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CAR-T cell immunotherapy is a highly efficacious treatment for CD19-positive hematological malignancies, however, some patients are non-responsive for reasons that are not well understood. Clinical efficacy has been correlated with long-term persistence, a propensity that can be predicted by the differentiation state of transplanted cells. Despite this, decades-old methods for expanding T cells have not been updated to prevent the deleterious effects of excessive differentiation in CAR-T cells. Uncoupling proliferation and differentiation is a long-held goal in the field of immunotherapy with both cytokines and pharmacological approaches being implemented to dissociate these parallel processes. Histone methyltransferases rewire transcriptional programs in T cells and simultaneously regulate multitudes of genes, making them attractive targets for modifying the proliferation-differentiation axis. Despite this, only a handful of studies have examined their role in regulating the transcriptional programs of human CD8+ T cells. MLL4 (encoded by KMT2B) belongs to the six-member group of MLL histone methyltransferases. MLL1, a paralog of MLL4, has been implicated in regulating the maintenance of IL-4 and GATA-3 expression in TH2 CD4 memory T cell populations, however the function of MLL4 in human CD8+ T cells is unknown. We report a critical role for MLL4 in the proliferation and differentiation of CD8+ T cells. CRISPR-Cas9-editing of MLL4 uncoupled the processes of proliferation and differentiation, increasing proliferation but maintaining central memory T cell (TCM)-like populations, allowing for the production of increased numbers of TCM-like CD62L+CD45RO+ cells. Pharmacologically inhibiting the MLL4-Menin complex with MI-2 during T cell expansion enriched the frequency of minimally differentiated TCM-like CD8+ T cells. TCM-associated CD62L, CCR7, CD122 and CD127 surface markers were upregulated and early memory-associated transcription factor TCF7, LEF1, EOMES, and FOXP1 transcripts were increased. CD8+ CAR-T cells expanded in the presence of MI-2 responded earlier, while improving both tumor burden and survival in a NSG xenograft model of human leukemia. This finding has important translational impact in improving the persistence and proliferative capacity of CD8+ CAR-T cells. / Infectious Disease & Immunity

This thesis outlines the design and implementation process of an electrified powertrain for use in an American muscle car. Designed as McMaster University's entrant to the EcoCAR 3 Advanced Vehicle Technology Competition (AVTC), an electrified powertrain was developed to provide a Chevrolet Camaro with the performance expected by the American muscle car market while maintaining ever increasing fuel economy regulations. A background of current trends in vehicle electrification, including the prominent market segments experiencing these trends, will be explored along with the history of the classic and modern American muscle car's technical specifications. Following an investigation into existing vehicle electrification trends, the selected hybrid architecture will be discussed. The process of converting a conventional combustion powertrain into a series-parallel hybrid electric powertrain will be explored from the component-level through to full system design. Following a review of the design process for the powertrain, a high-level testing plan will be proposed using a number of test cells available within the facility. This plan will begin at the component-level exploring specific areas of potential complication and move up to complete system-level testing of powertrain functionality. / Thesis / Master of Applied Science (MASc) / Until recently, hybrid electric vehicles have tended to be available in a fairly limited market segment with few offerings for performance-oriented vehicle customers. The introduction of high performance hybrid vehicles suggests that this trend is likely to change. Increasingly more stringent fuel economy and emissions standards means that performance vehicle segments such as American muscle cars must adopt new technologies to retain their performance characteristics. Hybrid powertrains are one solution to providing and improving on the iconic performance of American muscle while meeting future regulatory changes. The addition of a number of electrified components to a gasoline powertrain can assist in achieving desired performance while reducing fuel economy. This thesis investigates the detailed design process adopted to make these modifications while maintaining the functionality expected by muscle car owners. After the design and assembly of the hybrid muscle car powertrain, a specific testing plan was laid out to ensure that the system is capable of sustaining the expected power output. This design and testing process can help introduce new hybrid vehicles to the market which are capable of meeting both the upcoming fuel economy regulations as well as the ongoing performance expectations of the muscle car market.

Advances in our understanding of the molecular events leading to cancer have facilitated the development of next-generation targeted therapies. Among the most promising new approaches is immuno-oncology, where therapeutic agents engage the immune system to fight cancer. One exciting strategy therein is the adoptive transfer of ex vivo cultivated tumor-specific T lymphocytes into a cancer patient. Tumor-specific T cells can be produced by engineering a patient’s own T cells with synthetic receptors (e.g. chimeric antigen receptors (CARs)) designed to redirect T cell cytotoxicity against a tumor target. CAR-engineered T cells (CAR-T cells) were expected to be a non-toxic cellular therapy which would seek out and specifically eliminate disseminated tumors. The clinical experience supports the promise of CAR-T cell therapy (striking efficacy has been observed in the treatment of hematological malignancies), while highlighting areas for improvement; CAR-T cell use has been associated with a host of toxicities and robust clinical efficacy has yet to be replicated in solid tumors. This thesis uses pre-clinical models to describe previously unappreciated aspects of CAR-T cell-associated toxicity and novel synthetic receptor strategies, including: i. The capacity of NKG2D-based CAR-T cells to mediate toxicity. ii. The utility of designed ankyrin repeat proteins as CAR antigen-binding domains. iii. The discovery that variables intrinsic to human CAR-T cell products contribute to toxicity. iv. A novel synthetic receptor capable of redirecting T cell specificity against a tumor target – the T cell antigen coupler (TAC). Unlike equivalent CAR-T cells, TAC-T cells are capable of mediating efficacy against a solid tumor in the absence of toxicity. We anticipate that these results will contribute towards the development of next-generation synthetic receptor-engineered T cell products that can deliver upon the promise of safe, systemic cancer therapeutics. / Thesis / Doctor of Philosophy (PhD) / The human immune system has the unique capacity to “seek and destroy” tumor cells throughout the body. A novel class of drugs, immuno-oncology agents, harness this ability to fight cancer. Within this class is a new cellular drug where genetic engineering is used to create killer immune cells (called T cells) capable of recognizing and eliminating tumors. Two of these cellular drugs have recently received FDA approval, supporting the feasibility of this approach. However, further research is needed to improve the safety of engineered-T cells and increase the number of patients whom can benefit from their use. This thesis uses laboratory investigations to better understand the side-effects associated with anti-cancer engineered-T cells and evaluate new engineering strategies. We anticipate that these results will contribute towards the development of next-generation engineered-T cell drugs which retain the ability to function systemically against cancer but offer an enhanced safety profile.

