Yes / The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors. / Yorkshire Cancer Research, Wellcome Trust

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / African swine fever is a hemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV), a double-stranded DNA virus and the only member of the family Asfarviridae. The structure of this multilayer virion contains more than 34 proteins including the protein p72 which is the major capsid protein. A single conformational neutralizing epitope has been identified on p72, but information on the other antigenic regions (epitopes) is lacking. The objective of this study was to identify p72 epitopes using polyclonal swine sera and a panel of monoclonal antibodies with the ultimate goal being the development of a blocking ELISA assay for the detection of anti-ASFV antibodies. The segment of the p72 protein from amino acids 1 to 345 was divided into five overlapping fragments which were then commercially synthesized. These fragments were cloned into the pHUE expression vector and transformed into Escherichia coli competent cells. The recombinant proteins were expressed in vitro, purified, and used as antigens in indirect ELISAs and western blots to test monoclonal antibodies and polyclonal swine sera. The monoclonal antibodies were produced against the p72 protein based on the ASFV Georgia/07 strain. The polyclonal sera were obtained from pigs immunized with a defective alphavirus replicon particle, RP-sHA-p72, expressing a recombinant protein composed of the extracellular domain of the ASFV HA protein together with the whole p72 protein. The polyclonal sera reacted to p72 in two distinct regions: between amino acids 1 and 83 and between amino acids 250 and 280. The anti-p72 reactive monoclonal antibodies reacted with p72 in three regions: between amino acids 100 and 171, amino acids 180 and 250, and amino acids 280 and 345. Fine mapping with oligopeptides allowed for the identification of six different linear epitopes. Among the monoclonal antibodies selected for blocking assay development, two have been shown to be promising candidates for further evaluation using sera from ASFV-infected pigs.

No description available.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder, characterized by platelet destruction and/or underproduction. The pathophysiology is heterogeneous and can be mediated by autoantibodies and cytotoxic T lymphocytes (CTLs). While platelet destruction in ITP is well documented, there is little support for platelet underproduction due to the inhibition of megakaryocyte growth and considerably less support for CTL-mediated platelet underproduction. Our objective was to develop an assay that could test for CTL-mediated inhibition of megakaryocyte growth (megakaryopoiesis) in ITP, using healthy controls. Peripheral blood from healthy donors was used to prepare hematopoietic stem and progenitor cells (HSPCs). These cells were expanded with StemSpan to culture a large number of megakaryocytes for the CTL assay. Our studies show that CTLs can be stimulated in-vitro using anti-CD3 antibodies and that they can be used after freezing and thawing. We also assessed CTL stimulation via peptide presentation, using viral peptides whom almost 100% of the general population have memory CTL specificity to, in order to activate a lower frequency of CTLs and to model levels of CTL activation in autoimmune disease. Both stimulants were found to stimulate CTLs in healthy donors with donor variability in the IFN-γ ELISpot. The CTL assay was developed by co-culturing thrombopoietin (TPO) stimulated HSPCs with autologous CTLs for 7 days to observe inhibition of megakaryocyte growth. To induce CTL stimulation, CTLs were either incubated with anti-CD3 or HSPCs were incubated with viral peptides before co-culturing with CTLs. Results showed that while viral peptides can be used as an internal control for the CTL assay, it could not serve as a positive control as inhibition was donor dependent. Inhibition of megakaryocyte growth in the presence of anti-CD3 stimulated CTLs was observed in all donors, validating its use as an appropriate positive control to study CD8+ T cell pathophysiology in ITP. / Thesis / Master of Science (MSc)

