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Dementia causes an increasing social and economic burden worldwide, demanding action regarding its diagnosis, treatment and everyday management. Recent years have seen many advances in neurodegeneration research, but the search for new truly disease modifying therapies for Alzheimer's disease (AD) and frontotemporal dementia (FTD) has so far not been successful. This is mainly due to a lack of understanding of the precise intracellular events that lead up to neuronal dysfunction in early and in late stages of the disease. This thesis describes the approaches taken to extend the current knowledge about the intracellular effects of neuronal amyloid-beta and the signalling pathways causing neuronal death or disturbed synaptic function in dementia. Endophilin-1(Ep-1), amyloid-binding alcohol dehydrogenase (ABAD), peroxiredoxin-2 (Prx-2) and the EF-hand domain family, member D2 (EFHD2) have been found to be elevated in the human brain with dementia and in mouse models for frontotemporal lobar degeneration (FTLD) or AD. The expression of these proteins as well as the expression of c-Jun N-terminal kinase (JNK), c-Jun and APP were analysed by western blotting and real-time PCR in human brains affected by AD or FTLD as well as in mouse models for AD. This provided a new insight into the regulation of these proteins in relation to each other in the ageing brain and uncovered a new potential link between elevated levels of EFHD2, Prx-2 and APP in FTLD. By studying the effects of the overexpression of Ep-1 in neurons, this research has led to a better understanding of its role in JNK-activation. It furthermore verified a protective role for Prx-2 against neurotoxicity and pointed towards a new function for Prx-2 in the regulation of JNK-signalling. The analysis of the effect of increased levels of EFHD2 uncovered for the first time its involvement in the PI3K-signalling cascade in neuronal cells. The current work has therefore contributed to the knowledge about the cellular processes that are affected by Ep-1, Prx-2 and EFHD2 in different types of dementia and will greatly benefit future research into their actions in the neuronal network.

Currently the mammalian endoplasmic reticulum (ER) is known to contain at least 20 different protein disulphide isomerase (PDI) family members. The oxidoreductases in the PDI family are thought to catalyse the formation and rearrangement of disulphide bonds in newly synthesised proteins. The focus of this work was to characterise two of the PDI family members: PDI and ERp46. In vitro translation reactions of major histocompatibility complex (MHC), β1-integrin (β1-I), haemagglutinin (HA), procollagen α1(III) and preprolaction (pPL) were carried out in untreated or PDI-depleted cells. The depletion of PDI decreased the rate of folding of MHC and β1-I and also prevented the oligomerisation of HA, suggesting a role for PDI in folding these putative substrates. However, when PDI was depleted neither the folding of pPL or HA was affected, implying that they may not be substrates for PDI. To determine the role of ERp46 in the cell, a substrate-trapping approach was used. Here substrates interacting with ERp46 were trapped as mixed disulphides isolated by immunoprecipitation, separated by 2D SDS-PAGE and identified by mass spectrometry. It was demonstrated that ERp46 forms mixed disulphides with at least 23 proteins, including heavily secreted proteins such as laminins, integrins and collagens. In particular, interactions with Ero1, Prx IV, EDEM3 and ERAP2 were found and confirmed by immunoprecipitation of radiolabelled in vitro translated protein. Notably nine of these clients of ERp46 have previously been identified as substrates of ERp57 (Jessop, Watkins et al. 2009). This would support the hypothesis that several different oxidoreductases, working in concert, are required to fold certain substrate proteins. Also, it was confirmed that Prx IV and Ero1 each form a mixed disulphide with PDI. These results highlight the importance of PDI family members in recruiting co-factors to substrates. Additionally, the over-expression of ERp46 led to increased cell survival following DTT treatment, yet after depletion of ERp46, cells were less able to grow, perhaps suggesting a role for ERp46 in maintaining ER redox homeostasis and cell survival. This suggestion was supported by the finding that ERp46 is able to catalyse the reduction of Prx IV in the presence of glutathione. These results suggest that Prx IV provides a novel mechanism for the transfer of disulphide bonds to nascent proteins in the ER via PDI family members such as ERp46 and PDI.

