Phleboviruses are emerging zoonotic pathogens which constitute a global threat to human and animal health. The mosquito-borne Rift Valley fever virus (RVFV) is a widespread problem across the African continent and causes regular deadly outbreaks in ruminants. The recently emerged severe fever with thrombocytopenia syndrome virus (SFTSV) is a serious human public health concern in China which has rapidly spread to Japan and Korea with fatality rates as high as 16-30%. Phleboviral cell entry is mediated by two viral glycoproteins: the class II fusion protein Gc and the lesser known Gn. Initial cell attachment is glycan dependent and the penetration into the cell cytoplasm is mediated by the Gc fusion protein which catalyses viral and cell membrane merger. The entry mechanism is not well understood from a structural perspective which limits mechanistic insights. The purpose of this thesis is to further our understanding of the cell entry process by filling in the missing structural information on the phleboviral glycoprotein layer. To this end, an integrated structural approach using cryo-EM and X-ray crystallography was adopted. The crystal structure of the Gn ectodomain is presented which reveals an unprecedented structural relationship with seemingly unrelated viruses. Single-particle cryo-EM and localized reconstruction reveal the glycoprotein layer of the RVFV and a pseudo-atomic model of the RVFV is presented. The assembly shows the shielding of the Gc fusion protein and suggests that the Gn functions as a fusion chaperone. The post-fusion crystal structure of the Gc protein from SFTSV further consolidates a mechanism of membrane fusion by class II fusion proteins. Finally, preliminary data on receptor binding and mechanism of antibody mediated neutralization are presented. The work presented herein provides a novel platform for studying and understanding entry and assembly of phleboviruses as well as the design of novel therapeutics.

Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is one of the most successful pathogens, with much of its virulence predominantly associated with its thick, lipid rich cellular envelope that modulates the host immune response. Tuberculosis, having a long history, has proved to be a challenging disease to eradicate. In mycobacteria, trehalose occurs as a free sugar in the cytoplasm, as well as a constituent of cellwall glycolipids, such as cord-factor, sulfolipid-1 and lipooligosaccharides. M. tuberculosis possesses two pathways for synthesis of trehalose, the OtsA-OtsB pathway and the TreY-TreZ pathway. Multiple biosynthetic pathways underline the critical role that trehalose plays in the bacteria. OtsA-OtsB is the dominant pathway, required for bacterial growth in laboratory culture and for virulence in a mouse model. OtsA, a retaining glucosyltransferase encoded by the otsA (Rv3490) gene, catalyzes the synthesis of trehalose-6-phosphate from ADP-glucose and glucose- 6-phosphate, which is subsequently dephosphorylated by a functional homologue of OtsB (encoded by otsB2/Rv3372 gene) to yield trehalose. Both otsA and otsB2 gene have been shown to be essential for the growth of M. tuberculosis. The present investigation focuses on this pathway, more specifically on OtsA. The recombinant orthologous protein from M. thermoresistibile was structurally and functionally characterised. The crystal structure revealed that there are two domains, with the catalytic site at their interface. The biochemical and structural data indicated that the enzyme had high preference for ADP-glucose as a glucose-donor. The protein activity was also shown to be regulated by feedback inhibition. Furthermore, a structure-guided, fragment-based drug-discovery exercise was carried out against the target, which led to identification of fragment hits that have an inhibitory effect on the activity of the enzyme. Polymethylated polysaccharides (PMPSs) are complex intracellular polysaccharides, which are exclusive to mycobacteria and closely related species. PMPSs include methylglucose lipopolysaccharides (MGLPs) and methylmannose polysaccharides (MMPs). MGLPs, which are composed of acylated glucose and 6-O-methylglucose units, are found in all mycobacterial species, including the slow growing pathogenic M. tuberculosis. On the other hand, MMPs are linear polysaccharides that are not found in M. tuberculosis, but are found in non-pathogenic M. smegmatis and other fast growing mycobacteria. Three gene clusters (Rv1208-Rv1212c, Rv2418c-Rv2419c and Rv3030-Rv3037c), initially thought to coordinate MGLPs biosynthesis, contain several genes that are considered to be essential for M. tuberculosis growth. One of the genes Rv3030, encoding for a SAM-dependent 6-O-methyltransferase, is essential for survival of the bacilli and plays a pivotal role in the biosynthesis of MGLPs. In the present study, an orthologue of Rv3030 from M. smegmatis has been structurally characterized. To overcome a myriad of issues with crystallization, low resolution and lack of reproducibility of the crystals, an alternative approach was taken to determine the structural features of the protein by NMR spectroscopy using 15N, 13C labelled protein. The protein has the β-sheet topology of the classic Rossmann fold, and exists as a monomer in solution. OtsA and Rv3030, essential for survival of M. tuberculosis, represent attractive anti-tuberculosis drug targets, and this study focuses on understanding structural, biophysical and biochemical properties of these targets. This knowledge may then be used to identify lead chemical entities that bind to these proteins and modulate their function, providing new chemical tools that may be of use in designing antimicrobials for the fight against tuberculosis.

