Made available in DSpace on 2014-06-11T19:31:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T20:01:47Z : No. of bitstreams: 1 santos_clv_dr_araca.pdf: 3550795 bytes, checksum: fe4f97d551c4c15a44f2f46e8514ced2 (MD5) / Para um bom prognóstico no reimplante dentário, é necessário que o dente seja reposicionado o mais rápido possível em seu alvéolo de origem ou mantido em meio de conservação adequado. Quando o dente avulsionado é mantido em condições inadequadas de conservação, a remoção do ligamento periodontal desvitalizado é a conduta mais apropriada para minimizar a ocorrência da reabsorção radicular. Com o objetivo de prevenir e/ou retardar a instalação de reabsorções radiculares e preservar a integridade da camada de cemento propõe-se o estudo do processo de reparo em dentes de ratos reimplantados tardiamente, após a utilização da solução de papaína e fluoreto de sódio no tratamento de superfície radicular. Essa proposta tem como base as propriedades da papaína de remoção do tecido necrótico, ação antioxidante e antimicrobiana. Foram utilizados os incisivos superiores direitos de 40 ratos, divididos em 4 grupos, sendo que no Grupo I os dentes foram imediatamente reimplantados em seus respectivos alvéolos. Nos grupos II, III, IV, os dentes extraídos foram mantidos em meio seco pelo período de 1 hora. No grupo II, os dentes foram imersos em solução de papaína a 50% por 20 minutos, friccionados com gaze por 1 minuto e imerso em fluoreto de sódio fosfato acidulado a 2%, pH 5, pelo período de 20 minutos. No grupo III, os dentes foram mantidos em soro fisiológico por 20 minutos, tiveram a superfície radicular friccionada por 1 minuto e imerso em fluoreto de sódio fosfato acidulado a 2%, pH 5, por um período de 20 minutos. No grupo IV, os dentes foram reimplantados em seus respectivos alvéolos, sem nenhum tratamento a superfície radicular. Previamente ao reimplante, a polpa radicular foi removida e os canais preenchidos com Ca(OH)2 veiculado em propilenoglicol. Após o reimplante, os animais receberam antibioticoterapia sistêmica... / For a good prognostic in tooth replanting, it is necessary that the tooth be repositioned in its socket as quickly as possible or otherwise be properly conserved. When the avulsed tooth is improperly conserved, removing the devitalized periodontal ligament is the appropriate conduct to minimize the occurrence of radicular reabsorption. The aim of this study was to search for an effective form of the above process, while preserving the integrity of the cement layer. In this case, papain was used, due to its necrotic tissue removing proprieties and its microbacterial action. The research was conducted with 40 extracted rat incisives, divided into 4 groups. In group I the teeth were replanted immediately in their respective sockets. In groups II, III and IV the extracted teeth were kept in a dry environment for a period of 1 hour. In group II, the teeth were immersed in papain solution (50%) for 20 minutes, rubbed with gauze for 1 minute and immersed in sodium fluoride (2%, pH 5) for a period of 20 minutes. In group III, the teeth were kept in a saline solution for 20 minutes, the radicular surface was rubbed for 1 minute, and they were immersed in sodium fluoride (2%, pH5) for 20 minutes. In group IV, the teeth were replanted in their respective sockets without any treatment. Before replanting, the radicular pulp was removed and the root filled with Ca(OH)2 dissolved in propilenoglicol. Immediately upon replanting the rats received antibiotic systemic therapy and after 60 days they were euthanized. The pieces containing the replanted incisive were subjected to histological analysis. The results show a higher occurrence of ankylosis in group II when compared to group I (p<0.05). Reabsorption by substitution occurs more in group IV when compared to group I (p<0.05) and II (p<0.001). Considering all areas of reabsorption, it was observed that the teeth in group... (Complete abstract click electronic access below)

INTRODUÇÃO: O modelo eslastase-induzido de aneurismas tem se destacado, nos últimos anos, porque simula as características geométricas dos aneurismas intracranianos humanos. A elastase destrói as fibras elásticas e dilata as artérias. A papaína é uma enzima que ainda não foi usada com esta finalidade. Os objetivos deste estudo foram determinar se a papaína produz aneurismas saculares em coelhos, e comparar suas características macroscópicas e histológicas com as dos aneurismas elastase-induzidos. MÉTODO: Dezoito coelhos brancos Nova Zelândia (1,9-3,2 kg) foram divididos em 3 grupos: I- elastase (n=7), II- papaína (n=8) e III- cirurgia controle (n=3). Os animais foram submetidos à exposição cirúrgica do pescoço, sendo que a artéria