This work investigates the mechanisms of binding of T cell receptors (TCRs) to Class I MHC-peptide complexes (pMHC). The structure of a TCR specific for the Melan-A tumour antigen bound to its cognate pMHC was solved to a resolution of 2.5 Å which gives insight into how this TCR could be mutated to optimize binding and subsequently used as a cancer vaccine. Detailed sequence and geometric analyses of all currently available structures of Class I TCR-pMHC complexes revealed that TCRs can bind to pMHC with a range of orientations, yet always focus on the central portion of the peptide and use a specific subset of six residues on the MHC helices for binding. The most striking finding was the use of aromatic residues in the TCR CDR loops to bind to residue Q155 on the MHC α2 helix. Attempts were also made to express and purify Toll-like receptors (TLRs) with the aim of solving one or more of these structures. However, despite testing of over 50 different constructs from 12 different TLRs or associated proteins, insufficient soluble protein expression was obtained for crystallization trials. Finally, a protein disorder prediction tool was developed to aid construct design for structural biology studies and improve the chances of obtaining protein crystals. This tool is based on a novel type of neural network and blind tests comparing it to 8 other disorder prediction tools showed it is one of the best in the field. It is freely available at www.strubi.ox.ac.uk/RONN. Analysis of large datasets revealed that the position of order/disorder transitions is quite precisely defined in amino-acid sequences and that transition regions have an amino acid composition distinct from that of bulk ordered and disordered sequences. There is a steady decrease in order-promoting residues on the ordered side of boundaries as well as a weak sequence signal, both of which signify the approaching disorder and may prove useful for improving existing disorder prediction tools.

<p>Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. It was hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27 (IL-27)) could treat bone-metastatic prostate cancer and also exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. To test this hypothesis, <i>in vivo</i> experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), or a therapeutic gene (IL-27). Ultrasound was used to aid gene delivery. Various gene delivery methods were tested and optimized through <i>in vivo</i> studies, with microbubbles in combination with ultrasound (sonoporation) emerging as the best method. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing and cytokine arrays were used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone health by improving osteoblast differentiation and inhibiting osteoclast differentiation. Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach should enable a significant reduction in chemotherapy-associated toxicity, improving sensitivity to immunotherapy, and simultaneously improving bone quality.</p>

<p>Sea cucumbers have been found to contain bioactive compounds such as saponin, fucoidan, frondoside, and glycosides that have pharmacological properties like antitumor, antibacterial, anti-inflammation, and antihyperglycemic activity. Although several species of sea cucumbers have been studied and reared for the food and medicinal industries, not much research has been conducted on the species in the waters of the Eastern United States. In this research, physiological and immunological parameters of coelomic fluid from <em>Cucumaria</em> <em>frondosa</em>, <em>Isostychopus</em> <em>badionotus</em>, and <em>Pentacta</em> <em>pygmaea</em> were compared to find the most promising candidate with these properties and pharmacological benefits. We found that <em>C. frondosa</em> was the species with the best immunological and physiological parameters among the three studied. <em>C. frondosa</em> illustrated that its coelomic fluid contains the highest concentrations of cells and lysozymes that had the highest activity. Using <em>C. frondosa</em>’s tissue extracts and coelomic fluid, the ability of the extracts and coelomic fluid to inhibit murine melanoma cells (B16-F10) and modulate T-lymphocytes <em>in vitro</em> were investigated. Although no significant differences were seen statistically, the experiments illustrated that T-lymphocytes were highly activated at higher concentrations (0.001g/uL-0.0002g/uL) for tissue extracts and at lower concentrations (0.000008g/uL) for coelomic fluid. On the other hand, melanoma cells were inhibited highest at lower concentrations (0.000008g/uL-0.0000016/uL). In addition to these studies, the antibacterial activity of <em>C. frondosa</em> extract was tested on ten pathogenic bacterial species. Antibacterial activity of the <em>C. frondosa</em> extract was not seen in this experiment. However, hemolytic activity by compounds present in <em>C. frondosa</em> extracts was seen in blood agars culturing <em>Streptococcus pneumoniae</em> and <em>Enterococcus faecalis</em> in our experiment. Lastly, an <em>in vivo </em>study was conducted to see if <em>C. frondosa</em> extract can modulate stress in Nile tilapia. In our experiment, we observed that <em>C. frondosa</em> extract was able to enhance the activity of one of the parameters, phagocytic capacity significantly. However, we are not able to conclude that <em>C. frondosa</em> extract was able to mitigate chronic stress from the results obtained. Overall, observing the results from the projects, we cannot conclude that <em>C. frondosa</em> extracts illustrated pharmacological properties. Extensive studies are recommended and required to use <em>C. frondosa</em> extract for medicinal purposes. </p>

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.

