Biology Dissertations Copyright (c) 2013 Georgia State University All rights reserved. http://digitalarchive.gsu.edu/biology_diss Recent documents in Biology Dissertations en-us Mon, 06 May 2013 08:32:09 PDT 3600 Comparison of Nitrile Hydratases in Rhodococcus Rhodochrous DAP 96253 and DAP 96622 Growing on Inducing and Non-Inducing Media http://digitalarchive.gsu.edu/biology_diss/130 http://digitalarchive.gsu.edu/biology_diss/130 Fri, 26 Apr 2013 07:16:38 PDT Nitrile hydratase activity in Rhodococcus rhodochrous DAP 96253 can be induced with multiple inducers that include urea, cobalt (Co), iron (Fe) and nickel (Ni). When induced with Co/urea, cells of R. rhodochrous DAP 96253 expressed the highest level of nitrile hydratase activity (~200 units/min·mg-cdw) when compared with the other inducers tested. Cells induced with Co had the second highest nitrile hydratase activity (~7 units/min·mg-cdw), whereas in the uninduced cells, nitrile hydratase activity was lower than 1 unit/min·mg-cdw. Similarly in R. rhodochrous DAP 96622, when induced with Co/urea, the nitrile hydratase activity of R. rhodochrous DAP 96622 cells was around 50 units/min·mg-cdw which was the highest of all inducers tested. When induced with Co only, the nitrile hydratase activity of R. rhodochrous DAP 96622 was around 20 units/min·mg-cdw, and the nitrile hydratase activity of R. rhodochrous DAP 96622 uninduced was the same as the nitrile hydratase activity of uninduced R. rhodochrous DAP 96253.

When Co/urea induced R. rhodochrous DAP 96253 cell lysate was examined on gradient SDS-PAGE and analyzed by Image Quant TL, the nitrile hydratase bands (both α and β subunits) accounted for more than 55% of the total cytosolic proteins. Whereas in Co/urea induced R. rhodochrous DAP 96622, the nitrile hydratase bands accounted for around 25% of the total cytosolic proteins. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry results, amidase in R. rhodochrous DAP 96253 was approximately 38 kDa from the nitrilase/cyanide hydratase family and amidase in R. rhodochrous DAP 96622 was 55 kDa from the amidase signature family.

In addition, the nitrile hydratase regulation system in both R. rhodochrous DAP 96253 and DAP 96622 strains are different. Moreover, the nitrile hydratase regulation system in R. rhodochrous DAP 96253 is different from R. rhodochrous J1.

Purified nitrile hydratase from R. rhodochrous DAP 96253 may form a protein complex with glutamine synthetase, resulting in a nitrile hydratase activity of approximately 1500 units/mg-proteins, and nitrile hydratase from R. rhodochrous DAP 96622 is not a protein complex and results in a nitrile hydratase activity of 950 units/mg-proteins.

]]> Fengkun Du Investigation Of The Rescue Of The Rubella Virus P150 Replicase Protein Q Domain By The Capsid Protein http://digitalarchive.gsu.edu/biology_diss/129 http://digitalarchive.gsu.edu/biology_diss/129 Fri, 26 Apr 2013 07:16:36 PDT The rubella virus (RUB) capsid protein (C) is a multifunctional phosphoprotein with roles beyond encapsidation. It is able to rescue a large lethal deletion of the Q domain in the P150 replicase gene at a step in replication before detectable viral RNA synthesis, indicating a common function shared by RUB C and the Q domain. The goal of this dissertation was to use constructs containing the N-terminal 88 amino acids of RUB C, the region previously defined as the minimal region required for the rescue of Q domain mutants, to elucidate the function of RUB C in Q domain rescue and viral RNA synthesis. In the first specific aim, the rescue function of 1-88 RUB C and the importance of an arginine-rich cluster, R2, within 1-88 RUB C for rescue were confirmed. Rescue was not correlated with intracellular localization or phosphorylation status of RUB C. In the second specific aim, the involvement of RUB C in early events post-transfection with RUB RNA was analyzed. RUB C specifically protected RUB transcripts early post-transfection and protection required R2. However, it was concluded the protection observed was due to the encapsidation function of RUB C and not related to Q domain rescue. No differences in the translation of the RUB nonstructural proteins in the presence or absence of RUB C were observed. Interactions of RUB C with host cell proteins were analyzed. Although the interaction of RUB C with cellular p32 required the R2 cluster, both wild type (does not require RUB C for replication) and RQQ (requires RUB C for replication) Q domain bound p32, indicating interaction with this binding partner is not the basis of rescue. Using a human protein array phosphatidylinositol transfer protein alpha isoform (PITPα) was found to interact with RUB C but not its R2 mutant. However, co-immunoprecipitation experiments revealed that this protein binds both forms of RUB C. Although the mechanism behind the rescue of the RUB P150 Q domain by RUB C remains unknown, we propose a model that RUB C plays a role in generation of the virus replication complex in infected cells.

