Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A novel strategy for site-specific DNA transformation of plants has been proposed and the first component of the system developed. The proposed method overcomes the limitations of current techniques by providing a specific integration site for the insertion of transgenes using features of the adeno-associated virus (AAV) life cycle. In the absence of helper virus, AAV integrates into a specific location on human chromosome 19, the AAVS1 locus. The sequence for AAV integration was introduced into the model plant Arabidopsis thaliana using Agrobacterium tumefaciens-mediated transformation. A portion of the human AAVS1 sequence, including the Rep binding site (RBS) and terminal resolution site (TRS), was cloned between T-DNA borders of the Agrobacterium Ti plasmid. The reporter gene, b-glucuronidase (GUS) was inserted proximal to AAVS1 in the plasmid for use in screening for the presence of T-DNA. In addition, it will serve as an indicator of the expression level expected for transgene inserted into AAVS1 by recombinant AAV. PCR amplification, dideoxy sequencing, GUS expression assays and genomic Southern blots were performed to examine putative transgenic plants for the presence of the AAVS1 sequence. / Master of Science

Mitogen-activated protein kinases (MAPKs) are a family of highly conserved serine/threonine protein kinases which link upstream receptors to their downstream targets. These targets can be localized in the cytoplasm or the nucleus. Pathogens produce pathogen-associated molecular patterns (PAMPs) that are known to trigger the activation of MAPK cascades. In plants, MAPK signaling cascades regulate development and cellular processes such as stress responses, immunity, and apoptosis by means of the phosphorylation of specific targets. Phosphoproteomics analysis of PAMP-induced Arabidopsis plants led to the identification of several putative MAPK targets. USP1 (Universal Stress Protein A) (At1g11360) and WLIM2A (At2g39900) are two potential phosphorylation targets of MAPKs, and are the focus of this thesis. So far, little is known about their role in plant immunity. CRISPR-Cas9 generated knockout usp1 mutant lines enhanced resistance to infection by Pst DC3000, usp1 mutant showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. Although the physiological role of USP1 has been established, the biochemical and molecular functions are unknown. We discovered a new role for USP1, demonstrating that it functions as a molecular chaperone and is involved in thermal priming. Overall, these data show that phosphoprotein USP1 plays an important role in orchestrating plant immunity. CRISPR-Cas9 generated knockout wlim2a mutant showed susceptibility to infection by Pst DC3000. wlim2a mutants showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated in wlim2a mutants. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. wlim2a mutants show enhanced fungal infection by Botrytis cinerea. Overall, the data shows that WLIM2A phosphorylation is important during plant immunity.

No description available.