Thesis advisor: Tim van Opijnen / Thesis advisor: Michelle Meyer / Although bacteria are often studied as planktonic or free-living organisms, they frequently grow in complex surface-attached communities known as biofilms. Biofilms are communities of microorganisms attached to surfaces and embedded in a self-produced extracellular matrix. Biofilms are dynamic structures analogous to human settlements shaped by space and environment. These microbial communities fulfill critical roles in multiple infections in the human body. Streptococcuspneumoniae is a human pathogen that can cause biofilm-associated infections in various tissues and organs. This thesis offers a unique outlook for the study of S. pneumoniae biofilms by combining in vitro, genome-wide, and in vivo experiments to elucidate the complex population dynamics of S. pneumoniae biofilms. Existing methods to cultivate S. pneumoniae biofilms fail to fully capture the complexity of these communities, and most studies are limited to short periods of time. We developed a robust in vitro assay to grow S. pneumoniae biofilms. This assay can be maintained forever rather than days. We then use this robust assay to study their behavior in vivo and monitor disease outcomes. After establishing clear differences in biofilm and dispersal samples, we monitor population dynamics using genome-wide techniques (Tn-seq, RNA-seq and WGS) to provide some insights into this complex mode of growth. This work includes the first global identification of genetic requirements during biofilm establishment in two different S. pneumoniae strains using Tn-Seq. Coupled with our transcriptomic analysis, we found that genes involved in multiple pathways, such as capsule biosynthesis, nucleotide metabolism, and stress response, contributed to biofilm growth. Lastly, we studied the development of antibiotic resistance to three different types of antibiotics under S. pneumoniae biofilm conditions. We revealed common adaptive pathways to achieve biofilm growth and antibiotic resistance (antibiotic target genes), as well as novel routes of adaptation to develop resistance. Our findings add to the growing body of knowledge in the field of bacterial genetics and antimicrobial resistance, paving the way for future research and therapeutic advancement. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Clostridioides difficile is a Gram positive, spore-producing, anaerobic bacterium. It is considered a nosocomial pathogen due to high incidence rates of C. difficile infections (CDI) in hospitals. However, research reveals an increase in community-associated cases. CDI is most common in the elderly, immunocompromised, or those taking a course of antibiotics. These individuals are more vulnerable to experiencing gut dysbiosis, allowing C. difficile to colonize the colon. CDI are an urgent threat due to their ability to sporulate. Spores are hardy and not eradicated by common disinfectants. They persist on surfaces the patient may have contact with during CDI. Current decontamination methods include the utilization of bleach-based disinfectants followed by ultraviolet (UV) light and adherence to a strict quality control protocol. However, spores may remain in the environment even after this process, thus allowing the pathogen to spread through surface contact. Another problem is the cost incurred by the hospital over time in terms of inpatient and equipment turnover related costs. These reasons make it imperative a better means of disinfection is developed. Silver is a known antimicrobial agent and utilized in clinical settings for burn treatment. Cerium oxide nanoparticles (CNP) possess antioxidant properties and can be used for drug delivery. Preliminary studies show silver-modified CNP (AgCNP) possess antiviral activity against COVID-19 and rhinovirus. To study the antimicrobial effects of the AgCNP against vegetative cells, a three-day time-kill assay was performed. Two strains of C. difficile, R20291 and NAP1, were cultured in BHIS (brain heart infusion, supplemented) and grown overnight. Glass coupons coated with AgCNP1 or AgCNP3, CNP1 or CNP3, or left uncoated were used in this study. They were incubated with cell culture at a variety of time points. The results indicate AgCNP3 may possess bactericidal activity. Further research should be conducted to determine the extent of this activity.

<p>The obligate intracellular pathogen, Chlamydia trachomatis, is becoming an ever greater public health threat worldwide. Despite aggressive public health awareness campaigns and treatment with antibiotics, chlamydial infections continue to be the most frequently reported sexually transmitted infection in the United States and the cause of 3% of worldwide blindness. While research into understanding various mechanisms of chlamydial pathogenesis is ongoing, efforts to identify critical protein targets are hampered by the lack of facile genetic manipulation systems available for Chlamydia. Without the ability to perform genetic studies, researchers have employed chemical biology tools to close the gap in understanding how Chlamydia survives and thrives in the host cell.</p><p>Chlamydial protease-like activity factor (CPAF) has been identified as a central virulence factor in chlamydial pathogenesis. Several studies have indicated a role for CPAF-mediated degradation of host proteins in the late stages of infection. CPAF is hypothesized to interfere with myriad host cell processes, including inflammation, cell proliferation, cytoskeletal development, and immunity presentation. However, recent studies have called into question the methods used to previously identify bona fide in vivo CPAF targets, as CPAF has been shown to retain proteolytic activity even in the presence of broad spectrum protease inhibitors. As a result of these new finding, there is a renewed call to carefully identify CPAF substrates using methods that ensure total inhibition of post-lysis proteolysis.</p><p>This dissertation aims to clarify the role of CPAF in chlamydial pathogenesis and to identify mechanisms by which CPAF exhibits substrate specificity. Because enzymes can manifest specificity through kinetic mechanisms, sequence recognition, secondary site substrate binding, or protein structure level specificity, multiple methods of biochemical characterization were employed to distinguish between these modes of specificity. </p><p>Optimized HPLC-based and fluorescence quenching assays were developed and used to investigate the chemical and kinetic mechanism of CPAF proteolysis, as well as to characterize CPAF resistance to broad spectrum protease inhibitors. Peptide library proteomics were designed to probe active site sequence recognition of specific amino acids. Bioinformatic approaches were used to recognize and annotate a cryptic PDZ-like domain in CPAF, which bears strong structural similarity to human epithelial tight junction proteins. Using a new endocervical cellular model of infection, a recently developed C. trachomatis mutant lacking CPAF activity was investigated. Mass spectrometry proteomics analysis was employed to detect differential cleavage of host proteins in endocervical cells infected with CPAF+ and CPAF- strains of C. trachomatis. Lastly, methods for N-terminal labeling and enrichment were adapted for further identifying CPAF substrates in a cellular infection model. The subtiligase system for biotinylation of N-terminal amines was adapted for integration with C. trachomatis infection assays and downstream mass spectrometry proteomics. Ultimately, the dissertation offers clarification of the role of CPAF in chlamydial infection and provides chemical biology tools for further study of protease function in bacterial pathogenesis.</p> / Dissertation

