Genetic variability within both the HIV-1 coreceptor, CCR5, and its ligand, CCL3L, has been shown to contribute towards differences between individuals in their susceptibility to HIV-1 infection and rate of disease progression. In this study we investigated the extent of genetic variation within the CCR5 gene as well as CCL3L, CCL3La and CCL3Lb gene copy number distribution in two healthy HIV-1 uninfected South African populations, South African Africans (SAA) and South African Caucasians (SAC). The impact of variations within these genes on the expression of CCR5 and CCL3 was subsequently assessed. Furthermore, CCR5 genetic variability, CCL3L gene copy number distribution and the expression of CCR5 and CCL3, was assessed in a similar way in a small cohort of HIV-1 infected long term nonprogressors (LTNPs). Genotyping of the CCR5 gene in SAA (n=41) and SAC (n=46) HIV-1 uninfected individuals revealed a high degree of genetic variation between the two population groups, both in terms of single nucleotide polymorphism (SNP) profiles and CCR5 haplotype distribution. Seven complex putative haplotypes spanning the length of the sequenced region were identified with only one of the identified haplotypes, SAA/C-HHC, common to both study populations. The effect of genetic variability on promoter activity of four different CCR5 promoter regions for three CCR5 haplotypes,SAA-HHA, SAA/C-HHC and SAC-HHE, were evaluated. Results showed variability in (i) promoter activity between different promoter regions tested, (ii) results obtained with different cells used for analysis, and (iii) the haplotype being analysed, thereby highlighting that both the cellular environment as well as genetic variability within the promoter region, have the capacity to influence the efficiency of a promoter and consequently CCR5 expression levels. Haplotype-specific promoter analysis demonstrated the SAA-HHA haplotype to have the strongest promoter activity in THP-1 and K562 cells for both P1A (downstream) and P2 (upstream) promoter regions, while in the other cell lines tested (Jurkat and U937), HHA demonstrated intermediate promoter strength.Differences seen between the haplotypes tested in this study and other published studies may be attributable to additional SNPs being tested in the promoter constructs used in this study. The two population groups differed significantly with regards to cell activation levels, as measured by HLA-DR expression, in CD4+ T cell (P=0.002) and CD56+ NK cell subsets (P<0.001). CCR5 expression, determined both as the number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, was found to differ between SAA and SAC individuals across all peripheral blood cell types. SAA individuals had larger proportions of CCR5-expressing natural killer (NK) cell subsets (P<0.01) but lower CCR5 molecules per cell density on CCR5+CD8+ T cell and CCR5+ NK cell subsets (CD56+, CD16+CD56+ and CD56dim) (all P<0.05) compared to SAC individuals. These differences were maintained even after CCR3D32 heterozygous SAC individuals were included in the analyses. Furthermore, the previously described haplotypes, HHA and HHC, associated with differences in CCR5 expression on different cell subsets between individuals within the same population group. SAA individuals with the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells compared to SAA individuals that lacked the haplotype (P=0.001). SAC individuals with the HHC haplotype had significantly higher density on NK (CD56+) and CD16+CD56+ NK cell subsets (P=0.030 and P=0.024, respectively) compared to SAC individuals without this haplotype. The latter observation suggests that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals, thus highlighting the potential role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression. Despite significant differences in CCL3La (CCL3L chemokine coding) and CCL3Lb (nonchemokine coding) copy number between SAA and SAC populations, no difference in CCL3 production by peripheral blood mononuclear cells (PBMCs) was noted between the two study populations. Assuming equal contribution of CCL3 and each copy of CCL3La to CCL3 production,we found that SAC individuals produced higher levels of CCL3 per functional copy of CCL3La compared to SAA individuals (P<0.001). Although, when SAA and SAC individuals with comparable CCL3La and CCL3Lb gene copy numbers were compared, there was no difference in production per functional copy between the two groups (P=0.974). We also determined CCL3La and CCL3Lb gene copy number for a previously established cohort of HIV-1 intrapartum-infected (IP) and exposed uninfected (EU) infants and found that differences previously seen in cord blood mononuclear cell (CBMC) CCL3 production between IP and EU infants with comparable CCL3L copy numbers could not be attributed to differences in CCL3Lb copy number. The potential role of differences in CCR5 genotype, CCR5 expression, CCL3 genotypes and CCL3 production levels in the control of HIV-1 infection was then examined by comparing a small group (10 SAA and 4 SAC) of LTNPs to the respective background population. No polymorphisms in the CCR5 open reading frame were detected in these LTNP individuals. However, the HHA haplotype frequency was significantly higher in SAC LTNP individuals compared to SAC control individuals (P=0.010). Interestingly, CCR5 density on CD4+ T cells and monocytes was significantly lower in SAA LTNP individuals (P=0.025 and P=0.022, respectively) with a trend towards a similar relationship in CD8+ T cells (P=0.058), while the proportions of CCR5-expressing CD8+ T cells were elevated compared to SAA controls (P=0.043). This latter finding reflects the increased immune activation in these individuals compared to uninfected individuals, as evidenced by increased proportions of HLA-DR-expressing T cells (CD8+ and CD4+, P<0.0001). In addition,PHA-induced CCL3 production by PBMCs was significantly lower in LTNP (SAA and SAC combined) compared to control individuals (P=0.004). SAA LTNP individuals had higher proportions of CD8+ T cells (P<0.0001) and lower proportions of natural killer cells (CD56+,P=0.002) compared to control SAA individuals. Thus, CCL3 production differences may be partially explained by differences in the distribution of immune cell subsets between the two study groups.Furthermore, PBMCs of LTNP individuals with low viral loads (<400 copies/ml) produced CCL3 at lower levels than those from individuals with higher viral loads, irrespective of whether or not the cells were stimulated (P=0.005 and P=0.035, respectively). In summary, this study demonstrates that: (i) two ethnically divergent populations show marked differences in CCR5 genetic variability, cell activation and CCR5 expression which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression, (ii) both CCR5 genotypic differences and differences in baseline cellular activation levels appear to be contributing towards the observed differences in CCR5 protein expression, and (iii) the two study populations do not differ with respect to CCL3 production by PBMC cultures which suggests that either the two copy per diploid genome gene, CCL3, may play a significant role in CCL3 production and/or that as yet undefined mechanisms regulate production of CCL3 from variable CCL3L copy number. In addition, a pilot study conducted in a small group of LTNP individuals demonstrates that two major determinants of HIV-1 disease progression, CCR5 and CCL3, are both expressed at lower levels in LTNPs individuals compared to healthy uninfected controls and has identified CCR5 haplotypes which are potentially associated with disease progression.

Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of recurrent and metastatic (R/M) head and neck squamous cell carcinoma (HNSCC), only a small subset of patients responds to cetuximab, despite EGFR overexpression in virtually all of their tumors. At this time, there is a lack of validated predictive biomarkers to predict which patients will respond to cetuximab. Our previous work suggests that cetuximab activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the implications of activating this pathway are unclear. The IL-1 pathway plays a central role in immune response and displays both pro-tumor and anti-tumor activities. IL-1 may promote tumor growth by upregulating the secretion of pro-inflammatory mediators involved in angiogenesis and metastasis. On the other hand, IL-1 signaling may promote antitumor immunity via enhancement of natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell activity, which are important mechanisms of action of cetuximab. The goal of this work is to determine how modulation of the IL-1 pathway affects HNSCC tumor response to cetuximab and if IL-1 may serve as a predictive biomarker for patient response to cetuximab. Blockade of IL-1 signaling did not enhance the anti-tumor efficacy of cetuximab, while IL-1α overexpression and treatment with recombinant IL-1α and IL-1α nanoparticles increased HNSCC tumor response to cetuximab in immunodeficient and immunocompetent HNSCC mouse models. Mechanistically, these results appear to be due to activation of an anti-tumor NK and T cell-mediated immune response. Additionally, we found that both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and inducible nitric oxide synthase (iNOS) activity may be involved in the efficacy of IL-1-induced ADCC against cetuximab-coated HNSCC cells. Altogether, these results suggest that IL-1 signaling is necessary for HNSCC tumor response to cetuximab. Furthermore, we have shown that pre-treatment serum and tumor IL-1 ligands can predict progression-free survival of HNSCC patients treated with standard-of-care cetuximab and chemotherapy, cetuximab combined with other targeted therapies, and cetuximab monotherapy. Overall, we propose that IL-1α warrants further study as a novel therapeutic to enhance response to cetuximab and as a predictive biomarker for HNSCC response to cetuximab.