<p>Atherosclerosis is a multi-factorial disease and is the major cause of death in the western world. Numerous risk factors, including hyperlipidemia, obesity, diabetes, smoking, hypertension, and family history increase the risk of atherosclerosis and death from cardiovascular disease (CVD). Clinical and epidemiological studies have now shown that hyperhomocysteinemia (HHcy) is an independent risk factor for CVD. Further, we and others have demonstrated that HHcy accelerates atherosclerosis in apolipoprotein Edeficient ( apoff1-) mice. Although several studies have reported that homocysteineinduced endoplasmic reticulum (ER) stress causes growth arrest and programmed cell death (PCD) in cultured vascular endothelial cells, the cellular factors responsible for this effect and their relevance to atherosclerosis have not been completely elucidated.</p><p>Previously, we have demonstrated that homocysteine induces the expression of Tcell death associated gene 51 (TDAG51), a member of the pleckstrin homology-related domain family, in cultured human vascular endothelial cells. Transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion and promoted detachment-mediated PCD. In support of these in vitro findings, TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apoff1-mice fed hyperhomocysteinemic diets, compared to mice fed control diet. To investigate the in vivo significance of TDAG51 on atherosclerotic lesion development and progression, knockout mice deficient in both TDAG51 and apoE genes were generated. Our findings show that TDAG51-1-/apoff1-double knockout (DKO) mice fed control chow diet have significantly reduced atherosclerotic lesion size, compared to ageand sex-matched apoff1-control mice. Atheroprotective function of TDAG51 deficiency may be explained in part by the observation that there is a significant upregulation of peroxisome proliferator-activated receptor y (PPAR-y) in TDAG51-deficient (TDAG51 _1_) cells including mouse embryonic fibroblasts (MEFs), compared to control wildtype MEFs. Given that PPAR-y has both atheroprotective and anti-inflammatory properties, TDAG51 may represent a unique negative regulator of PPAR-y and its downstream gene targets. Taken together, my findings demonstrate that TDAG51 is a novel cellular mediator involved in the development and progression of atherosclerosis.</p><p>In addition to its anti-atherogenic properties, I have demonstrated that TDAG5 l _1_ MEFs have increased migratory properties following monolayer disruption or in response to chemotaxis on fibronectin-coated Boyden chambers, compared to wildtype control MEFs. Although TDAG51-induced cell migration could potentially affect atherosclerotic lesion development, our recent observations suggest that TDAG51 may also have a role in wound healing. Our studies have shown that dorsal skin wounds within TDAG5 l _ 1_ mice healed slowly, compared to those in control mice through a mechanism involving impaired myofibroblast differentiation. Since the underlying mechanisms of wound healing and fibrosis are similar, it is conceivable that TDAG51 may have role in fibrosis.</p><p>In summary, this thesis provides novel evidence that TDAG5 l is involved in the pathogenesis of atherosclerosis and wound healing. Furthermore, TDAG51 may represent a novel therapeutic target for attenuating atherosclerotic lesion development, thereby reducing the risk of cardiovascular disease and its complications.</p> / Thesis / Doctor of Philosophy (PhD)

<p> Samples from two granitic offshoot dikes from the Murray granite, and from the Murray granite itself were analysed for eight major elements using X-Ray Fluorescence, to determine whether or not the dikes represented remobilized parts of the Murray granite and as such could have a different chemical composition from the parent material.</p> <p> The analyses show that only very minor chemical differences exist between the two granitic dikes and the main Murray granite. However four samples of the dike rocks appear to show element exchange with the Sudbury norite surrounding them, resulting in increased Na2O and CaO contents and lower K2O contents, resulting in increased amounts of albitic plagioclase.</p> / Thesis / Bachelor of Science (BSc)

Medical implants constitute an essential advancement in modern medicine, often restoring or replacing functionality to failed organs. Whether a medical implant is temporary or permanent, medical implants carry the risk of implant failure due to an infection. Implant-associated infections (IAI) are challenging to treat and often result in increased medical costs, prolonged hospital stays, implant failure, and, in some instances, severe infections that can lead to amputations, sepsis, or mortality. Eradicating an IAI can be challenging since bacteria can form biofilms on the implant’s surface. The biofilms comprise an extracellular matrix protecting the bacterial cells against systemic antibiotics and the host’s immune system. Treating an IAI usually entails a broad range of antibiotic treatment and surgical procedures for tissue debridement or implant replacement. For the reasons stated above, scientists and engineers continue to develop technologies to protect the surface of medical implants against infections. Amongst the new technologies, Liquid-Infused Surfaces (LIS) are renowned for their repellent and anti-fouling properties created by tethering a stable liquid layer onto the surface. However, many challenges remain to adopt this technology for implantable devices. For instance, the high repellent properties can hinder implant-tissue interaction and discourage proper integration with the body. Furthermore, the stable liquid layer is contingent on the surface properties of the coated material. In other words, the long-term stability of these coatings may be compromised if the surface chemistry is covered by biological processes such as biofilm formation from adherent bacteria. This thesis aims to expand on the applications of LIS coatings and enhance their properties for implantable materials. This thesis reviews different types of antibiotic surface coatings and further examines LIS technologies as a viable antibacterial coating for medical implants. Then, three novel multifunctional LIS coatings are presented. The first developed coating enhanced the antibacterial properties of the coating by adding bactericidal agents within the LIS coating. The developed antibiotic liquid-infused coating not only repelled bacteria but also lysed bacteria upon contact. The second coating was designed to promote tissue integration. This multifunctional coating promoted cell deposition and proliferation while remaining repellent toward bacteria, while the conventional LIS coating displayed poor cell availability. Lastly, a collagen-bacteriophage conjugated liquid-infused coating was developed to promote tissue integration while having a two-tier layer of antibacterial protection. This coating was tested in a mouse sepsis model and prevented mortality of all mice, with other groups as high as 90% mortality. These coatings constitute essential steppingstones to bring LIS technology to medical implants. / Dissertation / Doctor of Philosophy (PhD) / Implant-associated infections (IAI) remain a significant problem in modern medicine. IAIs are challenging to treat and often result in increased medical costs, prolonged hospital stays, implant failure, and, in some instances, severe infections that can lead to sepsis or mortality. For these reasons, new technologies have been developed to protect the surface of medical implants against infections. Amongst the new technologies, Liquid-Infused Surfaces (LIS) are renowned for their repellent and anti-fouling properties created by tethering a stable liquid layer onto the surface. This thesis aims to expand on the applications of LIS coatings and enhance their properties for implantable materials. This thesis reviews different types of antibiotic surface coatings, examines LIS technologies, and presents three novel multifunctional LIS coatings. The newly developed coatings enhance the LIS coatings through the addition of antibacterial properties and biomolecules to promote tissue integration.