Melanoma incidence in the United States has grown continuously at a rate of 1.5% each year for the last decade. Disease detected early can be cured by surgery, but metastatic disease is lethal. Recent discoveries have led to promising-targeted MAPK-pathway inhibitors (MAPKih) and immunotherapies. However, low response rates and acquired drug resistance remain as significant challenges to improved outcomes. The mechanisms that drive resistance to MAPKih are elusive. On the other hand, our data suggests that drug-induced alterations in oxidative metabolism and cellular antioxidant systems (e.g., glutathione; GSH and superoxide dismutase; SOD) in melanoma cells play prominent roles in acquisition of resistance to melanoma drugs. Our studies further indicate a correlative relationship between changes in cellular/mitochondrial reactive oxygen species (superoxide, hydrogen peroxide) levels and adaptation of melanoma cells to MAPK pathway inhibition (MAPKi). Interestingly, our findings suggest that drug-induced alterations in the oxidized and reduced GSH balance facilitate acquisition of drug resistance by activating restorative pathways (e.g., the unfolded protein response; UPR and autophagy). Our data further show that inhibiting GSH synthesis using FDA-approved drug buthionine sulfoximine (BSO) in the presence of MAPKih prevents the acquisition of resistance to MAPKih in vitro. Further, simultaneously attenuating ER-stress responses using sodium 4-phenylbutyrate (PBA) and inhibiting autophagy using hydroxychloroquine (HCQ) in combination with MAPKih prevented adaptation to MAPKi in vitro and significantly improved tumor response and overall survival of mice bearing metastatic melanoma xenograft tumors (BRAFi-resistant and sensitive) in vivo. This thesis research focuses on exploring the role of MAPKi-mediated metabolic reprogramming and changes in the oxidative state in melanoma cells and tumors with the acquisition of resistance to MAPKih in metastatic melanoma.