]]> Heather Mousa Analysis of Simian Hemorragic Fever Virus Proteins and the Host Cell Responses of Disease Resistant and Susceptible Primates http://digitalarchive.gsu.edu/biology_diss/128 http://digitalarchive.gsu.edu/biology_diss/128 Fri, 26 Apr 2013 07:16:35 PDT African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication.

SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production.

The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

]]> Heather Vatter Molecular Mechanism of Heme Acquisition and Degradation by the Human Pathogen Group A Streptococcus http://digitalarchive.gsu.edu/biology_diss/127 http://digitalarchive.gsu.edu/biology_diss/127 Wed, 24 Apr 2013 11:41:36 PDT Heme is the major iron source for the deadly human pathogen, Group A Streptococcus (GAS). During infection, GAS lyses host cells releasing hemoglobin and other hemoproteins. This dissertation aims to elucidate the general mechanism by which GAS obtains and utilizes heme as an iron source from the host hemoproteins. GAS encodes a heme relay system consisting of Shr, Shp and the SiaABC transporter. We specifically determine the role of Shr in the heme uptake process, by conducting a detailed functional characterization of its constituent domains. We also undertake to solve the long-standing mystery surrounding the catabolism of heme in streptococci. The studies presented herein established Shr as a prototype of a new family of NEAT-containing hemoproteins receptors. They demonstrate its importance in heme acquisition by GAS and provide a molecular model for heme scavenging and transfer by the protein. We show that Shr modulates heme uptake depending on heme availability by a mechanism where NEAT1 facilitates fast heme scavenging and delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface. Finally, we identified and characterized for the first time, a heme oxygenase (HO) in the Streptococcus genus which was named HupZ. Sequence comparison between HupZ and several HOs from different structural families indicates that this enzyme is unrelated to any of the previously characterized HOs. However, orthologs of the protein are found in other important pathogens. The structure and the catalytic mechanism of HupZ suggest that it is the representative of a new family of flavoenzymes capable of degrading heme using their reduced flavin cofactor as a source of electrons. Overall, this work contributes significant knowledge to the topic of heme utilization by pathogens and importantly, provides new direct evidence that associates flavins with heme metabolism in bacteria. Thus it sets a new direction in the field and lays the ground for future fundamental and applied discoveries.

]]> Mahamoudou Ouattara Induction of Cyanide Metabolizing Enzymes and Production of Antifungal Compounds by Rhodococcus Species http://digitalarchive.gsu.edu/biology_diss/126 http://digitalarchive.gsu.edu/biology_diss/126 Wed, 24 Apr 2013 11:41:35 PDT Rhodococcus is a soil microbe known for its metabolic versatility. Cyanide is one of many compounds that Rhodococcus can detoxify. This work identified several inducers which affect the activity of two cyanide-metabolizing enzymes produced by R. rhodochrous DAP 96253, cyanidase and β-cyanoalanine synthase-like enzyme. Chemical inducers were added to growth media and cells were tested for the ability to transform cyanide to ammonia or hydrogen sulfide as a quantifiable measure of cyanidase or β-cyanoalanine synthase-like activity, respectively. Urea-supplemented YEMEA-grown R. rhodochrous DAP 96253 has been shown to inhibit germination of selected fungal spores. By varying supplements in growth media, several new compounds were identified which also enable Rhodococcus to inhibit germination. This inhibition is achieved through non-contact co-culture between organisms. Properties of anti-fungal activity were studied for each supplement. It was shown that supplemented R. rhodochrous DAP 96253 is able to inhibit germination of single or mixed cultures the selected fungi, Aspergillus niger, Aspergillus fumigatus, and Penicillium sp. Depending on the supplemental compound added to growth media, spores were unable to recover after a maximum 48 hour exposure to R. rhodochrous DAP 96253. Rhodococcus effectively inhibits germination of spores when actively growing on agar or when removed from the plate. Lastly, the effects of co-culturing three strains of YEMEA-grown Rhodococcus sp. with supplemented, YEMEA-grown R. rhodochrous DAP 96253 were examined to determine if supplemented R. rhodochrous DAP 96253 affected unsupplemented Rhodococcus sp. When co-cultured with cobalt- and urea-supplemented or just urea-supplemented YEMEA-grown R. rhodochrous DAP 96253, the three strains tested, R. rhodochrous DAP 96253, R. rhodochrous 96622, and R. erythropolis 47072 exhibited cyanidase enzyme levels comparable to those seen in directly induced cells. Additionally, after co-culture with urea-supplemented YEMEA-grown R. rhodochrous DAP 96253, YEMEA-grown cultures became inhibitory to germination of spores of A. niger. Overall, these findings showed that, with proper supplementation, R. rhodochrous DAP 96253 can be inhibitory to selected fungal species and is simultaneously able to stimulate antifungal activity in other, unsupplemented Rhodococcus sp.

