 5′ and 3′ LTR's are differentiated and regulated by the viral sequences adjacent to each. A positive regulatory element for transcription from the 3′ LTR lies within 53 bp upstream of the 3′ LTR, and deletion of this sequence from the myc-associated provirus substantially reduces transcription from the 3′ LTR. Similarly, a positive regulatory element for transcription from the 5′ LTR lies within 87 bp downstream of the 5′ LTR, and deletion of this sequence from a wild-type CSV provirus abolishes transcription from the 5′ LTR. This sequence is deleted in the myc-associated provirus, and restoration of it activates transcription from the 5′ LTR. This 87 bp sequence promotes transcription from the 5′ LTR in the sense orientation and inhibits it in the antisense orientation. Moreover, to fully activate transcription from the 5′ LTR, this 87 bp requires sequences in the R and/or U5 region of the 5′ LTR. Activation of 5′ LTR transcription by restoration of this 87 bp to the myc-associated provirus substantially reduces c-myc transcription from the 3′ LTR. This suggests that transcription from the 5′ LTR interferes with the transcription from the 3′ LTR and that the silencing of 5′ LTR transcription is an essential step in CSV activation of c-myc by 3′ LTR promoter insertion. Therefore, the oncogenic potential as well as the LTR transcriptional activity of CSV proviruses are modulated by viral sequences outside of the LTR's. Advisors/Committee Members: Kung, Hsing-Jien.

Subjects: Biology, Molecular

Keywords: CSV 5' 3'; LTR transcriptio n; c-myc activation

6. Burgoyne, Adam Michael. Protein Tyrosine Phosphatase Mu Regulates Glioblastoma Cell Migration And Dispersal.

Degree: PhD, Molecular Biology and Microbiology, 2010, Case Western Reserve University

► As the most common malignant primary brain tumor, GBM represents a significant… (more)

Subjects: Molecular biology

Keywords: glioblastoma; migration; tyrosine phosphorylation; tyrosine phosphatase

7. Cardaman, Richard C. Sporulation mutants of Myxococcus xanthus.

Degree: PhD, Molecular Biology and Microbiology, 1994, Case Western Reserve University

► Myxococcus xanthus strain DZF1 was mutagenized with transposon Tn5. Twenty-two hundred eighty-eight… (more)

Subjects: Biology, Molecular

Keywords: Sporulation mutants Myxococcus xanthus

8. Chang, Chi-Ming. Oncogenic specificity and domain interaction of the EGF receptor.

Degree: PhD, Molecular Biology and Microbiology, 1994, Case Western Reserve University

► Chicken EGF receptor (c-EGFR), which is encoded by the c-erbB gene, is… (more)

▼ Chicken EGF receptor (c-EGFR), which is encoded by the c-erbB gene, is highly homologous to human EGF receptor. This avian receptor can be preferentially activated by TGF-α stimulation leading to phosphorylation of its substrates and ultimately to cellular proliferation. Truncation of the ligand binding domain of c-EGFR results in a leukemogenic oncogene product encoded by IA c-erbB. Characterization of variants of IA c-erbB from different tumor types provides insight into the mechanism of growth control mediated by EGF receptor. TGF-α binds to c-EGFR with a 200-fold higher affinity than EGF does. To determine the regions on the ligands which determine binding specificity, chimeric molecules between EGF and TGF-α were studied. The five carboxyl-terminal amino acids (residues 46-50) of TGF-α played a major role in determining binding specificity. In addition, the four carboxyl-terminal amino acids (residue 50-53) of EGF and a region in the amino-terminal β-pleated sheet (residue 20-27) of TGF-α also had some minor effects on the binding specificity. The disease tropism of IA c-erbB can be expanded to include sarcomagenic potential by structural alterations in its kinase domain and/or carboxyl-terminal domain. To study the role of the kinase domain encoded IA c-er bB in regulating tissue-specific transformation, the erbB kinase was replaced with those of the src and trk oncogenes. The chimeric molecules remained functionally active tyrosine kinases and were strictly sarcomagenic. The carboxyl-terminus of IA c-erbB had an inhibitory effect on the in vitro transforming ability of the src kinase in the chimera. In addition, a novel tyrosine residue in erbB was found to be phosphorylated by the src kinase. To study the role of the autophosphotyrosine residues in mediating IA c-erbB transformation, they were individually mutated to phenylalanine residues. A mutant construct with the alteration of p5 phosphotyrosine residue displayed enhanced sarcomagenic potential. This is accompanied by increased association with the Grb2 protein and phosphorylation of the MAP kinase. These results are consistent with the deletion mutants that show that the p5 residue, which is located in a specific regulatory region (CAIN domain), is involved in interacting with negative regulators for fibroblast transformation. Advisors/Committee Members: Kung, Hsing-Jien.

Subjects: Biology, Molecular

Keywords: Oncogenic specificity domain interaction EGF receptor

9. Chang, Ji Suk. Phospho-Regulation of Actin Organization and Endocytosis in Yeast by the PP1 Targeting Protein Scd5p.

Degree: PhD, Molecular Biology and Microbiology, 2005, Case Western Reserve University

► Endocytosis is an essential process requiring a large number of factors that… (more)