Part I of this thesis describes the development of bioactive small molecules of relevance to the study of the apicomlexan parasite Toxoplasma gondii into useful chemical tools. The research includes the target identification and validation studies, using both chemical and biological methods. Chapter 1 provides an overview of chemical genetics with a particular emphasis on methods for the identification of the protein targets of bioactive small molecules. The concept of biochemical protein target identification techniques was introduced with a detailed discussion of interesting applications from the literature. Chapter 2 focuses on the development of a tetrahydro-β-carboline based lead molecule into a chemical tool through target identification studies. The structure activity relationship (SAR) data associated with this core structure, the design of a chemical inducer of dimerisation (CID) and the synthesis of this CID are discussed in detail. Chapter 3 described work done to identify the potential protein target(s) of Conoidin A. Experiments to assess whether Conoidin A can inhibit a proposed target in vitro are also included. Further optimisation of this structural class to develop more potent inhibitors is discussed in the second part of this chapter. Part II of this thesis describes the development of methods for the synthesis of medium-sized ring containing compounds using oxidative fragmentation and rearrangement strategies. Chapter 5 provides an overview of the existing oxidative fragmentation methodology, with an emphasis on the use of oxidative fragmentation reactions for the synthesis of medium-sized ring systems (8-11 ring atoms). Chapter 6 focuses on using the established oxidative fragmentation method in the oxizino carbazolone system to investigate the diasteroselectivity of this reaction. Possible mechanisms for this transformation are investigated and discussed using both chemical and computational methods. An interesting rearrangement reaction has also been observed during this study. Chapter 7 focuses on developing an asymmetric oxidative fragmentation method, for use in the diazabenz[e]aceathrylenes system. Asymmetric oxidative fragmentation reactions using [Ru(pybox)(pydic)] catalysts are discussed. Attempts to optimise the enantiomeric excesses of the reaction by varying reaction conditions and substituents in the substrate are also included.

Peroxiredoxins are a family of multifunctional enzymes that are able to protect the cell against oxidative stress. Peroxiredoxins form part of a recently discovered peroxide scavenging system along with thioredoxin, thioredoxin reductase and sulfiredoxin. This study describes the purification of a recombinant human peroxiredoxin II from human erythrocytes. The original recombinant clone contained a point mutation at the fourth residue from glycine to valine and a number of problems were encountered with aggregation during purification. Reverting back to the original amino acid sequence allowed the protein to be purified and concentrated without aggregation, as well as leading to over-expression in the same oligomeric state as the native sample from blood. This study also describes the over-expression and purification of the human peroxiredoxin II protein in the intermolecular disulfide form as well as the subsequent crystallisation and X-ray diffraction studies. The crystal structure for this form of the protein was obtained to 3.3 Å resolution revealing the peroxiredoxin to be in the decameric form. In addition conformational changes in the protein that are necessary for formation of the intermolecular disulfide between the peroxidatic (Cys52) and resolving cysteine (Cys172) have been observed. The structure also revealed that these movements did not interfere with the dimer:dimer interface as had been previously suggested. This then allows the disulfide to be seen within the decameric form of peroxiredoxin. The production of covalent complexes formed between peroxiredoxin and sulfiredoxin, and peroxiredoxin and thioredoxin was also investigated. Complexes were stabilised by using DTNB to form a covalent bond between specific cysteine residues. The complex binding results from size exclusion chromatography showed that decameric peroxiredoxin bound to sulfiredoxin in a 1:5 ratio and decameric peroxiredoxin bound to thioredoxin in a 1:10 ratio. Cloning, over-expression and purification of the selenocysteine containing enzyme thioredoxin reductase was achieved. A minimal selenocysteine insertion sequence was added to the 3’ end of the DNA sequence to drive selenocysteine insertion in place of the typical stop UGA codon. The activity of this protein was found to be low but was greatly increased when co-expressed with a plasmid containing the selA, selB and selC genes. Although the activity of this co-expressed thioredoxin reductase was ~20% of the native enzyme activity, it was comparable to the activity of other recombinant forms of the enzyme. These studies report the purification of all of the proteins necessary to reform the peroxiredoxin system and allow the production of a working assay for peroxiredoxin activity. Together with the first report for a structure of a decameric disulfide form of human peroxiredoxin II a greater insight into the peroxiredoxin system has been obtained.

