PFAS is a relatively new group of contaminants with unique characteristics, which in the early 21st century was understood being dangerous for both humans and the environment. In 2008, EFSA published a report on guidelines for human intake of PFAS. Target and limit values for ground and surface water around the world has been based on the information in the EFSA report. In the end of 2018, EFSA published a new preliminary report with new target values for PFAS, well below the target values published in 2008. Frösö Park in Östersund, Sweden, is polluted by PFAS from the time that the Swedish Armed Forces were active in the area. While the Swedish Armed Forces exercised their activities at Frösö Park, large amounts of aqueous fire-fighting foams were used, mainly for training purposes. AFFF at that time contained a mixture of many highly fluorinated chemicals known as PFAS, a collective name of more than 4,700 chemicals consisting of carbon-fluorine bonds. PFAS are, more or less, persistent, bioaccumulative and toxic. This study focuses on PFAS11, which Sweden has target and limit values for in respect of ground water and surface water (lake and sea). PFOS is the most common PFAS chemical and the most commonly occurring PFAS chemical at the Frösö Park area. Today, there is a combined urban runoff and waste water network at the Frösö Park area. The internal water conduit system is currently being examined in order to, eventually, disconnect the urban runoff from the waste water network in order to instead release the urban runoff to Storsjön in the immediate area. As the urban runoff has high levels of PFAS, it must be purified before it is discharged to the recipient. High levels of PFAS has been found in the sewage treatment plant. The sewage treatment plant is not able to purify the water from PFAS, which means that the pollution is discharged into Östersund’s drinking water source, Storsjön. The Municipality of Östersund wanted this thesis as the study will include newsworthy information and provide the municipality more knowledge about the PFAS issues in the Frösö Park area. The aim of this thesis is to examine how the urban runoff from the Frösö Park area can be handled to prevent PFAS leaking out in Östersund’s drinking water source, Storsjön. The thesis describes in a comprehensive way how different purification methods work and the function of the different methods based on the conditions that exist in the Frösö Park area. The purification methods for urban runoff are sedimentation methods, biofilters, and how additives with chemicals can affect the purification of urban runoff. After the urban runoff purification, the purification steps focused on PFAS are sorption methods, chemical redox methods, membrane methods and excavation methods. Based on previous reports for the Frösö Park area, existing data has been compiled into maps, figures and diagrams in order to clearly describe the current PFAS situation. The scientific literature presented herein has been selected by specific keywords in databases. The literature has been supplemented with materials provided by the municipality, tips from researchers and personal contact with other industry-related actors. In the purification steps focused on purifying particles, organic materials and metals in urban runoff, a barrier that restricts the flow of water is proposed, tentatively a dam, wetland or lamellar sedimentation, followed by sand filtration. A large advantage with a barrier restricting the flow of water is the possibility to control the water flow to the next purification step. In the purification steps focused on purifying the water from PFAS, purification with activated carbon, nanofiltration, ion exchange method or sonochemical oxidation are proposed. The purification methods are proposed because of the existing knowledge of the methods and the pollution situation for the Frösö Park area. The research for PFAS with new purification methods, target and limit values for humans and the nature as well as future costs for decontamination and health-related costs means that PFAS currently is a priority contaminant taken seriously. Advantages and disadvantages of the purification methods are presented herein, however, the issues with PFAS are very complex and the purification methods work differently depending on the conditions they are exposed to. In this thesis, the most interesting new information regarding PFAS has been compiled to show the current knowledge situation in order to facilitate for relevant actors to continue their work with the PFAS issues in the future.