carótida comum direita foi usada como teste e a artéria carótida comum esquerda como controle. No 21° dia após a cirurgia, os animais foram sacrificados para retirada das artérias, tomada de suas medidas e análise histológica. Considerou-se formação de aneurisma quando a artéria teste dilatou em relação ao seu controle. RESULTADOS: Não houve aneurismas no grupo cirúrgico controle. Houve formação de aneurismas nos grupos elastase (71,4%) e papaína (100%). A diferença do diâmetro das artérias testes e seus respectivos controles não foi significativa (p= 0,15) entre os grupos elastase (média= 1,2 ± 0,4mm) e papaína (média= 2,1 ± 0,4mm), embora houvesse tendência deste último à maior dilatação . A histologia demonstrou que a papaína produziu maior tendência à lesão endotelial, à trombose (p = 0,01) e à inflamação parietal do que a elastase. A análise da fibrose intimal foi prejudicada em 50% dos casos do grupo papaína devido à trombose acentuada. Não houve diferença significativa entre os grupos quanto ao espessamento parietal (p=0,81) e ao grau de destruição das fibras elásticas (p= 0,009). CONCLUSÕES: A papaína produz aneurismas com tamanhos semelhantes aos da elastase, contudo a papaína provoca maior lesão endotelial, maior trombose e maior inflamação do que a elastase / INTRODUCTION: The elastase-induced model of experimental saccular aneurysms has been relevant in the last years because it mimics the size and geometric features of human intracranial aneurysms. Elastase destroys the arterys elastic fibers and produces arterial enlargement. Papain enzyme is also elastolytic but it had not been tested on saccular aneurysms creation yet. The purpose of this study was determine if papain produces saccular aneurysms in rabbits and to compare its gross and microscopic features are with the elastaseinduced aneurysms. METHODS: Eighteen New Zealand white rabbits (1.9 kg 3,2 kg) were separated in 3 groups: 1) sham (n=3) 0,9% saline solution; papain (n=8) 17-40 U; elastase (n=7) 6-8 U. The animals underwent surgical exposure of the neck; the right common carotid artery (RCCA) was used as the test and the left common carotid artery (LCCA) as the control. On the 21st day after surgery, animals were sacrificed for removal of the arteries, measurements and histological analysis. We determine formation of aneurysm to occur when the test artery dilated compared to the control. RESULTS: The sham group didnt develop aneurysms. There was aneurysm formation in the elastase (71,4%) and papain (100%) groups. The difference of the diameter of the tests and their respective controls is not significant (p=0,15) between elastase (average = 1,2 ± 0,4 mm) and papain (average = 2,1 ± 0,4mm) groups although there was tendency of this last one to produce larger aneurysms. the and to thrombosis (p = 0,01) and to parietal inflammation than the elastase. The analysis of the intimal fibrosis was not possible in 50% of papain cases due to pronounced thrombosis. There was no significant difference between the groups regarding the parietal thickening (p = 0,81) and the degree of destruction of the elastic fibers (0,009). CONCLUSION: Papain creates saccular aneurysms with similar dimensions to elastase-induced aneurysms. The microscopic results indicated papain destroys more endothelial cells, produces more thrombosis and more inflammatory process than elastase

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-05-20T14:09:44Z No. of bitstreams: 1 Juliete Lira de Souza Lima.pdf: 1433864 bytes, checksum: 80b4978969bcd79b43a904e088a76553 (MD5) / Made available in DSpace on 2016-05-20T14:09:44Z (GMT). No. of bitstreams: 1 Juliete Lira de Souza Lima.pdf: 1433864 bytes, checksum: 80b4978969bcd79b43a904e088a76553 (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study aimed to evaluate and chemical castration with papain associated with lactic acid in adult Wistar rats. Twenty-five male Wistar rats at 90 days of age were divided into 5 groups. The animals of the groups G1, G2, G3, G4 received 0.1 mL of papain solution and lactic acid intratesticularly and they were euthanized respectively 24h, 48h, 72h and 96h after treatment, and the animals of the control group "CG" received 0.1ml of saline and they were euthanized 24h after treatment. Fragments of testis, epididymis, kidney, and liver were collected for histopathological evaluation and blood for analysis of testosterone. The daily monitoring of the animals showed no behavioral impairment, with physiological acts within the normal range, such as eating, defecation and urination, as well as socializing with the group. kidney and liver changes were observed in the histopathological study of these organs, suggesting that only a local effect