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<p>This work used biotechnology to invent new caloxins - allosteric peptide inhibitors of plasma membrane Ca²⁺pumps (PMCA) needed to understand the Ca²⁺signalling in coronary artery.</p> <p>PMCA are encoded by genes PMCA1-4. Defects in PMCA expression have been associated with several pathologies. The major objectives of my thesis were to determine the expression of PMCA isoforms in the smooth muscle and the endothelium of coronary artery and to invent high affinity and specificity caloxins for the isoforms present in these tissues.</p> <p>In Aim 1 it was determined that the total PMCA protein and activity was much greater in smooth muscle than in endothelium. Both tissues expressed only PMCA1 and PMCA4, with PMCA4 > PMCA1 in smooth muscle and PMCA1 > PMCA4 in endothelium. Therefore, the search for PMCA1 and 4 selective caloxins using phage display technique was conducted.</p> <p>Aim 2 was to invent PMCA1 selective inhibitors. Caloxin 1b3 was invented as the first known PMCA1 selective inhibitor. It inhibited PMCA1 Ca²⁺-Mg²⁺-ATPase with higher affinity than PMCA2, 3 or 4. Aims 1 and 2 were consistent with the greater potency of caloxin 1b3 than a known PMCA4 selective caloxin 1b1 in increasing cytosolic Ca²⁺ concentration in endothelial cells.</p> <p>Aim 3 was to obtain ultrahigh selectivity and affinity PMCA4 bidentate inhibitor using the previously invented PMCA4 selective caloxins 1c2 and 1b2. In the first step the affinity of caloxin 1b2 was improved by limited mutagenesis to obtain caloxin 1c4. Caloxin 1c4 had 5-6 times higher affinity than caloxin 1b2 for inhibiting PMCA4 activity. Optimization of the bidentate caloxins from caloxin 1c2 and 1c4 was also attempted.</p> <p>The novel caloxins may aid in elucidating the role of PMCA1 and PMCA4 in the physiology and pathophysiology of coronary artery and other tissues.</p> / Doctor of Philosophy (PhD)

Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA. We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3. The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.

Polyclonal antibodies against the first two enzymes of the Arabidopsis thaliana flavonoid biosynthetic pathway were developed using conventional and phage antibody technology. cDNAs from Arabidopsis coding regions of chalcone synthase (CHS) and chalcone isomerase (CHI) were sub-cloned in frame into a bacterial expression vector as fusions with glutathione Stransferase (GST) using standard directional cloning techniques. Analysis of crude extracts of Escherichia coli containing GST .. CHS or GST .. CHI fusion protein indicated that the cells expressed equivalent amounts per volume of culture. CHS and CHI were purified to near homogeneity, yielding approximately 100 micrograms of GST .. CHS and 1 milligram of GST-CHI per liter of culture. The purified fusion proteins were injected into chickens and polyclonal lgY·s were purified from egg yolk Accumulation of CHS and CHI, as well as products of the pathway, were compared during the first eight days of Arabidopsis development. CHS and CHI are sequentially induced and reach maximal accumulation levels by day 5. Anthocyanidin levels are offset by one reaching maximal levels at day 6. The fusion proteins were also used to screen a phage-display library for Fabl fragments that recognize CHS and CHI epitopes. Preliminary data indicated that enrichment of phage displaying antibodies against CHS and CHI was successful. Phage-derived antibodies against CHS and CHI provide valuable tools for future experiments addressing Western blot analysis, immunolocalization experiments, and disruption of the flavonoid biosynthetic pathway by introduction of the corresponding genes into transgenic Arabidopsis plants. / Master of Science