Esporos de Bacillus subtilis apresentam propriedades adjuvantes, sendo capazes de aumentar a resposta humoral após a sua coadministração com antígenos misturados ou adsorvidos à sua superfície. Mas, para isso, é necessária a produção de esporos altamente purificados e com rendimentos elevados. Neste trabalho, realizamos com sucesso uma análise quantitativa das condições de esporulação e dos métodos de purificação, o que melhorou a reprodutibilidade do processo e a obtenção de amostras com elevado grau de pureza e rendimento. Avaliamos também as propriedades imunomodulatórias destes esporos, utilizando como antígeno modelo a proteína recombinante Gag-p24 do HIV-1. A coadministração, mas não a adsorção à superfície do esporo, aumentou a imunogenicidade do antígeno sem induzir efeitos deletérios após a administração parenteral em camundongos BALB/c e C57BL/6. Além de promoveram a ativação das APCs, os esporos interagem com receptores relacionados à imunidade inata, devido à ausência do efeito adjuvante em camundongos nocautes para TLR2. Esses resultados abrem perspectivas interessantes para a utilização de esporos como adjuvantes vacinais. / Bacillus subtilis spores have been shown to behave as vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. Nonetheless, such specialized application requires highly purified spore preparations at high yields. In this work, we successfully performed a systematic quantitative analysis of sporulation conditions and spore purification methods, which improved the reproducibility of the process and the obtainment of samples with high purity and yield. Afterwards, we further evaluated the immune modulatory properties of these spores using a recombinant HIV-1 Gag-p24 protein as a model antigen. The co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen, without inducing deleterious effects, after subcutaneous administration to BALB/c and C57BL/6 mice. Besides promoting activation of antigen presenting cells, spores interact with receptors related to innate immunity, due to the absence of the adjuvant effect on TLR2 knockout mice. These results open interesting perspectives for the use of B. subtilis spores as vaccine adjuvants.

Esporos de Bacillus subtilis apresentam propriedades adjuvantes, sendo capazes de aumentar a resposta humoral após a sua coadministração com antígenos misturados ou adsorvidos à sua superfície. Mas, para isso, é necessária a produção de esporos altamente purificados e com rendimentos elevados. Neste trabalho, realizamos com sucesso uma análise quantitativa das condições de esporulação e dos métodos de purificação, o que melhorou a reprodutibilidade do processo e a obtenção de amostras com elevado grau de pureza e rendimento. Avaliamos também as propriedades imunomodulatórias destes esporos, utilizando como antígeno modelo a proteína recombinante Gag-p24 do HIV-1. A coadministração, mas não a adsorção à superfície do esporo, aumentou a imunogenicidade do antígeno sem induzir efeitos deletérios após a administração parenteral em camundongos BALB/c e C57BL/6. Além de promoveram a ativação das APCs, os esporos interagem com receptores relacionados à imunidade inata, devido à ausência do efeito adjuvante em camundongos nocautes para TLR2. Esses resultados abrem perspectivas interessantes para a utilização de esporos como adjuvantes vacinais. / Bacillus subtilis spores have been shown to behave as vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. Nonetheless, such specialized application requires highly purified spore preparations at high yields. In this work, we successfully performed a systematic quantitative analysis of sporulation conditions and spore purification methods, which improved the reproducibility of the process and the obtainment of samples with high purity and yield. Afterwards, we further evaluated the immune modulatory properties of these spores using a recombinant HIV-1 Gag-p24 protein as a model antigen. The co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen, without inducing deleterious effects, after subcutaneous administration to BALB/c and C57BL/6 mice. Besides promoting activation of antigen presenting cells, spores interact with receptors related to innate immunity, due to the absence of the adjuvant effect on TLR2 knockout mice. These results open interesting perspectives for the use of B. subtilis spores as vaccine adjuvants.