]]> Katharine M. Swensen Mechanistic Studies Of Drug Resistance Conferred By An ABC Transporter DrrAB http://digitalarchive.gsu.edu/biology_diss/125 http://digitalarchive.gsu.edu/biology_diss/125 Mon, 22 Apr 2013 10:31:41 PDT Multi-drug resistance (MDR) has become a serious clinical problem for both cancer and infectious disease treatment. One of the leading causes of MDR is the drug efflux mediated by the ATP-binding cassette (ABC) super-family of proteins which are found among diverse groups of organisms. In our laboratory, we are interested in studying an ABC transporter DrrAB from Streptomyces peucetius. It confers resistance to two anticancer antibiotics doxorubicin and daunorubicin by its drug efflux function. Insights into the function of the DrrAB complex are expected to facilitate a better understanding of mechanisms of MDR.

The normal function of the DrrAB complex depends on the cis expression of both subunits, DrrA and DrrB. DrrA forms the nucleotide binding domain (NBD) and provides energy for drug translocation, while the DrrB protein functions as the transmembrane domain (TMD) and forms the substrate translocation pathway. Studies on the detailed mechanisms of communication between DrrA and DrrB are critical for understanding the coupling of energy usage and substrate translocation. The present studies revealed the existence of two novel and functionally important modules in the C-terminal domain of DrrA that might be essential for conformational interplay between DrrA and DrrB during the catalytic cycle. One module present at the extreme C terminus of DrrA consists of two separate motifs, DEF and CREEM. CREEM motif together with its upstream region up to residue S319, interacts with the N-terminal cytoplasmic tail region of DrrB and forms an DrrA-DrrB interface, while the DEF motif regulates this interaction. The second novel module GATE is present 104 amino acids upstream of DEF. Based on our biochemical and structural analyses, we propose that GATE functions as a transducer of conformational changes (resulting from ATP binding) from DrrA to DrrB. Our studies provide important insights into the interplay between the NBD and TMD of the DrrAB drug transporter and their roles in communicating long range conformational changes during the catalytic cycle. These findings further deepen our understanding of the mechanisms of drug resistance.

]]> Han Zhang Structure Based Study of CA SPASE-3 and D-Arginine Dehydrogenase http://digitalarchive.gsu.edu/biology_diss/124 http://digitalarchive.gsu.edu/biology_diss/124 Tue, 04 Dec 2012 09:31:58 PST Caspases are important players in programmed cell death. Normal activities of caspases are critical for the cell life cycle and dysfunction of caspases may lead to the development of cancer and neurodegenerative diseases. They have become a popular target for drug design against abnormal cell death. In this study, the recognition of P5 position in substrates by caspase-3, -6 and -7 has been investigated by kinetics, modeling and crystallography. Crystal structures of caspase-3 and -7 in complexes with substrate analog inhibitor Ac-LDESD-CHO have been determined at resolutions of 1.61 and 2.45 Å, respectively, while a model of caspase-6/LDESD is constructed. Enzymatic study and structural analysis have revealed that Caspase-3 and -6 recognize P5 in pentapeptides, while caspase-7 lacks P5-binding residues.

D-arginine dehydrogenase catalyzes the flavin-dependent oxidative deamination of D-amino acids to the corresponding imino acids and ammonia. The X-ray crystal structures of DADH and its complexes with several imino acids were determined at 1.03-1.30 Å resolution. The DADH crystal structure comprises a product-free conformation and a product-bound conformation. A flexible loop near the active site forms an “active site lid” and may play an essential role in substrate recognition. The DADH Glu87 forms an ionic interaction with the side chain of iminoarginine, suggesting its importance for DADH preference of positively charged D-amino acids. Comparison of the kinetic data of DADH activity on different D-amino acids and the crystal structures demonstrated that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic D-amino acids bound within a flask-like cavity.

Understanding biology at the system level has gained much more attention recently due to the rapid development in genome sequencing and high-throughput measurements. Current simulation methods include deterministic method and stochastic method. Both have their own advantages and disadvantages. Our group has developed a deterministic-stochastic crossover algorithm for simulating biochemical networks. Simulation studies have been performed on biological systems like auto-regulatory gene network and glycolysis system. The new system retains the high efficiency of deterministic method while still reflects the random fluctuations at lower concentration.