This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.

Broadly speaking, the work herein discussed encompasses the development of biomolecular assays for biomarker detection. Specific to the assays in this thesis is the design of reaction schemes that consider the unique requirements of one class of single-molecule sensors in particular: solid-state nanopores formed using a novel fabrication and conditioning technique discovered during this research at the University of Ottawa. We present three unique assays for the detection of different biomolecular targets. The first uses a class of DNA origami structures termed nanoswitches to translate the presence of a short segment of single-stranded DNA Zika virus biomarker to a large configurational change in a double-stranded DNA scaffold. The signal amplification inherent in this topological change allowed us to a achieve a high degree of specificity for detecting a small nucleic acid target by requiring two separate binding events. Furthermore, through careful design of the configurational change, the number of topological states that a solid-state nanopore can sense is limited, providing unambiguous signals in ionic current recordings. Quantification of the Zika gene was performed by sensing the relative amounts of nanoswitches in looped and linear configurations from only hundreds of individual molecules. We then explored the sensitivity of solid-state nanopores for detecting small molecular features along short DNA scaffolds. Leveraging the ability of our nanopores to detect the presence of these protrusions, we present results in which ATP, a molecule significantly too small to be directly detected by the nanopore sensor, initiated an aptamer-based DNA displacement reaction to form a protrusion along scaffolds, producing measurable changes in ionic current signatures in nanopore recordings. Finally, we present an assay in which a microRNA, a biomarker linked to various cancers, was detected through the conjugation of two probes, each of which contained a binding site to different segments of the microRNA. In addition to examining different probe set structures for optimal performance, our two-probe design aimed to improve specificity over conventional single-probe-based assays which only require one recognition step, while still providing unambiguous signals due to the greater-than-doubling in molecular complex size upon conjugation. Furthermore, the use of two individual small probes, rather than one large nanoswitch, increased the resolution with which we could differentiate microRNA concentrations. The assay enabled the quantification of six concentrations of microRNA spanning a single order of magnitude, in only several hundred events, and allowed us to take advantage of the reduced cost, material and labour, as well as increased nanopore capture rates, associated with small assembled molecules.

