Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-05-23T17:25:42Z No. of bitstreams: 1 DIS_LENIO_TOGNI_PARCIAL.pdf: 1339619 bytes, checksum: f9a493b6c4f4be9a8d1569be186595f8 (MD5) / Made available in DSpace on 2017-05-23T17:25:42Z (GMT). No. of bitstreams: 1 DIS_LENIO_TOGNI_PARCIAL.pdf: 1339619 bytes, checksum: f9a493b6c4f4be9a8d1569be186595f8 (MD5) Previous issue date: 2017-01-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Objective: This study aimed to characterize the development of temporomandibular osteoarthritis (TMJ-OA) following the surgical anterior displacement of the articular disc (ADD) in rats, in comparison to the well-established model of OA induced by the intra-articular injection of the inflammatory agent CFA. Methods: Male Wistar rats (160-180 g) were randomly divided into two surgical groups, namely ADD (submitted to anterior disc displacement) and sham-operated (surgical access, without ADD). For purposes of comparison, two additional experimental groups received an intra-articular infiltration of complete Freund?s adjuvant (CFA; 50 ?l/site; 1:1 oil/saline emulsion), or the same volume of saline solution (0.9% NaCl). Different experimental subgroups (N=8 per group) were euthanized on 15, 30 or 60 days after procedures. The region corresponding to the left TMJ was collected for histological analysis of fibrocartilage thickness (hematoxylin-eosin staining), presence of proteoglycans (safranin-O and toluidine blue), subchondral bone metabolism (tartrate-resistant acid phosphatase; TRAP) and collagen levels (Masson's trichrome). The immunopositivity for the aggrecanase ADAMTS5 was examined in the fibrocartilage layers. Micro-CT images were acquired for morphological and microstructural evaluation (30 and 60 days; N=4 per group). The levels of inflammatory serum proteins (IL-1?, IL-6 and TNF) were assessed to evaluate the possible systemic outcomes of TMJ-OA induced by ADD or CFA. Results: The analysis of hematoxylin-eosin-stained sections revealed an increment of fibrocartilage thickness in the ADD group, on 15 and 30 days, mainly in the anterior third of the condyle. In the CFA group, a fibrocartilage thickening was seen in the posterior third, only at 15 days. Increased proteoglycan contents were present in the fibrocartilage of the ADD group, according to the qualitative analysis by safranin-O and toluidine blue staining. The ADD group also displayed an augmented immunopositivity for ADAMTS5 in the fibrocartilage cells, on 15 and 30 days, whereas the expression of this aggrecanase was subtly increased in the CFA group at 15 days. The histological evaluation of the subchondral bone did not reveal any evident difference in the collagen contents, or in the number of activated osteoclasts, in the ADD or CFA groups, as indicated by Masson?s trichrome and TRAP staining. However, the micro-CT analysis showed marked morphological and structural changes in condyles of the ADD group, with osteophyte formation on 30 days, and a flattening of condylar surface on 60 days. The anterior condyle third of the ADD group also presented an increment of trabecular separation and bone surface, associated to a reduction of trabecular thickness and bone volume. The serum levels of the pro-inflammatory cytokines, namely IL-1?, TNF or IL-6, were undetectable in all the experimental groups. Conclusion: The surgical model of ADD in rats led to long-term OA-like alterations, with the formation of osteophytes, followed by flattening of the condyle surface and trabecular derangements. This experimental model might represent a reliable strategy to investigate TMJ-OA-related mechanisms, allowing the identification of novel therapies for this condition. / Objetivo: Este trabalho teve por objetivo caracterizar o desenvolvimento de um modelo de osteoartrite, na articula??o temporomandibular (TMJ-OA), posteriormente ? cirurgia de anterioriza??o de disco articular em ratos, em compara??o a um modelo j? estabelecido de indu??o de osteoartrite (OA), pela infiltra??o intra-articular do agente inflamat?rio, CFA. M?todos: Ratos Wistar machos (160-180 g) foram randomicamente divididos em dois grupos, denominados ADD (submetidos ? anterioriza??o do disco articular) e falso-operados (acesso cir?rgico, sem a anterioriza??o do disco articular). Para fins de compara??o, dois grupos experimentais adicionais receberam a infiltra??o intra-articular do adjuvante completo de Freund (CFA; 50 ?l/s?tio; 1:1 ?leo/emuls?o salina) ou, o mesmo volume de solu??o salina (NaCl 0,9%). Diferentes subgrupos experimentais (n=8 por grupo) foram eutanasiados em 15, 30 ou 60 dias depois dos procedimentos. A regi?o correspondente ? articula??o temporomandibular (ATM) foi coletada para an?lises histol?gicas quanto ? espessura da fibrocartilagem (hematoxilina-eosina), ? presen?a de proteoglicanas (safranin-O e azul de toluidina), ao metabolismo ?sseo sub-condral (fosfatase ?cida tartarato-resistente; TRAP) e aos n?veis de col?geno (tricr?mico de Masson). A imunopositividade para a agrecanase, ADAMTS5, foi avaliada nas diferentes camadas de fibrocartilagem. Imagens de microtomografia foram adquiridas para avalia??o da morfologia e microestrutura (30 e 60 dias; N=4 por grupo). Os n?veis inflamat?rios das citocinas s?ricas, IL-1?, IL-6 e TNF, foram avaliados como indicativos dos poss?veis efeitos da indu??o de TMJ-AO, por ADD ou CFA. Resultados: A an?lise da colora??o por hematoxilina-eosina demostrou um aumento na espessura da fibrocartilagem no grupo ADD, em 15 e 30 dias, principalmente no ter?o anterior do c?ndilo. No grupo CFA, um espessamento