Streptococcus pneumoniae can be found in the normal flora of the throat and upper respiratory tract of humans. However, it can commonly become pathogenic causing diseases such as pneumonia and meningitis. S. pneumoniae is unique in that a large percentage of its genome encodes for proteins involved in carbohydrate metabolism. A number of these pathways are essential for full virulence of the bacterium, including a putative fucose utilization pathway. There are two strain-dependent varieties of fucose operons in S. pneumoniae. The type 1 operon consists of a putative extracellular galactosidase (Sp4GH98), intra-cellular fucosidase (GH95A), PTS relay system (EIIA, EIIB, EIIC, EIID), fucose mutarotase (FcsU), fucose isomerase (FcsI), fuculose kinase (FcsK), and fuculose 1-phosphate aldolase (FcsA). Alternatively, the type 2 operon consists of a putative extracellular galactosidase (Sp3GH98), intra-cellular fucosidase (GH29), two intra-cellular galactosidases (GH36A and B), ABC transporter system, and fucose processing enzymes (FcsI, FcsK, and FcsA). The objective of this research is to characterize individual components from both fucose operons ultimately to generate both pneumococcal fucose utilization pathways. Specific focus on the extracellular GH98 enzymes provided evidence that these fucose pathways are initiated by the depolymerization of specific histo-blood group antigens presented on host cells. It is then proposed that the products liberated from the complex carbohydrate degradation are transported into the bacterium for further cleavage by intracellular GH enzymes releasing fucose for processing. This process is critical for S. pneumoniae virulence and may be involved in bacterial internalization by host cells suggesting a novel role for this pathway in pneumococcal pathogenesis. / Graduate

The formation of amyloid fibrils is a key characteristic of many neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. Similarly prion diseases, those associated with the prion protein, are neurodegenerative disorders with characteristic protein aggregates accumulating in the brain of affected individuals. While fibrillar deposits of these disorders have long been associated with end-stage disease pathology, it is currently hypothesized that protein oligomers are the cytotoxic structural form of these systems. Residues 106-126 of the human prion protein have been found to form both amyloid fibrils, as well as toxic amyloid oligomers and thus provide a suitable model system. This thesis aims to describe the structures of the amyloid fibrils and oligomers formed by PrP(106-126), how they are interrelated as well as interaction with model membranes and cytotoxicity. Amyloid fibrils of PrP(106-126) contain long, unbranched filaments that contain β-sheet secondary structure and bind the amyloid-indicating dye, thioflavin-T. These fibrils are comprised of parallel β-sheets, stacked in an antiparallel fashion. The non-fibrillar amyloid oligomers are large, spherical structures that contain β-sheets but do not bind thioflavin-T. It was determined that these oligomers contain parallel β-sheets as well as the same intersheet packing as fibrils of PrP(106-126). Finally, the interaction of PrP(106-126) with lipid bilayers and cells was examined. Oligomers of PrP(106-126) were shown to affect model membranes; with anionic lipids losing integrity and cholesterol-containing lipid mixtures losing domain structure upon peptide addition. Additionally, amyloid oligomers of PrP(106-126) cause cell death across a number of cell lines as well as rat cerebellar slices. Overall, these results indicate that the conversion of oligomers to fibrils may be facilitated due to structural similarities between the two. Additionally, the toxicity of PrP(106-126) oligomers may be attributed to a loss of cholesterol domain structure causing subsequent cell death.

