Evaluation of Leishmania antigens preparation and storage for use in enzyme immunoassays

The influence of time and temperature on the storage of an alkaline antigen of L.major-like and L.(V.) braziliensis promastigotes added or not of a proteases inhibitor (PMSF) was evaluted by means of an IgG-ELISA. Antibodies in assays using L. major-like antigen stored at -20oC for 6 monsths had a statistically lower geometric mean titer (GMT) and different 95% confidence interval limits (CL) than antigens stored otherwise, as assessed by the "t" statistic. The PMSF L. major-like antigen after storage for 6 months at a temperature of 4oC had the same GMT and 95% CL displayed at time zero as well as when storage for 4 and 6 months at -20oC. Significant diferences were not found when L.(V.) braziliensis antigens were stored at times and temperatures mentioned; the PMSF antigen stored for 2 months at -70oC resulted in a lower serum GMT and 95% CL than any other, as assessed by the "t" statistic. Antigen performance did not show any statistical difference associated to the addition of PMSF within the same species; the largest difference between antigens was that between PMSF-L. (V.) braziliensis and L. major-like without PMSF.

Dot enzyme-linked immunosorbent assay (dot-ELISA) for schistosomiasis diagnosis using dacron as solid-phase

Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA) for schistosomiasis and compared to indirect immunofluorescence (IMF). Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP) was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05). The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05). In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.

Synthesis of new enzyme stabilisers inspired by compatible solutes of hyperthermophilic microorganisms

Dissertation presented to obtain the Ph.D degree in Engineering Sciences and Technology.

Study of novel energy metabolism pathways in anaerobic bacteria

Energy conservation in chemotrophic anaerobic bacteria is achieved by two possible processes, substrate level phosphorylation (SLP) and electron transfer phosphorylation (ETP). This second mechanism, also known as respiration, involves chemiosmotic coupling. However, a third mechanism for energy coupling was recently proposed: the flavin-based electron bifurcation (FBEB). (...)

Assembly and function of a protein cross-linking enzyme during bacterial spore morphogenesis

Sporulation in Bacillus subtilis culminates with the formation of a dormant endospore. The endospore (or spore) is one of the most resilient cell types known and can remain viable in the environment for extended periods of time. Contributing to the spore’s resistance and its ability to interact with and monitor its immediate environment is the coat, the outermost layer of B. subtilis spores. The coat is composed by over 70 different proteins, which are produced at different stages in sporulation and orderly assembled around the developing spore.(...)

Evaluation of an enzyme immunoassay for clinical diagnosis of neurocysticercosis in symptomatic patients

INTRODUCTION: Neurocysticercosis is an infection of the human central nervous system caused by the metacestode larvae of Taenia solium. Neurocysticercosis is the most common parasitic disease in developing countries. Epilepsy is the most common clinical manifestation. Difficulties in confirming the diagnosis motivated the evaluation of the enzyme-linked immunosorbent assay on cerebral spinal fluid (CSF). METHODS: Twenty-two patients with NCC and 44 control patients were studied. CSF was analyzed using a commercial ELISA kit developed for NCC. Sensitivity and specificity were measured and a multivariate logistic regression was performed. RESULTS: Sensitivity and specificity of ELISA were 31.8% and 100%, respectively, with accuracy of 77.3%. Only the size of the lesions proved to be important for performance of the test. CONCLUSIONS: The results showed that ELISA contributes to the diagnosis of neurocysticercosis if the result is negative or if the patient has a lesion of 2 cm or more.

Caracterização do papel dos genes TYMS e MTR no desenvolvimento de Cancro Colo-Rectal

