Serum C-reactive protein (CRP) is used as a marker of inflammation in several diseases including autoimmune disease and cardiovascular disease. CRP, a member of the pentraxin family, is comprised of five identical subunits. CRP has diverse ligand-binding properties which depend upon different structural states of CRP. However, little is known about the molecular dynamics and interaction properties of CRP. In this study, we used SAPS, SCRATCH protein predictor, PDBsum, ConSurf, ProtScale, Drawhca, ASAView, SCide and SRide server and performed comprehensive analyses of molecular dynamics, protein-protein and residue-residue interactions of CRP. We used 1GNH.pdb file for the crystal structure of human CRP which generated two pentamers (ABCDE and FGHIJ). The number of residues involved in residue-residue interactions between A-B, B-C, C-D, D-E, F-G, G-H, H-I, I-J, A-E and F-J subunits were 12, 11, 10, 11, 12, 11, 10, 11, 10 and 10, respectively. Fifteen antiparallel β sheets were involved in β-sheet topology, and five β hairpins were involved in forming the secondary structure. Analysis of hydrophobic segment distribution revealed deviations in surface hydrophobicity at different cavities present in CRP. Approximately 33 % of all residues were involved in the stabilization centers. We show that the bioinformatics tools can provide a rapid method to predict molecular dynamics and interaction properties of CRP. Our prediction of molecular dynamics and interaction properties of CRP combined with the modeling data based on the known 3D structure of CRP is helpful in designing stable forms of CRP mutants for structure-function studies of CRP and may facilitate in silico drug design for therapeutic targeting of CRP.

Serum C-reactive protein (CRP) is used as a marker of inflammation in several diseases including autoimmune disease and cardiovascular disease. CRP, a member of the pentraxin family, is comprised of five identical subunits. CRP has diverse ligand-binding properties which depend upon different structural states of CRP. However, little is known about the molecular dynamics and interaction properties of CRP. In this study, we used SAPS, SCRATCH protein predictor, PDBsum, ConSurf, ProtScale, Drawhca, ASAView, SCide and SRide server and performed comprehensive analyses of molecular dynamics, protein-protein and residue-residue interactions of CRP. We used 1GNH.pdb file for the crystal structure of human CRP which generated two pentamers (ABCDE and FGHIJ). The number of residues involved in residue-residue interactions between A-B, B-C, C-D, D-E, F-G, G-H, H-I, I-J, A-E and F-J subunits were 12, 11, 10, 11, 12, 11, 10, 11, 10 and 10, respectively. Fifteen antiparallel β sheets were involved in β-sheet topology, and five β hairpins were involved in forming the secondary structure. Analysis of hydrophobic segment distribution revealed deviations in surface hydrophobicity at different cavities present in CRP. Approximately 33 % of all residues were involved in the stabilization centers. We show that the bioinformatics tools can provide a rapid method to predict molecular dynamics and interaction properties of CRP. Our prediction of molecular dynamics and interaction properties of CRP combined with the modeling data based on the known 3D structure of CRP is helpful in designing stable forms of CRP mutants for structure-function studies of CRP and may facilitate in silico drug design for therapeutic targeting of CRP.