]]> Guoxing Fu Functional Study of the Threonine Phosphorylation and the Transcriptional Coactivator Role of P68 RNA Helicase http://digitalarchive.gsu.edu/biology_diss/123 http://digitalarchive.gsu.edu/biology_diss/123 Tue, 04 Dec 2012 09:31:57 PST P68 RNA helicase is a RNA helicase and an ATPase belonging to the DEAD-box family. It is important for the growth of normal cells, and is implicated in diverse functions ranging from pre-mRNA splicing, transcriptional activation to cell proliferation, and early organ development. The protein is documented to be phosphorylated at several amino-acid residues. It was previously demonstrated in several cancer cell-lines that p68 gets phosphorylated at threonine residues during treatments with TNF-α and TRAIL. In this study, the role of threonine phosphorylation of p68 under the treatment of anti-cancer drug, oxaliplatin in the colon cancer cells is characterized. Oxaliplatin treatment activates p38 MAP-kinase, which subsequently phosphorylates p68 at T564 and/or T446. P68 phosphorylation, at least partially, influences the role of the drug on apoptosis induction. This study shows an important mechanism of action of the anti-cancer drug which could be used for improving cancer treatment.

This study also shows that p68 is an important transcriptional regulator regulating transcription of the cytoskeletal gene TPPP/p25. Previous analyses revealed that p68 RNA helicase could regulate expression of genes responsible for controlling stability and dynamics of different cytoskeletons. P68 is found to regulate TPPP/p25 gene transcription by associating with the TPPP/p25 gene promoter. Expression of TPPP/p25 plays an important role in cellular differentiation while the involvement of p68 in the regulation of TPPP/p25 expression is an important event for neurite outgrowth. Loss of TPPP expression contributes to the development and progression of gliomas. Thus, our studies further enhance our understanding of the multiple cellular functions of p68 and its regulation of the cellular processes.

]]> Heena T. Dey Roles of TH2 and TH17 CD4+ T-Helper Cell Cytokines in the Pathogenesis of Experiemental Cytomegalovirus Retinitis http://digitalarchive.gsu.edu/biology_diss/122 http://digitalarchive.gsu.edu/biology_diss/122 Tue, 04 Dec 2012 09:31:56 PST Human cytomegalovirus (HCMV) is a betaherpesvirus that infects up to 80% of the population worldwide, and establishes latency in monocytes and bone marrow cells. Reactivated HCMV can become an opportunistic pathogen in individuals who are immunocompromised, such as those with acquired immunodeficiency syndrome (AIDS). HCMV infection of AIDS patients causes a sight-threatening retinitis that leads to vision loss and blindness in up to 46% of this population without antiretroviral treatment. Because untreated HIV-infected individuals exhibit the loss of cell-mediated immunity and alterations in CD4+ T-helper (Th) cell cytokines, including elevation of interleukin-4 (IL-4), IL-10, and IL-17, we sought to test the hypothesis that these cytokines play key roles in governing the susceptibility to AIDS-related HCMV retinitis. This hypothesis was tested utilizing a clinically relevant mouse model of experimental murine cytomegalovirus (MCMV) retinitis that occurs in C57BL/6 mice immunosuppressed by mouse retroviruses (MAIDS). Studies revealed that MAIDS progression was associated with increased levels of IL-4 and IL-10, cytokines whose production has been associated with diminished CD8+ T-cell-mediated immunity during HIV infection. However, MCMV–infected eyes of retinitis-susceptible IL-4-/- or IL-10-/- MAIDS mice exhibited frequency and severity of retinitis and viral titers equivalent to MCMV-infected eyes of wild-type MAIDS animals. These studies indicated that neither IL-4 nor IL-10 alone play key roles in increased susceptibility to MCMV retinitis. In comparison, IL-17, an inflammatory cytokine associated with the ocular autoimmune disease uveitis, was systemically increased during the progression of MAIDS, but MCMV-infected eyes of retinitis-susceptible MAIDS mice exhibited a significant reduction in IL-17. These findings suggested that IL-17 plays no direct role in the pathogenesis of experimental MCMV retinitis. However, these results also suggested the remarkable possibility that MCMV downregulates IL-17 production, a hypothesis supported by the observation that systemic MCMV infection of healthy and MAIDS mice resulted in the downregulation of IL-17. Mechanistic studies revealed that knockdown of IL-10 resulted in a partial recovery IL-17 levels during MCMV infection. We conclude that MCMV-induced IL-17 downregulation occurs via the stimulation of IL-10 and the suppressor of cytokine signaling (SOCS)-3. Taken together, our results add new information to the immunobiology of HCMV and to our basic understanding of the pathogenesis of AIDS-related HCMV retinitis.