RESUMO: O cancro colo-rectal (CCR) é um dos cancros que possui maior taxa de mortalidade a nível mundial. Em Portugal esta patologia é responsável pela morte de cerca de 3700 pessoas por ano, sendo que estes números aumentam de ano para ano. Ao longo das últimas décadas o papel das alterações genéticas na etiologia das patologias oncológicas tem vindo a ter cada vez mais um maior destaque. O número de estudos que avaliam a importância de polimorfismos, mutações, alterações na regulação génica e interacções entre genes no desenvolvimento destas patologias tem aumentado exponencialmente. Com o aumento do conhecimento da forma como estas alterações influenciam o desenvolvimento do cancro surgiram os primeiros meios de diagnóstico genético, levando assim a uma alteração da forma como são encarados o diagnóstico e a prevenção destas doenças. No CCR as formas hereditárias com alterações genéticas inequivocamente identificadas representam apenas 5% dos casos. Existem cerca de 25% que representam formas hereditárias para as quais ainda não foram estabelecidos os padrões de alterações genéticas subjacentes. Desta forma, estudos que venham contribuir para um maior conhecimento dos mecanismos moleculares responsáveis pelo aumento da susceptibilidade dos indivíduos para o desenvolvimento de CCR são extremamente importantes. O CCR é uma patologia multifactorial, onde factores genéticos interagem com factores ambientais no surgimento e desenvolvimento da doença. Assim, torna-se essencial integrar o estudo das alterações genéticas no contexto ambiental onde os indivíduos em estudo se encontram. No caso desta patologia um dos principais factores ambientais estudado é a nutrição. Vários estudos têm sido realizados ao longo dos últimos anos de forma a compreender como pode a ingestão dos nutrientes influenciar o desenvolvimento de CCR e de que forma interage com as alterações genéticas individuais. O ciclo do folato é um dos processos metabólicos onde o papel da nutrição em interacção com alterações genéticas mais tem sido estudado nos últimos anos. Deste cruzamento entre o estudo das alterações genéticas e ambientais surge a Nutrigenética. O conjunto de estudos da presente tese tem como objectivo aumentar o conhecimento do papel das alterações em genes do ciclo do folato, em interacção com factores nutricionais e de estilo de vida, não só no desenvolvimento de CCR, mas também de outra patologia do tracto gastrointestinal, a Doença de Crohn (DC), uma doença inflamatória muitas vezes associada como factor de risco para o desenvolvimento de CCR. Este estudo debruçou-se essencialmente no estudo dos genes timidilato sintetase (TYMS) e metionina sintetase (MTR) em populações com CCR e DC, bem como no padrão nutricional destas populações com particular incidência nos nutrientes envolvidos no ciclo do folato (folato, metionina, vitamina B6, vitamina B12). Analisando o conjunto de resultados obtidos para os estudos do CCR podemos concluir que quer a TYMS quer a MTR possuem um papel relevante na susceptibilidade para desenvolver esta patologia, assim como têm destaque no funcionamento do ciclo celular durante o processo oncogénico. Os resultados demonstram que os factores que levam a uma menor disponibilidade de grupos metil no ciclo de folato (baixos níveis de folato, alteração da actividade de MTR, elevada expressão de TYMS) constituem factores de risco, muito provavelmente por contribuírem para uma desregulação dos níveis de metionina disponível para a metilação do DNA da célula. Demonstram ainda que em células tumorais ocorrem alterações na regulação do ciclo do folato de forma a favorecer a síntese de DNA em detrimento da metilação do mesmo, alterando para isso a expressão dos genes de forma a que o fluxo de grupos metil provenientes do folato sejam encaminhados para a enzima TYMS. O polimorfismo de deleção 6pb da TYMS surge como um factor de diagnóstico e de prognóstico de CCR para a população portuguesa. Dos factores nutricionais analisados apenas o folato aparenta ter um papel relevante na modelação do risco de desenvolver CCR. Na doença de Crohn (DC) podemos verificar que a homocisteína e o seu metabolismo poderão contribuir para o aparecimento e desenvolvimento da patologia. O aumento da homocisteína poderá ser o responsável por um aumento da resposta auto-imune do organismo, promovendo o aparecimento da DC. O polimorfismo A2756G MTR desempenha um papel preponderante como factor de diagnóstico da DC, tendo sido associado pela primeira vez a esta patologia. Tem também um papel importante no desenvolvimento da doença, uma vez que está associado a uma idade de diagnóstico mais baixa, sugerindo assim que o desenvolvimento da doença ocorre de forma mais precoce. Concluindo, com este estudo pensamos ter contribuído para um melhor entendimento do papel do ciclo do folato no desenvolvimento de CCR e DC, sendo um ponto de partida para futuras investigações que possam revelar cada vez melhor as complexas interacções metabólicas desta via e a sua influência nas patologias estudadas. Do nosso estudo destacamos a importância de uma análise global das várias etapas do ciclo do folato para que se possa compreender a dinâmica que se estabelece no desenvolvimento destas patologias, podendo diversas alterações, quer a nível genético quer a nível nutricional, exercerem efeitos diferentes consoante o estado dos restantes intervenientes do ciclo do folato. Acreditamos que no futuro este estudo permitirá que o conhecimento do ciclo do folato tenha cada vez mais uma relevância fundamental a nível de diagnóstico e terapêutica destas patologias.