Human Immunodeficiency Virus (HIV) has become a global pandemic with at least 25 million deaths and no cure. One of the most important targets to inhibit this virus is HIV-1 protease (PR), which is required to cleave the viral proteins needed for maturation of the virus after it invades and replicates in the host cell. There are nine protease inhibitors that are used in AIDS treatment. The virus loses susceptibility to these inhibitors by drug resistance due to mutations. The goal of the project is to examine the highly drug resistant HIV PR P51 in its complex with inhibitors. In this experiment we expressed and purified HIV PR P51 protein. We performed protein crystallization with inhibitors Tipranavir, Amprenavir, Darunavir, and Saquinavir to obtain the structure of the protease and the inhibitors in their complexes. Future analysis of the crystal structures will help with the development of successful therapeutic inhibitors.

There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease. Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types. To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity. This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.

Trichostatin A (TSA) is a histone deacetylase inhibitor with beneficial effects in spinal muscular atrophy mouse models that carry the human SMN2 transgene. Whether TSA specifically targets the upregulation of the SMN2 gene or whether other genes respond to TSA and in turn provide neuroprotection in SMA mice is unclear. We have taken advantage of the Smn2B/- mouse model that does not harbor the human SMN2 transgene, to test the hypothesis that TSA has its beneficial effects through a non-Smn mediated pathway. Daily intraperitoneal injection of TSA from postnatal day 12 to 25 was performed in the Smn2B/- mice and littermate controls. Previous work from our laboratory demonstrated that treatment with TSA increased the median lifespan of Smn2B/- mice from twenty days to eight weeks. As well, there was a significant attenuation of weight loss and improved motor behavior. Pen test and righting reflex both showed significant improvement, and motor neurons in the spinal cord of Smn2B/-mice were protected from degeneration. Both the size and maturity of neuromuscular junctions were significantly improved in TSA treated Smn2B/- mice. Here, we have shown that TSA treatment does not increase the levels of Smn protein in mouse embryonic fibroblasts or myoblasts obtained from the Smn2B/- mice. Further, qPCR analysis revealed no changes in the level of Smn transcripts in the brain or spinal cord of TSA-treated SMA mice. Similarly, western blot analysis revealed no significant increase in Smn protein levels in the brain, spinal cord, hind limb muscle, heart muscle, or the liver of TSA treated Smn2B/- mice. However, TSA has beneficial effects in the muscles of Smn2B/- mice and improves motor behavior and myofiber size. TSA improves muscle development by enhancing the activity of myogenic regulatory factors independent of the Smn gene. The beneficial effect of TSA is therefore likely through an Smn-independent manner. Identification of these protective pathways will be of therapeutic value for the treatment of SMA.

Inhibitor of growth (ING) proteins have been classified as type II tumour suppressor proteins due to their ability to facilitate cellular events such as chromatin remodelling, apoptosis, angiogenesis, DNA replication, DNA repair, cell cycle progression, cell senescence and hormone response regulation. These processes are all associated with combating oncogenesis; conversely, recent evidence suggesting that ING proteins also function as oncogenes in certain cancers has spurred the investigation of ING proteins as potential anticancer targets. In order to better understand the complex role ING proteins play in the cell, the mechanisms that direct ING proteins to the chromatin template require extensive study. This dissertation investigates the role the chromatin environment plays in recruiting ING proteins by characterizing the interaction between ING proteins and chromatin. ING proteins have been shown to interact with the histone H3 lysine 4 trimethylated (H3K4me3) epigenetic mark through binding studies between peptides comprising the ING plant homeodomain (PHD) finger and the H3 N-terminal tail. However, these studies do not take into account the effect of organizing H3 into a nucleosome or the effect of the remaining ING protein structural domains. In order to address these elements, this dissertation describes binding studies between the PHD finger of Yng1 (Yng1PHD) and H3K4me3 in the context of a nucleosome, and between full-length Xenopus laevis ING1 (xING1) and H3K4me3 in the context of a nucleosome. A 6XHis tagged xING1 protein was purified, Yng1PHD was obtained from Dr. Leanne Howe, and an analog of H3K4me3 (H3KC4me3) was installed into recombinant H3 protein and used to reconstitute nucleosomes. Affinity-tag based anti-Yng1PHD and anti-xING1 pull-down assays were then used to display an in vitro H3K4 methylation-dependent interaction between Yng1PHD / xING1 and H3KC4me3 containing nucleosomes. In addition, analytical ultracentrifuge (AUC) analysis of the xING1 protein displayed the presence of 3 species containing sedimentation coefficients consistent with those that would be expected from monomeric, dimeric and tetrameric forms of xING1. Several studies have focused on the interaction between ING proteins and DNA binding proteins such as transcription factors and hormone receptors which recruit ING proteins to specific genes. However, little knowledge is available regarding the role chromatin plays in recruiting ING proteins with the exception of the interaction between the ING PHD fingers and H3K4me3. This dissertation addresses this gap in knowledge by investigating the nature of chromatin bound by the human ING1b (hING1b) protein. For this purpose, HEK293 cells were transfected with a Flag-hING1b construct. Upon fractionation of the HEK293 chromatin, Flag-hING1b was found to localize exclusively to the “Pellet” fraction. ChIP analysis of the HEK293 chromatin showed that Flag-hING1b bound nucleosomes were deprived of H3K9me3, H3K27me3 and H3S10P, contained no enrichment for H3K4me3 and H3K36me3, and were significantly enriched for H2A.Z. Lastly, a hING1b-GFP construct was transiently transfected into SKN-SH human neuroblastoma cells and found to be evenly distributed throughout the nucleus with moderate enrichment on chromatin and within the nucleolus. / Graduate