Breast cancer is one of the most common cancer types in women and its metastases cause most patient deaths in the advanced stage of this disease. 1,2,3 Unfortunately, metastases develop drug resistance to chemotherapy and impaired T lymphocyte infiltration into metastatic lesions by compressing blood vessels. 4,5,10,11,12 Although losartan decompressed vessels and increased the presence of T lymphocytes in the metastatic lesions, T-cells were not effective at eliminating tumors.10 In this thesis, we generated chimeric antigen receptor constructs that have specificity against epithelial cell adhesion molecule (EpCAM). After optimizing a retroviral transfection/transduction system, we successfully generated EpCAM CAR T-cells and tested their efficacy against tumor spheroids. We noticed a dramatic reduction of spheroids' area and spheroids' diameter after 36 hours of treatment and observed spheroids’ destruction and tumor cell elimination after 96 hours of treatment, compared to non-specific stimulated T-cells treatment on tumor spheroids. EpCAM CAR T-cells have been shown to be effective against cancer in vitro; therefore, injection of EpCAM CAR T-cells into mice with breast cancer will be conducted to determine whether losartan is able to improve infiltration. We expect that the use of losartan will improve the number of infiltrated CAR-T-cells and their efficacy against breast tumors.

The automobile is undeniably an icon of our modern era. Decades of accommodating the automobile have dramatically transformed the shape and the quality of our physical environment. The typical suburban retail parking lot is one symptom of our automobile dependence. Frequently the proposed solutions to sprawl development tend to minimize the economic and marketing appeal of a large surface parking lot. For many communities, the anticipated economic benefit and convenience of a suburban retail development overcomes any reservations about the appearance and effect of the associated sea of asphalt. A more achievable design goal might be a pedestrian-oriented parking lot. Is that feasible? What present and future advantages might be gained? What would it be like? This thesis studies the typical commercial surface parking lot through observation and analysis, leading to the design of an infrastructural system of elements to develop pedestrian-oriented parking lots. / Master of Architecture

This research is focused on driver behavior in traffic, especially during car-following situations and safety critical events. Driving behavior is considered as a human decision process in this research which provides opportunities for an artificial driver agent simulator to learn according to naturalistic driving data. This thesis presents two mechine learning methodologies that can be applied to simulate driver naturalistic driving behavior including risk-taking behavior during an incident and lateral evasive behavior which have not yet been captured in existing literature. Two special machine learning approaches Backpropagation (BP) neural network and Neuro-Fuzzy Actor Critic Reinforcement Learning (NFACRL) are proposed to model driver behavior during car-following situation and safety critical events separately. In addition to that, as part of the research, state-of-the-art car-following models are also analyzed and compared to BP neural network approach. Also, driver heterogeneity analyzed by NFACRL method is discussed. Finally, it presents the findings and limitations drawn from each of the specific issues, along with recommendations for further research. / Master of Science

Active suspension technologies are well known for improving ride comfort and handling of ground vehicles relative to passive suspensions. They are ideally suited for mitigating single-event road obstacles. The work presented in this thesis aims to develop a structured approach for finding the peak force and bandwidth requirements of actuators for active suspensions, to mitigate single-event road obstacles. The approach is kept general to allow for application to different vehicle models, ride conditions and performance objectives. The current state-of-art in active suspensions was first evaluated. Based on these findings, the objectives of the simulation models and approach was defined. A quarter-car model was developed in Matlab to simulate the behavior of active suspensions over unilateral boundary conditions due to different road obstacle profiles. The obstacle profiles were obtained from existing standards and literature and then processed to replicate the interaction of tires on road. A least-mean-squares (LMS) algorithm for adaptive filtering, with the help of look-ahead preview was used to determine the ideal control force profile to achieve the performance objective of the active suspension. A case study was conducted to determine the requirements of the actuator in terms of bandwidth and peak force for different single-event road obstacle profiles, vehicle speeds and look-ahead preview distances. The results of the study show that the vehicle velocity and type of road obstacle have a strong influence on the required peak force and bandwidth of the actuator, while look-ahead preview will be much more important for real time controller implementation. / Master of Science