No description available.

No description available.

<p>Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses in the Coronaviridae family. Within this family are four different genera, Alpha-, Beta-, Gamma-, and Deltacoronaviruses with human-infecting CoVs spanning the Alpha- and Beta-CoV genera. Most notably, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1) and SARS-CoV-2 are Betacoronaviruses that spread worldwide in their outbreaks from 2002-2003 (SARS-CoV-1) and 2019-2020 (SARS-CoV-2). Human-infecting Alphacoronaviruses, NL63-CoV and 229E-CoV, have caused milder infections involving respiratory disease, gastroenteritis, and in more severe cases, death. Despite milder disease, Alphacoronaviruses are the cause of 15-30% of severe upper and lower respiratory tract infections each year. There have been recent efforts in the development of potent, small-molecule inhibitors to treat SARS-CoV-2 infection but there is an ongoing need to develop new and effective anti-coronavirus therapeutics to treat other human-infecting CoVs circulating society. Coronaviruses encode two essential proteases, the papain-like protease (PLP) and the 3C-like protease. PLPs are cysteine proteases located in non-structural protein 3 (nsp3). PLPs processes the viral polyprotein, releasing the first three nonstructural proteins encoded in the virus, and also are involved in evading the innate immune response through deubiquitinating (DUB) and deISGylating activity. </p> <p><br></p> <p>This study compares the substrate specificity and catalytic function of multiple human-infecting PLPs from both Alpha- and Beta-CoVs including NL63-CoV PLP2, 229E-CoV PLP2, Canine-CoV PLP2, FIPV-CoV PLP2, PEDV-CoV PLP2, SARS-CoV-1 PLpro, and SARS-CoV-2 PLpro. Interestingly, Alphacoronavirus PLP2s have a >400-fold greater catalytic efficiency for ubiquitin compared to Betacoronaviruses PLpro. This work also identifies a non-covalent scaffold of inhibitors that has pan-CoV inhibition; however, the IC50 values are >30-fold higher for NL63-CoV PLP2 than for SARS-CoV-1 PLpro. The X-ray structures of NL63 PLP2 and 229E PLP2 were determined to 2.1 Å and 1.8 Å, respectively, and provide structural information about the substrate and inhibitor binding region that could be the result in the differences in Alpha- and Betacoronavirus PLP function. Since PLP does not function as a single-domain in vivo, it is critical to understand the function of PLP when tethered to other domains of nsp3. This study also investigates nine different constructs of SARS-CoV-2 nsp3 with increasing domains, ranging from the single PLpro domain to Ubl1-Ydomain ΔTM1-TM2. Interestingly, the longer constructs of SARS-CoV-2 nsp3 show less catalytic efficiency for Ub-AMC and greater affinity for ISG15-AMC, with 8-fold lower Km values compared to PLpro alone. Lastly, each SARS-CoV-2 nsp3 construct was inhibited by a known PLpro inhibitor, GRL-0617, with reported IC50 values ranging from 0.91 μM to 1.9 μM. These data show that GRL-0617 still remains a lead compound to be optimized for cellular potency. </p> <p><br></p> <p>Overall, this dissertation advances the understanding of the kinetic and structural differences between Alphacoronavirus PLP2 and Betacoronavirus PLpro enzymes in the efforts of developing a pan-CoV inhibitor. Additionally, these data provide initial kinetic and biophysical characterization of PLpro within the larger context of nsp3 to elucidate the function of PLpro in its most native context during coronaviral infection.</p>

<p> In an era of advancing printed electronics, solution-processable organic semiconductors continue to make significant strides in electronic and optoelectronic applications. Electrochromic (EC) technology, which encompass reversible optical modulation under electrochemical biasing, has progressed rapidly over the past half-century and developed into niche commercial-scale devices for auto-tinting glasses as well as low-power, non-emissive displays. To utilize the advantages of organic electrochromic materials in next-generation devices, it is imperative to understand their fundamental material properties, interactions with other device components, and the underlying electrochemistry that governs the overall optical and electrochemical response of the complete electrochromic device. This dissertation presents a discussion on the synergistic role of organic electrochromes, charge-balancing layers and electrolytes in determining two key performance metrics, namely the optical contrast and operational stability, of an electrochromic device (ECD). The absorption features of colored-to-transmissive switching conjugated polymers have been investigated by exploring material design strategies in conjunction with analytical approaches to optimize and enhance the optical contrast. In parallel, transmissive redox-active radical polymer counter electrodes have been developed as compatible charge-balancing layers and integrated into devices by pairing with electrochromic polymers (ECPs) to achieve stable and high-contrast optical modulation. Electrochemical activity of both conjugated and radical polymer electrodes in different ionic and solvent environments have been further examined to understand material-electrolyte interactions governing mixed ionic-electronic conduction. Finally, a small molecular approach to realizing transparent-to-colored electrochromism is discussed, where distinct substituent-induced degradation pathways of conjugated radical cations were revealed. Overall, this research aims to assist future development of robust, ultra-high contrast organic electrochromic platforms.  </p>