------------ ABSTRACT: Colorectal Cancer (CRC) is one of the cancers that have a higher rate of mortality worldwide. In Portugal this pathology is responsible for the deaths of about 3700 people per year, and these numbers increase each year. Over the past few decades the role of genetic changes in the etiology of oncological pathologies has had an increasingly greater emphasis. The number of studies that evaluate the importance of polymorphisms, mutations, changes in gene regulation and gene interactions in the development of these diseases has increased exponentially. With the increased knowledge of how these changes influence the development of cancer, appeared the first means for genetic diagnostic, leading to a change in the way diagnosis is seen and in the prevention of these diseases. In CRC the hereditary forms with clearly identified genetic changes represent only 5% of cases. There are about 25% representing hereditary forms for which the patterns of genetic changes haven’t been established. In this way, studies that will contribute to a greater understanding of the molecular mechanisms responsible for increased susceptibility of individuals to the CRC development are extremely important. CRC is a multifactorial pathology, where genetic factors interact with environmental factors in the emergence and development of the disease.Thus, it is essential to integrate the study of genetic changes in the environmental context of the individuals under study. In the case of this pathology one of the main environmental factors studied is nutrition. Several studies have been conducted over the past few years in order to understand how the intake of nutrients can influence the development of CRC and how nutrients interact with the individual genetic changes. The folate cycle is one of the metabolic processes where the role of nutrition in interaction with genetic alterations has been studied in recent years. This cross between the study of genetic and environmental changes developed Nutrigenetics. The set of studies of this thesis aims to increase awareness of the role of changes in genes of the folate cycle, in interaction with nutritional factors and lifestyle, not only in the development of CRC, but also of another pathology of the gastrointestinal tract, Crohn's disease (CD), an inflammatory disease often associated as a risk factor for the development of CRC. This study dealt mainly in the study of genes thymidylate synthase (TYMS) and methionine synthase (MTR) in populations with CRC and CD, as well as in the nutritional pattern of these populations with particular focus on nutrients involved in the folate cycle (folate, methionine, vitamin B6, vitamin B12). Analyzing the results obtained for the CRC studies we conclude that either the MTR TYMS have a relevant role in susceptibility to develop this pathology, and have an important role in the functioning of the cell cycle during oncogenesis. The results show that the factors that lead to a lower availability of methyl groups in folate cycle (low levels of folate, change the activity of MTR, high expression of TYMS) constitute risk factors, most likely by contribute to a dysregulation of methionine levels available for DNA methylation of the cell. Our results also demonstrate that in tumor cells occur changes in the regulation of the folate cycle in order to promote the synthesis of DNA, to the detriment of methylation of the same by changing the expression of genes so that the methyl groups from folate are forwarded to the TYMS enzyme reaction. The deletion polymorphism 6bp of TYMS emerges as a diagnostic and prognostic factor of CCR for the Portuguese population. Nutritional factors analyzed only folate appears to have a major role in modulating the risk of developing CCR.In Crohn’s disease (CD) we can check that homocysteine and its metabolism may contribute to the emergence and development of this pathology. Increased homocysteine may be responsible for an increase in the body's autoimmune response, promoting the emergence of CD. The polymorphism A2756G MTR plays a leading role as a factor of diagnosis of DC, having been associated with this pathology for the first time. It also has an important role in the development of the disease, since it is associated with a lower diagnostic age, suggesting that the development of the disease occurs earlier. In conclusion, our study has contributed to a better understanding of the role of folate cycle in the development of CRC and CD, being a starting point for future research that may prove increasingly complex metabolic interactions in this via and its influence on the pathologies studied. In our study we highlight the importance of a comprehensive analysis of the various steps of the folate cycle in order to understand the dynamics that settles in the development of these pathologies, and a number of amendments, whether at the genetic level or at the nutritional level, exercise different effects depending on the stage of the remaining participants in the folate cycle. We believe that in the future this study will allow the knowledge of folate cycle to have increasingly a fundamental relevance at the level of diagnosis and treatment of these diseases.

