Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Description based on contents viewed Oct. 14, 2008; title from PDF t.p. Includes bibliographical references (p. 53-59).

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Murthy, Niren, Committee Member ; Doyle, Donald, Committee Member ; Fahrni, Christoph, Committee Member ; May, Sheldon, Committee Member ; Powers, James, Committee Chair.

MEDEIROS, Melissa Soares. Genotipagem e perfil de resistência aos antiretrovirais do vírus da imunodeficiência tipo 1 em população com falha terapêutica no Ceará, Brasil : 2002 a 2004. 2006. 192 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2006. / Submitted by denise santos (denise.santos@ufc.br) on 2012-05-07T13:29:20Z No. of bitstreams: 1 2006_dis_msmedeiros.pdf: 2320693 bytes, checksum: e0ca832db9ac0a74bb96e8b202aefb31 (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2012-05-08T16:54:28Z (GMT) No. of bitstreams: 1 2006_dis_msmedeiros.pdf: 2320693 bytes, checksum: e0ca832db9ac0a74bb96e8b202aefb31 (MD5) / Made available in DSpace on 2012-05-08T16:54:28Z (GMT). No. of bitstreams: 1 2006_dis_msmedeiros.pdf: 2320693 bytes, checksum: e0ca832db9ac0a74bb96e8b202aefb31 (MD5) Previous issue date: 2006 / Genotypic testing for HIV-1 drug resistance is useful for selecting antiretroviral drugs for patients developing treatment failure. O melhor entendimento da sua interpretação facilitará sua utilização como ferramenta médica na terapêutica do HIV. The optimal understanding of its interpretation will give an important tool for HIV treatment. Objective: To identify common combinations of resistance mutations and antiretroviral resistance profile. Methods: Between April 2002 and March 2004, 101 protease and reverse transcriptase (RT) sequences were determined for HIV-1 isolates from patients who were failing antiretroviral therapy. Resistance profile was obtained by Stanford program. Results: male were 76.2%, median age 38 years, CD4 media was 279.21 cells/mm3 and Viral load 4.49 log. Total of 31 mutational patterns were detected to protease inhibitor (IP), 49 to nucleoside RT inhibitor (NRTI), and 17 to nonnucleoside RT inhibitor (NNRTI). K65R was detected in 5.9% isolates. The most frequent mutations were L90M, M184V and K103N to IP, NRTI and NNRTI respectively. The main mutational patterns accounted for 49% of mutant sequences to IP, 38.5% to ITRN accounted and 40,9% to NNRTI. Patients with three or more therapeutic failure had worst resistance profile to all IP except for Lopinavir, and NRTI except for Tenofovir. High resistance to Lamivudine and NNRTI were independent of failure quantity. Conclusion: The best susceptibility was found to Lopinavir at IP’s class and to Tenofovir at ITRN’s. The main mutational patterns to IP, ITRN and NNRTI represented almost half of all patterns found. / A Genotipagem está sendo usada como método para guiar a seleção de antiretrovirais em pacientes com falha terapêutica. O melhor entendimento da sua interpretação facilitará sua utilização como ferramenta médica na terapêutica do HIV. Objetivo: Avaliar o perfil de resistência aos antiretrovirais e identificar padrões mutacionais das seqüências de protease e TR do HIV-1. Métodos: Foram estudadas as sequências de genes da protease e TR isoladas de 101 amostras de pacientes com HIV-1 em falha terapêutica, entre abril/2002 a março/2004, através de Genotipagem realizadas no Ceará. O Banco de dados de Stanford foi utilizado para avaliação de resistência e SPSS versão 11 e Epi Info versão 6 para análise estatística. Resultados: Sexo masculino 76,2%, mediana de idade 38 anos, CD4 médio de 279,21 cells/mm3 e Carga Viral 4.49 log. Na classe de Inibidores de Protease (IP) 31 padrões mutacionais foram encontrados, nos inibidores da transcriptase reversa análogos de nucleosídeos (ITRN) 49 e para inibidores da transcriptase reversa não análogos de nucleosídeos (ITRNN) 17. As mutações mais frequentes foram L90M, M184V e K103N para IP, ITRN e ITRNN espectivamente. A K65R foi detectada em 5,9% dos isolados. Três ou mais falhas terapêuticas apresentaram maior perfil de resistência para todos os IPs exceto para Lopinavir, e para todos os ITRNs exceto para Tenofovir. Os seis principais padrões mutacionais para IPs equivaleram a 49% das sequências, para ITRNs a 38,5%, e para ITRNNs os dois principais padrões corresponderam a 40,9%. Foram encontrados altos índices de resistência para ITRNNs independente da quantidade de falhas terapêuticas. Conclusão: Nos IPs a menor resistência encontrada foi ao Lopinavir e nos ITRNs ao Tenofovir. Os principais padrões mutacionais para IPs, ITRNs e ITRNNs representaram quase metade de todos os padrões de resistência encontrados.