]]> Emily L. Blalock Molecular mechanisms of spermine on its synergistic effect with beta-lactams against Staphylococcus aureus http://digitalarchive.gsu.edu/biology_diss/121 http://digitalarchive.gsu.edu/biology_diss/121 Fri, 09 Nov 2012 08:51:41 PST Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) in a pH-dependent manner. At high pH (>8) Spm is a potent nucleophile, and able to form Spm-β-lactam adduct. At physiological pH (or lower), Spm carries positive charges, and can bind to DNA through charge interactions. The potential of Spm interfering with cell wall was first investigated. A spontaneous mutant of MRSA Mu50 selected for Spm resistance conferred resistance to Spm/β-lactam synergy. This phenotype was due to the presence of a 7-bp deletion in pbpB as identified by genome resequencing and confirmed by complementation. Analysis of cell wall composition by HPLC revealed the combination of Spm and β-lactam can reduce the cross-linkage of peptidoglycan. These two lines of evidence suggest Spm may perturb cell wall integrity in favor of β-lactam efficacy with PBPs as a promising target. However, from the results of microarray analysis and fluorescent Bocillin labeling, Spm did not appear to suppress the PBPs expression or alter their interactions with β-lactams. Next, transcriptome analyses reveal the genes responsive to the synergy effect overlap extensively with those to high Spm challenge, implying the enhanced detrimental effect of Spm facilitated by β-lactams in inhibition on cell growth. In particular the induction of iron transport and reduction of energy production under synergy were depicted in this study, and high dose Spm was found to turn off the SigB regulon. Of interest, the tetM gene encoding a ribosomal protection protein for tetracycline (Tc) resistance exhibited the most significant fold change and high signals by both high and low dose Spm. Further analysis by qRT-PCR demonstrated the tetM expression was specifically induced by Tc and Spm to a comparable level but not by other polyamines, suggesting a similar mode of action by Spm and Tc in interactions with the ribosome to initiate tetM induction. Collectively, these data indicated the role of Spm could be multifarious with more than one target, and a combination of Spm and β-lactams may inhibit growth of MRSA in a more complicated manner than just potentiating β-lactam inhibition on PBPs.

]]> Xiangyu Yao Development of a Novel Method for Biochemical Systems Simulation: Incorporation of Stochasticity in a Deterministic Framework http://digitalarchive.gsu.edu/biology_diss/120 http://digitalarchive.gsu.edu/biology_diss/120 Fri, 20 Jul 2012 09:23:02 PDT Heart disease, cancer, diabetes and other complex diseases account for more than half of human mortality in the United States. Other diseases such as AIDS, asthma, Parkinson’s disease, Alzheimer’s disease and cerebrovascular ailments such as stroke not only augment this mortality but also severely deteriorate the quality of human life experience. In spite of enormous financial support and global scientific effort over an extended period of time to combat the challenges posed by these ailments, we find ourselves short of sighting a cure or vaccine. It is widely believed that a major reason for this failure is the traditional reductionist approach adopted by the scientific community in the past. In recent times, however, the systems biology based research paradigm has gained significant favor in the research community especially in the field of complex diseases. One of the critical components of such a paradigm is computational systems biology which is largely driven by mathematical modeling and simulation of biochemical systems. The most common methods for simulating a biochemical system are either: a) continuous deterministic methods or b) discrete event stochastic methods. Although highly popular, none of them are suitable for simulating multi-scale models of biological systems that are ubiquitous in systems biology based research. In this work a novel method for simulating biochemical systems based on a deterministic solution is presented with a modification that also permits the incorporation of stochastic effects. This new method, through extensive validation, has been proven to possess the efficiency of a deterministic framework combined with the accuracy of a stochastic method. The new crossover method can not only handle the concentration and spatial gradients of multi-scale modeling but it does so in a computationally efficient manner. The development of such a method will undoubtedly aid the systems biology researchers by providing them with a tool to simulate multi-scale models of complex diseases.

]]> Amit Sabnis A Comparative Analysis of the Neural Basis for Dorsal-Ventral Swimming in the Nudipleura http://digitalarchive.gsu.edu/biology_diss/119 http://digitalarchive.gsu.edu/biology_diss/119 Fri, 20 Jul 2012 09:23:00 PDT Despite having similar brains, related species can display divergent behaviors. Investigating the neural basis of such behavioral divergence can elucidate the neural mechanisms that allow behavioral change and identify neural mechanisms that influence the evolution of behavior.

Fewer than three percent of Nudipleura (Mollusca, Opisthobranchia, Gastropoda) species have been documented to swim. However, Tritonia diomedea and Pleurobranchaea californica express analogous, independently evolved swim behaviors consisting of rhythmic, alternating dorsal and ventral flexions. The Tritonia and Pleurobranchaea swims are produced by central pattern generator (CPG) circuits containing homologous neurons named DSI and C2. Homologues of DSI have been identified throughout the Nudipleura, including in species that do not express a dorsal-ventral swim. It is unclear what neural mechanisms allow Tritonia and Pleurobranchaea to produce a rhythmic swim behavior using homologous neurons that are not rhythmic in the majority of Nudipleura species.