Degenerative disease of old age such as atherosclerosis and cerebral hemorrhage appear to be related to abnormal lipid metabolism. Many investigators have studied the role of steroid hormones on lipid and cholesterol metabolism in rats and chickens. The results have consistantly indicated that both corticosteroids and sex hormones play an important role in the regulation of lipid and cholesterol metabolism, in vivo. Serum cholesterol values in humans have been decreased by estrogens, while androgen administration tends to increase circulating cholesterol. Estrogens have been shown to decrease circulating cholesterol and prevent cholesterol-induced coronary atherosclerosis in chickens. A study of the relationships that exist between concentrations of serum lipids and the concentrations of urinary estrogens and degradation products of androgens and adrenal steroid hormones in normal human subjects should add to our understanding of lipid metabolism in humans. Observations in rats and chickens limited to meal eating rather than ad libitum feeding, have shown that serum cholesterol was significantly raised in these animals. The rats trained to eat their food in a short period each day also showed markedly increased lipid synthesis. The enhanced lipogenesis resulted in an increase in fat deposition in adipose tissue. These findings have been interpreted to suggest that using frequent small feedings might prove to be beneficial to people who have abnormal lipid metabolism. Steroids of the adrenal cortex contain cortisol, cortisone, and 17-hydroxy-11-deoxycorticosterone as well as their reduced derivatives. These corticosteroids have a feature which is a two-carbon-alpha-keto side chain at carbon 17. Cortisol which is the predominant product of the human adrenal can be detected by the Porter-Silber Method. This test is specific for a small group of closely related compounds referred to as 17, 21-dihydroxy-20-keto steroids and the 17-hydroxy-11-deoxycorticosterone as well as their derivatives. The neutral 17-ketosteroids are the catabolic end products of androgens, male hormones, which are produced by testes and adrenal cortex. The 17-keto steroids are excreted in the urine of both normal men and women. The purpose of the study reported herein was to determine the concentrations of neutral 17-ketosteroids and corticosteroid hormones end products in human urine, as influenced by the number of meals eaten per day. This study was part of a larger problem whose purpose was to determine the relationships that exist between concentrations of serum lipids (cholesterol, total lipids, and lipid phosphorus), and concentrations of estrogens and degradation products of the androgens and adrenal steroid hormones in urine as influenced by dietary factors. The research was based on a group of university students (four men and five women) maintained on self-chosen diets who were eating two meals per day (with breakfast but not lunch) or three meals a day. Urine specimens were collected for hormone estimation and finger-tip blood samples were also given by these subjects for cholesterol determination. The 17-ketosteroids and corticosteroids in urine were analyzed by chemical methods. The Zimmerman reaction, which involved coupling of the reactive 17-keto group of these compounds with m-dinitrobenzine, was employed in this experiment for analytical purposes. The intensity of color was measured in a Beckman DU spectrophotometer. A modification of the Porter-Silber reaction (Eik-Nes, 1961) was applied to determine the end products of adrenal steroid hormones in urine. This reaction is based on a color reaction between steroids and phenylhydrazine in an acidic condition.

17-Allylamino-17-demethoxygeldanamycin (17AAG) is an experimental chemotherapeutic agent, believed to form free radicals in vivo, and cancer cell resistance to 17AAG is believed to be a thiol-dependent process. Inhibitors of thiol-dependent hydroperoxide metabolism [L-buthionine-S,R-sulfoximine (BSO) and auranofin (AUR)] were combined with the inhibitor of glucose metabolism [2-deoxy-D-glucose (2DG)] to determine if 17AAG-mediated cancer cell killing could be enhanced. When 2DG (20 mM, 24 h), BSO (1 mM, 24 h), and auranofin (500 nM, 3 h) were combined with 17AAG, they significantly increased cell killing in three human cancer cell lines (PC-3, SUM159, MDA-MB-231), relative to 17AAG alone. Increases in toxicity seen with this drug combination also correlated with increased glutathione (GSH) and thioredoxin (Trx) oxidation and depletion. Furthermore, treatment with the thiol antioxidant, N-acetyl cysetine (NAC, 15 mM, 24 h), was able to significantly protect from drug-induced toxicity and ameliorate GSH oxidation, Trx oxidation, and Trx depletion. These data strongly support the hypothesis that simultaneous inhibition of GSH and Trx dependent metabolism is necessary to sensitize human cancer cells to 2DG + 17AAG-mediated cancer cell killing by enhancing thiol-dependent oxidative stress and suggest that simultaneous targeting of both GSH and Trx metabolism could represent an effective strategy for chemo-sensitization in human cancer cells.