O linfoma é uma doença neoplásica comum em cães, requerendo quimioterapia para aumentar a sobrevida dos pacientes. Durante o tratamento, são freqüentes as recidivas, que motivam alteração do protocolo medicamentoso. Proteína C-reativa e amilóide A sérica são mediadores de fase aguda produzidos no fígado que apresentam elevações de concentrações séricas em condições inflamatórias, infecciosas e neoplásicas de maneira geral. O objetivo do trabalho foi avaliar o papel dessas proteínas na monitorização da remissão e recidiva do linfoma em cães, utilizando 2 protocolos de tratamento. O protocolo COP (ciclofosfamida, vincristina e prednisona) caracterizou-se por fase de indução de 1 mês e ciclos de manutenção a cada 21 dias e o protocolo VCM (vincristina, ciclofosfamida, metotrexato e L- asparaginase) foi empregado em um regime semanal contínuo. Constituíram-se 5 grupos de estudo: Normal (20 cães hígidos), Controle COP (4 cães hígidos submetidos a quimioterapia com o protocolo COP), Controle VCM (4 cães hígidos submetidos a quimioterapia com o protocolo VCM), Linfoma COP (10 cães com linfoma multicêntrico tratados com o protocolo COP) e Linfoma VCM (10 cães com linfoma multicêntrico tratados com o protocolo VCM). Proteína C-reativa e amilóide A sérica foram determinadas pela técnica de Elisa e a eletroforese foi feita em tiras de acetato de celulose. Nos cães do grupo Normal, o estabelecimento das concentrações de proteína C-reativa, amilóide A sérica e as rações eletroforéticas ocorreu uma única vez; nos cães dos grupos Controle COP e Controle VCM na 1ª, 2ª, 3ª, 4ª, 7ª, 10ª, 13ª e 16ª semanas de quimioterapia e, nos cães dos grupos Linfoma COP e Linfoma VCM, na 1ª, 2ª, 3ª e 4ª semanas, bem como na recidiva e num momento de estabilidade da doença imediatamente antes da recidiva. Os resultados foram interpretados por análise de variância com medidas repetidas, tendo como fatores de controle os grupos e as semanas de observação, seguida de comparações múltiplas de Tukey, ao nível de significância de 5 %. Concluiu-se que: o linfoma induz a resposta de fase aguda em cães, sendo a intensidade da resposta amenizada durante o tratamento bem-sucedido dos pacientes; incrementos de proteína C-reativa e amilóide A sérica não estão relacionados à recidiva do linfoma; a quimioterapia do linfoma com os protocolos COP e VCM não altera a resposta de fase aguda, avaliada por meio dessas proteínas, nem existe diferença na resposta de fase aguda entre tais protocolos; existe relação direta entre os níveis de proteína C-reativa e amilóide A sérica no curso do linfoma e da quimioterapia; aumentos das concentrações séricas de proteína C-reativa são acompanhados de elevações da fração de β2-globulinas e aumentos de amilóide A sérica são acompanhados de elevações de β1-globulinas. / Lymphoma is a common neoplasm in dogs and chemotherapy is indicated to achieve long-term survivals. During the treatment, frequent relapses require drug regimen modifications. C-reactive protein (CRP) and serum amyloid A (SAA) are hepatic acute-phase mediators and usually are increased in inflammatory, infectious and neoplastic conditions. The aim of this study was to evaluate the role of these proteins in remission and relapse monitoring of dogs with lymphoma, under 2 chemotherapy protocols. COP protocol (cyclophosphamide, vincristine and prednisone) included an one-month induction period and maintenance cycles each 21 days and VCM protocol (vincristine, cyclophosphamide, methotrexate and L-asparaginase) was administered in a continuous weekly schedule. Five groups were composed: Normal (20 healthy dogs), COP Control (4 healthy dogs submitted to chemotherapy with COP protocol), VCM Control (4 healthy dogs submitted to chemotherapy with VCM protocol), COP Lymphoma (10 dogs with multicentric lymphoma treated with COP protocol) and VCM Lymphoma (10 dogs with multicentric lymphoma treated with VCM protocol). CRP and SAA were determined by Elisa tests and the electrophoresis was performed in cellulose acetate strips. In Normal dogs, CRP and SAA levels, as well the electrophoretic fractions, were measured only one time; in COP Control and VCM Control groups of dogs at the chemotherapy weeks 1, 2, 3, 4, 7, 10, 13 and 16; in dogs from groups COP Lymphoma and VCM Lymphoma, at the weeks 1, 2, 3 and 4, beside the relapse and a called stability moment immediately before the relapse. Results were compared by means of repeated measures variancy analyses, considering the groups and the observation weeks as the control factors, followed by Tukey´s multiple comparisons, at 5 % of significance level. It was concluded that: lymphoma induces an acute phase response in dogs and the intensity of response declines along the disease remission; increases of CRP and SAA are not related to lymphoma relapse; neither COP nor VCM chemotherapy changes the acute-phase response, when CRP and SAA are taken in account, and there is not difference on acute-phase response between both regimens; there is a positive correlation between CRP and SAA levels during lymphoma assessment and chemotherapy; increases of CRP levels are followed by β2-globulin elevations and increases of SAA levels are related to β1-globulin elevations.