Viruses have huge impact on controlling diseases and regulating many key ecosystem processes. As metagenomic data can contain many microbiomes including many viruses, by analyzing metagenomic data we can analyze many viruses at the same time. The first step towards analyzing metagenomic data is to identify and quantify viruses present in the data. In order to answer this question, we developed a computational pipeline, FastViromeExplorer. FastViromeExplorer leverages a pseudoalignment based approach, which is faster than the traditional alignment based approach to quickly align millions/billions of reads. Application of FastViromeExplorer on both human gut samples and environmental samples shows that our tool can successfully identify viruses and quantify the abundances of viruses quickly and accurately even for a large data set. As viruses are getting increased attention in recent times, most of the viruses are still unknown or uncategorized. To discover novel viruses from metagenomic data, we developed a computational pipeline named FVE-novel. FVE-novel leverages a hybrid of both reference based and de novo assembly approach to recover novel viruses from metagenomic data. By applying FVE-novel to an ocean metagenome sample, we successfully recovered two novel viruses and two different strains of known phages. Analysis of viral assemblies from metagenomic data reveals that viral assemblies often contain assembly errors like chimeric sequences which means more than one viral genomes are incorrectly assembled together. In order to identify and fix these types of assembly errors, we developed a computational tool called VirChecker. Our tool can identify and fix assembly errors due to chimeric assembly. VirChecker also extends the assembly as much as possible to complete it and then annotates the extended and improved assembly. Application of VirChecker to viral scaffolds collected from an ocean meatgenome sample shows that our tool successfully fixes the assembly errors and extends two novel virus genomes and two strains of known phage genomes. / Doctor of Philosophy / Virus, the most abundant micro-organism on earth has a profound impact on human health and environment. Analyzing metagenomic data for viruses has the beneFIt of analyzing many viruses at a time without the need of cultivating them in the lab environment. Here, in this dissertation, we addressed three research problems of analyzing viruses from metagenomic data. To analyze viruses in metagenomic data, the first question needs to answer is what viruses are there and at what quantity. To answer this question, we developed a computational pipeline, FastViromeExplorer. Our tool can identify viruses from metagenomic data and quantify the abundances of viruses present in the data quickly and accurately even for a large data set. To recover novel virus genomes from metagenomic data, we developed a computational pipeline named FVE-novel. By applying FVE-novel to an ocean metagenome sample, we successfully recovered two novel viruses and two strains of known phages. Examination of viral assemblies from metagenomic data reveals that due to the complex nature of metagenome data, viral assemblies often contain assembly errors and are incomplete. To solve this problem, we developed a computational pipeline, named VirChecker, to polish, extend and annotate viral assemblies. Application of VirChecker to virus genomes recovered from an ocean metagenome sample shows that our tool successfully extended and completed those virus genomes.

The CRISPR-Cas system is a bacterial adaptative immune system which protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers — fragments of the invading nucleic acids — into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, phages quickly take over the metabolism of the bacteria, leaving no time for the bacteria to acquire new spacers, transcribe them and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases eDNA which could be involved in the development of CRISPR immunity. Using S. thermophilus and phages 2972 and 858 as a model for CRISPR immunity, I show that eDNA is crucial to the development of optimal CRISPR immunity, as generation of phage-immune bacterial colonies decrease with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since its addition increases the generation of phage-immune colonies. I also show that the effect of eDNA is phage-specific, sequence specific and can even be traced to a region of the genome covering the early-expressed genes which differ between phages 2972 and 858. While the acquisition of CRISPR spacers is not random and while the supplementation of eDNA influences that bias, eDNA is not used as a source of genetic information for spacer acquisition. This suggests that the effect of eDNA involves a new mechanism of phage resistance. Moreover, the effect of eDNA is highly dependent on environmental conditions as variation in media suppliers are sufficient to interfere with this effect. These results link environmental conditions, specifically eDNA, to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect the presence of phages before infection. / Thesis / Doctor of Philosophy (PhD) / Phages are viruses that can infect and kill bacteria with a 99.9999% success rate. To defend themselves, bacteria have evolved an adaptive immune system called the CRISPR-Cas system. This system uses a piece of DNA, called a spacer, that matches the phage to destroy it. However, in order to use their CRISPR-Cas system, they need to obtain this spacer. Given how dangerous phages are, how bacteria acquire this spacer is a mystery. My project investigates the possibility that bacteria use DNA floating in the environment to vaccinate themselves against phages before ever encountering them. In this thesis I show that DNA floating in the environment helps bacteria acquire these spacers. I also show that it is specific sections of phage DNA that helps bacteria. This shows that bacteria can use their environment to defend themselves against threats before they even happen.