The formation of amyloid fibrils is a key characteristic of many neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. Similarly prion diseases, those associated with the prion protein, are neurodegenerative disorders with characteristic protein aggregates accumulating in the brain of affected individuals. While fibrillar deposits of these disorders have long been associated with end-stage disease pathology, it is currently hypothesized that protein oligomers are the cytotoxic structural form of these systems. Residues 106-126 of the human prion protein have been found to form both amyloid fibrils, as well as toxic amyloid oligomers and thus provide a suitable model system. This thesis aims to describe the structures of the amyloid fibrils and oligomers formed by PrP(106-126), how they are interrelated as well as interaction with model membranes and cytotoxicity. Amyloid fibrils of PrP(106-126) contain long, unbranched filaments that contain β-sheet secondary structure and bind the amyloid-indicating dye, thioflavin-T. These fibrils are comprised of parallel β-sheets, stacked in an antiparallel fashion. The non-fibrillar amyloid oligomers are large, spherical structures that contain β-sheets but do not bind thioflavin-T. It was determined that these oligomers contain parallel β-sheets as well as the same intersheet packing as fibrils of PrP(106-126). Finally, the interaction of PrP(106-126) with lipid bilayers and cells was examined. Oligomers of PrP(106-126) were shown to affect model membranes; with anionic lipids losing integrity and cholesterol-containing lipid mixtures losing domain structure upon peptide addition. Additionally, amyloid oligomers of PrP(106-126) cause cell death across a number of cell lines as well as rat cerebellar slices. Overall, these results indicate that the conversion of oligomers to fibrils may be facilitated due to structural similarities between the two. Additionally, the toxicity of PrP(106-126) oligomers may be attributed to a loss of cholesterol domain structure causing subsequent cell death.

Two research questions are investigated here: the first, major, section addresses the problem of uneven distributions of dose (absorbed energy per unit mass) in crystals used for macromolecular crystallography (MX), and the second presents the develop- ment of a high-throughput metalloprotein characterisation technique, HT microPIXE. In MX, the advent of X-ray microbeam data collection has led to uneven distributions of dose within the crystal volume becoming increasingly common. In these cases, the rotation method creates a highly damaged central region of crystal that stays within the beam throughout exposure, and less damaged outer regions, which are introduced during rotation. This thesis presents a new software program, raddose-3d, which performs a full 3D simulation of the profile of absorbed energy (the dose state) within a crystal during X-ray exposure. In order to utilise this time resolved, 3D picture of the dose state of the crystal, a new metric – Diffraction Weighted Dose – is proposed. This metric is then experimentally validated, and is found to summarise the dose state into a single dose value, which reflects the damage state of the crystal. Simulations are performed using raddose-3d and Diffraction Weighted Dose to compare possible dose spreading strategies, and generalised recommendations for MX experimentalists are offered. Uniquely identifying the species and stoichiometry of bound metals in protein sam- ples is a significant challenge for biophysical characterisation. Low throughput mi- crobeam Proton Induced X-ray Emission (microPIXE) provides an unambiguous anal- ysis of these properties, but has a limited throughput of ∼10 samples per day. As a consequence, its applicability has been restricted to niche cases. This thesis presents significant progress, including proof of principle experiments, on developing sample preparation methods, data acquisition systems, and data analysis protocols to increase this throughput by an order of magnitude, opening up major new applications for the technique.

Marek's Disease Virus (MDV) induces a wide range of neurological syndromes in susceptible hosts; however, the mechanisms behind the MDV-induced neuropathology are still poorly understood. The immediate-early 14kDa phosphoprotein, pp14, is associated with the neurovirulence phenotype of the virus. Yeast-two-hybrid screening identified the ER-bound transcription regulator, human CREB3 (cAMP Response Element-Binding protein), as an interacting partner of pp14, and fluorescence colocalisation between pp14 and chicken CREB3 (chCREB3) in MDV infected cells suggested an interaction between these proteins. The primary focus of this DPhil project was to further investigate this putative interaction using in vitro studies, with a view to determining if the interaction is linked to the neurovirulence of MDV. This investigation, which employed a combination of biochemical, cellular, and functional assays, found no conclusive evidence in support of the predicted interaction. In addition, this project aimed to gain structural and functional insights into the MDV neurovirulence factor pp14 and the host transcription factor, chCREB3. Biophysical characterisation of recombinant pp14B identifies pp14 as a molten globule. The results reveal the protein, while possessing substantial secondary structure, is largely disordered lacking a stable tertiary structure. Multiple lines of evidence from this study also indicate pp14 is a putative zinc-binding protein. Moreover, phosphorylation analysis of recombinant pp14B, extracted from DF1 cells, by mass spectrometry provides conclusive evidence for the presence of two phosphorylation sites in the shared C-terminal region of pp14 - serines 72 and 76 of pp14B. Structural flexibility, through a lack of a definite ordered tertiary structure, and functional features that can induce structural modifications indicate pp14 might interact with a number of binding partners and therefore could play multiple roles during MDV infection - a strong possibility due to the expression of the protein in all the different stages of virus infection. Furthermore, this thesis presents the crystal structure of the homodimeric chCREB3 bZIP. The chCREB3 bZIP possesses a structured DNA binding region even in the absence of DNA, a feature that could potentially enhance both the DNA-binding specificity and affinity of chCREB3. Significantly, chCREB3 has a covalent intermolecular disulphide bond in the hydrophobic core of the bZIP, which may play a role in promoting stability. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily possess this cysteine residue, indicating it could act as a redoxsensor. These results indicate members of the CREB3 subfamily, by possessing a putative redox-sensitive cysteine with the capacity to form an intermolecular disulphide bond, may be activated in response to oxidative stress.