Long Interspersed Element 1(LINE1 or L1), along with the parasitic Short Interspersed Element (SINE), are the only two currently active retrotransposons in the human genome. These genetic elements are capable of causing DNA damage through their mobilization and the enzymatic activities of the L1-encoded Open Reading Frame 2 (ORF2) protein (ORF2p). The L1 ORF2p contains four annotated domains important to retrotransposition. These include the endonuclease (EN) and reverse transcriptase (RT) domains, as well as the Z domain and the cysteine rich domain (Cys). While much is known about the enzymatic activities of the EN and RT domains, and individual amino acids important to retrotransposition have been identified in the Z and Cys domains, more than 50% of the 150kDa ORF2p amino acid sequence serves no known function. I hypothesized that the unannotated areas of the ORF2p, specifically the sequence C-terminal to the EN domain and N-terminal to the Z domain as well as the sequence C-terminal to the Cys domain, contained amino acids important to the retrotransposition process. Specifically, I hypothesized that they contained amino acids involved in the activity of the EN domain, RT domain, or interaction with the L1 ORF1p. To test this hypothesis, I developed a technique termed Bipartile Alu Retrotransposition (BAR) that utilized EN and RT-containing ORF2 fragments combined with the Alu retrotransposition reporter construct. This system allowed me to define a new ORF2p region, which I termed Cryptic. This region contains an essential WD pair important for cDNA syntheses by the ORF2p. This WD pair is also involved in the regulation of the EN domain activity. I also discovered a putative PCNA binding domain that is essential for retrotransposition. Additionally, I identified the region in Cryptic that is involved in the previously reported differences in subcellular localization and cytotoxic potential of EN-containing ORF2p fragments. Using truncated ORF2p fragments generated for use in BAR, I also made several discoveries concerning the extreme C-terminus of the ORF2p. Notably, I discovered that the extreme C-terminal end of the ORF2p is dispensable for retrotransposition. I also identified a human-specific Y residue that is important for Alu retrotransposition driven by the ORF2p. / 1 / Claiborne Magnant Christian

No description available.

The Human Immunodeficiency Virus -1 (HIV-1) has been the source of substantial human misery since it was first discovered in the early 1980s. Despite remarkable progress that has been made towards understanding HIV- 1, there is no cure, no vaccine and life-prolonging therapies are beyond the reach of millions in need. Our research sought to both gain insight into potential therapeutic targets as well as the preclinical testing of drug candidates. We demonstrate that a RhoA derived peptide is an effective inhibitor of HIV-1 entry. Although this peptide inhibits HIV-1 due to its poly-anionic nature, it nevertheless demonstrates that endogenous host proteins may be repurposed for novel therapeutic uses. We also characterized the mechanism and effectiveness of Basant, a polyherbal topical microbicide candidate for the prevention of HIV-1 transmission. Our data demonstrate that it inhibits the entry of both CCR5 and CXCR4 tropic HIV-1 at non-toxic concentrations. Finally, data is presented on the characterization of a novel HIV-1 protein expressed from an alternative reading frame of the HIV-1 polymerase gene. We demonstrate that this protein is localized to the nucleolus and is likely expressed from a novel HIV-1 transcript. This work lays the foundation for further studies to target this protein for drug development. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from May 9, 2011 - May 9, 2012

This thesis examines the interconnection between the in vitro and in vivo phosphorylation of rabbit muscle phosphofructokinase. The first goal of the project was to show whether a novel site of rabbit muscle phosphofructokinase that is subject to in vitro phosphorylation, serine 376, may also become phosphorylated in vivo. Evidence obtained through iron chelate chromatography, amino acid analysis, gas phase sequencing, ammonium sulfate reversed phase high pressure liquid chromatography (HPLC), and matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) spectroscopy of cyanogen bromide digests of the enzyme purified from epinephrine-stimulated rabbit hearts demonstrate the in vivo phosphorylation of serine 376. Parallel experiments with phosphofructokinase isolated from unstimulated rabbit hearts show no detectable phosphorylation of serine 376. The second part of the thesis examines the effects of alterations in experimental conditions on the in vitro phosphorylation of rabbit muscle phosphofructokinase that is catalyzed by the cAMP-dependent protein kinase (cAMP-dPK). Significant phosphorylation of serine 376 takes place in the presence of specific proteins, calmodulin-calcium and troponin C-calcium, that are known to stabilize the catalytically inactive phosphofructokinase dimer. Conditions that stabilize the catalytic activity of phosphofructokinase generally inhibit the in vitro phosphorylation reaction. / Graduation date: 1999