▼ Endocytosis is an essential process requiring a large number of factors that are recruited to the plasma membrane for membrane invagination and endocytic vesicle formation. A major mechanism for regulation of endocytosis is to control interactions by reversible phosphorylation of these endocytic factors. In budding yeast, where actin is crucial for internalization, many endocytic/cortical actin factors localize to spots at the cell periphery. A novel actin regulating serine/threonine kinase family (ARKs) including Prk1p, Ark1p, and Akl1p was shown to phosphorylate many cortical actin patch/endocytic factors such as Pan1p, Sla1p, Ent1/2p, and YAP1801p. However, an opposing phosphatase has not been discovered or analyzed. Interestingly, Scd5p, which plays an essential role in endocytosis and actin organization, binds to protein phosphatase-1 (PP1) and PP1 binding is important for Scd5p function. Scd5p also has a three repeat region of 20 amino acids (3R) that is phosphorylated by Prk1p, and a nine repeat region of 12 amino acids (9R). Thus the importance of PP1 binding could be to reverse the Prk1p-mediated phosphorylation on Scd5p. Alternatively, Scd5p could target PP1 to dephosphorylate other cortical actin patch/endocytic components. Here I provide evidence that Scd5p is regulated by PP1 and also targets PP1 to other endocytic/cortical actin factors phosphorylated by Prk1p, such as Pan1p, Sla1p, Ent1/2p, and Yap1801/2p. Thus I suggest that Scd5p targets PP1 to regulate cortical actin/endocytic complex components by reversing Prk1p-mediated phosphorylation. Like other endocytic proteins, Scd5p localizes to cortical patches and its 9R region is critical for cortical localization; however, cortical recruitment of Scd5p is not essential for Scd5p’s function in cortical actin organization and endocytosis. I propose that this role of Scd5p may be performed in the cytoplasm rather than at the cell cortex probably by targeting PP1 to endocytic factors that have been disassembled and/or inactivated by phosphorylation. Furthermore, similar to some endocytic factors in animal cells, Scd5p shuttles between the cytoplasm and the nucleus in a Crm1p-dependent manner, suggesting that Scd5p has a nuclear function. Advisors/Committee Members: Lemmon, Sandra K.

Keywords: Endocytosis; Actin organization; Protein phosphatase-1; nuclear-cytoplasmic shuttling

10. Cirino, Nick Mario. Structure-function analysis of the ribonuclease-H domain of human immunodeficiency virus type-1 reverse transcriptase.

Degree: PhD, Molecular Biology and Microbiology, 1995, Case Western Reserve University

► The structure-function relationships of the RNase H domain of HIV-1 reverse transcriptase… (more)

▼ The structure-function relationships of the RNase H domain of HIV-1 reverse transcriptase (RT) have been probed by examining several forms of this domain. These include; (i) an isolated 15 kDa RNase H which approximates that present in virions following RT maturation (p15 RNase H), (ii) amino-terminally extended, isolated RNase H (p20 RNase H), (iii) point mutants in isolated RNase H or p66/p51 RT which affect substrate binding (His539 → Phe539) or catalysis (Glu478 → Gln478), and (iv) chimeric HIV-1 RT containing the RNase H domain from related lentiviruses. The 15 and 20 kDa RNase H, as well as two point mutants of each, were examined by intrinsic fluorescence quenching in order to assess substrate binding independent of catalytic activity. This analysis indicates that; (i) p15 RNase H can bind RNA/DNA substrate, (ii) the 42-residue amino terminal extension increases the intrinsic affinity for substrate 33 fold, (iii) p20 RNase H can bind RNA/RNA substrates as well as RNA/DNA substrates, and (iv) this analysis can differentiate binding and catalytic mutants. Although p20 E478Q RNase H bound substrate as effectively as wild type p20, it was catalytically inert under standard assay conditions. When the RNase H activity of p66/p51 RT carrying the same mutation (p66 E478Q/p51) is assayed with Mn2+ as the divalent cation activator instead of Mg2+, specific endoribonuclease activity is restored. Endoribonuclease activity is cleavage of non-nicked RNA in an RNA/DNA hybrid, whereas exoribonuclease activity is directional release of ribonucleotides from nicked RNA/DNA hybrids. The lack of exoribonuclease activity in this mutant correlates with reduced strand transfer activity in vitro. The results of these experiments are discussed in relation to the proposed catalytic mechanism of the RNase H domain. While HIV-1 RT can accomodate an HIV-2 RNase H domain, it cannot tolerate an equivalently sized portion of the EIAV RNase H domain. The HIV-1/EIAV chimera was dimerization defective and therefore catalytic activities could not be characterized. Chimeric HIV-1/HIV-2 RT had comparable DNA polymerase and RNase H activity as wild type HIV-1 Advisors/Committee Members: Le Grice, Stuart F. J.

Subjects: Biology, Molecular

Keywords: HIV-1 reverse transcriptase; Ribonuclease-H domain

11. Conlin, Christopher Arthur. Characterization of the Salmonella typhimuriumopdA gene, encoding oligopeptidase A: Nucleotide sequence; identity with the Escherichia coliprlC gene; and its role in bacteriophage P22 development.

Degree: PhD, Molecular Biology and Microbiology, 1992, Case Western Reserve University

► The Salmonella typhimurium opdA gene, encoding oligopeptidase A, was mapped to 76… (more)

Keywords: Salmonella typhimuriumopdA gene oligopeptidase A Nucleotide Escherichia coliprlC gene bacteriophage P22

12. Davis, Nicole J. Investigating the Involvement of C. crescentus TipF in Flagellar Biogenesis.

Degree: PhD, Molecular Biology and Microbiology, 2011, Case Western Reserve University

► In Caulobacter crescentus TipN, TipF and PflI establish the placement of the… (more)