SARS-CoV-2, the coronavirus responsible for the CoVID-19 syndrome, continues to be a major public health crisis worldwide. While the ongoing vaccination development and deployment efforts are a critical first line of defense, small-molecule therapeutics are needed to treat those who are infected and in desperate need of medical attention. Perhaps the most studied coronaviral target is the 3CL protease enzyme responsible for the proteolytic processing of the viral polyproteins pp1a and pp1ab which is essential for viral replication. We evaluated a series of five new compounds with four containing an (acyloxy)-methyl ketone reactive group including clinical candidate Pf-00835231 for their inhibitor potencies against SARS-CoV23CL protease. All five compounds exhibit remarkable potencies with Kivalues in the high picomolar to low nanomolar range against SARS-CoV-2. The X-ray structures of all five compounds were determined in complexes with SARS-CoV-2 3CLpro to between 1.4 Å and 1.6 Å resolution. All five compounds are observed to form a covalent bond with catalytic Cysteine 145 with four compounds forming adducts with the expected tetrahedral geometry. Compound 4 however, which contains an (acyloxy)-methyl ketone warhead, was found to form an adduct with bond geometries similar to an episulfonium cation. Despite possessing similar chemistry and scaffolds, inhibitor binding to the SARS-CoV-2 3CLpro induced a variety of subtle active site conformational differences, particularly in the S2/S4 separating strand and connecting strands.<div><br></div><div>Understanding substrate specificity in coronaviral main protease is essential for designing competitive inhibitors. While first principles are already established, including a Q/X cleavage-site (where X is either Alanine or Serine), differences exist between α, β, γand δ clades that are important for recognition, and ultimately inhibitor design. Covalent complexes of SARS-CoV-2 and avian infectious bronchitis virus (IBV) covalently bound, in trans, to the C-terminus of the same enzyme have been crystallized and modeled at 2.6 and 2.2 Å, respectively. The similarities and differences in their binding are described in chapter 6.<br></div><div><br></div><div>Middle-East Respiratory Syndrome Coronavirus is a re-emergent zoonotic pathogen with a 30% mortality rate in humans. The positive-sense single-stranded RNA genome is translated by the into polyproteins 1a (pp1a) and polyprotein 1ab (pp1ab). Pp1ab contains the constituents of the viral RNA-dependent RNA polymerase complex. These must be cleaved by 3CLpro, which is contained within this pair of polyproteins, before viral replication and transcription may occur. Attempts to drug this cysteine protease have proven difficult since competitive inhibitors activate the Wild-Type protease at low concentrations via a non-competitive binding mechanism. This mechanism is mediated by non-conserved residues in regions that are distal to the catalytic site. During the viral life-cycle these residues modulate recognition This study focuses on determining the identities of these residues and their effects on the dose-response curves of established competitive inhibitors of Coronaviral 3CLpro. It also explores how the residues in these regions synergize to effect intrinsic kinetic parameters of this family of enzymes including turnover (kcat) and dimer dissociation constant (KD). Additionally, a rapid-equilibrium kinetic model was developed to rationalize this unique phenomenon where competitive inhibitors cause significant activation of the enzyme’s activity.<br></div>

