Neisseria gonorrhoeae requires iron for survival and establishment of infection in the human host. Pathogenic Neisseriae have evolved a repertoire of high-affinity iron acquisition systems to facilitate iron uptake in the human host. This requires specific outer membrane receptors and energy-harnessing cytoplasmic membrane proteins. The transferrin receptor proteins of Neisseria gonorrhoeae are necessary for iron uptake from transferrin in the host. The iron uptake system consists of two transferrin binding proteins, (Tbp) A and B. TbpA is an integral outer membrane, TonB-dependent transporter that forms the pore for iron internalization. TbpB is a surface exposed lipoprotein that makes the iron internalization process more efficient. TbpA is proposed to consist of two distinct domains: a β barrel and an N-terminal plug domain. Previous studies have shown that the EIEYE sequence in the TbpA plug domain plays an important role in iron internalization. We undertook a collaborative project to test the hypothesis that the conserved EIEYE sequence in the wild-type TbpA plug binds Fe3+ during the outer membrane iron transport process. CD spectra analysis and fluorescence emission titration spectra of purified recombinant wildtype and mutated plug proteins revealed that Fe3+ is sequestered by the wildtype TbpA plug protein, unlike the mutated plug protein. Modeling data with the wild-type plug predicts the EIEYE sequence is part of a flexible loop structure and acts as an Fe3+ binding site. Characterization of the Tbps constituting the gonococcal receptor is important to understanding how the gonococcus survives within its host. TbpA and TbpB act together to acquire iron from human transferrin. We hypothesize that the presence of TbpA impacts the exposure or conformation of TbpB. In this study, we have utilized photoactivable cross-linkers to assess the effect of TbpA on TbpB in live gonococcal cells and studied it in presence of ligand and TonB derived energy. We employed insertion mutants, in which TbpA and TbpB contained the hemagglutinin (HA) epitope tag, to probe for impact of TbpA on TbpB. Our results demonstrate that photo-cross-linking altered TbpB size and migration and was dependent on the presence of TbpA. HA epitope insertion mutants in surface exposed loops of TbpA and TbpB did not impact the mobility of cross-linked TbpB. Addition of human transferrin to the de-energized mutant caused a change in TbpB migration after cross-linking. This result indicates that when ligand is bound tightly and irreversibly to de-energized TbpA, the surface accessibility and perhaps conformation of TbpB is altered and TbpA does not interact with TbpB. Our findings were confirmed with recent structural studies of TbpA-TbpB-ligand triple complex, which illustrate that Tbps bind ligand through unique, non-overlapping binding sites such that TbpA and TbpB do not interact. TbpB is not an essential member of transferrin-iron acquisition pathway. It is surface exposed and tethered to the outer membrane by a lipid moiety. The role of TbpB has not been clearly outlined in the transferrin iron acquisition system. The last objective of this study was to look at the significance of two specific conserved regions of TbpB and its importance in transferrin iron utilization and TbpA-TbpB interaction. Using site-directed mutagenesis we created two mutants, in the first mutant the conserved lipobox of TbpB was replaced with a signal I peptidase cleavage site, and the second mutant contained a deletion of the conserved poly glycine residue stretch, immediately downstream of the lipobox. Our results indicate that lipobox is required for lipidation of TbpB, both the mutants were impaired for transferrin-iron utilization, and neither of the mutations altered TbpA-TbpB interaction. Overall, these studies help elucidate the functional importance of the specific regions in TbpA and TbpB in Neisseria gonorrhoeae, thereby adding to our understanding of the process of iron acquisition through the transferrin binding proteins.

The genetic disease Hutchinson-Gilford Progeria Syndrome (HGPS) arises from a de novo single nucleotide mutation (1824CàT) in the LMNA gene. As a result, the mutated lamin A protein (progerin) remains farnesylated and permanently attached to the nuclear membrane. Progerin accumulates and deforms the nuclear membrane leading to an array of cellular abnormalities driving the cells to enter a state of permanent cell-cycle arrest early on in replicative age i.e. premature cellular senescence. Cellular senescence has been extensively studied as one of the contributing factors to aging in HGPS patients and other age-related diseases. There has also been evidence to show that aging is accompanied by epigenetic changes and that epigenetic manipulation can incite progeroid syndromes in mice. It has been found in this study that HGPS fibroblasts express distinctly lower levels of SIRT6, a member of the sirtuin family of NAD-dependent protein deacetylases/ADP-ribosyltransferases, than normal fibroblasts. Findings from this study demonstrate that overexpression of SIRT6 prevents a decrease in replicative capacity and the onset of premature senescence in HGPS fibroblasts. Thus, SIRT6 may have promising therapeutic implications for improving HGPS age-related pathologies.