▼ In Caulobacter crescentus TipN, TipF and PflI establish the placement of the single polar flagellum. TipN localizes to the flagellar pole followed by TipF and, subsequently, PflI. The TipF EAL domain, linked to cyclic-di-GMP phosphodiesterase activity, is necessary for proper function. To understand the role TipF plays in flagellar biogenesis in C. crescentus, we investigated the transcriptional activity of flagellar genes in the absence of TipF. We found that tipF mutant had upregulated transcription of class II flagellar genes and downregulated transcription of class III flagellar genes. Class IV flagellar genes; however, were transcribed near WT levels in the absence of TipF. Chromatin immunoprecipitation (ChIP) assays confirmed the status of FlbD; a transcriptional regulator, at the promoters of Class II/III/IV flagellar genes and correlated with the transcriptional activity observed. To characterize the role of TipF in flagellar biogenesis, we investigated the contribution TipF makes to the localization of the flagellar positioning factor PflI. In the absence of TipF, PflI did not localize, yet, TipF did not require PflI to aggregate at the flagellar pole. TipF, therefore, dictates the localization of PflI to enable the proper position of the flagellum and is localized prior to PflI localization at the flagellar pole. TipF has an EAL domain linked to cyclic-di-GMP phosphodiesterase activity and essential for function. We investigated the necessity of cellular levels of cyclic-di-GMP for TipF function and the ability of the TipF EAL to bind and hydrolyze cyclic-di-GMP. TipF required cyclic-di-GMP to localize and recruit PflI to the flagellar pole. The TipF-EAL bound cyclic-di-GMP, however, lacked phosphodiesterase activity. These results demonstrate that TipF EAL does not hydrolyze cyclic-di-GMP as homologous EAL enzymes have been shown to do. It further suggests that the binding of cyclic-di-GMP to TipF is the regulatory function required to ensure proper TipF activity. We isolated second-site suppressors of the tipF phenotype which restored motility to the C. crescentus cell. These mutants were motile, restored class III/IV flagellar gene expression to wild-type, and localized TipF to the site of flagellar biogenesis. Additional analyses demonstrate that these mutants localize to the flagellar pole independent of cyclic-di-GMP. The transcription of class III and class IV flagellar genes in the absence of cyclic-di-GMP, however, was still downregulated in the tipF suppressors similar to WT cells. This suggests that the tipF suppressors possess a constitutively active TipF not dependent on cyclic-di-GMP for localization, yet, these suppressors transcribe enough class III/IV flagellar genes to make a flagellum in the absence of the second messenger. We identified that TipN, PflI, and TipF are present in a complex together. In addition, TipF was found to be in a complex with a component of the flagellar switch, FliM. Together, these experiments propose that TipF, present in a complex with TipN, PflI, and FliM, binds cyclic-di-GMP to orchestrate the placement and construction of the polar flagellum of C. crescentus. Advisors/Committee Members: Viollier, Patrick.

Subjects: Microbiology; Molecular Biology

Keywords: TipF; TipN; flagellar transcription, flagellar biogenesis; FlbD; FliX; PflI

13. Del Rio, Samuel. Structural and functional studies of Xenopus laevis transcription factor IIIA zinc finger mutants.

Degree: PhD, Molecular Biology and Microbiology, 1992, Case Western Reserve University

► Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is… (more)

▼ Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III. We describe the purification and characterization of recombinant TFIIIA and TFIIIA zinc finger mutants expressed in E. coli. We have analyzed a set of TFIIIA mutants in which each mutant protein contains a single amino acid substitution in one of the putative zinc-binding histidine residues. We were able to assign specific functions, 5S DNA interactions and kinetic and equilibrium parameters to the individual zinc fingers of TFIIIA. The ability to analyze TFIIIA zinc finger mutants with an intact C-terminal domain made it possible to determine which TFIIIA zinc fingers were important for transcriptional activation. Chromatographic properties of the mutant proteins and the results of partial proteolysis studies indicate that the mutation in each case results in structural disruption of the zinc finger domain containing the mutation with no apparent effect on adjacent fingers. While all fingers contribute to the binding energy when TFIIIA interacts with the intragenic control region (ICR) of the 5S rRNA gene, some fingers are more importan t than others, with mutations in fingers 3 and 4 resulting in the largest decreases in binding affinity. Though the first six N-terminal fingers of TFIIIA contribute greater than 70% of the relative binding energy to the 5S DNA, mutations in any one of these six N-terminal fingers did not affect the transcriptional activity of TFIIIA. The three C-terminal fingers of TFIIIA, fingers 7-9, though contributing less than 30% of the relative binding energy to the 5S DNA, are essential for the transcriptional activity of TFIIIA. This demonstrates that the C-terminal fingers of TFIIIA are important not only in binding to the 5′ end of the 5S gene ICR, but also in mediating transcriptional activation through interactions with other components of the RNA polymerase III transcription machinery. Advisors/Committee Members: Setzer, David R.

Keywords: Xenopus laevis transcription; factor IIIA; zinc; finger mutants

14. Dirksen, Wessel Peter. Determination of CIS-acting signals that control alternative splicing of bovine growth hormone pre-mRNA.

Degree: PhD, Molecular Biology and Microbiology, 1995, Case Western Reserve University

► Bovine growth hormone (bGH) pre-mRNA contains five exons and four introns. A… (more)

Subjects: Biology, Molecular

Keywords: Alternative splicing; CIS-acting signals; Growth hormone pre-mRNA

15. Dudley, Dawn M. HIV-1 ENV: IMPACTING HIV-1 FITNESS, ENTRY INHIBITOR DRUG SENSITIVITY, AND IN VIVO SELECTION OF A RESISTANT VIRUS TO THE MICROBICIDE PSC-RANTES.