Here, C2 homologues were also identified in species that do not express a dorsal-ventral swim. We found that certain electrophysiological properties of the DSI and C2 homologues were similar regardless of swim behavior. However, some synaptic connections differed in the non-dorsal-ventral swimming Hermissenda crassicornis compared to Tritonia and Pleurobranchaea. This suggests that particular CPG synaptic connections may play a role in dorsal-ventral swim expression.

DSI modulates the strength of C2 synapses in Tritonia, and this serotonergic modulation appears to be necessary for Tritonia to swim. DSI modulation of C2 synapses was also found to be present in Pleurobranchaea. Moreover, serotonergic modulation was necessary for swimming in Pleurobranchaea. The extent of this neuromodulation also correlated with the swimming ability in individual Pleurobranchaea; as the modulatory effect increased, so too did the number of swim cycles produced. Conversely, DSI did not modulate the amplitude of C2 synapses in Hermissenda. This indicates that species differences in neuromodulation may account for the ability to produce a dorsal-ventral swim.

The results indicate that differences in synaptic connections and neuromodulatory dynamics allow the expression of rhythmic swim behavior from homologous non-rhythmic components. Additionally, the results suggest that constraints on the nervous system may influence the neural mechanisms and behaviors that can evolve from homologous neural components.

]]> Joshua L. Lillvis The Role of Rankl in Prostate Cancer Progression and Bone Metastasis http://digitalarchive.gsu.edu/biology_diss/118 http://digitalarchive.gsu.edu/biology_diss/118 Thu, 12 Jul 2012 11:47:49 PDT This study focused on the role of RANKL in prostate cancer EMT progression and metastasis. Activation of RANK, a receptor activator of NF-kB, by its ligand RANKL, in a paracrine manner is responsible for osteoclast differentiation and bone remodeling. RANK activation in cancer cells, however, is thought to be promoted by both autocrine and paracrine mechanisms because RANKL has been shown to be derived from either tumor or its microenvironment, such as osteoblasts, infiltrating inflammatory cells and stromal fibroblasts. In the present study, we demonstrated that autocrine and paracrine RANKL-RANK signaling could be responsible for driving prostate cancer bone metastasis by promoting epithelial to mesenchymal transition (EMT). We further characterized a novel converging RANKL-c-Met signaling network in which the activation of RANKL was found to promote the expression of both RANKL and c-Met in an autocrine manner in prostate cancer cells. The induced RANKL and c-Met in prostate cancer cells is biologically functional and contributes to increased osteoclastogenesis, epithelial to mesenchymal transition (EMT), cell motility, migration and invasion and conferred bone and soft tissue metastases. Remarkably, RANKL expression by 1,000 prostate cancer cells can provoke bone and soft tissue metastases of a “dormant” population of prostate cancer cells which by themselves failed to form tumors and colonize mouse skeleton, suggesting RANKL can serve as a factor in “reawakening” cancer dormancy to initiate the re-growth and metastasis of cancer cells. We also showed that RANKL-induced RANKL feed-forward autocrine regulation is mediated through cMyc transactivation, allowing the establishment of a “vicious cycle” further promoting prostate cancer growth and metastasis. The converging RANKL-c-Met signaling network is therefore a novel target that could be further manipulated for delaying the lethal progression of castration-resistant human prostate cancer bone metastasis.

]]> Chia-Yi Chu Tyrosine Phosphorylation of p68 RNA Helicase Promotes Metastasis in Colon Cancer Progression http://digitalarchive.gsu.edu/biology_diss/117 http://digitalarchive.gsu.edu/biology_diss/117 Tue, 19 Jun 2012 12:54:51 PDT The initiation of cancer metastasis usually requires Epithelial-Mesenchymal Transition (EMT), by which tumor cells lose cell-cell interactions and gain the ability of migration and invasion. Previous study demonstrated that p68 RNA helicase, a prototypical member of the DEAD-box RNA helicases, functions as a mediator to promote platelet-derived growth factor (PDGF)-induced EMT through facilitating nuclear translocation of β-catenin in colon cancer cells. In this context, p68 RNA helicase was found to be phosphorylated at the tyrosine 593 residue (referred as phosphor-p68) by c-Abl kinase, and this phosphorylation is required for the activation of β-catenin signaling and the consequent EMT. The phosphor-p68 RNA helicase-mediated EMT was characterized by the repression of an epithelial marker, E-cadherin, and the upregulation of a mesenchymal marker, Vimentin. E-cadherin, a major cell-cell adhesion molecule that is involved in the formation of adherens junctions, has been shown to sequester β-catenin at the cell membrane and thus inhibit its transcriptional activity. The functional loss of E-cadherin is the fundamental event of EMT. Despite the role of phosphor-p68 RNA helicase in regulating nuclear translocation of β-catenin, whether phosphor-p68 is involved in the regulation of E-cadherin remains unknown. Here, our data indicated that phosphor-p68 RNA helicase initiated EMT by transcriptional upregulation of Snail1, a master transcriptional repressor of E-cadherin. The data suggest that phosphor-p68 RNA helicase displaced HDAC1 from the chromatin remodeling MBD3:Mi-2/NuRD complex at the Snail1 promoter, thereby activating the transcription of Snail1. In the xenograft tumor model, abolishing the phosphorylation of p68 RNA helicase by the expression of Y593F mutant resulted in a significant reduction of metastatic potential in human colon cancer cells. Analyses in the colon cancer tissues also revealed that the tyrosine 593 phosphorylation level of p68 RNA helicase is substantially enhanced in the tumor tissues comparing to that in the corresponding normal counterparts, suggesting a correlation of phosphor-p68 and tumor progression. In conclusion, we showed that tyrosine phosphorylation of p68 RNA helicase positively correlated to the malignant status of colon cancer progression. The molecular basis behind this correlation could be partly through the transcriptional regulation of Snail1.