Purpose: The purpose of this study was to test whether children who have undergone cancer chemotherapy have a higher prevalence of enamel abnormalities of the second mandibular premolars than age matched controls. Methods: This study was a case-control design where the case group involved 26 subjects that had received chemotherapy treatment between the ages of 2 to 6 and at the time of the study were between the ages of 9 to 18. The control group consisted of 26 subjects matched for age and sex to the case group that had not received chemotherapy. The second mandibular premolars were assessed based on the types of defects, their number and location according to a modified DDE index. A secondary examiner, blinded to the results of the primary, analyzed photographs taken at examination and provided their own assessment in order to calibrate results. Result: Nominally there were more normal surfaces in the case group than in the control group (81% vs 70%) and fewer hypoplasias in the case group (5% vs 13%). There was no statistical difference between the buccal and occlusal surfaces. For the buccal surfaces, the cases were nominally lower but not statistically significant (P=0.0680) and there is no evidence for a case-control difference on the lingual surfaces (P>0.9). Conclusions: In this study developmental defects of the enamel organ were not observed to be statistically different between the case and control groups, although previous studies have shown otherwise.

The multiplicity of glucocorticoid-responsive hepatic cytochromes P-450 in the rat was investigated by developing a series of monoclonal antibody probes directed against purified steroid-inducible isozymes from the rat (P-450p) and man (HLp). Two antibodies, termed 1G8 and 13-7-10, showed unique specificity for purified cytochromes P-450 upon immunoblot analyses. These antibodies were further characterized in terms of their reactivity toward proteins in liver microsomes from untreated and xenobiotic-treated rats. Three P-450p-related proteins were identified using 1G8 and 13-7-10 to monitor the expression of P-450p family subtypes. In untreated rats, members of this family exhibited sex-specific expression. Treatment with various chemicals resulted in differential induction of the 1G8- and 13-7-10-reactive proteins, demonstrating distinct regulatory features of these immunochemically-related proteins. While searching for novel inducers of the P-450p family, the imidazole antifungal drug clot rimazole was found to significantly increase total cytochrome P-450. Detailed studies were undertaken to investigate the mechanism by which clotrimazole and other antifungal drugs induce hepatic cytochrome P-450. The effect of treatment of rats with the antifungal drugs clotrimazole, miconazole, or ketoconazole on the expression of three distinct P-450 gene families (glucocorticoid-responsive P-450p, phenobarbital-responsive P-450b/e , and ethanol-responsive P-450j) was determinded by measuring: 1) microsomal enzymatic activities with marker substrates, 2) the microsomal content of immunoreactive cytochromes P-450 with specific antibodies, and 3) the amounts of liver RNA hybridizing to cloned P-450 cDNAs. These studies establish that P-450p is the predominant P-450 isozyme induced by clotrimazole, miconazole, and ketoconazole. Two of these drugs, clotrimazole and miconazole also induced P-450b/e , whereas ketoconazole induced P-450j. Differential increases in specific P-450 mRNAs and proteins resulted from treatment of rats with these imidazol e antifungal drugs, suggesting that multiple cellular events are involved t heir mechanism of P-450 induction. In conclusion, expression of the P-450p family of cytochromes P-450 in rat liver is under complex regulatory control and is subject to modulation by clinically useful drugs.