Altered signaling pathways, which are mediated by post-translational modifications and changes in protein expression levels, are key regulators of cancer initiation, progression, and therapeutic escape. Many aspects of cancer progression, including early carcinogenesis and immediate response to drug treatment, are beyond the scope of genetic profiling and non-invasive monitoring techniques. Global protein profiling of cancer cell line models, tumor tissues, and biofluids (e.g. serum or urine) using mass spectrometry-based proteomics produces novel biological insights, which support improved patient outcomes. Recent technological advances resulting in next-generation mass spectrometry instrumentation and improved bioinformatics workflows have led to unprecedented measurement reproducibility as well as increased depth and coverage of the human proteome. It is now possible to interrogate the cancer proteome with quantitative proteomics to identify prognostic cancer biomarkers, stratify patients for treatment, identify new therapeutic targets, and elucidate drug resistance mechanisms. There are, however, numerous challenges associated with protein measurements. Biological samples have a high level of complexity and wide dynamic range, which is even more pronounced in samples used for non-invasive disease monitoring, such as serum. Cancer biomarkers are generally found in low abundance compared to other serum proteins, particularly at early stages of disease where cancer detection would make the biggest impact on improving patient survival. Additionally, the large-scale datasets generally require bioinformatics expertise to produce useful biological insights. These difficulties converge to create obstacles for down-stream clinical translation. This dissertation research demonstrates how proteomics is applied to develop new resources and generate novel workflows to improve protein quantification in complex biosamples, which could improve translation of cancer research to benefit patient care. The studies described in this dissertation move from assessment of quantitative mass spectrometry platforms, to analytical assay development and validation, and ending with personalized biomarker development applied to patient samples. As an example, four different quantitative mass spectrometry acquisition platforms are explored and comparisons of their ability to quantify low abundance peptides in a complex background are explored. Lung cancers frequently have aberrant signaling resulting in increased kinase activity and targetable signaling hubs; kinase inhibitors have been successfully developed and implemented clinically. Therefore, changes in amounts of kinase peptides in the complex background of peptides from all ATP-utilizing enzymes in a lung cancer cell line model after kinase inhibitor treatment was selected as a model system. Traditional mass spectrometry platforms, data dependent acquisition and multiple reaction monitoring, are compared to the two newer methods, data independent acquisition and parallel reaction monitoring. Relative quantification is performed across the four methods and analytical performance as well as downstream applications, including drug target identification and elucidation of signaling changes. Liquid chromatography – multiple reaction monitoring (LC-MRM) was selected for development of multiplexed quantitative assays based on superior sensitivity and fast analysis times, allowing for larger peptide panels. Method comparison results also provide guidelines for quantitative proteomics platform selection for translational cancer researchers. Next, a multiplexed quantitative LC-MRM assay targeting a panel of 30 RAS signaling proteins was developed and described. Over 30% of all human cancers have a RAS mutation and these cancers are generally aggressive and limited treatment options, leading to poor patient prognosis. Many targeted inhibitors have successfully shut down RAS signaling, leading to tumor regression, however, acquired drug resistance is common. The multiplexed LC-MRM assays characterized and validated are a publically available resource for cancer researchers to interrogate the RAS signal transduction network. Feasibility has been demonstrated in cell line models in order to identify signaling changes that confer BRAF inhibitor resistance and biomarkers of sensitivity to treatment. This analytical LC-MRM panel could support meaningful development of new therapeutic options and identification of companion biomarkers, with the end goal of improving patient outcomes. Multiplexed LC-MRM assays developed for personalized disease biomarkers using an integrated multi-omics approach are described for Multiple Myeloma, an incurable malignancy with poor patient outcomes. This disease is characterized by clonal expansion of the plasma cells in the bone marrow, which secrete a monoclonal immunoglobulin, or M-protein. Clinical treatment decisions are based on multiple semi-quantitative assays that require manual evaluation. In the clinic, minimal residual disease quantification methods, including multi-parameter flow cytometry and immunohistochemistry, are applied to bone marrow aspirates, which is a highly invasive technique that does not provide a systemic evaluation of the disease. To address these issues, we hypothesized that unique variable region peptides could be identified and LC-MRM assays developed specific to each patient’s M-protein to improve specificity and sensitivity in non-invasive disease monitoring. A proteogenomics approach was used to design personalized assays for each patient to monitor their disease progression, which demonstrate improved specificity and up to a 500-fold increase in sensitivity compared to current clinical methods. Assays can be developed from marrow aspirates collected when the patient was at residual disease stage, which is useful if no sample with high disease burden is available. The patient-specific tests are also multiplexed with constant region peptide assays that monitor all immunoglobulin heavy and light chain classes, which could reduce analysis to a single test. In conclusion, highly sensitive patient-specific assays have been developed that could change the paradigm for patient evaluation and clinical decision-making, increasing the ability of clinicians to continue first line therapy in the hopes of achieving a cure, or to intervene at an earlier time point in disease recurrence. This study also provides a blueprint for future development of personalized diagnostics, which could be applied to biomarkers of other cancer types. Overall, these studies demonstrate how quantitative proteomics can be used to support translational cancer research, from the impact of different mass spectrometry platforms on elucidating signaling changes and drug targets to the characterization of multiplexed LC-MRM assays applied to cell line models for translational research purposes and in patient serum samples optimized for clinical translation. We believe that mass spectrometry-based proteomics is poised to play a pivotal role in personalized diagnostics to support implementation of precision medicine, an effort that will improve the quality and efficiency of patient care.