Restriction enzyme analysis of the human cytomegalovirus genome in specimens collected from immunodeficient patients in Belém, State of Pará, Brazil

INTRODUCTION: Human cytomegalovirus is an opportunistic betaherpesvirus that causes persistent and serious infections in immunodeficient patients. Recurrent infections occur due to the presence of the virus in a latent state in some cell types. It is possible to examine the virus using molecular methods to aid in the immunological diagnosis and to generate a molecular viral profile in immunodeficient patients. The objective of this study was to characterize cytomegalovirus genotypes and to generate the epidemiological and molecular viral profile in immunodeficient patients. METHODS: A total of 105 samples were collected from immunodeficient patients from the City of Belém, including newborns, hemodialysis patients, transplant recipients and HIV+ patients. An IgG and IgM antibody study was completed using ELISA, and enzymatic analysis by restriction fragment length polymorphism (RFLP) was performed to characterize viral genotypes. RESULTS: It was observed that 100% of the patients had IgG antibodies, 87% of which were IgG+/IgM-, consistent with a prior infection profile, 13% were IgG+/IgM+, suggestive of recent infection. The newborn group had the highest frequency (27%) of the IgG+/IgM+ profile. By RFLP analysis, only one genotype was observed, gB2, which corresponded to the standard AD169 strain. CONCLUSIONS: The presence of IgM antibodies in new borns indicates that HCMV continues to be an important cause of congenital infection. The low observed genotypic diversity could be attributed to the small sample size because newborns were excluded from the RFLP analysis. This study will be continued including samples from newborns to extend the knowledge of the general and molecular epidemiology of HCMV in immunodeficient patients.

Evaluation of Leishmania (Leishmania) chagasi strains isolated from dogs originating from two visceral leishmaniasis-endemic areas in Brazil using multilocus enzyme electrophoresis

INTRODUCTION: Domestic dogs are the most important reservoir in the peridomestic transmission cycle of Leishmania (Leishmania) chagasi. The genetic variability of subpopulations of this parasite circulating in dogs has not been thoroughly analyzed in Brazil, even though this knowledge has important implications in the clinical-epidemiological context. METHODS: The objective of this study was to evaluate and compare the phenotypic variability of 153 L. chagasi strains isolated from dogs originating from the municipalities of Rio de Janeiro (n = 57) and Belo Horizonte (n = 96), where the disease is endemic. Strains isolated only from intact skin were selected and analyzed by multilocus enzyme electrophoresis using nine enzyme systems (6PG, GPI, NH1 and NH2, G6P, PGM, MDH, ME, and IDHNADP). RESULTS: The electrophoretic profile was identical for all isolates analyzed and was the same as that of the L. chagasi reference strain (MHOM/BR/74/PP75). Phenetic analysis showed a similarity index of one for all strains, with the isolates sharing 100% of the characteristics analyzed. CONCLUSIONS: The results demonstrate that the L. chagasi populations circulating in dogs from Rio de Janeiro and Belo Horizonte belong to a single zymodeme.

The role of pneumococcal carbon metabolism in colonisation and invasive disease

Streptococcus pneumoniae is a common asymptomatic commensal of the human nasopharynx. However, it is better known as a threatening pathogen that causes serious diseases such as pneumonia, meningitis and sepsis, as well as other less severe but more prevalent infections (e.g. otitis media). With the increase of antibiotic resistance and the limited efficacy of vaccines, pneumococcal infections remain a major problem. Therefore, the discovery of new therapeutic targets and preventive drugs are in high demand.(...)