Biomarker validation remains one of the most important constraints to development of new clinical diagnostic assays. To address this challenge, an immuno-mass spectrometric assay known as SISCAPA has been developed for quantitation of protein biomarkers in human blood. The SISCAPA assay overcomes the sensitivity barrier facing most mass spectrometric approaches by utilizing high affinity antibodies for enrichment of specific surrogate peptide analytes from complex mixtures such as trypsin-digested human plasma. However, several technological barriers remain before the SISCAPA technology gains widespread use for biomarker validation. Improvements are required in areas such as selection of high affinity anti-peptide antibodies, peptide detection sensitivity and increasing sample throughput to allow biomarker validation on large sample sets. The work presented in this dissertation describes the development of new methods for antibody selection and for high-throughput application of SISCAPA technology to biomarker measurement in human plasma. Specifically, two technological developments are described: 1) an assay called MiSCREEN was developed, which allows high-throughput screening of anti-peptide antibodies, enabling selection of high affinity reagents for de novo SISCAPA assays and 2) a liquid chromatography (LC)-free SISCAPA assay was developed that enables quantitation of surrogate peptides using both MALDI-TOF and RapidFire/MS platforms. Taken together, these technological advances provide a meaningful solution to the biomarker validation dilemma and allow a unified system for biomarker qualification, verification, validation and development of clinical assays for diagnosis and monitoring of a variety of diseases. To demonstrate the utility of the unified SISCAPA system for biomarker measurement, an assay was developed for protein C inhibitor (PCI) as a marker for prediction of biochemical recurrence in prostate cancer patients. The PCI-specific analyte was shown to predict biochemical recurrence of prostate cancer after radiation/hormone treatment. Early stage detection of recurrence was achieved, when compared to the ‘gold standard’ marker for prostate cancer, prostate specific antigen (PSA). Two-dimensional gel electrophoresis studies on PCI, revealed unique protein spots in a serum sample from a biochemically recurrent patient. Studying such alterations at the protein level may enable understanding of the molecular mechanisms by which PCI is involved in prostate cancer progression. / Graduate

This research examined the impacts of water quality, phosphate-based corrosion inhibitors and pipe wall exposure on free chlorine (HOCl)/chloramine (NH2Cl) degradation and haloacetic acid (HAA)/N-nitrosodimethylamine (NDMA) formation in simulated distribution system water mains and household plumbing at bench-scale and pilot scale. In bench-scale bottle tests, the reactivity of fresh/pre-corroded pipe materials with HOCl/NH2Cl in decreasing order was: ductile iron, copper, lead. The addition of phosphate-based corrosion inhibitors generally increased HOCl/NH2Cl degradation for fresh iron coupons, but decreased HOCl/NH2Cl decay only for fresh copper coupons. Generally, these corrosion inhibitors did not impact HAA formation. Copper corrosion products, including Cu(II), Cu2O, CuO and Cu2(OH)2CO3, catalyzed HAA and NDMA formation. For HAAs, copper catalysis increased with increasing pH from 6.6 to 8.6 and/or increasing concentrations of these copper corrosion products. Interactions of copper with natural organic matter (NOM), likely by complexation, and the subsequent increase in the reactivity of NOM were proposed to be the primary reason for the increased HAA formation.NDMA formation increased with increasing Cu(II) concentrations, DMA concentrations, alkalinity and hardness but was inhibited by the presence of NOM. The transformation of NH2Cl to dichloramine (NHCl2) and complexation of copper with DMA were proposed to be involved in elevating the formation of NDMA at pH 7.0. Finally, in pilot-scale modified pipe loop tests, copper catalysis of NDMA formation was confirmed, especially under laminar flow conditions, and iron was shown to possibly catalyze NDMA formation under turbulent conditions. Orthophosphate increased the catalytic effects of iron but decreased copper catalysis on NDMA formation by either modifying the properties of the iron-associated suspended particles or reducing the dissolved metal concentrations. Orthophosphate increased chloramine decay when in contact with iron, likely by promoting nitrite formation, but orthophosphate decreased chloramine decay for copper and lead by reducing the availability of metal corrosion products.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Carboxypeptidase N (CPN) is a plasma carboxypeptidase that was discovered in the 1960s as a regulator of inflammation and vascular tone. Through the removal of carboxy-terminal basic residues, CPN alters the activity or binding specificity of inflammatory mediators and vasoactive peptides. CPN shares significant homology with carboxypeptidases known to mediate antifibrinolysis through the removal of basic residues from fibrin clots, which would otherwise stimulate fibrinolysis. Despite the similarity of these enzymes, CPN is generally regarded as lacking a role in fibrinolysis. This thesis demonstrates that CPN is indeed a capable antifibrinolytic enzyme, and that the antifibrinolytic activity of CPN was previously undisclosed due to the presence of a circulating CPN inhibitor, which is likely the free CPN2 subunit. This inhibitor is described for the first time here. Furthermore, potential mechanisms of inhibition and mechanisms of enhancing activity of CPN are proposed based upon the additional structural characterization of CPN presented here.

No description available.