solution. Already in the testis and epididymis changes were characterized by inflammatory infiltrate, vacuolation and necrosis of germ cells. On testosterone levels G1 and G2 had values below 1 ng / ml, which technique was effective in reducing testosterone levels within 48 hours. Given the histopathological findings in the testis and epididymis, papain and lactic acid solution did not affect the kidney and liver and did not affect the behavior of animals. The solution was shown to reduce the activity of Leydig cells in the first 48 hours. But further studies are needed on the long-term effects and the application of higher concentrations of this solution in rats and the development of new projects that could cover all species. / Este trabalho teve como objetivo avaliar a castração química com papaína associada ao ácido lático em ratos Wistar adultos. 25 ratos Wistar com 90 dias de idade foram divididos em 5 grupos. Os animais dos grupos G1, G2, G3, G4 receberam 0,1mL da solução papaína e ácido lático por via intratesticular e foram eutanasiados respectivamente em 24h, 48h, 72h e 96h após o tratamento, os do grupo controle "GC" receberam 0,1mL de solução fisiologica a 0,9% e foram eutanasiados 24h após o tratamento. Foram coletados fragmentos de testículo, epidídimo, rim e fígado para avaliação histopatológica e sangue para análise dos níveis de testosterona. O acompanhamento diário dos animais não evidenciou comprometimento comportamental, apresentando atos fisiológicos dentro da normalidade, tais como, alimentação, defecação e micção, assim como socialização com o grupo. Não foram observadas alterações renais e hepáticos no estudo histopatológico desses órgãos, sugerindo ser uma solução de efeito apenas local. Já nos testículos e epidídimos as alterações foram caracterizadas por infiltrado inflamatório, vacuolização citoplasmática e necrose de células germinativas. Sobre os níveis de testosterona os grupos G1 e G2 apresentaram valores abaixo de 1 ng/mL, cuja técnica mostrou ser eficaz em diminuir esses níveis em 48 horas. Tendo em vista os achados histopatológicos no testículo e epidídimo, a solução papaína e ácido lático não afetaram o rim e o fígado e não comprometeram o comportamento dos animais. A solução mostrou-se capaz de reduzir a atividade das células de Leydig nas primeiras 48 horas. Porém são necessários novos estudos sobre os efeitos prolongados e na aplicação de maiores concentrações desta solução em ratos Wistar além da elaboração de novos projetos que se possam abranger outras as espécies.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-17T17:34:41Z No. of bitstreams: 1 Nicoletti, Caroline Deckmann [Dissertação, 2015].pdf: 2492191 bytes, checksum: 2cc1df517b27092f16baf567910a3c1a (MD5) / Made available in DSpace on 2017-03-17T17:34:41Z (GMT). No. of bitstreams: 1 Nicoletti, Caroline Deckmann [Dissertação, 2015].pdf: 2492191 bytes, checksum: 2cc1df517b27092f16baf567910a3c1a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A papaína é uma enzima proteolítica utilizada no tratamento de feridas. É parcialmente solúvel em água e tem baixa estabilidade em preparações farmacêuticas, dessa forma os géis com concentrações de papaína acima de 1% podem apresentar precipitados. O presente trabalho teve como objetivo o desenvolvimento de géis de papaína a 4% (p/p) usando tensoativos como agente solubilizante. Como fase inicial do estudo realizou-se a determinação da Concentração Micelar Crítica (CMC) por meio da medida de tensão superficial de geis de papaína a 4,0% (p/p) com e sem L-cisteína e diferentes concentrações de polissorbato 80. Novas preparações foram desenvolvidas usando-se concentrações de polissorbato e L-cisteína que resultavam na completa solubilização da papaína. O estudo de estabilidade dos géis de papaína a 4,0% (p/p) com polissorbato 80 foi planejado e executado a partir de desenho experimental fatorial 33 em amostras armazenadas sob refrigeração por 30 dias. As variáveis independentes estipuladas foram concentração de L-cisteína, concentração de polissorbato 80 e o tempo de armazenagem; a variável dependente avaliada foi a concentração de papaína ativa. Todas as amostras foram analisadas quanto à manutenção das características sensoriais e físico-químicas. Durante o período de avaliação as preparações conservaram-se como géis homogêneos, mantendo-se estáveis quanto aos valores de pH, de viscosidade e de estabilidade termodinâmica. Estas amostras não apresentaram mudanças no seu comportamento reológico. As preparações estudas apresentaram concentrações de papaína ativa entre 90 e 110% apos 24 horas de preparo, mas não foi possível a manutenção desta atividade durante o período de estudo. As avaliações estatísticas decorrentes do desenho