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<p>  </p> <p>Cancer immunotherapies have revolutionized anticancer treatment, saving lives by utilizing patients’ immune systems. Immunogenic cell death inducing chemotherapies have recently gained interest as they can not only inhibit the growth of the tumor but also allows the patient to develop a long-lasting immune response to the tumor. However, due to the poor retention of chemotherapies in the tumor and immunosuppressive tumor microenvironment, the activity of immunogenic cell death inducing chemotherapy is limited. To overcome the limitations, I have developed immunofunctional poly(lactic-co-glycolic acid) nanoparticles to enhance the retention of immunogenic cell death inducers at the tumor and increase the recruitment of antigen-presenting cells to the tumor.</p> <p><br></p> <p>In our study, paclitaxel and carfilzomib were determined as immunogenic cell death inducers, supported by in vitro screening of damage-associated molecular patterns and in vivo vaccination study. Both drugs were identified as immunogenic cell death inducing chemotherapy agents. To deliver immunogenic cell death inducers, immunofunctional poly(lactic-co-glycolic acid) nanoparticles were developed by modifying the surface with adenosine triphosphate. The coating of adenosine triphosphate attracted dendritic cells in a concentration gradient manner and improved the stability of adenosine triphosphate against its degrading enzyme. Both paclitaxel and carfilzomib were successfully encapsulated into the developed nanoparticle formulation. Paclitaxel encapsulated nanoparticles were chosen as a lead candidate due to the inherent immunotoxicity of carfilzomib.</p> <p>Paclitaxel encapsulated nanoparticles coated with ATP effectively suppressed tumor growth in CT26 murine carcinoma and B16F10 murine melanoma. The formulation also increased the immune cell infiltration into the tumor, which may explain the enhanced efficacy of the nanoparticle formulation. Combinational therapy of nanoparticles with anti-PD-1 antibodies significantly increased the complete regression rate in tumor-bearing mice by invigorating the immunosuppressive environment. </p> <p><br></p> <p>In summary, paclitaxel (an immunogenic cell death inducer) encapsulated in adenosine triphosphate-coated poly(lactic-co-glycolic acid) nanoparticles attracted dendritic cells in a concentration gradient manner and effectively suppressed tumor. Additional anti-PD-1 antibodies further improved the antitumor effect, inducing complete tumor regression in 75% of CT26-bearing mice, by inhibiting the interactions between T cells and immunosuppressive cells (regulatory T cells and myeloid-derived suppressor cells). </p> <p><br></p> <p>Chapter 1 discusses the current understanding of immunotherapy and delivery systems to enhance immunotherapy. Chapter 2 describes the determination of immunogenic cell death inducers and the development of immunofunctional nanocarrier. The in vivo antitumor efficacy of the nanocarrier was tested in Chapter 3. </p>

Escherichia coli transcription termination factor Rho is a hexameric RNA/DNA helicase that terminates transcription using energy derived from the hydrolysis of ATP. The ATP binding sites of Rho are located at the interfaces of adjoining subunit Cterminal domains and have the Walker A and B motifs, characteristic of many ATPases (Skordalakes & Berger, 2003; Richardson 2002). Available Rho crystal structures capture the protein with its active site in an open configuration that must close to permit ATP hydrolysis. Because of this, the identities of active site residues predicted to mediate ATP hydrolysis are uncertain. To determine which amino acids activate water, stabilize transition state, sense the γ- phosphoryl group, and coordinate the magnesium ion of MgATP, we have carried out site-specific mutagenesis on candidate residues which are conserved across bacterial species, and characterized the relevant properties of the mutant proteins. The residues chosen were E211 as the water activator, R212 as the γ sensor, R366 as the arginine finger, and D265 as the residue that coordinates Mg2+. Each mutant protein was investigated for its ability to oligomerize as hexamers, assayed for ATPase activity, ATP and RNA binding, and pre-steady-state kinetics. The results show that the mutant proteins form hexamers similarly as to wild type Rho. The RhoE211 mutants display at least a 200-fold lower activity as ATPases, bind both ATP and RNA with similar affinities as the wild type protein, and display no burst in pre-steady-state kinetics. RhoR212A protein has 20-fold lower activity as an ATPase compared to wild type Rho, binds ATP with at least a 50-fold weaker affinity, and RNA with a 2-fold higher KD compared to wild type Rho. RhoR366A functions as an ATPase with 50-fold lower activity, binds RNA with similar affinity as wild type Rho and binds ATP with a 5- fold weaker affinity. RhoD265N displays 150-fold lower ATPase activity compared to the wild type enzyme, binds ATP with a 10-fold weaker affinity, and binds RNA with similar affinity as wild type Rho. Pre-steady-state kinetics studies indicate that the mutant proteins investigated