Prostate cancer is the most common and second leading cause of cancer death in men. Prevailing theories state that inflammation may drive prostate carcinogenesis via oxidative stress and the generation of reactive oxygen species (ROS) that induce somatic mutagenesis. Since high proliferating cells or cancer cells exhibit aberrant metabolism to satisfy the needs of energy for rapid cell proliferation, they also generate high levels of ROS which stimulates the expression of cellular antioxidants to overcome the dysregulation of redox homeostasis through the regulation of redox-sensitive transcription factors. Among different antioxidants, peroxiredoxin (Prx) family proteins are the highly conserved and mostly abundant family of peroxidases that scavenge hydrogen peroxide as well as mediate redox signaling under both physiological and pathological conditions. Peroxiredoxin IV (Prx4) is one of Prx family members that mainly localizes in the endoplasmic reticulum (ER), but can also be found extracellular space. But, the absence of mechanistic input and experimental evidence compromise the understanding of role and significance of Prx4 in the pathogenesis of human diseases, especially in prostate cancer. In this study, our goal is to evaluate the expression of Prx4 in human prostate cancer, to study whether and how Prx4 contributes to prostate cancer malignancy, as well as to understand its secretion mechanism. Prx4 is highly expressed in patient specimens of prostate cancer as well as established prostate cancer cell lines, and its levels can be further stimulated through the activation of androgen receptor (AR) signaling. To study the mechanism of Prx4 in regulating prostate cancer malignancy, we used two different strategies to deplete the endogenous Prx4, including short hairpin RNA and CRISPR/Cas9 techniques. Compared with control cells, Prx4-depleted cells show delayed cell cycle progression, and reduced rate of cell proliferation. We found that loss of Prx4 results in insufficient phosphorylation of both Akt and its downstream kinase GSK3α/β via Proteome profiler phospho-kinase arrays. Moreover, Prx4-depleted cells are more sensitive to ionizing radiation with the observation of compromised ability to scavenge ROS and increased accumulation of DNA damage. We also found loss of Prx4 shows reduced the population of migrating cells and stem cells, ability of migration and invasion. By performing RNA sequencing and Gene Set Enrichment Analysis, we found depletion of Prx4 reduces the expression of NF-κB2, and inhibits Epithelial-Mesenchymal transition (EMT). All the conclusions were further confirmed using xenograft mouse model. Because of its important role in prostate cancer growth and metastasis, we further investigate the secretion mechanism of Prx4. We first confirmed the secretion capacity of Prx4 via western blot analysis of human/mouse plasma and cell-cultured medium. By manipulating the N-terminal hydrophobic region, we further confirmed that Prx4 has its signal peptide which drives its secretion. Through the use of inhibitors, downregulation of key regulatory genes, and fluorescence imaging, we targeted classical and unconventional protein secretion pathways and investigated the colocalization of Prx4 with different subcellular organelles. Our results revealed that Prx4 secretion depends on the Endo-lysosomal mediated Type III protein secretion pathway, with Prx4 being present in exomeres or distinct nanoparticles (DNPs) rather than EVs. The secreted Prx4 retained its antioxidant function with reduced cysteine residues. Moreover, we observed upregulated Prx4 expression in all three types of human cancers, with varying secretion levels among different cell lines. We also observed the accumulation of Prx4 in mouse plasma in both PTEN/Pb-Cre transgenic prostate cancer mouse model as well as AOM/DSS induced colorectal cancer mouse model. In conclusion, our results show the importance and function of Prx4 in contributing to prostate cancer growth, metastasis, and stemness via different signaling pathways. Our study indicates that targeting Prx4 may be utilized as a potential strategy to control tumor growth and metastasis in prostate cancer patients. The absence of a dedicated inhibitor holds its utilization to improve current therapies. Therefore, strategies targeting Prx4 are urgent to develop and use to improve current clinical therapeutic treatments in prostate cancer. Aside as a therapeutic target, Prx4 holds its potential as a biomarker whether alone or combined with PSA to help early diagnosis and monitor prostate cancer relapse.