Degree: PhD, Molecular Biology and Microbiology, 2008, Case Western Reserve University

► In the advent of the promise for HIV-1 entry inhibitors to treat… (more)

▼ In the advent of the promise for HIV-1 entry inhibitors to treat patients with drug resistance to available antiretroviral therapy, there has been a surge in studies related to HIV-1 entry. It is clear that the intrinsic susceptibility of primary virus isolates to entry inhibitors varies, which indicates a greater probability for intrinsic resistance to this class of drugs. The studies presented here provide tools and insights into the impact of the envelope (env) gene on HIV-1 replicative capacity, how that capacity influences the emergence of drug resistance, and how easily drug resistance is selected in vivo to a microbicide entry inhibitor. The fitness of a virus is a marker of its replication capability in a given environment. The specific impact of HIV-1 entry on the overall fitness of a virus was tested by cloning a region of the env gene into a common backbone and comparing the resultant recombinant fitness to that of the wildtype virus. The env gene was sufficient to determine the overall fitness of these viruses, which also correlated with sensitivity to entry inhibitors. Specifically, the binding avidity of a virus to the host cell coreceptor contributed the most to fitness. PSC-RANTES is an entry inhibitor that acts on the coreceptor CCR5 to block HIV-1 binding and down-regulate coreceptor expression. When used as a microbicide in a rhesus macaque model, PSC-RANTES failed to block transmission of SHIV¬¬¬SF162 at high doses in some animals. Two mutations were identified in isolates from one of these animals and were cloned into a common HIV-1 backbone to create a chimeric virus. The chimeric virus exhibited resistance to PSC-RANTES and an increase in fitness over that of the wildtype virus used to infect the rhesus macaques. This study showed for the first time the selection of drug resistant viruses to a microbicide in an HIV-1 animal model system. Lastly, a cloning strategy was developed to quickly create replication-competent, fully infectious HIV-1 chimeric viruses. This system eliminates the need for common endonuclease sites among diverse primary isolates of HIV-1 and can be used to clone any gene region from any HIV-1 isolate. Advisors/Committee Members: Arts, Eric J.

Keywords: HIV-1; SHIV; microbicide; chimeric virus; HIV-1 fitness; HIV-1 entry; HIV-1 drug resistance; Rhesus macaque; PSC-RANTES; HIV-1 cloning

16. Flickinger, Thomas Walter. Characterization of a secreted, truncated receptor encoded by the avianc-erbB gene.

Degree: PhD, Molecular Biology and Microbiology, 1992, Case Western Reserve University

► At least four major transcripts, 12-, 8.6-, 5.8-, and 2.6-kilobases (kb) are… (more)

▼ At least four major transcripts, 12-, 8.6-, 5.8-, and 2.6-kilobases (kb) are produced by the avian c-erbB (EGF/TGFα receptor) gene. The three largest transcripts are detected on Northern transfers by hybridization probes from the intracellular domain of the avian receptor. In addition to the three large transcripts, a small 2.6-kb transcript is detected by hybridization probes from the extracellular ligand-binding domain (LBD) of the avian receptor. cDNAs corresponding to this 2.6-kb transcript were isolated from an adult chicken liver cDNA library. Sequence analysis revealed that the 3′ end of one cDNA clone diverged from the known sequence of the extracellular LBD of the full-length receptor encoded by the avian c-erbB gene. A chicken genomic DNA clone that contained this unique 3′ divergent end was isolated. Sequence analysis of a genomic DNA subfragment revealed that the 2.6-kb transcript is an alternatively processed mRNA that utilizes a consensus polyadenylation signal between exons 12 and 13 of the avian c-erbB gene. Translation of this 2.6-kb transcript would produce a secreted, truncated receptor molecule which contains the amino-terminal three-fourths of the extracellular LBD of the native receptor. COS1 cells and primary chicken embryo fibroblast (CEF) cells were transfected with expression vectors that contained the 2.6-kb c-erbB cDNA. Conditioned medium from these transfected cells contained a 70-kilodalton (kDa) protein that was specifically immunoprecipitated by a polyclonal antiserum directed against the LBD of the avian c-erbB gene product. The 70-kDa truncated receptor could be co-immunoprecipitated from conditioned medium of transfected COS1 cells that was supplemented with recombinant human transforming growth factor alpha (TGFα) by a monoclonal antibody against human TGFα. Additionally, transfected CEF cells that overexpressed the 70-kDa truncated receptor were blocked in their ability to form TGFα-dependent colonies in soft agar. These data suggest that the secreted, truncated receptor encoded by the 2.6-kb c-erbB transcript can bind to TGFα and may play an important growth regulatory function in vitro Advisors/Committee Members: Kung, Hsing-Jien.

Subjects: Biology, Molecular

Keywords: receptor avianc erbB gene

17. Funderburg, Nicholas Thomas. Human Beta Defensin 3: Linking Innate and Adaptive Immune Responses.

Degree: PhD, Molecular Biology and Microbiology, 2008, Case Western Reserve University

► Human Beta defensins (hBDs) are cationic antimicrobial peptides that can be produced… (more)

▼ Human Beta defensins (hBDs) are cationic antimicrobial peptides that can be produced in response to microbial challenge at mucosal sites. Acting as effector molecules of the innate immune system, hBDs can bind to and lyse microorganisms. Defensins have also been shown to bridge the innate and adaptive immune systems through the chemoattraction of immune cells. Here, we provide evidence that hBD-3 also activates antigen presenting cells. Following overnight exposure, hBD-3 can induce expression of the co-stimulatory molecules CD80, CD86, and CD40 on monocytes and myeloid dendritic cells. Activation of monocytes by hBD-3 is mediated by interaction with TLRs 1 and 2, resulting in signaling that requires MyD88 and results in IRAK-1 phosphorylation. Cell lines (HEK and CHO) that have been engineered to express Toll-like Receptors (TLRs) 1 and 2 could be activated by hBD-3, but cells that express other individual TLRs, or TLRs 1 and 6, could not. Antibodies to TLR 1 and TLR 2 were also able to inhibit the hBD-3 mediated induction of CD80 on monocytes. HBD-3 can also induce expression of inflammatory cytokines (IL-8, IL-6, and IL-1β) from purified monocytes. Exposure to hBD-3 induces the expression of the homing receptors CCR7, CD62L, and a key antigen-presenting molecule - HLA-DR on the surfaces of monocytes. Comparisons of the TLR1, 2 binding elements hBD-3 and Pam3CSK4, demonstrate that cell stimulation by these molecules results in differential outcomes. Exposure to Pam3CSK4 results in heightened production of IL-10 and decreased surface expression of CD86. HBD-3 does not stimulate IL-10 production and induces heightened levels of CD86 on monocytes. Also, induction of co-stimulatory molecule expression (CD80) on monocytes by hBD-3 and Pam3CSK4 is regulated differentially by the MAP kinase pathways. This work demonstrates for the first time, that a human antimicrobial peptide has the potential to stimulate cells in a TLR dependent manner; and that stimulation of TLRs 1 and 2 by unique ligands results in qualitative differences in protein expression. By activating antigen presenting cells through TLRs 1 and 2, HBD-3 may be playing a key role in bridging the innate and adaptive immune systems at mucosal sites. Advisors/Committee Members: Lederman, Michael.