show no burst kinetics, indicating a failure or a significantly slower rate of the hydrolysis (chemistry) step. It is possible that the rate-limiting step is the chemistry step in these mutant proteins, contrary to the wild type protein where the chemistry step is much faster (300/s). Together, the results obtained are consistent with the proposed roles for these residues: E211 is involved in activating a water molecule, R212 functions as the γ sensor, R366 functions as the arginine finger and D265 is involved in coordination of the Mg2+ ion. This study has elucidated the mechanism of ATP hydrolysis, by determining some of the key residues involved in the hydrolysis reaction. This study is only a part of the characterization of the active site residues. There might be other residues involved in one or all of the functions proposed. Utilizing the findings from this study, other experiments and models can be implemented to understand how Rho hydrolyzes ATP and utilizes the energy to move along the RNA molecule and functions as a helicase. / Biochemistry

Cholesterol and fatty acid homeostasis is maintained by a complex network of regulatory mechanisms that control the biosynthesis and deposition of lipids over diverse physiological conditions. However, these processes can become dysregulated and uncoupled from energy metabolism by metabolic stress such as a hyper-caloric diet and physical inactivity; eventually manifesting as risk factors associated with atherosclerotic cardiovascular disease (ASCVD), Type 2 diabetes (T2D), and/or non-alcoholic fatty liver disease (NAFLD). AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that promotes metabolic homeostasis by mediating effects on multiple cellular processes including cholesterol and fatty acid synthesis biosynthesis. However, the mechanisms linking AMPK to lipid metabolism under normal and pathological conditions, remain undefined. In these studies, we identify a novel nutrient sensing mechanism whereby the coenzyme A (CoA) activated esters of long-chain fatty acids (LCFA-CoA) directly activate AMPK via specific interactions within the β1-regulatory subunit involving a Ser108 residue previously shown only with synthetic activators. We demonstrate the physiological relevance for this mechanism in an acute setting by showing that fatty acid oxidation was attenuated in mice harboring an AMPKβ1-S108A knock-in mutation compared to WT mice. We then demonstrated that β1-selctive AMPK activation is mimicked by the CoA conjugated form of bempedoic acid, a synthetic small molecule lipid synthesis inhibitor in clinical development for lowering elevated levels of low-density lipoprotein cholesterol (LDL-C). The importance of this mechanism was determined by assessing multiple disease outcomes in Ampkβ1-/-/Apoe-/- double knockout (DKO) mice fed a high fat-high cholesterol (HFHC) diet ± bempedoic acid. In these studies, bempedoic acid treatment reduced plasma LDL-C and atherosclerosis in both Apoe-/- and DKO mice, while no differences in disease outcomes was detected between the two genotypes in response to HFHC feeding. Further mechanistic investigations in rodent and primary human hepatocytes, revealed that the CoA conjugate of bempedoic acid suppressed lipid synthesis via competitive inhibition of ATP-citrate lyase (ACL), which promoted LDL receptor upregulation and associated reductions in LDL-C. We then integrate these findings with published literature in a written synthesis aimed to evaluate the role of ACL in metabolism, and its potential utility as a therapeutic target to treat ASCVD and metabolic disorders in humans. Although several questions remain regarding the metabolic role of AMPK activation by LCFA-CoAs, these studies have expanded our understanding of how cells acutely integrate lipid and energy signals to maintain lipid homeostasis, and identified ACL as a promising strategy to treat hypercholesterolemia, ASCVD, and associated metabolic disorders. / Thesis / Doctor of Philosophy (PhD) / The dysregulation of cholesterol and triglyceride metabolism can manifest as risk factors for life-threating diseases such as atherosclerotic cardiovascular diseases (ASCVD), Type-2 diabetes (T2D), and nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms controlling lipid homeoastasis in health and disease are not completely understood. ATP-citrate lyase (ACL) and AMP-activated protein kinase (AMPK) are emerging as key nodes in metabolism that integrate lipid metabolism with signals of nutrient availability and cellular energy status, respectively. These strategic positions in metabolism suggest that both these enzymes could play an important role in the underlying pathophysiology of lipid-related diseases, and are therefore, prime candidates for therapeutic intervention. In these studies, we expand our understanding of the role of AMPK in metabolism beyond energy sensing by identifying specific lipid metabolites as direct allosteric activators of kinase activity. We also evaluate the therapeutic utility of targeting both AMPK and ACL in novel models of hypercholesterolemia and metabolic disease, and demonstrate that ACL inhibition offers a promising strategy to address multiple unmet medical needs.