Reactive oxygen species (ROS) are oxygen-containing free radicals and/or molecules that are more reactive than O2. ROS such as hydroxyl radical (•OH) and hydrogen peroxide (H2O2) are produced naturally in the body as a result of reactions such as aerobic respiration and oxidative protein folding. ROS undergo reduction-oxidation (redox) reactions and mediate cell signaling. Accumulation of excessive ROS can damage DNA, RNA, protein and lipids. Antioxidants are enzymes and small molecules that react with ROS to modulate redox signaling and to prevent and repair oxidative damage. Examples of antioxidants include glutathione, thioredoxin, superoxide dismutase, catalase, vitamin C and peroxiredoxin (Prx). In mammals, there are six Prxs that reduce hydrogen peroxide, alkyl hydroperoxide and peroxynitrite to maintain redox balance and protect against oxidative stress. Different Prxs have similar functions but different subcellular distribution. Peroxiredoxin IV (Prx4) scavenges ROS and participates in oxidative protein folding in the endoplasmic reticulum. We hypothesized that Prx4 plays an oncogenic role in colorectal cancer. To understand the significance of the Prx4 in colorectal cancer formation, wildtype and Prx4-/- mice of FVB/N background were subjected to a standard protocol of colorectal carcinogenesis induced by sequential exposure to Azoxymethane and Dextran sulfate sodium (AOM/DSS). Compared with wildtype littermates, Prx4-/- mice had significantly fewer and smaller tumors. Histopathological analysis revealed that loss of Prx4 leads to increased cell death through lipid peroxidation and lower infiltration of inflammatory cells in the knockout tumors compared to wildtype. We also examined the role of Prx4 in the progression of colorectal cancer. Loss of Prx4 reduced migration and invasion of colon cancer cell lines in vitro. Additionally, orthotopic implantation of HCT116 cells into immunodeficient NSG mice resulted in significantly lower metastasis after Prx4 knockdown. Mechanistic studies showed the involvement of tumor suppressor DKK1 in reducing migration and invasion in Prx4 knockdown cells. Thus, Prx4 plays a pro-tumorigenic role in colorectal cancer initiation and progression. This dissertation identifies Prx4 as a promising therapeutic target for prevention and treatment of colorectal cancer and highlights the need for further research to bridge the gap to clinical application.

Lung cancer is a leading cause of cancer-related mortality irrespective of gender. The Sulfiredoxin (Srx) and Peroxiredoxin (Prx) are a group of thiol-based antioxidant proteins that plays an essential role in non-small cell lung cancer. Understanding the molecular characteristics of the Srx-Prx interaction may help design the strategies for future development of therapeutic tools. Based on existing literature and preliminary data from our lab, we hypothesized that the Srx plays a critical role in lung carcinogenesis and targeting the Srx-Prx axis or Srx alone may facilitate future development of targeted therapeutics for prevention and treatment of lung cancer. First, we demonstrated the oncogenic role of Srx in urethane-induced lung carcinogenesis in genetically modified FVB mice. The Srx-null mice showed resistance to urethane-induced lung cancer. Second, we demonstrated the Srx and Prx sites important for Srx-Prx interaction. The orientation of this arm is demonstrated to cause some steric hindrance for the Srx-Prx interaction as it substantially reduces the rate of association between Srx and Prx. Finally, we carried out virtual screening to identify molecules that can successfully target Srx-Prx interaction. Multiple in-silico filters were used to minimize the number of chemicals to be tested. We identified ISO1 as an inhibitor of the Srx-Prx interaction. KD value for Srx-ISO1 interaction is calculated to be 42 nM. Together, these data helps to identify an inhibitor (ISO1) of the Srx-Prx interaction that can be further pursued to be developed as a chemotherapeutic tool.

Atherosclerosis is a type of cardiovascular disease that is characterized by localized thickening of the arteries at sites referred to as atheromas. The earliest events leading to atheroma formation involve the oxidative modification of low-density lipoprotein in the arterial intima, and the uptake of oxidatively-modified low-density lipoprotein by intimal macrophages. This unregulated uptake results in lipid-laden macrophage-derived foam cells, which aggregate at potential sites of atheroma development known as fatty streaks. We hypothesized that the transition from macrophage to foam cell is characterized by differential expression that, as a whole, is pro-atherogenic. A proteomic and transcriptomic analysis identified several proteins and mRNA transcripts that were differentially-regulated following acute and chronic exposure to oxLDL, including mediators of antioxidant and immune response. Further, chronic oxLDL exposure led to a decrease in oxLDL-induced toxicity, which coincided with an increased antioxidant response when compared to macrophages treated with a single exposure to oxLDL. Data obtained from the analysis of differential expression in foam cells provided a foundation for the second stage of this project. Peroxiredoxin I was identified as a protein that is upregulated in macrophages following exposure to oxLDL, which led to the hypothesis that the upregulation of peroxiredoxin I could protect against toxicity induced by oxLDL-generated reactive oxygen species. It was determined that peroxiredoxin I can decrease oxLDL-induced toxicity when induced prior to oxLDL-exposure. This effect coincides with a decrease in reactive oxygen species, verifying the antioxidant role of peroxiredoxin I. Additionally, alternative functionality for peroxiredoxin I was tested, and it was determined that the activation of p38 MAPK is dependent on peroxiredoxin I expression. The sum of this data suggests that peroxiredoxin I contributes to cell survival and signaling, which affects macrophage foam cell function and survival.