Breast cancer (BC) is one of the deadliest forms of cancers with high incidence and mortality rates, especially in women. Encouragingly, targeted therapies have improved the overall<br>survival and quality of life in patients with various subtypes of BC. Unfortunately, these first-line therapies often fail due to inherent as well as acquired resistance of cancer cells. Treatment evading cancer cells can exhibit systemic dormancy in patients over a long period of time without manifesting any symptoms. In a suitable environment, these undetected disseminated tumor cells can relapse in the form of metastasis. Therefore, it is essential to understand the mechanisms of<br><div>BC recurrence and to develop durable therapeutic interventions to improve patient’s survival. In this dissertation work, we studied fibroblast growth factor receptors (FGFR), as therapeutic targets to treat the recurrence of drug-resistant and immune-dormant BC metastasis. <br></div><div><br></div><div>The HER2 subtype of BC is characterized by the overexpression of human epidermal growth factor receptor 2 (HER2), which drives elevated downstream signaling promoting tumorigenesis. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in which an anti-HER2 antibody targets HER2 overexpressing tumor cells and delivers a highly potent microtubule inhibitor. Using novel models of minimal residual disease (MRD) following T-DM1 treatments, we found that epithelial to mesenchymal transition is a critical process for cells to persist the TDM1 treatments. The upregulation of FGFR1 may facilitate insensitivity to T-DM1. Our data also showed that FGFR1 overexpression in HER2+ tumors leads to a higher incidence of recurrence, and these recurrent tumors show sensitivity towards covalent inhibition of FGFR. <br></div><div><br></div><div>In addition to drug-induced MRD in the primary tumor sites, disseminated tumor cells (DTCs) can demonstrate dormant phenotype via maintaining an equilibrium with immunemediated tumor clearance. Factors affecting such equilibrium may contribute to the recurrence of breast cancers metastasis. We show that such immune-mediated dormancy can be modeled with the 4T07 tumors. These tumors display immune-exclusion phenotypes in metastatic pulmonary organs. The inhibition of FGFR modulates the immune cell compositions of pulmonary organs favoring anti-tumor immunity. However, inhibition of FGFR may also affect T cell receptor downstream signaling, resulting in the inhibition of cytolytic T cell’s function. Finally, we report that combination therapy using the FGFR kinase inhibitor and an immune checkpoint blockade showed effective targeting of metastatic 4T07 tumors. <br></div><div><br></div><div>FGFR signaling as a therapeutic target in various tumors has been an active focus of cancer research. In this dissertation work, we have expanded our understanding of the role of FGFR in the recurrence of drug-resistant breast cancers as well as in the maintenance of an immune evasive microenvironment promoting pulmonary growth of tumors. Moreover, we presented evidence that it is possible to repurpose FGFR targeted therapy alone or in combination with checkpoint blockades to target recurrent metastatic BCs. In the future, our novel models of minimal residual diseases and systemic immune dormancy may act as valuable biological tools to expand our understanding of the minimal residual disease and dormant tumor cells.</div>

<p>  </p> <p>The phosphatases of regenerating liver are a subfamily of protein tyrosine phosphatases that consist of PRL1, PRL2 and PRL3. The overexpression of PRLs promote cell proliferation, migration and invasion and contribute to tumorigenesis and metastasis to aggravate survival outcome. Although there is increasing interest in understanding the implication of these phosphatases in tumor development, currently, limited knowledge is available about their mechanism of action and the efficacy of PRL inhibition in <em>in vivo</em> tumor models, the tumor extrinsic role of PRLs that allow them to impact tumor development, as well as <em>in vivo</em> physiological function of PRLs that could implicate them in diseases other than cancer. The work presented here aims to address these limitations.</p> <p><br></p>