Keywords: Antimicrobial peptides; Toll-like Receptors; Antigen Presenting cells

18. Goodwin, Edward Culver. The dissection of the bovine growth hormone polyadenylation signal reveals a complex element within the 3' flanking sequence.

Degree: PhD, Molecular Biology and Microbiology, 1993, Case Western Reserve University

► In addition to the conserved AAUAAA hexanucleotide, GU and U-rich sequences in… (more)

Subjects: Biology, Molecular

Keywords: polyadenylation flanking sequence

19. Guo, Chunguang. Ugene, a Newly Identified Protein that is Commonly Over-Expressed in Cancer, and that Binds to Uracil DNA-Glycosylase.

Degree: PhD, Molecular Biology and Microbiology, 2009, Case Western Reserve University

► Expression microarrays identified a novel transcript, designated as Ugene, whose expression is… (more)

Subjects: Molecular biology

Keywords: colon cancer, uracil DNA-glycosylase, base excision repair

20. Hannon, Gregory James. Trans-splicing of nematode pre-messenger RNA.

Degree: PhD, Molecular Biology and Microbiology, 1992, Case Western Reserve University

► We have used the parasitic nematode, Ascaris, as a model system for… (more)

▼ We have used the parasitic nematode, Ascaris, as a model system for the biochemical analysis of nematode gene expression. Cell free extracts derived from synchronously developing Ascaris embryos have proven remarkably versatile for reproducing a variety of cellular processes in vitro. These extracts catalyze accurate transcription of RNA polymerase II and RNA polymerase III genes as well as cis- and trans-splicing. Nematodes are among the few organisms which are known to carry out intermolecular (trans) RNA splicing. Ascaris extracts are unique in their ability to catalyze trans-splicing in vitro. In these extracts, the spliced leader sequence is donated to a synthetic pre-mRNA acceptor from either endogenous SL RNA or from synthetic SL RNA. Detailed analysis of reaction intermediates and products revealed that trans-splicing is mechanistically analogous to cis-splicing. However, these reactions differ in their cofactor requirements. Cis-splicing requires intact U1, U2 and U4/U6 snRNPs. Trans-splicing, however, does not require an intact U1 snRNP. Our analyses have revealed that the SL RNA participates in trans-splicing as an Sm snRNP. However, much of the primary sequence of the SL RNA is dispensable for its function. In particular, intramolecular base-pairing between the nematode spliced leader and its 5' splice site is not essential for trans-splicing in vitro. This interaction was proposed to substitute in trans-splicing for the base pairing between U1 snRNA and conventional cis-splice sites. Furthermore, the 22 nucleotides SL sequence is dispensable for trans-splicing and can be replaced by artificial exons of 2 to 246 bases. Strikingly, the essential properties of the SL RNA are encoded within a short sequence surrounding the SL RNA Sm binding site. This sequence alone can convert a fragment of the U1 snRNA into a functional SL RNA. Ascaris extracts are also unusual in their ability to efficiently transcribe the U-snRNA genes. We have analyzed the transcriptional control elements for one U-snRNA gene, the SL RNA gene, in detail. Remarkably, the sequences which signal termination of SL RNA transcription reside within the SL RNA coding sequence, and internal elements, particularly the SL sequence, are also essential for transcription initiation. Advisors/Committee Members: Nilsen, Timothy W.

Subjects: Biology, Molecular

Keywords: RNA splicing; Nematodes

21. Hershberger, Richard Paul. Regulation of expression of alternatively-spliced human fibronectin IIICS mRNA variants.

Degree: PhD, Molecular Biology and Microbiology, 1991, Case Western Reserve University

► Fibronectin polypeptide diversity can be generated by alternative splicing of fibronectin primary… (more)

▼ Fibronectin polypeptide diversity can be generated by alternative splicing of fibronectin primary transcripts at three sites, including a unique region termed the type-III connecting segment (IIICS). A novel double primer extension assay was developed to measure each of the five human IIICS mRNA splicing variants. Expression of IIICS mRNAs was analyzed in a variety of human normal and tumor cells, and in liver tissue. Differences in IIICS expression patterns were observed among different cell types, among fibroblasts of different tissue origins, and between normal and transformed cells. The most predominant differences were in the abundance of the one IIICS- mRNA variant relative to the four IIICS+ variants. The expression of the IIICS- variant was elevated in liver tissue relative to cultured cells, indicative of differences in subunit composition between cellular and plasma fibronectins. Additional changes in expression levels of the four IIICS+ variants are consistent with a model in which regulation of IIICS mRNA expression occurs predominantly at the level of alternative 3′ splice site selection. The degree to which the IIICS alternative splicing process is subject to ongoing regulation within individual cell types was experimentally ex amined. Possible alterations in IIICS mRNA expression patterns during in vitro or in vivo aging of human dermal fibroblasts were tested. The possible effects on IIICS expression of factors that regulate matrix synthesis (TGF-β, glucocorticoids) were studied. As a prelude to studies on the sequences and factors required to mediate regulated IIICS alternative splicing, several plasmids were constructed directing synthesis of a human IIICS pre-mRNA splicing substrate. This work was designed to provide insights into the regulation of alternative splicing of human fibronectin pre-mRNAs in both generating and responding to adhesive preferences, and also addresses the possible regulation of the process of pre-mRNA splicing. Advisors/Committee Members: Culp, Lloyd A.