Cheng Yee Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 95-112). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Figures --- p.xi / List of Plates --- p.xiv / List of Tables --- p.xvi / Abbreviations --- p.xviii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Legionella pneumophila --- p.1 / Chapter 1.1.1 --- Bacterial morphology and ultrastructure --- p.2 / Chapter 1.1.2 --- Microbial ecology and natural habitats --- p.4 / Chapter 1.1.2.1 --- Association with amoeba --- p.5 / Chapter 1.1.2.2 --- Association with biofilm --- p.5 / Chapter 1.2 --- Legionnaires' disease and clinical significance --- p.6 / Chapter 1.2.1 --- Epidemiology --- p.6 / Chapter 1.2.1.1 --- Worldwide distribution --- p.6 / Chapter 1.2.1.2 --- Local situation --- p.7 / Chapter 1.2.2 --- Clinical presentation --- p.7 / Chapter 1.2.3 --- Route of infection and pathogenesis --- p.8 / Chapter 1.2.4 --- Diagnosis --- p.10 / Chapter 1.2.4.1 --- Culture of Legionella --- p.10 / Chapter 1.2.4.2 --- Direct fluorescent antibody (DFA) staining --- p.13 / Chapter 1.2.4.3 --- Serologic tests --- p.13 / Chapter 1.2.4.4 --- Urine antigen testing --- p.14 / Chapter 1.2.4.5 --- Detection of Legionella nucleic acid --- p.15 / Chapter 1.2.5 --- Risk factors --- p.15 / Chapter 1.2.6 --- Treatment for Legionella infection --- p.16 / Chapter 1.3 --- Detection of Legionella in environment --- p.16 / Chapter 1.4 --- Disinfection methods --- p.17 / Chapter 1.4.1 --- Physical methods --- p.19 / Chapter 1.4.1.1 --- Filtration --- p.19 / Chapter 1.4.1.2 --- UV-C irradiation --- p.20 / Chapter 1.4.1.3 --- Thermal eradication (superheat-and-flush) --- p.21 / Chapter 1.4.2 --- Chemical methods --- p.21 / Chapter 1.4.2.1 --- Chlorination --- p.21 / Chapter 1.4.2.2 --- Copper-silver ionization --- p.22 / Chapter 1.4.3 --- Effect of biofilm and other factors on disinfection --- p.23 / Chapter 1.5 --- Photocatalytic oxidation (PCO) --- p.24 / Chapter 1.5.1 --- Generation of strong oxidants --- p.24 / Chapter 1.5.2 --- Disinfection mechanism(s) --- p.27 / Chapter 1.5.3 --- Major factors affecting the process --- p.28 / Chapter 2. --- Objectives --- p.30 / Chapter 3. --- Materials and Methods --- p.31 / Chapter 3.1 --- Chemicals --- p.31 / Chapter 3.2 --- Bacterial strains and culture --- p.31 / Chapter 3.3 --- Photocatalytic reactor --- p.33 / Chapter 3.4 --- PCO efficacy tests --- p.33 / Chapter 3.5 --- PCO sensitivity tests --- p.35 / Chapter 3.6 --- Optimisation of PCO conditions --- p.35 / Chapter 3.6.1 --- Optimization of TiO2 concentration --- p.36 / Chapter 3.6.2 --- Optimization of UV intensity --- p.36 / Chapter 3.6.3 --- Optimization of depth of reaction mixture --- p.36 / Chapter 3.6.4 --- Optimization of stirring rate --- p.37 / Chapter 3.6.5 --- Optimization of initial pH --- p.37 / Chapter 3.6.6 --- Optimization of treatment time and initial cell concentration --- p.37 / Chapter 3.6.7 --- Combinational optimization --- p.37 / Chapter 3.7 --- Transmission electron microscopy (TEM) --- p.38 / Chapter 3.8 --- Fatty acid profile analysis --- p.40 / Chapter 3.9 --- Total organic carbon (TOC) analysis --- p.42 / Chapter 3.10 --- UV-C irradiation --- p.44 / Chapter 3.11 --- Hyperchlorination --- p.44 / Chapter 3.12 --- Statistical analysis and replication --- p.45 / Chapter 3.13 --- Safety precautions --- p.45 / Chapter 4. --- Results --- p.46 / Chapter 4.1 --- Efficacy test --- p.46 / Chapter 4.2 --- PCO sensitivity --- p.47 / Chapter 4.3 --- Optimization of PCO conditions --- p.48 / Chapter 4.3.1 --- TiO2 concentration --- p.48 / Chapter 4.3.2 --- UV intensity --- p.48 / Chapter 4.3.3 --- Depth of reaction mixture --- p.51 / Chapter 4.3.4 --- Stirring rate --- p.56 / Chapter 4.3.5 --- Effect of initial pH --- p.56 / Chapter 4.3.6 --- Effect of treatment time and initial concentrations --- p.56 / Chapter 4.3.7 --- Combinational effects --- p.63 / Chapter 4.4 --- Transmission electron microscopy (TEM) --- p.66 / Chapter 4.4.1 --- Morphological changes induced by PCO --- p.66 / Chapter 4.4.2 --- Comparisons with changes caused by UV-C irradiation and chlorination --- p.67 / Chapter 4.5 --- Fatty acid profile analysis --- p.71 / Chapter 4.6 --- Total organic carbon (TOC) analysis --- p.73 / Chapter 4.7 --- UV-C irradiation --- p.74 / Chapter 4.8 --- Hyperchlorination --- p.74 / Chapter 5. --- Discussion --- p.76 / Chapter 5.1 --- Efficacy test --- p.76 / Chapter 5.2 --- PCO sensitivity --- p.76 / Chapter 5.3 --- Optimization of PCO conditions --- p.77 / Chapter 5.3.1 --- Effect of TiO2 concentration --- p.77 / Chapter 5.3.2 --- Effect of UV intensity --- p.78 / Chapter 5.3.3 --- Effect of depth of reaction mixture --- p.79 / Chapter 5.3.4 --- Effect of stirring rate --- p.79 / Chapter 5.3.5 --- Effect of initial pH --- p.80 / Chapter 5.3.6 --- Effect of treatment time and initial concentrations --- p.81 / Chapter 5.3.7 --- Combinational effect --- p.82 / Chapter 5.4 --- Transmission electron microscopy (TEM) --- p.83 / Chapter 5.4.1 --- Morphological changes induced by PCO --- p.83 / Chapter 5.4.2 --- Comparisons with changes caused by UV-C irradiation and chlorination --- p.85 / Chapter 5.5 --- Fatty acid profile analysis --- p.85 / Chapter 5.6 --- Total organic carbon (TOC) analysis --- p.86 / Chapter 5.7 --- Comparisons of the three disinfection methods --- p.88 / Chapter 6. --- Conclusion --- p.91 / Chapter 7. --- References --- p.95 / Chapter 8. --- Appendix --- p.113

Philosophiae Doctor - PhD / Tumour-specific protein markers are usually present at elevated concentrations in patient biopsy tissue; therefore tumour tissue is an ideal biological material for studying cancer proteomics and biomarker discovery studies. To understand and elucidate cancer pathogenesis and its mechanisms at the molecular level, the collection and characterisation of a large number of individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived, FFPE tissues are important collections of pathology material, often accompanied by important metadata, such as patient medical history and treatments. FFPE tissue blocks are conveniently stored under ambient conditions for decades, while retaining cellular morphology due to the modifications induced by formalin. / 2022