O linfoma é uma doença neoplásica comum em cães, requerendo quimioterapia para aumentar a sobrevida dos pacientes. Durante o tratamento, são freqüentes as recidivas, que motivam alteração do protocolo medicamentoso. Proteína C-reativa e amilóide A sérica são mediadores de fase aguda produzidos no fígado que apresentam elevações de concentrações séricas em condições inflamatórias, infecciosas e neoplásicas de maneira geral. O objetivo do trabalho foi avaliar o papel dessas proteínas na monitorização da remissão e recidiva do linfoma em cães, utilizando 2 protocolos de tratamento. O protocolo COP (ciclofosfamida, vincristina e prednisona) caracterizou-se por fase de indução de 1 mês e ciclos de manutenção a cada 21 dias e o protocolo VCM (vincristina, ciclofosfamida, metotrexato e L- asparaginase) foi empregado em um regime semanal contínuo. Constituíram-se 5 grupos de estudo: Normal (20 cães hígidos), Controle COP (4 cães hígidos submetidos a quimioterapia com o protocolo COP), Controle VCM (4 cães hígidos submetidos a quimioterapia com o protocolo VCM), Linfoma COP (10 cães com linfoma multicêntrico tratados com o protocolo COP) e Linfoma VCM (10 cães com linfoma multicêntrico tratados com o protocolo VCM). Proteína C-reativa e amilóide A sérica foram determinadas pela técnica de Elisa e a eletroforese foi feita em tiras de acetato de celulose. Nos cães do grupo Normal, o estabelecimento das concentrações de proteína C-reativa, amilóide A sérica e as rações eletroforéticas ocorreu uma única vez; nos cães dos grupos Controle COP e Controle VCM na 1ª, 2ª, 3ª, 4ª, 7ª, 10ª, 13ª e 16ª semanas de quimioterapia e, nos cães dos grupos Linfoma COP e Linfoma VCM, na 1ª, 2ª, 3ª e 4ª semanas, bem como na recidiva e num momento de estabilidade da doença imediatamente antes da recidiva. Os resultados foram interpretados por análise de variância com medidas repetidas, tendo como fatores de controle os grupos e as semanas de observação, seguida de comparações múltiplas de Tukey, ao nível de significância de 5 %. Concluiu-se que: o linfoma induz a resposta de fase aguda em cães, sendo a intensidade da resposta amenizada durante o tratamento bem-sucedido dos pacientes; incrementos de proteína C-reativa e amilóide A sérica não estão relacionados à recidiva do linfoma; a quimioterapia do linfoma com os protocolos COP e VCM não altera a resposta de fase aguda, avaliada por meio dessas proteínas, nem existe diferença na resposta de fase aguda entre tais protocolos; existe relação direta entre os níveis de proteína C-reativa e amilóide A sérica no curso do linfoma e da quimioterapia; aumentos das concentrações séricas de proteína C-reativa são acompanhados de elevações da fração de β2-globulinas e aumentos de amilóide A sérica são acompanhados de elevações de β1-globulinas. / Lymphoma is a common neoplasm in dogs and chemotherapy is indicated to achieve long-term survivals. During the treatment, frequent relapses require drug regimen modifications. C-reactive protein (CRP) and serum amyloid A (SAA) are hepatic acute-phase mediators and usually are increased in inflammatory, infectious and neoplastic conditions. The aim of this study was to evaluate the role of these proteins in remission and relapse monitoring of dogs with lymphoma, under 2 chemotherapy protocols. COP protocol (cyclophosphamide, vincristine and prednisone) included an one-month induction period and maintenance cycles each 21 days and VCM protocol (vincristine, cyclophosphamide, methotrexate and L-asparaginase) was administered in a continuous weekly schedule. Five groups were composed: Normal (20 healthy dogs), COP Control (4 healthy dogs submitted to chemotherapy with COP protocol), VCM Control (4 healthy dogs submitted to chemotherapy with VCM protocol), COP Lymphoma (10 dogs with multicentric lymphoma treated with COP protocol) and VCM Lymphoma (10 dogs with multicentric lymphoma treated with VCM protocol). CRP and SAA were determined by Elisa tests and the electrophoresis was performed in cellulose acetate strips. In Normal dogs, CRP and SAA levels, as well the electrophoretic fractions, were measured only one time; in COP Control and VCM Control groups of dogs at the chemotherapy weeks 1, 2, 3, 4, 7, 10, 13 and 16; in dogs from groups COP Lymphoma and VCM Lymphoma, at the weeks 1, 2, 3 and 4, beside the relapse and a