Subjects: Biology, Molecular

Keywords: expression human fibronectin IIICS mRNA

22. Jadlowsky, Julie Kendal. Dual Control of HIV Transcription Elongation: Virus-Specific Negative Control by NELF-E is Counterbalanced by Positive Transcription Factor P-TEFb.

Degree: PhD, Molecular Biology and Microbiology, 2009, Case Western Reserve University

► Expression of the HIV provirus is regulated at the level of transcription… (more)

▼ Expression of the HIV provirus is regulated at the level of transcription by an intricate interplay between positive and negative factors. In the presence of positive transcription elongation factors such as the cellular cofactor Positive Transcription Elongation Factor b (PTEF-b), transcription proceeds efficiently, resulting in full-length HIV mRNA. Alternatively, paused elongation produces an accumulation of short, abortive transcripts. Non-processive transcription occurs in the presence of negative factors, such as Negative Elongation Factor (NELF), which has been shown to suppress elongation through direct interaction between its NELF-E subunit and the HIV TAR. This work is intended to ascertain whether these factors can potentially be manipulated to benefit treatment of HIV, either through therapeutics to target active, ongoing infection, or by manipulating the latent viral reservoir which currently prevents eradication of virus from the body.This study used the development of dominant negative CycT1 mutants to attempt to specifically inhibit positive regulation of HIV transcription. Two separate CycT1 mutants were found to interfere with successful transcription of HIV mRNA by distinct mechanisms. One mutant forms a kinase-inactive complex with Tat, while the other causes specific degradation of Tat. On the opposing side of regulation, shRNA-expressing lentiviruses targeting NELF were applied to a re-activatable model of post-integration latency to better understand the associated transcriptional suppression. These results suggest the NELF-E subunit is essential for the suppression of transcription associated with latency, and that this NELF-E effect is specific to the Human Immunodeficiency Virus. These investigations demonstrate that the regulation of HIV transcription is a critical stage in the life cycle of the virus which can be exploited to not only gain a better understanding of the virus itself, but to possibly open new avenues for the future treatment of AIDS. Advisors/Committee Members: Karn, Jonathan.

Subjects: Biology; Biomedical research; Molecular biology

Keywords: HIV Transcription

23. Jones, Daniel Morgan. Retroviral insertion into herpesvirus.

Degree: PhD, Molecular Biology and Microbiology, 1993, Case Western Reserve University

► Retroviruses and herpesviruses are common viral pathogens in humans and animals which… (more)

Subjects: Biology, Molecular

Keywords: Retroviral insertion into herpesvirus.

24. Kehres, David George. Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein.

Degree: PhD, Molecular Biology and Microbiology, 1993, Case Western Reserve University

► The surface of the Escherichia coli L-arabinose-binding protein, ABP, that interacts with… (more)

▼ The surface of the Escherichia coli L-arabinose-binding protein, ABP, that interacts with the AraG:AraH complex, the cytoplasmic membrane component of the L-arabinose periplasmic transport system, has been defined by saturation and site-specific mutagenesis. A set of ABP mutants with single or multiple amino acid substitutions has been generated. The transport phenotypes of these mutants, when expressed with wild-type AraG and AraH in whole cells, suggest that the surface containing the mouth of the arabinose-binding cleft is responsible for most if not all of ABP's interactions with AraG:AraH. Most mutations which alter transport involve replacement of one or more charged residues on this surface with neutral amino acids. In most instances charge neutralization reduces uptake, but in some cases it increases uptake. The saturating initial uptake velocity Ven and the half-saturating substrate concentration Ken vary in proportion to each other in most of these mutants. This observation has led to the development of a simple first-order kinetic model, applicable to periplasmic transport in general, and to the proposal that wild-type arabinose periplasmic transport represents a limiting case of the model in which kfor, the forward rate of transport for a docked complex between liganded ABP and AraG:AraH, greatly exceeds kund, the rate at which the liganded binding protein dissociates. Genetic dominance patterns in cells expressing two different forms of ABP indicate that the Km of translocation at the cytoplasmic membrane surface is lower for "down" mutants and higher for "up" mutants than it is for wild-type ABP, and vesicle reconstitution studies suggest that the equilibrium binding affinity of liganded wild-type ABP for AraG:AraH is between 0.025 μM and 1.0 μM, much smaller than the expected Km. Both of these results are consistent with the kinetic limit predicted by the model. Vesicle-binding studies also suggest that at least one of the transport mutants has an altered equilibrium binding affinity for AraG:AraH, which supports the contention that the ABP cleft mouth surface contacts the membrane complex directly. Advisors/Committee Members: Hogg, Robert W.

Keywords: Mutational; kinetic; analysis; Escherichia coli; L-arabinose binding; protein

25. Kiss, Daniel L. The Exozyme Model: A New Paradigm of Exosome Subunit Activity Revealed by Diverse and Distinct Substrate Specificities of Exosome Subunits In Vivo.