]]> Chia Yi Liu The Influences of LuxS in Escherichia coli Biofilm Formation and Improving Teacher Quality through the Bio-Bus Program http://digitalarchive.gsu.edu/biology_diss/116 http://digitalarchive.gsu.edu/biology_diss/116 Tue, 24 Apr 2012 12:19:22 PDT The objectives of this work are: 1) to agarose-stabilize fragile biofilms for quantitative structure analysis; 2) to understand the influences of LuxS on biofilm formation; 3) to improve teacher quality by preparing Georgia’s middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative digital image analysis demonstrated the effectiveness of the agarose stabilization technique for generating reproducible measurements of three dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging. AI-2-dependent and independent effects of LuxS on biofilm-related phenotypes were revealed, suggesting that LuxS is a versatile enzyme, possessing multiple functions in E. coli ecology that could assist E. coli in adapting to diverse conditions. Overall, the work presented in this dissertation supported the concept that quorum sensing, biofilm formation, and cell adhesion are largely related. Additionally, through this project, teachers enhanced content knowledge and confidence levels, mastered innovative teaching strategies and integrated inquiry-based, inter-disciplinary, hands-on activities in the classroom. As a result, student learning was enhanced, and teachers are better equipped to give Georgia’s students a solid foundation in the sciences.

]]> Chandan Morris Robbins LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis Through HB-EGF Shedding and EGFR-mediated ERK Signaling http://digitalarchive.gsu.edu/biology_diss/115 http://digitalarchive.gsu.edu/biology_diss/115 Tue, 24 Apr 2012 12:19:17 PDT LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there is no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In this study, we found increased LIV-1 expression in a progresssive manner in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives prostate cancer EMT in an androgen-refractory human prostate cancer cell (ARCaP) bone metastasis model. LIV-1, when overexpressed in ARCaPE cells (derivative cells of ARCaP with epithelial phenotype), promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells, eliciting constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. Further investigation of the HB-EGF promoter revealed that both Stat3 and AP-1 controlled HB-EGF promoter activity. Ectopic LIV-1 overexpression induced AP-1 and Stat3 activation. Blockade of both Stat3 and AP-1 by specific inhibitors or dominant negative expression vectors diminished the HB-EGF promoter activity induced by LIV-1 overexpression. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promotes EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

]]> Hui-wen Lue Analysis of the Streptococcal Hemoprotein Receptor: A Role in Virulence and Host Defense http://digitalarchive.gsu.edu/biology_diss/114 http://digitalarchive.gsu.edu/biology_diss/114 Tue, 24 Apr 2012 12:19:13 PDT Group A streptococcus (GAS) is an important pathogen that produces a wide spectrum of suppurative infections and autoimmune sequelae in humans, ranging from less complex pharyngitis, impedigo to more severe manifestations such as necrotizing fasciitis, toxic shock syndrome, rheumatic fever and glomerulonephritis. The worldwide burden of GAS infections and sequelae is considerable, but an immunization program that defends against the hyper-variable GAS is missing. The streptococcal hemoprotein receptor (Shr), is an iron-regulated protein involved in heme acquisition. An unspecified region in the amino terminus of Shr mediates the interactions with hemoglobin and two protein modules named NEAT1 and NEAT2 bind heme. In this study, we analyzed the molecular structure and function of Shr, investigated its antigenic properties and role in GAS disease production. We demonstrated that Shr is a new type of GAS adhesin that contributes to the pathogen interactions with extracellular matrix (ECM) proteins. Shr enabled bacterial adherence to host cells and was important for GAS virulence in vivo. Immunizations with Shr protein by intraperitoneal or intranasal administration conferred resistance to systemic GAS challenge in mice. Shr antiserum allowed bacterial opsonization and defended against GAS diseases in a murine model for passive vaccination. Studies with isolated Shr domains localized ECM-binding to the NEAT domains and showed that most of the protein is exposed on the bacterial surface. In addition, Shr N-terminal region and both of the NEAT modules elicited strong antibody response in rabbits. In conclusion, Shr is a protective antigen that contributes to GAS pathogenesis by facilitating both heme uptake and bacterial adherence. Since Shr is conserved among GAS strains and other pyogenic streptococci, this study demonstrates that Shr may be used to develop a vaccine against GAS strains and related pathogens.