Radiotherapy is the clinical application of the ionizing radiation to treat cancer. Ionizing radiation causes multiple modes of damage to the DNA damage such as SSBs, DSBs and modified bases such as thymine glycol. These lesions can exist as clusters in one or two helical turns of DNA. DNA double-strand breaks (DSBs) are extremely toxic to cells because they can lead to genomic rearrangements and even cell death. If base lesions accompany these DSBs, there will be a substantial hindrance for repair. NHEJ is the primary DSB repair pathway in mammalian cells. HRR repairs single strand breaks (SSBs) or Double strand breaks (DSBs), during late S phase and G2 phase of the cell cycle, by using an undamaged copy of the DNA sequence, and is therefore largely error-free. The NHEJ pathway repairs DSBs without the requirement for sequence homology and can be error-free or error-prone, and is most active during G1 phase. Thymine glycol (Tg) is the most common oxidation product of thymine. It is produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as ionizing radiation (IR); it is one of the predominant types of base modifications produced by ionizing radiation. Due to clustering of radiation-induced damages, many DSBs are accompanied by damaged bases such as Tg at or near the DSB ends that may interfere with subsequent gap filling and ligation. The base excision repair pathway plays a major role in removal of thymine glycol from the damaged DNA strand. During NHEJ, after synapsis by Ku and DNAPKcs and processing of the DNA ends, XRCC4/Ligase IV complex ligates the DNA. This ligase activity is promoted by the interaction of XLF/XRCC4 filament with Ligase IV. Linearized plasmids with Tg at the 5th -Tg5 positions from the broken end were subjected to a repair assay using XRCC4-like factor (XLF)-deficient cell extracts, with or without the addition of XLF and Endonuclease III and/or ddTTP and Klenow fragments. End joining of Tg5 was compared to plasmid with Tg at third position-Tg3 in extracts. In addition, the ability of purified NHEJ proteins Ku, DNAPKcs and XRCC4/Ligase IV, to repair the Tg1 (Tg at the end), Tg2 (Tg at the second position), Tg3 and Tg5 in the presence and absence of XLF was assessed. The data indicated that the cell extract could ligate the Tg5 plasmids only in the presence of XLF. End joining of the Tg5 was less in comparison to Tg3 with base excision repair being more active in Tg5 competing with the joining. Plasmids with Tg were treated with Endonuclease III and ddTTP to test whether the end joining occurred before or after Tg removal. Endonuclease III and ddTTP treatment showed reduced intensity of the joined fragment suggesting that end joining occurred without removal of Tg in some cases. The extracts were not able to fill in the processed end by the BER though it has a 3′-OH which was filled in by the treatment with Klenow enzymes derived from E.coli DNA Polymerase I, following removal of extract proteins by proteolysis. When purified proteins were used to treat the plasmids, it is observed that there was increased efficiency of repair with increased distance of the Tg from the end may be due to less distortion of the ends as Tg is away from the end. While Tg1 and Tg2 required XLF presence for the repair, Tg3 and Tg5 could show a small amount of repair in its absence. XLF enhanced the repair of Tg3 and Tg5. While the repair by extract showed no repair in the same substrates without XLF, there was repair by purified proteins without XLF, suggesting there is some competition with the XRCC4/Ligase for the DSB by other proteins and XLF is required to overcome this. In conclusion, cell extract was able to ligate the plasmid with Tg located at fifth position from the DSB but with lower efficiency compared to Tg3 plasmids. The base excision repair pathway is more functional if the modified base is far from the DSB. End joining by the purified proteins was proportional to the thymine glycol position from the DSB end. XLF was mandatory for repair of Tg1 and Tg2 by the purified proteins, and not for Tg3 and Tg5.

Staphylococcus aureus is one of the leading causes of nosocomial and community acquired infections. Many of the virulence factors are encoded on staphylococcal mobile genetic elements. Members of the SaPI family of S. aureus mobile elements encode superantigens and are mobilized at high frequency by specific helper bacteriophages. One remarkable feature of helper phage exploitation by SaPIs is remodeling of the normal T=7 bacteriophage capsid to produce smaller T=4 phage-like particles. These particles, composed entirely of helper phage proteins, can accommodate the smaller SaPI genome while excluding that of a complete helper phage. This study was designed to understand the mechanism of capsid size redirection and high frequency mobilization of SaPIs. A multipronged approach employing cryo-EM analysis, protein profile comparison and genetic analysis was used to study the capsid size redirection. Two proteins encoded by the prototype element SaPI1, gp6 and gp7, have been identified in SaPI1 procapsids but not in mature SaPI1 particles. These proteins are sufficient and required to direct small capsid formation, which involves alteration of an internal scaffold. While many phages use internal scaffolding proteins, the involvement of an internal scaffold in capsid size redirection is novel.

Despite the widespread use of Highly Active Anti-retroviral Therapy (HAART) to combat Human Immunodeficiency Virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), HIV-Associated Neurocognitive Disorders (HANDs) still remain a dire issue. HIV can enter the brain through infected monocytes and infect microglia, the resident macrophages within that compartment. Due to its pro-inflammatory properties, HIV regulatory protein Tat (Transactivating protein) may play a role in the progression of neurocognitive disorders. The aim of this project was to determine whether the select cannabinoids THC and CP55,940 could ablate the inflammatory response on BV-2 mouse microglia cells caused by Tat. Within the constraints of the experiment, no major effects of cannabinoid treatment were observed at the level of the proteome as tested by two-dimensional gel electrophoresis . In contrast, these cannabinoids ablated the inflammatory response caused by the HIV protein Tat at the level of the secretome and at the level of gene expression. These collective observations suggest that select cannabinoids have the potential to down-regulate the elicitation of proinflammatory gene products that are engendered by the HIV protein Tat. Furthermore, the results suggest a potential for cannabinoid agonists at cannabinoid receptors to serve as adjunct ablative agents in the treatment of HIV-associated neuropathological processes.