Zeolites are crystalline, microporous aluminosilicates that have been extensively used in heterogeneous catalysis, separations, and ion-exchange operations. It has long been understood that particle size and morphology play a central role in the successful application of zeolites. This dissertation reports on controlling the morphology of all-silica zeolite, silicalite-1, made in nonionic/ionic microemulsions under conventional synthesis conditions. Silicalite-1 materials formed in microemulsion-mediated syntheses possess different morphological properties as compared to samples grown using the same synthesis mixture in the absence of the microemulsion. The work presented here is a systematic study showing how parameters such as synthesis temperature, microemulsion composition, silica precursor, alkali content, presence of salt, and the surfactant identity impact the material properties, most notably crystal morphology. In the nonionic microemulsion mediated synthesis, the work demonstrates the possibility of using microemulsions to manipulate the shape and size of silicalite-1 materials, growing both spheres and high-aspect ratio platelets. In both cases these large particles are robust aggregates of small submicron particles. Based on the results presented, a mechanism is proposed illustrating the role of both the confined space presented by the microemulsion as well as the importance of the surfactant-silicate interactions leading to the formation of the large aggregates. In the cationic microemulsion mediated synthesis, it is concluded that the surfactant??silicate interactions are primarily responsible for the modulation of crystal morphology observed. The results indicate that surfactant adsorption on the growing crystal surface, not the confined space afforded by the microemulsion, is essential. The results suggest that this may be a versatile and useful approach to controlling zeolite crystal morphology and growth of crystals obtained from conventional high-silica zeolite synthesis procedures.

The serotonin-1A (5-HT1A) receptor plays an important role in the regulation of the serotonin (5-HT) system as an autoreceptor on 5-HT neurons. The transcription factor CC2D1A/Freud-1 is a potent repressor of the 5-HT1A promoter in neuronal but not in non-neuronal cells. The clinical relevance of Freud-1 is evident in a naturally occurring mutation, resulting in a truncated form of Freud-1 lacking its C-terminal half, that is associated with non syndromic mental retardation in humans. Thus, it is of interest to clarify the structure and function of Freud-1. As Freud-1 was shown to interact with the transcriptional regulator Brg1 at the 5-HT1A promoter, identification of the structural domains mediating the Brg1/Freud-1 interaction is required to assess the role of Brg1 in Freud-1 repression. In this study, I used pull-down assays with recombinant proteins, co-immunoprecipitation studies and immunofluorescent staining with confocal microscopy to show that Freud-1 interacts directly with the C-terminus of Brg1 and that the C-terminal domain of Freud-1 is required for this interaction.

The incretin hormone glucagon-like peptide-1 enhances glucose-dependent insulin secretion and is therefore a most attractive therapeutic approach for the treatment of Type 2 Diabetes Mellitus. The anti-diabetic drug, metformin, has previously been shown to increase circulating levels of GLP-1, although its mechanism of action is currently unknown. Neither metformin nor AICAR (activators of AMPK) directly stimulated GLP-1 secretion from the L-cell in vitro. However, oral treatment of rats with metformin enhanced plasma levels of active and total GLP-1, independent of GLP-1 degradation. Furthermore, pre-treatment with the general muscarinic antagonist, atropine, or the M3 antagonist, 4-DAMP, decreased metformin–induced GLP-1 secretion, while M1 and M2 antagonists did not. Chronic bilateral subdiaphragmatic vagotomy had no effect, while the GRP antagonist, RC-3095, reduced metformin-induced GLP-1secretion. Therefore, I conclude that metformin-induced GLP-1 secretion occurs in part through the parasympathetic nervous system, the M3 and GRP receptors, but is independent of the vagus nerve.

The incretin hormone glucagon-like peptide-1 enhances glucose-dependent insulin secretion and is therefore a most attractive therapeutic approach for the treatment of Type 2 Diabetes Mellitus. The anti-diabetic drug, metformin, has previously been shown to increase circulating levels of GLP-1, although its mechanism of action is currently unknown. Neither metformin nor AICAR (activators of AMPK) directly stimulated GLP-1 secretion from the L-cell in vitro. However, oral treatment of rats with metformin enhanced plasma levels of active and total GLP-1, independent of GLP-1 degradation. Furthermore, pre-treatment with the general muscarinic antagonist, atropine, or the M3 antagonist, 4-DAMP, decreased metformin–induced GLP-1 secretion, while M1 and M2 antagonists did not. Chronic bilateral subdiaphragmatic vagotomy had no effect, while the GRP antagonist, RC-3095, reduced metformin-induced GLP-1secretion. Therefore, I conclude that metformin-induced GLP-1 secretion occurs in part through the parasympathetic nervous system, the M3 and GRP receptors, but is independent of the vagus nerve.