The dysregulation of numerous genes has been associated with the development and progression of cancer, many of which are being investigated as potential therapeutic targets. Previous work in our laboratory and others reported the elevated expression of the nuclear import protein Karyopherin Beta 1 (Kpnβ1) in various cancers. The inhibition of Kpnβ1 by siRNA silencing inhibited the proliferation of cancer cells and induced cell death via apoptosis while having little effect on non-cancer cells. These findings suggested that Kpnβ1 has potential as an anti-cancer therapeutic target. Using an in silico screening approach to identify small molecule inhibitors of Kpnβ1 with anti-cancer activity, a number of compounds were selected for further investigation in our laboratory. The aim of this study was to investigate a novel small molecule, Compound 60 (9-[(1-methyl-3-piperidinyl)methoxy]-4-[(6-methyl-2-pyridinyl)methyl]-7-(5-methyl-2-thienyl)-2,3,4,5-tetrahydro-1,4-benzoxazepine); by monitoring (i) its effect on cancer cell biology using cervical and oesophageal cancer cell lines, (ii) its effect on nuclear import activities associated with Kpnβ1, (iii) its in vitro ADME pharmacokinetics and in vivo anti-cancer properties and (iv) performing biophysical analysis of Kpnβ1:C60 interactions. Cervical and oesophageal cancer cells were found to be more sensitive to C60 treatment compared to non-cancer epithelial cell. C60 treatment resulted in the inhibition of cancer cell proliferation, colony formation, migration and invasion. G1/S cell cycle arrest and a reduced expression of cell cycle regulatory proteins such as Cyclins D1, B1 and A as well as CDK4 was observed on treatment with C60. C60 induced cell death via apoptosis as observed PARP cleavage. These results suggest that C60 has an inhibitory effect on cancer cell biology. Immunofluorescent analysis and nucleo-cytoplasmic western blot analysis showed that C60 treatment resulted in the cytoplasmic retention of Kpnβ1. Immunofluorescent analysis and luciferase reporter assays showed that C60 inhibited the nuclear entry and transcriptional activity of a Kpnβ1 cargo, NFκB. Similarly, the transcriptional activity of cargo proteins NFAT and AP-1 were also inhibited. This suggests that C60 inhibits the nuclear entry of Kpnβ1 and thus its function as a nuclear importer of cargo proteins. In vitro ADME pharmacokinetics analysis found C60 to have high solubility, permeability and plasma protein binding properties and a short half-life. These findings suggest that C60 may have good oral absorption but rapid clearance in living systems. In vivo toxicology studies showed that C60 is tolerable, allowing for its testing in a xenograft nude mouse model. Intraperitoneal injection of C60 selectively inhibited the growth of oesophageal tumour cells with a significant effect observed on KYSE 30 oesophageal tumours. To investigate Kpnβ1:C60 binding interactions, Kpnβ1 was purified using the GST-tagged purification technique. Purified Kpnβ1:C60 interactions were monitored using the Bio-layer interferometry technique. Our preliminary data suggest an interaction between C60 and Kpnβ1 with Kd values in the micromolar range. We obtained varying Kd values, hence further optimisation is required to arrive at a conclusive Kd value. In conclusion, this study is a first to show that Compound 60 (9-[(1-methyl-3-piperidinyl)methoxy]-4-[(6-methyl-2-pyridinyl)methyl]-7-(5-methyl-2-thienyl)-2,3,4,5-tetrahydro-1,4-benzoxazepine), a small molecule identified to bind Kpnβ1 in an in silico screening approach interferes with the subcellular localisation of Kpnβ1, inhibits the localisation and transcriptional activity of Kpnβ1 cargo proteins and inhibits cancer cell biology in vitro and in vivo. In vitro ADME pharmacokinetics shows that C60 has tolerable drug properties. Biophysical analysis shows that C60 appears to bind Kpnβ1 in the micromolar range. Together, these results provide evidence for C60 as a compound that warrants further investigation as an anti-cancer agent.