]]> Ya-Shu Huang The Nucleocytoplasmic Shuttling Functions of P68 in Cancer Cell Migration and Proliferation http://digitalarchive.gsu.edu/biology_diss/113 http://digitalarchive.gsu.edu/biology_diss/113 Tue, 24 Apr 2012 12:19:08 PDT P68 RNA helicase (p68), as a DEAD family protein, is a typical RNA helicase protein. P68 functions in many other biological processes, which include the regulations of the gene transcription, cell proliferation and cell differentiation. In our group, Y593 phosphorylated p68 was found to have a function in the epithelial mesynchymal transition, which is an important process for cancer metastasis. In the present study, we found that p68 is a nucleocytoplasmic shuttling protein. The protein carries two functional nuclear exporting signal sequences and two nuclear localization signal sequences. Calmodulin, a calcium sensor protein, is well known to play roles in cell migration by regulating the activities of its target proteins at the leading edge. Calmodulin interacts with p68 at the IQ motif of p68. However, the biological function of this interaction is not known. In this study, we found that the p68/calmodulin protein complex functions as a microtubule motor in migrating cells. The shuttling function of p68 along with the motor function of p68/calmodulin causes the leading edge distribution of calmodulin in migrating cells. Disruption the interaction between p68 and calmodulin inhibits cancer cell metastasis in an established mouse model. On the other hand, Y593-Y595 double phosphorylated p68 were found to interact with PKM2, an important tumor isoform of pyruvate kinase. The shuttling function of p68 is reasoned to promote the dimer formation of PKM2 and transport the PKM2 to the cell nucleus. The nuclear PKM2 was found to function as a protein kinase to promote cell proliferation. In specific, the nuclear PKM2 phosphorylates and activates Stat3, an important transcription factor functions in cell proliferation. Overall, p68 is found to have functions in both cell migration and cell proliferation, and these two functions depend on the nucleocytoplasmic shuttling activity and the post-translational modification of p68.

]]> Haizhen Wang The Reorganization of Primary Auditory Cortex by Invasion of Ectopic Visual Inputs http://digitalarchive.gsu.edu/biology_diss/112 http://digitalarchive.gsu.edu/biology_diss/112 Tue, 24 Apr 2012 12:19:04 PDT Brain injury is a serious clinical problem. The success of recovery from brain injury involves functional compensation in the affected brain area. We are interested in general mechanisms that underlie compensatory plasticity after brain damage, particularly when multiple brain areas or multiple modalities are included. In this thesis, I studied the function of auditory cortex after recovery from neonatal midbrain damage as a model system that resembles patients with brain damage or sensory dysfunction. I addressed maladaptive changes of auditory cortex after invasion by ectopic visual inputs. I found that auditory cortex contained auditory, visual, and multisensory neurons after it recovered from neonatal midbrain damage (Mao et al. 2011). The distribution of these different neuronal responses did not show any clustering or segregation. As might be predicted from the fact that auditory neurons and visual neurons were intermingled throughout the entire auditory cortex, I found that residual auditory tuning and tonotopy in the rewired auditory cortex were compromised. Auditory tuning curves were broader and tonotopic maps were disrupted in the experimental animals. Because lateral inhibition is proposed to contribute to refinement of sensory maps and tuning of receptive fields, I tested whether loss of inhibition is responsible for the compromised auditory function in my experimental animals. I found an increase rather than a decrease of inhibition in the rewired auditory cortex, suggesting that broader tuning curves in the experimental animals are not caused by loss of lateral inhibition.

These results suggest that compensatory plasticity can be maladaptive and thus impair the recovery of the original sensory cortical function. The reorganization of brain areas after recovery from brain damage may require stronger inhibition in order to process multiple sensory modalities simultaneously. These findings provide insight into compensatory plasticity after sensory dysfunction and brain damage and new information about the role of inhibition in cross-modal plasticity. This study can guide further research on design of therapeutic strategies to encourage adaptive changes and discourage maladaptive changes after brain damage, sensory/motor dysfunction, and deafferentation.

]]> Yuting Mao Immunoglobulin Gamma Subclasses and Corresponding Fc Receptors in Rhesus Macaques: Genetic Characterization and Engineering of Recombinant Molecules http://digitalarchive.gsu.edu/biology_diss/111 http://digitalarchive.gsu.edu/biology_diss/111 Tue, 17 Apr 2012 09:31:12 PDT Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions.

We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.

]]> Doan C. Nguyen