Borrelia burgdorferi and Borrelia hermsii cause Lyme disease and relapsing fever, respectively. These spirochetes are maintained in an enzootic cycle, involving tick vectors and mammalian hosts. Differential gene expression is central in their survival in various environmental conditions. C-di-GMP has been demonstrated to be important in bacterial adaptation. Borrelia deletion mutant phenotypes have shown that c-di-GMP regulates motility, infectivity, and enzootic cycle progression. As the only known receptors encoded by Borrelia, PlzA and PlzC characterization is necessary in delineating c-di-GMP roles within the cell. In this study, biochemical, biophysical, and FRET methods demonstrated that these proteins exhibit a structural rearrangement when binding c-di-GMP likely significant to downstream activities. Substitution of a highly conserved residue within PlzA altered the structure and charge of the PilZ domain, leading to abolished binding. PlzA and PlzC functionality studies are vital to discover mechanisms of c-di-GMP-mediated regulation of motility and host invasion by the Borrelia.

Opioids are excellent pain relievers. A major side-effect of chronic opioid treatment is constipation whereas withdrawal following chronic exposure leads to diarrhea and increased gastrointestinal motility. These effects of chronic opioids are mediated by μ-opioid receptors expressed on enteric neurons. Previous studies have shown that chronic opioids enhance sensitivity to nicotine in the gastrointestinal tract. This suggested that prokinetic effects of nicotine mediated through the activation of nicotinic acetylcholine receptors (nAChRs) may be useful in reversing opioid-induced constipation. The goal of this dissertation was to investigate the nAChR subtype expressed on enteric neurons and their role in reversing opioid-induced constipation. The effect of nicotine on small intestinal transit and fecal pellet output were determined in-vivo in morphine-pelleted mice xiii (75 mg for 4 days). Nicotine-induced currents were measured by whole-cell voltage clamp in isolated adult mouse myenteric neurons treated with morphine over short term (10 mins) and long term (16-20 hrs). Following long term morphine exposure in-vivo (morphine pellet – 4 days), nicotine increased fecal pellet output, and enhanced small intestinal transit. The prokinetic effect of nicotine was not seen in placebo pelleted mice or after acute morphine (10 mg/kg, 30 min). Peak-amplitude of nicotine-induced inward currents in isolated neurons was also enhanced after long-term but not short term exposure to morphine. Nicotine-induced currents were inhibited by mecamylamine (10 μM) and α-conotoxin AUIB (3 μM), suggesting the expression of α3β4 subtype of nAChRs on enteric neurons. Conversely, NS3861, a partial agonist at α3β4 nAChR enhanced fecal pellet expulsion in a dose-dependent manner in chronic but not acute morphine treated mice. Overall, our findings suggest that the efficacy of nAChR agonists on enteric neurons is enhanced after chronic morphine exposure and activation of α3β4 subtype of nAChR reverses chronic but not acute morphine induced constipation. In conclusion, development of peripherally selective α3β4 partial agonists may be of therapeutic benefit in treatment of chronic opioid-induced constipation.

Risperidone is an ‘atypical’ antipsychotic and is approved by the USFDA mainly for the treatment of schizophrenia and symptoms of bipolar disorder. Risperidone (an SDA or serotonin-dopamine antagonist) has ~20-fold higher affinity at 5-HT2A receptors over dopamine D2 receptors, which makes it more efficacious against the negative symptoms of schizophrenia and less liable to causing extrapyramidal side effects than ‘typical’ antipsychotics. The major goal of the current investigation was to study the structure of risperidone and to identify the minimum structural features required for 5-HT2A receptor affinity that retain antagonist action. The structure of risperidone was systematically deconstructed, and functional activity studies using calcium imaging in HEK293 cells and a two-electrode voltage clamp (TEVC) assay in a Xenopus laevis heterologous system were coupled with radioligand binding affinity studies to achieve this goal. The biological studies showed that the entire structure of risperidone was not required for activity or affinity at the receptor, as 6-fluoro-[3-(1-methylpiperidin-4-yl)]benz[d]isoxazole was comparable to risperidone in both affinity and activity. Next, the structure of risperidone was elaborated to determine the importance of its left and right “halves” in its actions. The left and the right halves of risperidone were substituted with those of another antagonist, ketanserin, to give structural hybrids. Biological studies suggested that the right half of risperidone [i.e., the 6-fluoro-(3-piperidin-4-yl)benz[d]isoxazole moiety] might be important for affinity. In order to assess how the biologically-active compounds interact at the receptor, homology models of the human 5-HT2A receptor were developed, and docking and Hydropathic INTeraction studies were conducted. Risperidone seemed to form a bifurcated hydrogen bond with S159 (TM3), which ketanserin was unable to form. This interaction might account for high binding affinity at the receptor as it is common to both, risperidone and 3-[2-(4-(6-fluorobenz[d]isoxazol-3-yl)piperidin-1-yl)ethyl]-2,4-(1H,3H)quinazolinedione. With the data currently in hand, we can conclude that the entire structure of risperidone is not required for activity or affinity, and that the right “half” (i.e. the benzisoxazolyl portion) of risperidone might be influencing activity and affinity at 5-HT2A receptors.