8-hydroxyguanine (8-oxoG) is one of the most important products resulting from the oxidation of guanine in DNA. 8-oxoG is known as a biomarker of oxidative stress such as lung cancer. 2,5-diamino-4H-imidazol-4-one (Iz) and its hydrolysis product 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)-amino]-2,5-dihydrooxazol-5-one (Oz) are also important products of oxidation of guanine in DNA. Recently, novel types of dimer intermediates related to 8-oxoG and Iz have been identified in our research lab. Using high performance liquid chromatography (HPLC), LC-MS, and NMR coupled with educated hypotheses, it is possible to identify the products, X1 and X2, resulting from the oxidation of guanine and decipher the mechanisms leading to the products. Gaining a better understanding of these mechanisms could potentially lead to more effective medical treatment of conditions resulting from oxidative stress. X1 and X2 were isolated in large amounts from deoxyguanosine (dGuo) via photochemical oxidation. Subsequent testing included a comparison of the effects of different pH values as well as determination of the lifetimes for the X2 product during hydrolysis and reactions with amines. It was shown from the results that X1 undergoes further oxidation to produce X2.

Endophilin B1 (EnB1) is a BAR protein located in the cytosol that controls membrane dynamics of different organelles such as the mitochondria and the Golgi, as well as autophagosomes. It has been suggested that this protein coordinates membrane remodeling events during essential cell death processes. For instance, previous studies show that knockdown of EnB1 leads to dysregulation of mitochondrial dynamics and inhibition of apoptosis. This protein could thereby have a critical tumor suppressor role in the cell. Despite the important role of EnB1 in many intracellular signaling processes, some parts of its underlying mechanisms of function are still unknown, more specifically, what drives the protein to bind to the membrane and what the protein structure looks like when bound.  Since EnB1 plays an important role in many intracellular trafficking events, it is of interest to obtain more information about this protein, both about its structure and membrane binding interactions. New information on this subject will contribute to a better understanding of how EnB1 modulates intracellular membranes to control several critical trafficking processes that contribute to neuron degradation and carcinogenesis.  This specific project aims at designing membrane templates that support EnB1 membrane- binding and bending for evaluation of binding capacity and for structural characterization by cryo-EM, and other associated methods.  To study the binding interactions of EnB1, different protein constructs were first expressed and purified. Membrane templates (liposomes and nanodiscs) were then created to enable structural characterization as well as studying the binding capacity of EnB1 to different lipids. A lipid binding assay with multiple variants of liposomes were created to study the biding capacity of EnB1, and negative stain transmission electron microscopy, as well as cryo-electron microscopy was used for visualization of the templates. By analyzing the data from the lipid binding assay, it can be concluded that both lipid composition and membrane curvature affects EnB1 membrane binding. The cryo-EM visualization also confirm that EnB1 is involved in membrane remodeling.