called stability moment immediately before the relapse. Results were compared by means of repeated measures variancy analyses, considering the groups and the observation weeks as the control factors, followed by Tukey´s multiple comparisons, at 5 % of significance level. It was concluded that: lymphoma induces an acute phase response in dogs and the intensity of response declines along the disease remission; increases of CRP and SAA are not related to lymphoma relapse; neither COP nor VCM chemotherapy changes the acute-phase response, when CRP and SAA are taken in account, and there is not difference on acute-phase response between both regimens; there is a positive correlation between CRP and SAA levels during lymphoma assessment and chemotherapy; increases of CRP levels are followed by β2-globulin elevations and increases of SAA levels are related to β1-globulin elevations.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-04-28T15:21:16Z No. of bitstreams: 1 karineandradeoliveirazanini.pdf: 564422 bytes, checksum: 95992f692adb8715af66d371f3987b2f (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-05-02T01:12:26Z (GMT) No. of bitstreams: 1 karineandradeoliveirazanini.pdf: 564422 bytes, checksum: 95992f692adb8715af66d371f3987b2f (MD5) / Made available in DSpace on 2016-05-02T01:12:26Z (GMT). No. of bitstreams: 1 karineandradeoliveirazanini.pdf: 564422 bytes, checksum: 95992f692adb8715af66d371f3987b2f (MD5) Previous issue date: 2016-03-04 / Introdução: Apesar dos avanços recentes no tratamento de pacientes com doença de Crohn (DC), os sintomas oclusivos e suboclusivos observados na presença de estenoses clinicamente significativas permanecem um problema clínico desafiador. Na DC, a identificação de fatores que se associam à redução do risco de cirurgia é importante. Materiais e Métodos: Neste estudo retrospectivo, avaliamos os possíveis fatores preditivos, incluindo os marcadores inflamatórios associados à redução da necessidade de intervenção cirúrgica em pacientes com DC que apresentaram o primeiro episódio de suboclusão intestinal clinicamente resolvido e tratados, subsequentemente, com azatioprina (AZA) durante três anos. Resultados: Trinta e seis pacientes com DC suboclusiva foram incluídos, dos quais, 24 não necessitaram de ressecção intestinal. Nenhum dado demográfico ou clínico associou-se com a resposta à AZA. Apenas a proteína C reativa (PCR) apresentou correlação com a eficácia da AZA. Para cada aumento de 1 mg na PCR, houve uma redução do risco de cirurgia em 8% (RR 0,92; IC 0,86-0,98; p=0,008). O grupo PCR>6 (elevada) apresentou 81% de redução de risco de cirurgia em relação ao grupo PCR<6 (OR 0,19 IC 0,05-0,64; p=0,008). Conclusões: Os pacientes que apresentaram PCR elevada tiveram uma menor taxa de cirurgia a médio e longo prazos durante a terapia com AZA. A PCR pode identificar pacientes com estenoses predominantemente inflamatórias e responsivas ao tratamento clínico. / Background: Despite recent advances in the treatment of patients with Crohn's disease (CD), occlusive and subocclusive symptoms observed in the presence of clinically significant stenosis remains a challenging clinical problem. In inflammatory bowel diseases (IBD), the identification of factors associated with reduced risk of surgery in this context is important. Materials and methods: In this retrospective study, we evaluated the possible predictive factors, including inflammatory markers associated with reduced need for surgical intervention in patients with CD who presented the first episode of clinically solved subocclusion and treated subsequently with azathioprine (AZA) for three years. Results: Thirty-six patients with subocclusive CD were included, of these, 24 has not required bowel resection. No demographic or clinical data associated with the response to AZA. Only C reactive protein (CRP) was correlated with the effectiveness of AZA. For each increase of 1 mg CRP, there was a reduction of surgery risk in 8% (RR 0.92, CI 0.86-0.98; P = 0.008). The CRP group> 6 (elevated) had 81% of surgery risk reduction compared to PCR group <6 (OR 0.19 CI 0.05-0.64; P = 0.008). Conclusions: Patients with elevated CRP has a lower rate of surgery in the medium and long term during therapy with AZA. CRP can identify patients with inflammatory stenosis and responsive to clinical treatment.