The metastatic process requires that tumour cells are capable of traversing various micro-environmental barriers, such as the basement membrane. There are various proteolytic mechanisms which could contribute to the process, plasminogen activation by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is one such mechanism. Extensive reports in the literature (reviewed in the introduction) indicate that most tumour cells synthesize uPA and that it is this enzyme, particularly when receptor-bound, which plays a role in invasion. UCT-Mel 3 is a human malignant melanoma cell line which was established in our laboratory, and has been shown to be highly metastatic in the nude mouse. This cell line is typical of many melanomas in that it synthesizes only tPA and not uPA. In part 1 of this thesis I further investigated the plasminogen activator production by these cells (at the level of mRNA as well as activity) as well as expression of plasminogen activator inhibitor PAl-1 and receptors for tPA and uPA (uPAR). UCT-Mel 3 cells expressed uPAR although uPA was not detected. I also examined cells cultured from two metastatic deposits. Interestingly, the metastatic cells produced PAl-1 which was undetected in the parent cells. After confirming that UCT-Mel 3 do not express detectable levels of uPA, I attempted (in part 2) to determine whether tPA could play a comparable role to that of uPA in the invasive process. My strategy was to inhibit the expression of tPA via two different methods, namely the use of antisense RNA and ribozyme. I then hoped to isolate clones producing no tPA, which would have been injected into nude mice in order to assay for metastasis. Unfortunately, neither of these methods proved to be successful in abrogating tPA expression. I was thus unable to achieve the ultimate aim of the project. However, during the course of the study a number of unforeseen problems arose. Firstly, the clonal variation within the cell population, and secondly, my inability to obtain antisense transfectants. I have speculated that a possible reason for the latter may be that the cells are in fact unable to grow in the absence of tPA.

Coronaviruses (CoVs) comprise a family of enveloped viruses that cause respiratory disease in humans, including CoV disease 2019 (COVID-19), caused by severe-acute respiratory syndrome CoV-2 (SARS-CoV-2). For CoV infection to occur, the CoV spike (S) protein must mediate fusion between the viral and host membranes. This entry process can also be repurposed during infection to promote cell-to-cell fusion, further contributing to viral spread. To trigger fusion, S must bind its cognate receptor and be cleaved by host proteases. Identifying cellular proteases capable of triggering CoV fusion is critical to understand CoV entry, tropism, and cell-cell spread, however the range of proteases capable of promoting CoV fusion has not been fully explored. Here, using fusion and entry assays, I provide evidence implicating matrix metalloproteinase-9 (MMP-9) as a fusion trigger for SARS-CoV-2 and HCoV-229E. Additionally, I show MMP-9 expression is upregulated during CoV infection, highlighting its potential relevance as a CoV triggering factor.

A series of experiments were conducted to evaluate the effects of dietary enzyme supplementation in broilers exposed to a live coccidia oocyst vaccine. In each experiment, Cobb 500 broilers were obtained at day of hatch from a commercial hatchery. Half of the chicks were sprayed with Coccivac BTM and housed in battery brooders in experiment (EXP) 1 and 2 or floor pens (EXP 3). Experimental diets were formulated according to Cobb 500 nutrient recommendations (positive control; PC) with the exception of Ca and available P (aP), which were reduced in the negative control (NC) diets approximately 0.1% (EXP 1), 0.11 and 0.13%, respectively (EXP 2), and 0.13% (EXP 3). Negative control diets in EXP 1 were supplemented with phytase (PHY), protease (PRO), xylanase (XYL), and the combination of PHY+PRO, PHY+XYL, and PHY+PRO+XYL. Negative control diets in EXP 2 were supplemented with PHY A, PHY B, and PHY C. In EXP 3, PC diets were supplemented with PHY at 1000 FTU/kg, and NC diets were supplemented with PHY at 1000 or 5000 FTU/kg. In all three experiments PHY supplementation generally improved (P ≤ 0.05) broiler performance and bone ash. Vaccination reduced (P ≤ 0.05) broiler performance in EXP 1 and 3, but increased (P ≤ 0.05) broiler performance in EXP 2. Xylanase and/or PHY supplementation tended to improve (P = 0.10) ileal amino acid digestibility (IAAD) in vaccinated broilers in EXP 1 and EXP 3. Phytase supplementation improved (P ≤ 0.05) IAAD and vaccination reduced (P ≤ 0.05) IAAD in EXP 3. Small intestinal morphology and goblet cell numbers were affected by enzyme supplementation and vaccination, which resulted in significant (P ≤ 0.05) interactions. In general, vaccination increased (P ≤ 0.05) small intestinal crypt depth and reduced (P ≤ 0.05) goblet cell numbers in EXP 3. Phytase supplementation of the NC diets fed to vaccinated broilers ameliorated the reduction in ileal goblet cells associated with vaccination (EXP 3). Enzyme supplementation tended to improve nutrient digestibility and altered small intestinal morphology in vaccinated broilers. Vaccination reduced broiler performance but nutrient digestion and dietary enzymes may improve nutrient utilization during a coccidia vaccination. / Ph. D.