Gene therapy has emerged as a revolutionary approach for treating genetic disorders, holding great promise for improving patient outcomes. Among the various viral vectors used for delivery of therapeutic transgenes, Adeno-Associated Viruses (AAVs) have gained prominence due to their favorable characteristics including low immunogenicity, long-term gene expression, and the ability to target both dividing and non-dividing cells. However, AAV’s are associated with the high costs of production and challenges with production of a high-quality virus, limiting AAV’s utilization and widespread use. In this study, we aimed to develop a high-throughput screening assay targeting AAV production enhancers, thus addressing the manufacturing obstacles and advancing the affordability and accessibility of gene therapies. To help overcome the limitations and expenses associated with AAV manufacturing, an innovative high-throughput screening assay was developed with the intent to identify cell culture additives/conditions which maximize AAV production. We optimized various parameters, including the transgene, producer and reporter cell lines, harvest timings and methods, and transduction techniques. The optimized screening assay was employed to evaluate novel compounds across several timings of addition, for their ability to enhance AAV production. Notably, several compounds indicated transfection enhancing capabilities up to 3.4-fold and the developed assays final variability was below 14%. Additionally, compound combinations were assessed to uncover potential additive and synergistic effects that could further enhance AAV productivity. In conclusion, our study presents a significant advancement in targeting the manufacturing challenges associated with AAV. By utilizing an optimized high-throughput screening assay, researchers and manufacturers can identify compounds that enhance AAV production, paving the way for cost-effective and scalable manufacturing processes. Ultimately, this progress holds the potential to improve the affordability, accessibility, and impact of gene therapies for patients worldwide.