Objectives. Evaluate C-reactive protein (CRP) levels according to weight and physical activity. The study explored how changes in CRP were associated with baseline CRP, weight, and physical activity and changes in these variables. Methods. A randomized controlled study design assigned 348 individuals to the intervention or control group with measurements taken at baseline, 6 weeks, and 6 months of body weight, physical activity, and serum CRP levels. Participants attended an intensive 40-hour educational course delivered over a four-week period. Results. At baseline, CRP was negatively associated with total steps/week, and positively associated with weight, BMI, percent fat, and saturated fat at baseline. CRP significantly decreased through 6 weeks and also through 6 months for only those with high CRP at baseline. For those with high CRP at baseline, the decrease was significant for normal, overweight, and obese groups of people. Changes in weight or physical activity were not significantly associated with changes in CRP. Conclusions. Over 6 week and 6 month follow-up periods, the intervention failed to discriminate changes in CRP. Changes in CRP were only associated with baseline levels of CRP and BMI and were not associated with changes in any of the selected variables considered.

Electrochemical biosensors are renowned for their ability to detect a wide range of analytes in biological fluids for clinical diagnosis. The implementation of biomarkers in electrochemical biosensors for clinical diagnosis is essential for the specific and accurate diagnosis of the disease with high sensitivity and selectivity. Therefore, this thesis evaluates the challenges pertaining to the stability, reproducibility, and obtaining a low limit of detection for the internal/external biomarkers associated with two distinct electrochemical biosensors. The first study tackles the challenge of detecting low analyte concentrations in a label-free biosensor. It introduces an innovative label-free electrochemical biosensing method for the detection of glycosylated hemoglobin (HbA1c) and C-reactive protein (CRP) to predict Coronary Heart Disease (CHD) progression using tailored redox probes, proposing a dual biomarker biosensing platform for future research. Calibration curves reveal an LOD of 5 mg/mL in PBS (8) FeCN (II) and 6 mg/mL in SB for a linear range of 0 – 30 mg/mL of HbA1c. Similarly, an LOD of 0.007 mg/mL and 0.008 mg/mL in PBS (7.4) PcA-NO2 and SB, respectively, is reported for a linear range of 0 – 0.05 mg/mL of CRP. The second study focuses on stabilizing a biomolecule-free sensor for the ultra-low detection of Δ9-tetrahydrocannabinol (THC) in roadside testing. Pre-depositing THC, an external biomarker for drug-impaired driving, onto the biosensor's working electrode enhances its interaction with analytes. However, THC's oxidative nature compromises sensor stability during manufacturing. Consequently, optimal electrode storage conditions were explored, indicating frozen storage as ideal for up to six months, effectively preventing THC oxidation at -18°C, while degradation occurs at 4°C. Modified electrodes stored under optimal conditions exhibit improved calibration curves when exposed to various THC samples. / Thesis / Master of Applied Science (MASc) / An electrochemical biosensor is a sensing device with the ability to detect biological species via the transduction of a specific biological event into electrochemical signals. These sensors are extremely useful for the detection of analytes in biological fluids for clinical diagnostics, to determine the presence or absence of diseases. This manuscript addresses the challenges associated with the stability, reproducibility, and the low limits of detection associated with screen-printed carbon electrodes used in electrochemical biosensing. Subsequently, due to the strong correlation between glycated hemoglobin (HbA1c) and C-reactive protein (CRP) to connote the risk of contracting coronary heart disease (CHD), the manuscript presents a novel label-free electrochemical biosensing method for the detection of HbA1c and CRP with low detection limits. Secondly, the manuscript identifies ambient storage conditions for the long-term stability of a biomolecule-free sensing device for the roadside detection of ultra-low concentrations of Δ9-tetrahydrocannabinol (THC).