<p dir="ltr">This dissertation delves into two research projects that aim to characterize post-translational modifications in two distinct proteins, each originating from a different species – one from the eukaryotic sea slug Aplysia californica and the other from the bacterial pathogen Bordetella bronchiseptica.</p><p dir="ltr">Aplysia have an unusually large neuron and therefore serve as an excellent model for studying cell signaling regulating neuronal chemotaxis. Cortactin is an actin binding protein that is regulated by post-translational modifications, including acetylation and phosphorylation. Studies have shown that Src2 tyrosine kinase phosphorylates cortactin to regulate lamellipodia protrusion and filopodia formation in Aplysia bag cell neurons. However, these in vivo phenotypes have not been tested mechanistically in vitro. To this end, the goal of my thesis work was to validate in vivo observations. The following work describes the methodology we developed to purify homogenous non-phosphorylated proteins. Our collaborative results show that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro.</p><p dir="ltr"> Filamentation induced by cAMP (Fic) proteins constitute a recently characterized family of enzymes that are being recognized to regulate diverse cellular processes in bacteria and metazoans. While Fic proteins predominantly utilize adenosine triphosphate (ATP) to post-translationally modify target proteins via a covalent addition of AMP, two Fic proteins have been reported that utilize uridine triphosphate (UTP) and cytidine diphosphate-choline (CDP-choline) to alter the activity of their target. In this dissertation, we report the discovery of the first guanosine triphosphate (GTP) specific Fic protein – BB0907 (BbFic) from Bordetella bronchiseptica. BbFic displays weak to no binding to ATP; instead has a 10-fold increased preferential usage for GTP. We identify key residues involved in GTP recognition. Additionally, sequence similarity network (SSN) analyses reveal that BbFic represents a distinct clade of Fic proteins, highlighting BbFic as a representative new class of guanylyltransferase. Our discovery adds to the functional diversity of the growing Fic protein family and frames the groundwork for understanding Fic-mediated GMPylation as a novel signaling paradigm. </p><p dir="ltr">Taken together, my thesis work provides novel insights into biological consequences of Fic-mediated GMPylation in bacteria and Src-mediated phosphorylation in filopodia formation.</p><p><br></p>

No description available.

<p>Systemic fungal infections brought about by <em>Cryptococcus</em> species are associated with some of the highest mortality rates of any infectious disease. Alarmingly these pathogens have overtaken tuberculosis as the second greatest killer among Sub-Saharan AIDS patients and are an emerging disease among immunocompetent populations on the Pacific Coast of North America. This clinical threat has been exacerbated by our inability to discover novel compounds that specifically target fungal cellular architecture at the genus level. To confront this challenge, we have made a concerted effort to biologically prospect the vast chemical potential of Actinomycete bacteria isolated from diverse and underexplored niches around the world. A novel phenotypic screen was developed whereby bacterial small molecule producers were co-cultured on agar plates in an intimate setting with evolutionary distant fungal pathogens <em>Candida albicans</em> and <em>Cryptococcus neoformans</em>. Diffusible small molecules released by the organisms created a signaling environment that stimulated profound phenotypic changes both in the Actinomycetes and the pathogens. We were able to discern a unique relationship whereby the growth of <em>C. neoformans</em> was specifically inhibited by Nigerian soil Actinomycete isolate curated as WAC 2288. Further bioactivity guided purification and chemical analysis lead to the identification of ibomycin, a previously undescribed 34 membered macrolactone decorated with seven sugar moieties. A draft genome of WAC 2288 revealed a 140kb gene cluster containing 12 type I PKS modules and downstream capacity to generate rare sugars are responsible for ibomycin biosynthesis. Purification of ibomycin analogs has revealed that the terminal vancosamine on the molecule is dispensable for bioactivity, establishing a chemical antecedent for target identification through affinity chromatography. Throughout these studies the unprecedented anticryptococcal activity of ibomycin is consistently recapitulated. Future work on the molecule may validate ibomycin as an effective antifungal therapy.</p> / Master of Science (MSc)