Thesis advisor: Abhishek Chatterjee / Directed evolution is a powerful technique to expand chemical space in biological systems. In particular, this method has been used to develop cellular machinery to enable genetic code expansion (GCE), the incorporation of unnatural amino acids (UAAs) into proteins during the translation process. GCE relies on evolving an aminoacyl tRNA synthetase (aaRS) and tRNA pair from a different domain of life to incorporate a UAA into proteins in their new host, as these evolutionarily distant pairs are less likely to be cross-reactive with host pairs. The aaRS and tRNA must meet a number of conditions to be useful for GCE: the pair must be orthogonal (non-cross-reactive) to the host’s native aaRS/tRNA pairs in order to ensure site-specific UAA incorporation; the aaRS must have an active site suited to accept the shape of the UAA; and the tRNA must cooperate with the host ribosome, elongation and release factors, and other translational machinery to efficiently incorporate the UAA into the protein. Numerous aaRS/tRNA pairs have been evolved to allow incorporation of diverse UAAs in bacteria due to the tractable nature of these organisms for directed evolution experiments. While an aaRS evolved in bacteria to charge a novel UAA can be used in eukaryotes, tRNAs cannot be evolved for GCE in bacteria and then used in eukaryotes because they will not have evolved in the presence of the correct translational machinery. It is necessary to evolve tRNAs directly in their host cells. Unfortunately for researchers working on GCE in mammalian cells, it is difficult to perform directed evolution on small gene products in these hosts. Transformation efficiency in mammalian cells is poor, and transient transfection yields heterogeneous DNA distribution to target cells, making selection based on performance of individual library members impossible. Viruses are an ideal DNA delivery vector for mammalian cells, as production of recombinant viruses allows control over library member generation, and viruses can be delivered with exquisite copy number control. The Chatterjee lab recently developed a platform, Virus-Assisted Directed Evolution of tRNAs (VADER), using adeno-associated virus (AAV) to evolve tRNAs for GCE directly in mammalian cells. While VADER is the first directed evolution platform that allows the evolution of small gene products in mammalian cells, its efficiency is limited by its continued reliance on transient transfection to deliver non-library DNA that is necessary for the production of rAAV. To overcome this limitation, baculovirus delivery vectors were developed to boost DNA delivery and AAV capsid production to improve virus production efficiency during selections. VADER allows the evolution of tRNAs to incorporate certain UAAs, but the technique relies on installing a UAA into the AAV capsid, which is sensitive to disruption caused by slight modifications in structure. To expand the scope of VADER to evolve tRNAs for UAAs that cannot be incorporated into the AAV capsid, an alternate selection handle (Assembly Activating Protein, or AAP) was deleted from the genome and provided in trans to incorporate 5-hydroxytryptophan (5HTP). Incorporating the UAA into this flexible protein allows UAA-dependent production of AAV and expands the scope of tRNAs that can be evolved in mammalian cells. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Background: Hypercholesterolemia is a leading risk factor for cardiovascular diseases, with an average of 102.3 million American diagnosed with Hypercholesterolemia. Medical conditions, lifestyle habits, hereditary factors, and psychiatric illnesses have been associated with Hypercholesterolemia. A few studies postulate that Hypercholesterolemia leads to brain changes that underlie depressive illness. Depression is anticipated to overtake chronic diseases like hypertension in developed nations by 2030, and over 17 million US adults suffer from depression. Tennessee ranks 9th (22.36%) out of 50 states with the highest depression rates, with the incidence rising steadily. As a major public health concern needing urgent attention, it is imperative to establish the association between depression and Hypercholesterolemia among adults in Tennessee. Methods: We used cross-sectional data from the 2021 Behavioral Risk Factor Surveillance System a nationally representative U.S. telephone-based survey of adults aged 18 years, and extracted data for Tennessee (n=4,788). Logistic regression analyses were conducted to test the association between diet, physical activity, depression, no past month exercise, high body mass index, substance use, and high cholesterol (outcome). We controlled for income, race, educational status, health insurance status, and age. Results: Overall, 36.1% (n=1,726) of participants in our sample reported high cholesterol. Results of our logistic regression model revealed that depression (OR =1.37, 95% CI, 1.19, 1.58), High body mass index (OR=1.75, 95% CI, 1.52,1.99), no past month exercise (OR=1.45, 95% CI, 1.27,1.66), male gender (OR =1.16, 95% CI, 1.03, 1.32) and low income (OR =1.33, 95% CI, 1.15, 1.53) were associated with high cholesterol. Furthermore, participants with high cholesterol are 37% more likely to report depression. On the other hand, cigarette use, e-cigarette use, alcohol use, no insurance, and marijuana use were not significantly linked. Conclusions: There is a need for awareness and prevention of Hypercholesterolemia in patients managed for depression, as they might die from the complications of high cholesterol rather than the psychiatric illness itself. It is important to intensify the existing programs and interventions for the prevention of Hypercholesterolemia, which would favorably impact on the burden of depression among adults in Tennessee. Also, studies should be done on the outcomes of cholesterol-reducing medications to prevent Hypercholesterolemia in patients with chronic diseases and depressed patients.