A number of research studies have shown that aging is a continuous and cumulative process of biological changes which frequently result in cardiovascular diseases in the older age group. Degenerative diseases of old age such as atherosclerosis and coronary artery diseases appear to be related to abnormal lipid metabolism. The findings which indicate serum cholesterol values rise with age are by no means unanimous. The combined evidence in the literature points out that serum cholesterol of newborn infants is low, then rises steadily with age in both sexes from adolescence through middle age with a higher value in males than in females. The serum cholesterol level reaches the maximum in the sixth decade of life and then drops for men while for women it rises. Also, the changes in serum phospholipid and total lipid levels with age were similar to the changes in serum cholesterol levels in both sexes. Several factors such as age, sex, metabolism, heredity, environment, and endocrine functions have been found to alter serum lipid levels. These factors appear to be associated to a complex pattern; the precise proportions of which have not as yet been clearly defined. The environmental factor plays an important role in altering the serum lipid valued and various aspects of this factor can easily be studied and modified by workers. One of the components of the environmental factor, dietary fat has been studied extensively, yet, the exact relation which it has to the development of atherosclerosis has not been settled. The amount and kind of fat and the nature of constituent fatty acids in the diet are considered to be the influential factors directly affecting serum lipid levels. Most of the workers have indicated that the unsaturation of the fatty acids or the ratio between polyunsaturated and saturated fatty acids might best explain the serum cholesterol-regulating activity of the various fats. Many investigators have demonstrated that the substitution of certain vegetable oils for certain animal fats in human diets leads to a decrease of serum cholesterol and phospholipid values. Of the vegetable oils which were studied, corn oil, due to its high content of plant sterols and unsaturated fatty acids, has the most effective hypocholesterolemic activity. However, this is no proof that the lowering of serum cholesterol levels decreases coronary heart diseases, but there is circumstantial evidence that lowering is desirable. Serum lipid is present as many fractions, namely, free cholesterol, cholesterol esters, triglycerides or neutral fat, free fatty acids and phospholipids. More than one-third of the total lipids usually are cholesterol esters, about one-third phopholipids, one-fifth triglycerides, and about one-tenth is free cholesterol. Although the absolute concentration of lipid values varies, the ratio between the free and esterified cholesterol is constant and is approximately 1:3. Considerable variation in the concentration of serum lipids is found among different individuals, but the lipid in serum appears to be maintained within a range which is characteristic for each individual and from which large deviations do not ordinarily occur in the absence of disease or therapeutic treatment. There are relatively few studies of the blood lipid levels of older people. Hence, there is limited information on the kind and amount of different lipids in the serum of normal, senior citizens who are 65 to 90 years of age and less information on studies of the effect of diet on their blood levels. Further work with the older age people is desirable. The objectives of this study were to determine the normal levels of serum cholesterol, total lipids, and phospholipids of older people on the usual diet and to determine the effect of the substitution of corn oil for all other fats used in cooking on these serum lipids. The research was based on a group of forty older, institutionalized subjects, men and women, ranging from 62 to 95 years of age, in the Sunshine Terrace home. Studies were made on serum lipids while the subjects were on the usual house diet and after corn oil was used as the only source of cooking fat in the same kind of diet. Venous blood samples were taken in each period for serum cholesterol, total lipid and phospholipid determinations. Dietary records were also collected for dietary calculation.

A study was made of the urinary excretion of estrogens of healthy university students consuming self-selected diets living under home conditions (4 men and 5 women). Two 31-day test periods were used. During the two test periods, two meal patterns were used; three meals a day or two meals a day with no lunch. Weight was kept constant. Calories, protein, carbohydrate, and fat of their diets were calculated. Urine specimens were collected twice a week, and additional samples were also collected on specified days for female subjects. Urinary estrogens were determined by using Brown's method (1955, 1957) with some modifications. The excretion values of the three individual estrogens, by both men and women showed that 17 B-estradiol was usually present in least amounts and estriol in greatest amounts. The total estrogen value for the women was much higher than that for the men. The mean values were 33.5 and 15.4 meg per day for women and men, respectively. The menstrual cycle did affect the excretion of estrogen which showed higher estrogen values during the middle of the cycle and again between the third and fourth week. However, this increase was less than the first time. There was no definite evidence that the meal frequency had any effect on the excretion of estrogens in human urine. Further work including a greater number of subjects, daily analyses during the menstrual cycle, and increasing the meal frequency is desirable and is recommended to obtain more reliable basic data on young adults maintained in their usual home living conditions.

Structural biology lays the molecular foundation for the modern field of life sciences. In this thesis, X-ray crystallography is the primary resource for atomic detail structural information and is the major technology employed in our research. Three examples show how structural biology addresses the basic processes of life. Firstly, two crystal structures of R21, corresponding to two biological states, reveal a new activation mechanism of SAR-endolysin, which not only complements the previous model, but is also more generally applicable to the endolysin family. The structural information was further corroborated by NMR data in solution. The second example is the crystal structure of mycobacteriophage lysin B, which identified the function of the protein, and tackles the unique problem of how mycobacteriophage circumvent the mycolic acid-rich outer membrane of mycobacterium. The last example is the homology modeling of the Plasmodium ribosomal L4 protein. The action mode for the drug in Plasmodium was proposed based on that, which accounts for the anti-malaria effect of azithromycin.