Wong Man Yung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 106-120). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Figures --- p.xi / List of Plates --- p.xiii / List of Tables --- p.xv / Abbreviations --- p.xvi / Equations --- p.xviii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Water disinfection --- p.1 / Chapter 1.2 --- Bacterial species --- p.2 / Chapter 1.2.1 --- Staphylococcus saprophyticus --- p.2 / Chapter 1.2.2 --- Enterobacter cloacae --- p.3 / Chapter 1.3 --- Disinfection methods --- p.4 / Chapter 1.3.1 --- Physical methods --- p.4 / Chapter 1.3.1.1 --- UV-C irradiation --- p.4 / Chapter 1.3.1.2 --- Solar disinfection --- p.5 / Chapter 1.3.2 --- Chemical methods --- p.6 / Chapter 1.3.2.1 --- Chlorination --- p.6 / Chapter 1.3.2.2 --- Ozonation --- p.7 / Chapter 1.3.2.3 --- Mixed disinfectants --- p.8 / Chapter 1.3.3 --- Other disinfection methods --- p.8 / Chapter 1.4 --- Advanced oxidation processes (AOPs) --- p.9 / Chapter 1.5 --- Photocatalytic oxidation (PCO) --- p.10 / Chapter 1.5.1 --- PCO process --- p.12 / Chapter 1.5.2 --- Photocatalysts --- p.14 / Chapter 1.5.2.1 --- Titanium dioxide (P25) --- p.15 / Chapter 1.5.2.2 --- Silver sensitized P25 (Ag/P25) --- p.16 / Chapter 1.5.2.3 --- Silicon dioxide doped titanium dioxide (SiO2-TiO2) --- p.17 / Chapter 1.5.2.4 --- Copper(I) oxide sensitized P25 (Cu2O/P25) --- p.18 / Chapter 1.5.3 --- Irradiation sources --- p.19 / Chapter 1.5.4 --- PCO disinfection mechanisms --- p.20 / Chapter 1.6 --- Bacterial defense mechanisms against oxidative stress --- p.22 / Chapter 2. --- Objectives --- p.25 / Chapter 3. --- Materials and Methods --- p.26 / Chapter 3.1 --- Chemicals --- p.26 / Chapter 3.2 --- Bacterial culture --- p.26 / Chapter 3.3 --- Photocatalytic reactor --- p.27 / Chapter 3.4 --- PCO efficacy test --- p.30 / Chapter 3.5 --- Optimization of PCO conditions --- p.31 / Chapter 3.5.1 --- Effect of P25 concentrations --- p.31 / Chapter 3.5.2 --- Effect of UV intensities --- p.32 / Chapter 3.5.3 --- Combinational study of P25 concentrations and UV intensities --- p.32 / Chapter 3.5.4 --- Effect of stirring rates --- p.32 / Chapter 3.5.5 --- Effect of initial cell concentrations --- p.33 / Chapter 3.6 --- PCO disinfection using different photocatalysts --- p.33 / Chapter 3.6.1 --- Effect of CU2O/P25 concentrations --- p.33 / Chapter 3.6.2 --- Effect of CU2O powder on the two bacterial species --- p.33 / Chapter 3.7 --- Transmission electron microscopy (TEM) --- p.34 / Chapter 3.8 --- Catalase (CAT) test --- p.37 / Chapter 3.9 --- Superoxide dismutase (SOD) activity assay --- p.39 / Chapter 4. --- Results --- p.40 / Chapter 4.1 --- Efficacy test --- p.40 / Chapter 4.2 --- PCO disinfection under UV irradiation --- p.40 / Chapter 4.2.1 --- Control experiments --- p.40 / Chapter 4.2.2 --- Optimization of PCO conditions using P25 as a photocatalyst --- p.42 / Chapter 4.2.2.1 --- Effect of P25 concentrations --- p.42 / Chapter 4.2.2.2 --- Effect of UV intensities --- p.45 / Chapter 4.2.2.3 --- Combinational study of P25 concentrations and UV intensities --- p.48 / Chapter 4.2.2.4 --- Effect of stirring rates --- p.54 / Chapter 4.2.2.5 --- Effect of initial cell concentrations --- p.57 / Chapter 4.2.3 --- Comparison of PCO inactivation efficiency between S. saprophyticus and E. cloacae --- p.60 / Chapter 4.2.4 --- PCO disinfection using different photocatalysts --- p.62 / Chapter 4.2.4.1 --- Control experiments --- p.62 / Chapter 4.2.4.2 --- Ag/P25 --- p.62 / Chapter 4.2.4.3 --- SiO2-TiO2 --- p.64 / Chapter 4.2.4.4 --- Cu2O/P25 --- p.64 / Chapter 4.3 --- PCO