<p>Monoclonal antibodies (mAbs) as therapeutic proteins have shown great potential in treatment of various human diseases because of their highly specific nature. This has attracted worldwide attention leading to increased demand for such mAb products. To meet this demand large scale manufacturing is carried out using recombinant mammalian cell culture techniques for high yields and faster production. mAb products are worth the investment if produced in their native state. The quantity of mAb present in such cell cultures is very less and therefore special care is needed while handling them. Purifying antibody molecules from heterogeneous cell culture impurities and maintaining their native functional state is a critical task mainly because these antibodies are labile in nature. Care also need to be exercised during processing because mAbs have inherent tendancy to aggregate which is undesirable since such aggregates in antibody formulation produces immunogenic reaction when injected in humans. The other important factor in mAb purification is the processing cost involved since majority of the total production cost is utilized for purification of mAb. Protein-A chromatography is the first choice for purifying antibodies and is widely adopted. However failure in distinguishing between monomer and aggregate antibody molecules along with harsh acidic processing conditions necessitates the use of further purification steps.</p> <p>In this work various techniques for mAb processing are discussed and are outlined below:</p> <p>Removal of impurities from mAbs is a major challenge and this thesis discusses various processing options available to purify these mAbs. Impurities in mAb products are usually the aggregate byproducts formed due to unfolded monomer antibody molecules. These molecules are naturally hydrophobic in nature and display great differences in hydrophobicity on aggregation. Hydrophobic interaction membrane chromatography (HIMC) makes use of this hydrophobicity difference and helps in removal of aggregate impurities from monomer antibody.</p> <p>Heavy chain mAbs (hcmAbs) are promising new developments in the area of biopharmaceuticals because of their unique structural composition. Similar to conventional mAbs these hcmAbs are also rapidly finding their way into therapeutic markets. Purifying hcmAbs will be an important step in their development and for this purpose we use HIMC technique for removing impurities and obtain pure product.</p> <p>Antibody molecules are almost always lost as aggregates which leads to great economic losses and the ability to disaggregate these mAb oligomers would be of significant practical and scientific interest. In this work a novel thermalcycling technique is discussed to disaggregate such mAb oligomers and potentially recover functional monomer mAb molecules.</p> / Master of Applied Science (MASc)

<p>This dissertation describes how the surface chemistries of 2D nanomaterials can be modified to alter overall material properties. Specifically, through a focus of the ligand-surface atom bonding in addition to the overall ligand structure we highlight the ability to direct morphological outcomes in lead free halide perovskites, maximize optoelectronic responses in substoichiometric tungsten oxide, and alter physicochemical properties titanium carbide MXenes.   </p>

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques. / Fructose-1,6-bisphosphate aldolases (FBPA) are glycolytic enzymes (EC 4.1.2.13) that catalyze the reversible cleavage of fructose-1,6-bisphosphate (FBP) into the triose phosphates, glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). There are two classes of FBPAs that differ at the level of their mechanism. Class I FBPAs form a covalent iminium intermediate whereas class II FBPAs, being metalloenzymes, generally use a catalytic zinc in their reaction mechanism. In contrast to the mechanism of the class I FBPAs, which has been thoroughly studied, there are several unresolved inquiries as to the mechanism of class II FBPAs. We have therefore pursued a detailed analysis of the reaction mechanism using as a primary tool the elucidation of crystallographic structures. Several high resolution complexes have been resolved and have provided critical evidence to help us suggest the implication and role of several key residues in the active site. Consequently, we have correctly identified the residue which is responsible for the abstraction of the O4 proton from FBP, a vital step in the reaction mechanism. The residue responsible for this abstraction, which had incorrectly been assigned to Asp82 (in Helicobacter pylori), has been appropriately consigned to His180, a residue which is involved in coordinating the zinc molecule. Nevertheless, Asp82 remains an important residue as it orients, activates and stabilizes substrates. Finally, our study brings to evidence the dynamic character of our enzyme in which catalysis entails the relocalization of the catalytic zinc and several residues. The complexity of this reaction, notably one of the proton exchanges in the mechanism, could not be resolved solely by crystallographic means. In fact, the residue responsible for the stereospecific transfer of the pro(S) proton on carbon 3 (C3) of DHAP is situated on a loop that was not resolved in any of our structures. We therefore developed a molecular dynamics approach to study this intricate movement. After preliminary validation by inhibitor studies with class I FBPAs, the protocol was applied to class II FBPAs and several remarkable observations emerged: the residue responsible for this abstraction in Escherichia coli is Glu182 whereas a different residue, Glu149 (instead of Glu142) appears to assume this role in H. pylori. Our preliminary validations have confirmed this observation and shall be further consolidated in the future. Class II FBP aldolases, although absent from the human proteome, are prevalently found in several pathogens, and have further been found to be essential to a number of these organisms. As such, they are ideal targets for the development of novel anti-microbial agents. Developing new analogues of the cognate substrates of these enzymes is therefore not only advantageous for mechanistic studies, but has endless pharmacological potential. In the context of a collaborative effort involving a group of chemists, a compound that initially had an inhibition constant in the millimolar range was optimized and produced a series of compounds that inhibit in the nanomolar range.