Degree: PhD, Molecular Biology and Microbiology, 2010, Case Western Reserve University

► The RNA processing exosome was originally defined as an evolutionarily conserved multi-subunit… (more)

▼ The RNA processing exosome was originally defined as an evolutionarily conserved multi-subunit complex of ribonucleases (RNases) responsible for the processing and/or turnover of stable RNAs and mRNAs. Results from several in vivo systems challenge the prevailing model of exosome complex function, which relies heavily on in vitro reconstructions of the complex. Further, the detailed mechanisms for how individual exosome subunits participate in each of these RNA metabolic pathways remains unclear. Here, I use RNA interference to deplete most exosome subunits in Drosophila melanogaster S2 tissue culture cells. Surprisingly, neither depletion nor over-expression of most exosome subunits greatly hinders the proliferation of S2 cells. I assayed the effects of the depletions on global mRNA levels using gene expression microarrays. Consistent with the RNase activities ascribed to the exosome complex, most affected mRNAs are increased. However, my results revealed that the pools of mRNAs affected by exosome subunit depletion vary significantly in both the number and identity of the transcripts affected. Notably, the altered mRNAs possess both 5’ and 3’ untranslated regions that are longer than the representative transcriptomic average. The data also show a subunit specific preference for certain mitochondrial-targeted transcripts and for transcripts surveyed by the nonsense-mediated decay (NMD) pathway. In particular, a very large portion of transcripts affected by Rrp6 depletion were NMD substrates, suggesting that Rrp6 may have a core exosome-independent function in NMD. Since exosome subunit depletion had different effects on one class of known exosome substrates (NMD-surveyed mRNAs) in my microarray studies, I wanted to determine if another class of mRNA would be affected in a similar manner. I selected two heat shock-inducible transcripts, hsp70 and hsp26, which contain AU-rich elements (AREs) for further study. Consistent with the microarray results, I observed distinct effects on hsp70 and hsp26 mRNA levels when different subunits are depleted. In this work, I propose and test the validity of the exozyme model, which is a new model for exosome subunit complex assembly and function. Together, these studies provide compelling evidence for multiple, independent functions for individual exosome subunits and suggests a larger population of exosome subunit assemblages than heretofore anticipated. Advisors/Committee Members: Andrulis, Eirk.

Subjects: Molecular biology

Keywords: RNA turnover; exosome; exozyme; RNA metabolism; heat shock; microarray

26. Lackner, Laura L. Investigating the Mechanism of Escherichia coli Min Protein Dynamics.

Degree: PhD, Molecular Biology and Microbiology, 2006, Case Western Reserve University

► In Escherichia coli the pole-to-pole oscillation of the MinC division inhibitor is… (more)

▼ In Escherichia coli the pole-to-pole oscillation of the MinC division inhibitor is required for proper placement of the division machinery. The membrane association/dissociation cycle of MinC is driven by the MinD ATPase and the MinE topological specificity factor, which themselves undergo a coupled oscillatory localization cycle. During this cycle, MinD and a portion of MinE assemble on the membrane along one cell half in a pattern resembling a test tube (the MinD/E tube), while another portion of MinE assembles at the rim of this tube forming a ring-like structure (E-ring). To understand the biochemical mechanisms behind Min protein dynamics, we investigated the interactions of purified Min proteins with ATP and phospholipid vesicles. We found that the ATP-bound form of MinD binds phospholipid vesicles in a cooperative fashion and recruits both MinC and MinE to the vesicles. In addition, we found that MinE stimulates the dissociation of MinC, MinD, and itself from the vesicles. Thus, ATP and MinE play critical roles in regulating the association and dissociation, respectively, of the Min proteins and the membrane. MinE has two known functional domains: the N-terminal anti-MinCD domain (DMinE) and the C-terminal topological specificity and dimerization domain (TSMinE). We investigated the contributions of each domain of MinE in the regulation of Min protein-membrane dissociation. We found that DMinE is necessary and sufficient to stimulate dissociation of the Min proteins from the membrane. However, TSMinE is required to establish Min protein oscillation in a majority of cells. In addition, TSMinE is also required for E-ring formation. These results suggest that TSMinE and perhaps E-ring formation play a critical role in Min protein oscillation by helping to initiate and/or sustain Min protein dynamics. Characterization of the MinD mutant MinDK16R revealed an additional role for MinE. While MinE does not induce MinDK16R oscillation, MinE does promote the accumulation of MinDK16R into static membrane-associated spiral-like structures. We propose that these structures may represent a stage in the dynamic Min protein oscillation cycle, therefore, suggesting that MinE has at least two roles in Min protein dynamics, regulating Min protein-membrane dissociation and regulating Min protein spiral formation. Advisors/Committee Members: de Boer, Piet.

Keywords: Min system; MinD; MinE; cell division; E.coli cell division

27. Lancy, Edward Donald Jr. Genetic requirements for growth of Salmonella typhimurium lacking the proofreading subunit of DNA polymerase III.

Degree: PhD, Molecular Biology and Microbiology, 1990, Case Western Reserve University

► DnaQ (mutD) encodes the editing exonuclease subunit (epsilon) of DNA polymerase III.… (more)

▼ DnaQ (mutD) encodes the editing exonuclease subunit (epsilon) of DNA polymerase III. Deletion-substitution alleles of the dnaQ gene were constructed in vitro and then introduced into the Salmonella chromosome to yield dnaQ null mutants. These null mutants exhibited a growth defect and as a consequence of the poor growth of dnaQ mutants, these strains were replaced within single colonies by derivatives carrying an extragenic suppressor mutation that compensated the growth defect. Sixteen independently derived suppressors mapped in the vicinity of dnaE, the gene for the polymerization subunit (alpha) of DNA polymerase III. Using a combination of nucleotide sequencing and marker rescue experiments, the alteration in one such suppressor was localized to the dnaE gene and results in a valine to glycine substitution at amino acid 832 of the 1,160 amino acid alpha polypeptide. Partially purified DNA polymerase III containing this altered alpha subunit was active in polymerization assays and lacked any detectable editing activity. A two hundred nucleotide region encompassing the site of the valine to glycine substitution mutation was analyzed in fourteen other suppressor mutants using a combination of the polymerase chain reaction and DNA sequencing. Three of the fourteen suppressor mutants contained the valine to glycine substitution at position 832. The other eleven contained valine (wild type) at position 832 and did not contain any other changes from the wild type sequence. In addition to their dependence on a suppressor mutation affecting alpha for healthy growth, the dnaQ null mutants strictly required DNA polymerase I for viability. All three activities of DNA polymerase I were required in the dnaQ null mutants when a suppressor mutation was not present. In the presence of a suppressor mutation, the dnaQ null mutants no longer required the 3′ → 5′ exonuclease of DNA polymerase I. These studies indicate that in the absence of epsilon, DNA replication falters unless secondary mechanisms come into play, including a genetically-coded alteration in the intrinsic replication capacity of alpha and an increased use of DNA polymerase I. Thus, epsilon plays a role in DNA replication distinct from its known role in controlling spontaneous mutation frequency. Advisors/Committee Members: Maurer, Russell A.