Development of 3D in vitro models for prediction of hepatic metabolism and toxicity

The development of human cell models that recapitulate hepatic functionality allows the study of metabolic pathways involved in toxicity and disease. The increased biological relevance, cost-effectiveness and high-throughput of cell models can contribute to increase the efficiency of drug development in the pharmaceutical industry. Recapitulation of liver functionality in vitro requires the development of advanced culture strategies to mimic in vivo complexity, such as 3D culture, co-cultures or biomaterials. However, complex 3D models are typically associated with poor robustness, limited scalability and compatibility with screening methods. In this work, several strategies were used to develop highly functional and reproducible spheroid-based in vitro models of human hepatocytes and HepaRG cells using stirred culture systems. In chapter 2, the isolation of human hepatocytes from resected liver tissue was implemented and a liver tissue perfusion method was optimized towards the improvement of hepatocyte isolation and aggregation efficiency, resulting in an isolation protocol compatible with 3D culture. In chapter 3, human hepatocytes were co-cultivated with mesenchymal stem cells (MSC) and the phenotype of both cell types was characterized, showing that MSC acquire a supportive stromal function and hepatocytes retain differentiated hepatic functions, stability of drug metabolism enzymes and higher viability in co-cultures. In chapter 4, a 3D alginate microencapsulation strategy for the differentiation of HepaRG cells was evaluated and compared with the standard 2D DMSO-dependent differentiation, yielding higher differentiation efficiency, comparable levels of drug metabolism activity and significantly improved biosynthetic activity. The work developed in this thesis provides novel strategies for 3D culture of human hepatic cell models, which are reproducible, scalable and compatible with screening platforms. The phenotypic and functional characterization of the in vitro systems performed contributes to the state of the art of human hepatic cell models and can be applied to the improvement of pre-clinical drug development efficiency of the process, model disease and ultimately, development of cell-based therapeutic strategies for liver failure.

Laboratory diagnosis of amebiasis in a sample of students from southeastern Brazil and a comparison of microscopy with enzyme-linked immunosorbent assay for screening of infections with Entamoeba sp.

Introduction: Epidemiological studies on amebiasis have been reassessed since Entamoeba histolytica and E. dispar were first recognized as distinct species. Because the morphological similarity of these species renders microscopic diagnosis unreliable, additional tools are required to discriminate between Entamoeba species. The objectives of our study were to compare microscopy with ELISA kit (IVD®) results, to diagnose E. histolytica infection, and to determine the prevalence of amebiasis in a sample of students from southeastern Brazil. Methods: In this study, diagnosis was based on microscopy due to its capacity for revealing potential cysts/trophozoites and on two commercial kits for antigen detection in stool samples. Results: For 1,403 samples collected from students aged 6 to 14 years who were living in Divinópolis, Minas Gerais, Brazil, microscopy underestimated the number of individuals infected with E. histolytica/E. dispar (5.7% prevalence) compared with the ELISA kit (IVD®)-based diagnoses (15.7% for E. histolytica/E. dispar). A comparison of the ELISA (IVD®) and light microscopy results returned a 20% sensitivity, 97% specificity, low positive predictive value, and high negative predictive value for microscopy. An ELISA kit (TechLab®) that was specific for E. histolytica detected a 3.1% (43/1403) prevalence for E. histolytica infection. Conclusions: The ELISA kit (IVD®) can be used as an alternative screening tool. The high prevalence of E. histolytica infection detected in this study warrants the implementation of actions directed toward health promotion and preventive measures.

Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI) in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs) and one nucleic acid amplification test (NAAT) were evaluated against a cytotoxicity assay (CTA) and toxigenic culture (TC). Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2%) of 92 samples were positive according to the CTA, and 23 (25%) were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

Effects of ocean acidification on the swimming ability, development and biochemical responses of sand smelt larvae

Ocean acidification, recognized as a major threat to marine ecosystems, has developed into one of the fastest growing fields of research in marine sciences. Several studies on fish larval stages point to abnormal behaviours, malformations and increased mortality rates as a result of exposure to increased levels of CO2. However, other studies fail to recognize any consequence, suggesting species-specific sensitivity to increased levels of CO2, highlighting the need of further research. In this study we investigated the effects of exposure to elevated pCO2 on behaviour, development, oxidative stress and energy metabolism of sand smelt larvae, Atherina presbyter. Larvae were caught at Arrábida Marine Park (Portugal) and exposed to different pCO2 levels (control: ~600μatm, pH=8.03; medium: ~1000μatm, pH=7.85; high: ~1800μatm, pH=7.64) up to 15days, after which critical swimming speed (Ucrit), morphometric traits and biochemical biomarkers were determined. Measured biomarkers were related with: 1) oxidative stress - superoxide dismutase and catalase enzyme activities, levels of lipid peroxidation and DNA damage, and levels of superoxide anion production; 2) energy metabolism - total carbohydrate levels, electron transport system activity, lactate dehydrogenase and isocitrate dehydrogenase enzyme activities. Swimming speed was not affected by treatment, but exposure to increasing levels of pCO2 leads to higher energetic costs and morphometric changes, with larger larvae in high pCO2 treatment and smaller larvae in medium pCO2 treatment. The efficient antioxidant response capacity and increase in energetic metabolism only registered at the medium pCO2 treatment may indicate that at higher pCO2 levels the capacity of larvae to restore their internal balance can be impaired. Our findings illustrate the need of using multiple approaches to explore the consequences of future pCO2 levels on organisms.