The etiology of anemia is characterized by abnormal hemoglobin synthesis. Iron deficiency is characterized by microcytic and hipochromic red cells and low serum ferritin, being the most prevalent nutritional deficiency worldwide, responsible for iron deficiency anemia (FA). Anemia of chronic disease (ACD) is considered a clinical syndrome associated with chronic inflammation, infectious disease, neoplastic or traumatic, being the second most frequent cause of anemia. The severity of anemia correlates with the degree of pathology. Both have functional iron deficiency. The objective of this study was to evaluate hematological and inflammatory, as well as the presence of oxidative stress in patients with anemia. The blood analyzer was done by the CBC, automated hematology analyzer processed, Sysmex® (Automated Hematology Analyzer). The quantitative determination of ferritin is serum was done in IMMULITE analyzer. Levels of CRP and AOPP were performed in serum by automated Cobas MIRA® (Roche Diagnostics). Statistical analysis was performed using GraphPad Prism 5. We analyzed 70 patients with microcytic and hypochromic anemia. Of these, 29 (41.43%) were diagnosed as iron deficiency anemia and 41 (58.57%) with anemia of chronic disease. As a control group, we used samples from 44 patients with hematological parameters, serum ferritin, CRP and AOPP normal. The values of MCV, MCH and MCHC significantly lower in iron deficiency anemia. Ferritin levels showed that it can be considered both a measure of iron store as an inflammatory marker. In ACD there is increased production of inflammatory cytokines, which, in turn, increases the concentration of C-reactive protein (CRP). The results indicate that AOPP in both groups with anemia showed increased levels of this marker, which indicates the presence of oxidative stress, probably caused by increased production of free radicals and decreases in enzyme activities of the antioxidant defense system of erythrocytes. / A etiologia das anemias caracteriza-se pela síntese anormal de hemoglobina. A deficiência de ferro é caracterizada por eritrócitos microcíticos e hipocrômicos e por ferritina sérica baixa, sendo a carência nutricional mais prevalente em todo o mundo, responsável pela Anemia Ferropriva (AF). A Anemia de Doença Crônica (ADC) é considerada uma síndrome clínica, associada à inflamação crônica, doença infecciosa, traumática ou neoplásica, sendo a segunda causa mais freqüente de anemia. Ambas apresentam deficiência funcional de ferro. O objetivo deste trabalho foi avaliar parâmetros hematológicos e inflamatórios, bem como a presença de estresse oxidativo em pacientes com anemia. A análise hematológica foi feita através do hemograma, processado em analisador hematológico automatizado, Sysmex® (Automated Hematology Analyzer). O doseamento quantitativo da ferritina no soro foi feito em analisador IMMULITE. A dosagem de Proteína C-Reativa (PCR) e de Produtos da Oxidação Avançada de Proteínas (AOPP) foram realizadas no soro através do sistema automatizado Cobas MIRA® (Roche Diagnostics). A análise estatística foi realizada através do programa GraphPad Prism 5. Foram analisados 70 pacientes portadores de anemia microcítica e hipocrômica. Destes, 29 (41,43%) foram diagnosticados como anemia ferropriva e 41 (58,57%) com anemia de doença crônica. Como grupo controle, foram utilizadas amostras de 44 indivíduos com parâmetros hematológicos, níveis de ferritina, PCR e AOPP dentro da normalidade. Os valores de VCM, HCM e CHCM foram significativamente menores na anemia ferropriva. Os níveis de ferritina revelaram que ela pode ser considerada tanto uma medida das reservas de ferro quanto um marcador inflamatório. Na ADC há aumento da produção de citocinas inflamatórias, que, por sua vez, aumenta também a concentração de PCR. Os resultados do AOPP indicam que ambos os grupos com anemia apresentaram níveis aumentados deste marcador, o que indica a presença de estresse oxidativo, provavelmente causado por aumento na produção de radicais livres e declínio das atividades das enzimas do sistema de defesa antioxidante dos eritrócitos.