Keywords: Genetic requirements; Salmonella typhimurium; polymerase III

28. Lee, Pei-Chung. Effector Secretion Control by the Pseudomonas aeruginosa Type III Secretion System.

Degree: PhD, Molecular Biology and Microbiology, 2011, Case Western Reserve University

► P. aeruginosa uses a type three secretion system (T3SS) to inject effectors… (more)

▼ P. aeruginosa uses a type three secretion system (T3SS) to inject effectors into host cells. The T3SS is important for the virulence of P. aeruginosa. The T3SS forms a needle protruding from the bacterial surface and a basal apparatus across the envelope. Effector secretion is triggered by host cell contact; however, the mechanism of effector secretion control is still unclear. The needle tip protein, PcrV, and its chaperone, PcrG, are negative regulators of effector secretion. My thesis study focused on how PcrV and PcrG regulate effector secretion. My data demonstrate that PcrV needs to be secreted and properly assembled at the needle tip in order to control effector secretion. I constructed PcrV and PcrG mutants that abolish the PcrG/PcrV interaction and demonstrated that the PcrG/PcrV interaction is dispensable for effector secretion control. I showed that PcrG regulates effector secretion in the cytoplasm in a PcrV independent manner and the C-terminus of PcrG contains most of the regulatory activity. I identified the apparatus component, PcrD, is the interaction partner of the C-terminus of PcrG and PcrG regulates effector secretion by interacting with PcrD. In addition, I found that the center region of PcrG interacts with PscO, a component of the T3SS ATPase complex. The PcrG/PscO interaction may stabilize the PcrG/PcrD interaction and control secretion activity of the apparatus. My data support the model that PcrV and PcrG allosterically regulate effector secretion. Assembly of PcrV at the needle tip and the interaction between PcrG and PcrD stabilize the apparatus in an effector secretion “off” conformation. Once the bacterium contacts with the host cell, PcrV changes its conformation and PcrG is released from the apparatus; therefore, the apparatus switches to an effector secretion “on” conformation. Furthermore, I isolated the mutations in the needle protein, PscF, that cause de-regulated effector secretion, suggesting that the needle can be locked in effector secretion “on” conformation. Therefore, effector secretion is controlled by conformational changes of the apparatus in P. aeruginosa. Advisors/Committee Members: Rietsch, Arne.

Subjects: Microbiology

Keywords: Pseudomonas; type III secretion

29. Lin, Wen-chang. Histochemical expressing genes as ultrasensitive markers for micrometastasis studies.

Degree: PhD, Molecular Biology and Microbiology, 1992, Case Western Reserve University

► Evaluation of micrometastasis formation at secondary organs has been an important issue… (more)

▼ Evaluation of micrometastasis formation at secondary organs has been an important issue in cancer treatment, as well as in understanding metastasis mechanisms. A model system, with bacterial β-galactosidase (lacZ) gene as an ultrasensitive histochemical marker for identifying transfected metastatic tumor cells, was developed to overcome the inability of identifying single metastatic tumor cells in situ. With a chromogenic substrate (X-gal), the lacZ-bearing EJ Ha-ras oncogene-transformed tumor cells stain intensely blue and can be easily distinguished from the host tissue cells at minutes to weeks post-injection. The lacZ marker gene appears to be an ultrasensitive marker for analyzing the earliest stages in micrometastasis development. In order to study interactions of different tumor cell clones, as well as between tumor cells and host cells, alternative histochemical marker genes (human placental alkaline phosphatase and Drosophila alcohol dehydrogenase) were developed for multiple-color marker tagging systems. Cells carrying different marker genes can easily be differentiated with double-staining protocols. Co-injection or sequential-injection experiments with two classes of transformed cells carrying different oncogenes (ras or sis) and alternative histochemical markers (β-galactosidase or alkaline phosphatase) demonstrate cooperation among different oncogene-transformed cells. The ultrasensitivity of these histochemical markers provides opportunities to evaluate quantitatively the metastatic potential of different tumor cell lines, examine the genetic instability as well as clonal dominance/evolution of metastatic cells, follow the development of micrometastasis at multiple organs in situ, and study expression of growth-related genes in micrometastatic foci at the single-cell level. Therefore, these marker genes allow us to analyze qualitatively and quantitatively not only the development of micrometastases at their earliest stages, but also their interaction with host cells and clonal progression within metastatic tumor cells Advisors/Committee Members: Culp, Lloyd A.

Subjects: Biology, Molecular

Keywords: Histochemical marker genes; Micrometastasis

30. Lobritz, Michael Andrew. VARIATIONS IN THE V3 CROWN OF HIV-1 ENVELOPE IMPACT AFFINITY FOR CCR5 AND AFFECT ENTRY AND REPLICATIVE FITNESS.

Degree: PhD, Molecular Biology and Microbiology, 2007, Case Western Reserve University

► Entry inhibitors represent a new class of antiretroviral agents for the treatment… (more)

Keywords: HIV-1; Entry; Replicative Fitness; Entry Inhibitors

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