experimental fatorial resultaram em gráficos de superfície de resposta e de contorno, que demonstraram que o tempo é o fator de maior influência sobre a perda da atividade da papaína nas preparações em gel. A L-cisteína interfere favoravelmente para a manutenção da atividade, mas sua ação isolada é incapaz de sobrepor-se ao efeito do tempo. A associação da L-cisteína e do polissorbato 80 também apresenta ação favorável a manutenção da atividade enzimática, que se torna evidente somente nas maiores concentrações estudadas para estes adjuvantes / Papain is a proteolytic enzyme used in the treatment of wounds. It is partially soluble in water and has low stability in pharmaceutical preparations thus gels with papain above 1% concentration may have precipitated. This study aimed to the development of papain gels at 4% (w / w) using surfactants as solubilizing agent. As the initial phase of study was carried out to determine the critical micellar concentration (CMC) by action of surface tension of papain gels at 4.0% (w / w) with and without L-cysteine and different concentrations of polysorbate 80. New preparations were developed using concentrations of polysorbate and Lcysteine resulted in complete solubilization of papain. The stability study of papain gels at 4.0% (w / w) polysorbate 80 was planned and carried out from 33 factorial experimental design for samples stored under refrigeration for 30 days. The independent variables were prescribed concentration of L-cysteine, concentration of polysorbate 80 and the storage time; the evaluated dependent variable was the concentration of active papain. All samples were analyzed for the maintenance of sensorial and physicochemical characteristics. During the evaluation period preparations presented as homogeneous gels, remaining stable as the pH, viscosity and thermodynamic stability. These samples showed no changes in their rheological behavior. The preparations studied showed ative papain concentrations between 90 and 110% after 24 hours of preparation, but it was not possible to maintain this activity over the study period. The statistical evaluations resulting from the factorial experimental design resulted in response surface graphs and contour, showing that time is the most influential factor on the loss of papain activity in preparations gel. L-cysteine favorably affects the maintenance of the activity, but its action alone is unable to override the effect of the time. The combination of Lcysteine and polysorbate 80 also presents favorable action to maintain the enzyme activity, which becomes evident only at the highest concentrations investigated for these adjuvants

Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.

The papain-like cysteine protease family (C1 proteases) is highly important because of its involvement in research and industrial applications and its role in various human diseases. Protein inhibitors are an important aspect of C1 protease biology and are relevant to its clinical, industrial and research importance. To study the interaction between the proteases and the inhibitors it is very useful to have accurate structural models of the protease-inhibitor complexes. To this end, a high-throughput pipeline for modelling complexes of papain-like cysteine proteases and protein inhibitors was implemented and tested (Tastan Bishop & Kroon, 2011). The pipeline utilizes a novel technique for obtaining modelling templates by using superpositioning to combine coordinates from separate experimental structures. To test the pipeline, models of complexes with known structures (test set) were modelled using many different templates and the resultant models evaluated to compare the quality of the different templates. It was found that use of the new technique to obtain templates did not introduce significant errors, while allowing closer homologs to be used for modelling - leading to more accurate models. The test set models were also used to evaluate certain steps of the modelling protocol. The effect of Rosetta energy minimization on model accuracy and the use of Rosetta energy and DOPE Z-score values to identify accurate models were investigated. Several complexes were then modelled using the best available templates according to criteria informed by the previous results. A website was built that allows a user to download any of the metrics or models produced in the study. This website is accessible at http://rubi.ru.ac.za/cpmdb