Both vertebrates and anopheline mosquitoes inhibit Plasmodium spp. (malaria parasite) development via induction of nitric oxide (·NO) synthase. Expression of Anopheles stephensi ·NO synthase (AsNOS) is induced in the midgut epithelium beginning at 6 h following a Plasmodium berghei-infected blood meal. ·NO reacts readily with other biocompounds forming a variety of reactive nitrogen intermediates (RNIs) that may impose a nitrosative stress. These RNIs are proposed to be responsible for the AsNOS-dependent inhibition of Plasmodium development. In my studies, I identified several RNIs that are induced in the blood-filled midgut in response to Plasmodium infection. Stable end products of ·NO (NO₃⁻ and NO₂⁻), measured using a modified Griess assay, are elevated in infected midguts at 24 h post-blood meal (pBM). Further studies using chemical reduction-chemiluminescence with Hg displacement showed that infected midguts contained elevated levels of potentially toxic higher oxides of nitrogen (NOx), but S-nitrosothiol (SNO) and nitrite levels did not differ between infected and uninfected midguts at 12.5 and 24 h pBM. Thus, nitrates contributed to elevated NOx levels. SNO-biotin switch westerns indicated that S-nitrosated midgut proteins change over the course of blood meal digestion, but not in response to infection. Photolysis-chemiluminescence was used to release and detect bound ·NO from compounds in blood-filled midguts dissected from 0-33 h pBM. Results showed increased ·NO levels in Plasmodium-infected midgut lysates beginning at 8 h, with significant increases at 12.5-13.5 h and 24-25.5 h pBM and peak levels at 20-24 h. Photolyzed ·NO is derived from SNOs and metal nitrosyls. Since SNO concentrations did not change in response to infection, I proposed that metal nitrosyls, specifically Fe nitrosyl hemoglobin (nitrosylHb) based on the concentration of hemoglobin, were elevated in the infected midgut. At 12-24 h pBM, levels of midgut RNIs in infected mosquitoes were typical of levels measured during mammalian septic inflammation. The inverse relationship between AsNOS activity and parasite abundance indicates that nitrosative stress has a detrimental effect on parasite development. However, nitrosative stress may impact mosquito tissues as well in a manner analogous to mammalian tissue damage during inflammation. Elevated levels of nitrotyrosine (NTYR), a marker for nitrosative stress in many mammalian disease states, were detected in tissues of parasite-infected A. stephensi at 24 h pBM. Greater nitration of tyrosine residues was detected in the blood bolus, midgut epithelium, eggs and fat body. In the midgut, Hb remained in an oxygenated state for the duration of blood digestion. The reaction between ·NO and oxyhemoglobin (oxyHb) can result in the formation of nitrate and methemoglobin (metHb). Although nitrate levels were elevated in response to parasite infection, there was little to no metHb present in the mosquito midgut. The simultaneous presence of nitrates, nitrosylHb, oxyHb, and NTYR, together with a lack of elevated nitrites and metHb, suggested that alternative reaction mechanisms involving â ¢NO had occurred in the reducing environment of the midgut. In addition, I proposed that nitroxyl and peroxynitrite participated in reactions that yielded observed midgut RNIs. To cope with the parasite-induced nitrosative stress, cellular defenses in the mosquito may be induced to minimize self damage. I proposed that peroxiredoxins (Prx), enzymes that can detoxify peroxides and peroxynitrite, may protect A. stephensi from nitrosative stress. Six Prx genes were identified in the A. gambiae genome based on homology with known D. melanogaster Prxs. I identified one A. stephensi Prx, AsPrx, that shared 78% amino acid identity with a D. melanogaster 2-Cys Prx known to protect fly cells against various oxidative stresses. AsPrx was expressed in the midgut epithelium and is encoded by a single-copy, intronless gene. Quantitative RT-PCR analyses confirmed that induction of AsPrx expression in the midgut was correlated with malaria parasite infection and nitrosative stress. To determine whether AsPrx could protect against RNI- and ROS-mediated cell death, transient transfection protocols were established for AsPrx overexpression in D. melanogaster (S2) and A. stephensi (MSQ43) cells and for AsPrx gene silencing using RNA interference in MSQ43 cells. Viability assays in MSQ43 cells showed that AsPrx conferred protection against hydrogen peroxide, ·NO, nitroxyl and peroxynitrite. These data suggested that the ·NO-mediated defense response is toxic to both host and parasite. However, AsPrx may shift the balance in favor of the mosquito.