McMaster University MASTER OF SCIENCE (2019) Hamilton, Ontario (Medical Sciences) TITLE: Modulation of cyclic adenosine monophosphate for potentiation of long-acting β2-agonist and glucocorticoids in human airway epithelial cells AUTHOR: Yechan Kim, B.HSc. (McMaster University) SUPERVISOR: Dr. Jeremy Alexander Hirota NUMBER OF PAGES: xiv, 81 / In Canada, asthma is the third most common chronic disease resulting in 250 premature deaths annually and related healthcare expenses exceeding $2.1 billion/year. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Despite an advanced understanding on how to treat and manage the symptoms of asthma, current therapy is sub-optimal in 35-50% of moderate-severe asthmatics around the world resulting in lung inflammation, persistent impairment of lung function, and increased risk of mortality. Combination of long-acting β2 agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma. Increasing intracellular cyclic adenosine monophosphate (cAMP) beyond existing combination LABA/GCS are likely to be beneficial for the management of difficult to control asthmatics that are hypo-responsive to mainstay therapy. In human airway epithelial cells (HAEC), cAMP is either exported by transporters or broken down by enzymes, such as phosphodiesterase 4 (PDE4). We have demonstrated that HAEC express ATP Binding Cassette Transporter C4 (ABCC4), an extracellular cAMP transporter. We also show that ABCC4 and PDE4 inhibition can potentiate LABA/GCS anti-inflammatory responses in a human epithelial cell line in a cAMP-dependent mechanism validating the pursuit of novel ABCC4 inhibitors as a cAMP elevating agent for asthma. / Thesis / Master of Science in Medical Sciences (MSMS) / Asthma is a common chronic lung disease characterized by narrow and inflamed airways that cause breathing difficulties. Current management includes the combination of bronchodilators, to relax the airway, and steroids, to decrease inflammation. Unfortunately, this combination therapy is suboptimal in 35-50% of users, increasing the risk of asthma attacks, hospitalization rate, and health care costs. Recently, there have been studies theorizing that we can improve the therapy’s ability to decrease inflammation by increasing cAMP, an important molecule for biological activities. We tested this claim by blocking the breakdown and export of cAMP to increase its levels and measured inflammatory cytokines, molecules that direct the action of immune cells. Our results show that in a model of viral infection, administering the combination therapy while increasing cAMP levels can further decrease inflammatory cytokines prompting further investigation for its potential implication in the clinic.

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This dissertation presents a new class of engineered devices, fabricated from synthetic materials and protein transporters extracted from cell membranes of plants, that use chemomechanical and chemoelectrical energy conversion processes to perform mechanical and electrical work. The chemomechanical energy conversion concept is implemented in a protein based actuator. The chemical energy is applied as an electrochemical gradient of protons across a membrane assembly formed from phospholipids and SUT4 -a proton-sucrose cotransporter. The membrane assembly forms a physical barrier between two chambers in the actuator. The SUT4 proteins in the membrane assembly balances the applied electrochemical gradient by a concentration gradient of sucrose across the membrane. The sucrose gradient simultaneously generates an osmotic flow which deforms a flexible wall in a constrained chamber of the actuator, thus exhibiting mechanical strain. The sucrose concentration balanced by the protein transporter is used as the control variable for fluid flow through the membrane. The transport properties of the membrane assembly has been characterized for the control variable in the system. The reaction kinetics based model for solute transport through the cotransporter is modified to compute the equilibrium constant for solute binding and fluid translocation rate through the membrane. The maximum initial flux rate through the membrane is computed to be 2.51+/-0.6 ul/ug.cm^2.min for an applied pH4.0/pH7.0 concentration gradient across the membrane. The flux rate can be modulated by varying the sucrose concentration in the actuator. The prototype actuator has been fabricated using the characterized membrane assembly. A maximum deformation of 60microns at steady state is developed by the actuator for 20 mM sucrose concentration in the system. The chemoelectrical energy conversion concept is based on the electrogenic proton pumps in plasma and vacuolar membranes of a plant cell. A prototype device referred to as a BioCell demonstrates the chemoelectric energy conversion using V-type ATPase extracted from plant cell membranes. The enzyme in the bilayer lipid membrane hydrolyzes ATP and converts the chemical energy from the reaction into a charge gradient across the membrane. Silver-silver chloride electrodes on both the sides of the membrane convert the charge established by the proton pumps into cell voltage. The redox reactions at the surface of the electrodes result in a current through the external load connected to the terminals of the BioCell. The single cell behaves like a constant current power source and has an internal resistance of 10-22kOhms. The specific power from the cell of the membrane assembly is estimated to be around 2microwatts/sq/cm. The demonstration of chemoelectrical energy conversion shows the possibility to use ATP as an alternative source of electrical power to design novel chemo-electro-mechanical devices. / Ph. D.