The purpose of this dissertation is to characterize the transcriptome of Borrelia (Borreliella) burgdorferi to discover novel transcripts, important for pathogenesis. As a spirochete and the etiological agent of Lyme disease, the foremost vector-borne bacterial infection in the world, B. burgdorferi fulfills a distinctive niche among bacterial pathogens. Persisting in the disparate environments of a tick vector and mammalian reservoirs, it is absolutely dependent on its hosts for transmission and nutrient acquisition. B. burgdorferi harbors a complex fragmented genome which is largely linear, unlike that of most prokaryotes, lacks an array of classically described metabolic genes, and contains an unusually large percentage of unique genomic sequences specific to Borrelia (Borreliella) species. To date, few regulatory mechanisms have been identified which contribute to the ability of the spirochete to sense and respond to its environment. Efforts to use global transcript analysis to elucidate the molecular mechanisms of B. burgdorferi host adaptation have proven challenging due to the low numbers of the pathogen present during infection. Previously, our laboratory successfully developed an in vivo expression technology based approach for B. burgdorferi (BbIVET) to identify spirochete promoter sequences that are active during a murine infection. This screen identified 233 unique putative promoters which mapped to locations across the entire genome. These putative infection-active B. burgdorferi promoters were not only located at the 5' end of annotated open reading frames (ORFs), but also mapped to unannotated locations antisense, intergenic, and intragenic to ORFs. Given the limited characterization of the B. burgdorferi transcriptome, this dissertation applies an RNA sequencing approach (5'RNA-seq) to globally annotate the transcriptional start sites (TSSs) and 5' processed ends of the spirochete's RNA during in vitro cultivation. This resulted in the discovery of numerous novel internal, intergenic, and antisense transcripts. Synergistic analysis combining Northern blotting techniques, alignments of these transcripts to BbIVET proposed promoters, and interrogation of promoter activity via in vivo live imaging of mice, confirmed the expression of a variety of RNAs during laboratory culture and mammalian infection. Further, as a means to improve quantitation of the expression of these transcripts, a new methodology was developed and applied to measure B. burgdorferi promoter activity during tick-pathogen interactions, in a strand specific manner. Finally, because the Lyme disease spirochete harbors many unclassified and unique genomic sequences, the mammalian infection-expressed gene bb0562, identified through BbIVET and 5'RNA-seq, was selected for targeted deletion and evaluation throughout B. burgdorferi's infectious cycle. This demonstrated that gene bb0562 encodes a membrane associated protein, whose presence is critical for establishing murine infection through the bite of an infected tick. In sum, this work contributes significant insight into the transcriptome of B. burgdorferi, provides an innovative approach for the analysis of RNA transcripts at the tick-pathogen interface, and identifies a novel gene critical for Lyme disease pathogenesis.

Malaria kills over 500,000 people each year and over a third of the global population is at risk of infection. Though the human race has been fighting the malaria war for over 4,000 years and we have made great strides in eliminating malaria from many countries, we are treading on the edge of what could be another malaria epidemic primarily due to widespread drug resistance. There are documented cases of resistance for every known antimalarial in use today, including Artemisinins. It is critical that we open a new window of discovery in development of next generation antimalarials that circumvent current resistance paradigms. These compounds must attack new targets, have different speeds of action, and ideally possess powerful transmission blocking potential if they are to be successful antimalarial candidates. Screening endeavors historically focused on either synthetic or natural product libraries. Recent efforts have focused on combining privilege elements of natural products into synthetically tractable compounds to create hybrid libraries. To discover novel antimalarial pharmacophores, we have screened natural products derived from marine biodiversity as well as natural product-inspired synthetic libraries. Our phenotypic screening of 3,164 marine natural products from the Harbor Branch Oceanographic Institute, 56 high density combinatorial natural product based libraries from the Torrey Pines Institute for Molecular Studies, alkaloid, terpene, and macrocyclic libraries from Memorial Sloan Kettering Cancer Center, and 594 natural product inspired compounds from Asinex have identified several new selective antiplasmodial hit chemotypes. In this study, we have focused on compounds that exhibit cellular actions differing from current antimalarials. Two of the scaffolds, UCF 201 and 501, a spirocyclic chromane and a nitroquinoline, respectively, act early in the development cycle and block invasion. The alkaloid derived compound M03 blocks egress. UCF 501 cures malaria in the rodent model and significantly inhibits stage V gametocytogenesis. Given that discovery of transmission blocking agents are a priority in the malaria elimination strategies, this result is significant. This work is of high impact as it addresses a critical need in the field- next generation antimalarial scaffolds for malaria therapy and elimination campaign.