disinfection under visible light irradiation --- p.66 / Chapter 4.3.1 --- Effect of Cu2O/P25 concentrations --- p.67 / Chapter 4.3.2 --- Effect of CU2O powder on the two bacterial species --- p.70 / Chapter 4.4 --- Feasibility use of indoor light (fluorescent lamps) for PCO disinfection --- p.71 / Chapter 4.5 --- Transmission electron microscopy (TEM) --- p.74 / Chapter 4.5.1 --- Morphological changes induced by PCO using P25 as a photocatalyst --- p.74 / Chapter 4.5.2 --- Morphological changes induced by PCO using Cu2O/P25 as a photocatalyst --- p.77 / Chapter 4.6 --- Catalase (CAT) test --- p.80 / Chapter 4.7 --- Superoxide dismutase (SOD) activity assay --- p.82 / Chapter 5. --- Discussion --- p.83 / Chapter 5.1 --- Efficacy test --- p.83 / Chapter 5.2 --- PCO disinfection under UV irradiation --- p.83 / Chapter 5.2.1 --- Optimization study --- p.84 / Chapter 5.2.1.1 --- Effect of P25 concentrations --- p.84 / Chapter 5.2.1.2 --- Effect of UV intensities --- p.85 / Chapter 5.2.1.3 --- Combinational study of P25 concentrations and UV intensities --- p.86 / Chapter 5.2.1.4 --- Effect of stirring rates --- p.86 / Chapter 5.2.1.5 --- Effect of initial cell concentrations --- p.87 / Chapter 5.2.2 --- Comparison of PCO inactivation efficiency between S. saprophyticus and E. cloacae --- p.88 / Chapter 5.2.3 --- PCO disinfection using different photocatalysts --- p.89 / Chapter 5.2.3.1 --- Ag/P25 --- p.89 / Chapter 5.2.3.2 --- SiO2-TiO2 and Cu2O/P25 --- p.90 / Chapter 5.3 --- PCO disinfection under visible light irradiation --- p.90 / Chapter 5.3.1 --- Effect of Cu20/P25 concentrations --- p.91 / Chapter 5.3.2 --- Effect of CU2O powder on the two bacterial species --- p.92 / Chapter 5.4 --- Feasibility use of fluorescent lamps for PCO disinfection --- p.93 / Chapter 5.5 --- Transmission electron microscopy (TEM) --- p.95 / Chapter 5.5.1 --- Morphological changes induced by PCO using P25 as a photocatalyst --- p.95 / Chapter 5.5.2 --- Morphological changes induced by PCO using CU2O/P25 as a photocatalyst --- p.96 / Chapter 5.6 --- Catalase (CAT) test --- p.98 / Chapter 5.7 --- Superoxide dismutase (SOD) activity assay --- p.99 / Chapter 6. --- Conclusion --- p.101 / Chapter 7. --- References --- p.106 / Chapter 8. --- Appendix --- p.121

<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>

Soil aquifer treatment was simulated in 1 m laboratory soil columns containing silica sand under saturated and unsaturated soil conditions to examine the effect of travel length through the unsaturated zone on the removal of wastewater organic matter, the effect of soil type on dissolved organic carbon removal and also the type of microorganisms involved in the removal process. Dissolved organic carbon removal and nitrification did enhance when the wastewater travelled a longer length through the unsaturated zone. A similar consortium of microorganisms was found to exist in both saturated and unsaturated columns. Microbial concentrations however were lowest in the soil column containing silt and clay in addition to silica sand. The presence of silt and clay was detrimental to DOC removal efficiency under saturated soil conditions due to their negative effect on the hydraulic performance of the soil column and microbial growth.