För att förhindra transfusionsreaktioner utförs antikroppsidentifieringar vid misstanke om att en patient bildat förvärvade antikroppar. Förvärvade antikroppar bildas efter att en individ blivit immuniserad, genom en tidigare transfusion eller vid graviditet.Rh-systemet är ett av de kliniskt viktigaste blodgruppssystemen man tar hänsyn till vid transfusion. De viktigaste blodgrupperna inom detta system är D, c, C, e och E. Den metod som främst används vid antikroppsidentifieringar är indirekt antiglobulintest (IAT), men ibland behöver denna kompletteras med ytterligare metoder, såsom papain-gelteknik eller polyetylenglykol-IAT (PEG-IAT). Papain verkar genom att spjälka bort sialinsyrainnehållande grupper på erytrocytens yta medan PEG är en vattenlöslig polymer som konkurrerar bort vattenmolekyler. Detta gör att Z-potentialen minskar och chansen för antigen-antikroppsreaktion ökar. Syftet med studien var att studera detektionen av erytrocytantikroppar inom Rh-systemet med PEG-IAT. För att undersöka metodens prestanda jämfördes PEG-IAT med resultat från antikroppsidentifieringar som utfördes med IAT och papain-gelteknik.  Under studien genomfördes antikroppsidentifieringar på 34 avidentifierade plasmaprover med metoderna IAT, papain-gelteknik och PEG-IAT. Detta resulterade i att antikropparna D, c, C, e, E detekterades som förväntat för de flesta proverna.  PEG-IAT hittade även ytterligare antikroppar i två av proverna. Vid en jämförelse mellan de olika metoderna detekterade PEG-IAT flest antikroppar följt av IAT och papain-gelteknik. Det finns även en signifikant skillnad i reaktionsstyrka mellan de olika metoderna för en testerytrocyt som är heterozygot för samtliga testade antigen i Rh-systemet.  Slutsatsen med studien är att PEG-IAT visar lovande resultat men behöver testas mot fler kliniskt viktiga antikroppar innan det kan implementeras i verksamheten.

The proteolytic actjvity of papain was investigated with respect to its action on both natural substrates (native papaya protein and bovine serum albumin) and artificial synthetic substrate (benzoyl~l-arginine ethyl ester). Following removal of 30 percent and 40 percent of the amino acid residues from the amine terminus, the activity of the degraded papain was determined with respect to the above substrates, and compared to the activity of the intact papain molecule. Native papaya protein showed resistance to digestion by both intact papain and 30 percent 'degraded papain. This resistance to digestion persisted for time periods ranging from three to six hours and was not found with the other substrates. Papaya protein did not show this resistance to digestion by 40 percent degraded papain. Digestion began immediately upon addition of 40 percent degraded papain to papaya protein. In contrast to this, papaya protein showed no resistance to digestion by other proteolytic enzymes, both of plant and animal origin. These investigations clearly showed an interaction between native papaya protein and papain, which serves to prevent digestion of the native papaya protein by papain. This interaction was shown to be with a region of the papain between amtno acid residue 62 (30 percent degraded papain) and amino acid residue 84 (40 percent degraded papain). It is suggested that this type of interaction may be a general mechanism for the prevention of digestion of native proteins by the enzymes of the host. If this is the case, then the papain-papaya protein system could serve as an easily controlled model system for the investigation of other such enzyme systems.

No / The oligomerisation of L-amino acids by papain was studied in a mixer ball mill and in a planetary ball mill. The biocatalyst proved stable under the ball milling conditions providing the corresponding oligopeptides in good to excellent yields and with a variable degree of polymerisation. Both parameters were found to be dependent on the reaction conditions and on the nature of the amino acid (specifically on its side-chain size and hydrophobicity). In addition, the chemoenzymatic oligomerisation was demonstrated by utilising twin-screw extrusion technology, which allowed for a scalable continuous process. Finally, the synthesised oligo(L-Leu) 2b proved to be active as a catalyst in the Juliá–Colonna enantioselective epoxidation of chalcone derivatives. / We acknowledge RWTH Aachen University for support by the Distinguished Professorship Program funded by the Excellence Initiative of the German federal and state governments. We kindly acknowledge Marcus Frings and Plamena Staleva for the HPLC analysis of products 4a–c (RWTH Aachen University) and ASEP for the TGA analysis (Queen’s University Belfast). D. E. C. and S. L. J. acknowledge the agency EPSRC, grant no. EP/R019655/1. Part of this work was performed at the Center for Chemical Polymer Technology (CPT) unit of DWI, which was supported by the EU and the federal state of North Rhine-Westphalia (grant EFRE 30 00 883 02).

<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>