Metalloproteins represent a large portion of the total proteome. When bound to a protein a metal ion influences both protein stability and function through structural, catalytic or regulatory roles. Discovery of a metal ion cofactor presents new insight into both the structural and functional aspects of a protein and can be essential for the elucidation of the functional and mechanistic details of a protein of interest. The cupin, 2-oxoglutarate/Fe2+-dependent oxygenases (2OG oxygenases) and the di-iron oxygenase families of metalloproteins exemplify the diversity and catalytic potential of a metal ion cofactor, as well as the conservation of 3-dimensional fold and structural features in proteins with similar functions. The structural and biochemical characterization of three novel microbial metalloenzymes are presented; two Escherichia coli hypothetical proteins of previously unknown function, E. coli cupin sugar isomerase (EcSI) and a 2OG oxygenase, YcfD, and the novel microbial carbon-phosphorus (C-P) bond cleavage enzyme, PhnZ, are presented. In each case the identification of a metal ion cofactor and structure determination led to important functional insights. EcSI is encoded by a gene that is highly conserved among pathogenic bacteria. It has been identified as a sugar isomerase with specificity for the rare sugar D-lyxose as well as D-mannose based on structural homology to the cupin phosphoglucose isomerases, suggesting a role for EcSI in metabolism of alternative carbon sources. Structural homology of YcfD, the second metalloenzyme, to the 2OG oxygenase family, particularily human proteins involved in ribosome assembly, combined with evidence that YcfD interacts with the essential ribosomal protein L-16 provides the first evidence of translational regulation by a 2OG oxygenase in E. coli. The third metalloenzyme, PhnZ, was previously identified as an iron dependent oxygenase. Structural characterization revealed that PhnZ possesses a di-iron cofactor and shows significant structural homology to a di-iron oxygenase, providing structural evidence for its novel mechanism of C-P bond cleavage. Combined, these three structures also highlight several features of metal ion-enzyme interaction and regulation mechanisms employed by metalloenzymes as well as the importance of structure in the elucidation of functional and mechanistic characteristics of a protein. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-01-24 21:21:36.195

The adhesion GPCRs are a poorly understood and evolutionarily ancient family of cell surface receptors, several of which have emerging functions in the development of the nervous system. aGPCRs comprise a large extracellular domain, providing binding sites for a variety of ligands, alongside a seven transmembrane domain characteristic of GPCRs. It has been proposed that aGPCRs may function as "context-recognisers", using their large ectodomains to bind different combinations of ligands depending on the molecular make-up of the environment. However there is a lack of direct evidence for this at a molecular level. The ectodomain of one subfamily of aGPCRs, the Latrophilins (Lphns) has been shown to directly interact with several ligands with roles in synaptogenesis and neural guidance. The best-validated of these interactions are those with Fibronectin Leucine-Rich Transmembrane (FLRT) proteins and the Teneurins. In addition, FLRT proteins, also interact with Uncoordinated5 (Unc5) proteins, mediating cell repulsion. Here I reveal that the FLRT-binding site of Lphn is bifunctional, mediating both cell adhesion and repulsion, and that Unc5 is capable of influencing the functional outcome of this interaction. Biophysics and structural studies show that fragments of the Lphn, FLRT and Unc5 ectodomains interact in an unusual and homologue-dependent stoichiometry. Despite the fact that Teneurin interacts with Lphn at a distinct site, Teneurin seems incapable of interacting with the Lphn-FLRT-Unc5 complex, but can form a ternary complex with Lphn and FLRT in the absence of Unc5. Alongside this I present a crystal structure of a large portion of a Teneurin ectodomain, revealing the ancient evolutionary origins of this receptor. Together these data provide strong molecular evidence for a role of Lphns as context-recognisers, by their abilities to bind diverse ligands in distinct combinations and variable stoichiometries.