The bacterium Burkholderia strain 2.2 N is a soil isolate and a member of a group of non-obligative predator bacteria that can prey on other microorganisms or grow saprophytically. The bacterium has anti-bacterial, anti-fungal, anti-yeast and anti-protozoan activities. Burkholderia strain 2.2 N culture shows hydrolysis on Milk Casein Agar, indicating the bacterium also produces a protease. Azocoll hydrolysis was used to detect and measure protease activity. Protease activity was two-fold higher at pH of 7.5 than pH 9.0 and 25-fold at pH 4.0. Cultures grown in media containing 1.0 % yeast extract (YE), tryptic soy, tryptone or beef extract had protease activity, whereas activity was absent in cultures grown in media containing peptone, soytone, casitone, or tryptone as sole protein source. Addition of 1.0 % sucrose or glucose to 1.0 % YE medium increased protease activity 1.8-fold and 1.4-fold, respectively. Protease activity was 2-fold higher in cultures grown in media containing 1.0 % YE and 10 mM MgCl₂ or FeCl₂, than in 1.0 % YE medium lacking metals or containing 10 mM MnCl2 or CaCl2. The 1.0 % YE medium containing either ZnCl₂ or CuCl₂ lacked protease activity (< 5.0 %). In cultures grown in 1.0 % YE at 30°C with rotation at 120 rpm, protease activity was higher in stationary phase (0.38 units /mg protein) than in exponential phase (0.04 units/mg protein). The Burkholderia strain 2.2 N protease is evidently exported from cells because 86 % of the total proteolytic activity of cells was found in the cell-free culture medium. The cell free filtered culture supernatant medium assayed at 4°C had protease activity, however at a three-fold lower specific activity compared to the same supernatant assayed at 3°C. Protease activity was lower in filtered culture supernatants stored at 4°C, room temperature, and 30°C. Protease activity in samples stored at 4°C was only 40 % (24 hours) and 15 % (48 hours) of activity at time zero. Protease activity in samples stored at room temperature was only 45 % (24 hours) and 35 % (48 hours) of activity at time zero. Protease activity in samples stored at 30°C was only 78 % (24 hours) and 9 % (48 hours) of activity at time zero. Purification of the protease from filtered culture supernatant medium by ammonium sulfate precipitation, increased the protease activity 20-fold. An eluted protein fraction from DEAE-Sepharose column chromatography had 50-fold higher protease activity. Protease activity was inhibited by 10 mM 1-10-phenathroline, EDTA and EGTA, all metalloprotease inhibitors. Purified protease activity inactivated with 10 mM 1-10-phenanthroline or 10 mM EGTA was regained through the addition of Ca2+ or Mg2+. Protease activity was reduced by exposure to dithiothreitol (29 % with 1 mM and 84 % with 10 mM), a disulfide bond inhibitor. Protease activity was not inhibited by leupeptin or phenylmethylsulphonyl flouride. Casein polyacrylamide zymography revealed a band of hydrolysis at approximately 60,000 Da. SDS-PAGE resolved a doublet band present at 60,000 Da present in both the filtered culture supernatant sample and the ammonium sulfate / DEAE-Shepharose column chromatography purified protease sample. Burkholderia strain 2.2 N protease is a metalloprotease with a broad temperature range of activity. It has a molecular weight of approximately 60,000 Da. / Master of Science