Osteoclastogenesis, inflammatory cytokines and biomarkers of bone metabolism in psoriatic arthritis

RESUMO:A artrite psoriática (AP) é uma doença inflamatória crónica caracterizada por várias manifestações nas articulações, nas enteses e na pele. A formação de novo osso após inflamação nas enteses é um dos aspetos mais intrigantes desta doença. Os mecanismos celulares e moleculares deste processo ainda não são completamente conhecidos. Este estudo tem como objetivo compreender melhor os mecanismos subjacentes à formação e reabsorção óssea, bem como o efeito de anti-inflamatórios não esteroides (AINEs) nestes processos. Para atingir este objetivo foram quantificados biomarcadores do metabolismo ósseo e citocinas inflamatórias em doentes AP, antes e após terapêutica com AINEs. Os biomarcadores selecionados foram marcadores de remodelação óssea como CTX-I e P1NP, fatores de diferenciação e ativação de osteoclastos como o RANKL e a OPG, inibidores da via de sinalização Wnt, nomeadamente o DKK-1 e a SOST e ainda citocinas pro-inflamatórias como a IL-22 e a IL-23 e a prostaglandina PGE2. Neste contexto foram também estabelecidas culturas celulares de monócitos, isoladas de doentes AP e de controlos saudáveis. Os monócitos foram cultivados in vitro em condições não estimuladas e estimuladas e realizados dois ensaios funcionais: coloração com TRAP e ensaio de reabsorção. Foi observada uma diminuição nos níveis séricos de CTX-I e OPG em doentes AP em relação aos controlos. De igual forma os níveis séricos de SOST encontram-se significativamente mais baixos, em comparação com os controlos saudáveis. Estes valores de SOST são semelhantes aos dos doentes com espondilite anquilosante (EA), documentados anteriormente. Os ensaios com osteoclastos confirmaram a necessidade da presença de RANKL para estimulação da osteoclastogénese e que o celecoxib parece ter um papel inibitório neste processo. Os resultados obtidos sugerem que a população de doentes com AP analisados têm baixos níveis de reabsorção óssea e alguma atividade na formação óssea. --------------------------- ABSTRACT: Psoriatic arthritis (PsA) is a chronic inflammatory disease characterized by several manifestations involving the joints, enthesis and the skin. New bone formation after inflammation at enthesis site has been one of the most intriguing aspects of the disease. Cellular and molecular mechanisms in this process are still not completely understood. This study aims to understand better the mechanisms underlying bone formation and resorption and the effect of non-steroid anti-inflammatory drugs (NSAIDs) in these processes. To access that, biomarkers of bone metabolism and inflammatory cytokines were measured in PsA patients’ serum before and after NSAID therapy. These selected biomarkers were bone turnover markers such as CTX-I and P1NP, osteoclast differentiation and activation factors RANKL and OPG, Wnt pathway inhibitors DKK-1 and SOST and pro-inflammatory cytokines IL-22, IL-23 and prostaglandin PGE2. In this context monocyte cell culture was also established after PBMC isolation from PsA patients and healthy controls. Monocytes were cultured in vitro under unstimulated and stimulated conditions and two functional assays were performed: TRAP staining and resorption pit assay. It was demonstrated that CTX-I and OPG serum levels in PsA patients were lower than controls. SOST levels were extremely decreased in comparison with controls, resembling the ankylosing spondylitis patients results already documented. Osteoclast assays confirmed the need of RANKL in stimulating osteoclastogenesis and that celecoxib seems to have an inhibitor role in this process. The results obtained suggest that PsA patient population analyzed in this study have low bone resorption levels and some bone formation activity.