da fibrocartilagem foi encontrado no ter?o posterior, somente em 15 dias. Um aumento da presen?a de proteoglicanas foi encontrado na fibrocartilagem do grupo ADD, de acordo com a an?lise qualitativa pelas colora??es de safranin-O e de azul de toluidina. O grupo ADD tamb?m apresentou um aumento da imunopositividade para ADAMTS5 nas c?lulas da fibrocartilagem, em 15 e 30 dias, enquanto que a express?o dessa agrecanase, no grupo CFA, apresentou-se discretamente aumentada em 15 dias. A avalia??o histol?gica do osso subcondral n?o revelou nenhuma diferen?a em rela??o ao conte?do de col?geno ou, ao n?mero de osteoclastos ativos, nos grupos ADD ou CFA, como indicado pelas colora??es de tricr?mico de Masson e TRAP. Contudo, a an?lise microtomogr?fica demostrou mudan?as marcantes na morfologia e na estrutura ?ssea dos c?ndilos do grupo ADD, com forma??o de oste?fitos em 30 dias e, achatamento da superf?cie articular em 60 dias. O ter?o anterior do c?ndilo do grupo ADD tamb?m apresentou um aumento na separa??o trabecular e na superf?cie ?ssea, associada com uma redu??o da espessura trabecular e do volume ?sseo. Os n?veis s?ricos de citocinas pr?-inflamat?rias (IL-1?, IL-6 e TNF) foram indetect?veis em todos os grupos experimentais. Conclus?o: O modelo cir?rgico ADD em ratos levou a altera??es cr?nicas compat?veis com OA, com forma??o de oste?fitos, seguida por achatamento da superf?cie condilar e altera??es trabeculares. Este modelo experimental pode representar uma nova estrat?gia para investigar os mecanismos relacionados ? TMJ-OA, permitindo a identifica??o de novas terapias para esta condi??o.

Die juvenilen rheumatischen Erkrankungen sind häufiger, als von Laien angenommen wird. In der vorliegenden Studie werden Patienten der Kinderklinik und Polyklinik der Universität Würzburg genauer betrachtet um mögliche Verlaufsparameter retrospektiv auswerten zu können. Es zeigt sich, dass weibliche Patienten als häufiger von JIA-Erkrankungen betroffene länger therapiert wurden als die männlichen Patienten. Dabei erhielten sie länger NSAR und Steroide sowie häufiger MTX. Die Wahrscheinlichkeit eine Remission zu erreichen war im untersuchten Kollektiv nicht per se vom Geschlecht abhängig, jedoch geschätzt nach Kaplan und Meier zu Ungunsten der Mädchen, wie laut Literatur zu erwarten. Nicht bestätigen ließ sich ein negativer Zusammenhang zwischen erhöhten Entzündungswerten zu Beginn der Erkrankung und einer Remission im Verlauf. Weiterhin nicht bestätigen ließ sich eine generell geringere Remissionswahrscheinlichkeit bei Polyarthritis. Als prädiktive Marker für eine häufigere Remission können nach unseren Ergebnissen nun theoretisch das männliche Geschlecht (nach Kaplan und Meier) sowie erhöhte Entzündungswerte zu Beginn der Erkrankung angenommen werden. Wobei eine Remission häufiger auftrat, je schneller nach Krankheitsbeginn der Patient „an der richtigen Adresse“ vorgestellt wurde. / The frequency of juvenile idiopathic arthritis is bigger than most non-professional may expect. This trial analyzes retrospective e.g. the outcome of patients from the children's hospital of the University of Wuerzburg. In our cohort it is seen, that female patients are more often affected with rheumatism and need longer medication with NSAR and steroids, also they need more often MTX. The likelihood to reach remission did not differ between boys and girls at first sight, but rated by Kaplan and Meier the girls come out badly - like other references say, too. In our cohort there was no correlation between high inflammation values at the beginning of the disease and remission at a later time. Also it could not be seen, that children with Polyarthritis reached less often remission in general. Only regarding our data one could use male gender and higher inflammation values at the beginning of the disease as a predictive marker for a more frequent remission at a later time. Whereas the remission appeared more often in children who were presented to the "Specialist" early after the beginning of the disease.

This thesis explored the development of a faster and more efficient means of qualitative and quantitative analysis of the products of the nitric acid oxidation of D-glucose and other simple sugars, for the Shafizadeh Rocky Mountain Center for Wood and Carbohydrate Chemistry. During the research, analysis was carried out based on previous work completed in a similar area using two Aminex HPX-87H+ cation-exchange columns at different temperatures, and plumbed in series. Standards were filtered and injected on to the columns, then eluted with 5 mM sulfuric acid. A total run time of 33 minutes enabled the elution of all products and by-products of the reaction. Retention times of standards and the use of spiking helped specify and quantify unknowns in samples from a series of oxidation reactions involving D-glucose and other aldoses. The PrevailTM Organic acid (OA) column was said to provide 'unsurpassed resolution of organic acids'. It was therefore investigated, and a method was developed and refined in order to optimise conditions enabling the column's use for the required analyses. The optimised parameters were established as: ambient temperature with an eluent of 10 mM KH2PO4 adjusted to a pH of 2.1 with phosphoric acid. The sample size was 5 uL with a flow rate of 0.3 mL/min, giving a total run time of approximately 13 minutes. The Aminex HPX-87H+ column method and the PrevailTM OA method were compared to determine the superior method for the analyses intended. While some improvements were made for detection in the PrevailTM OA method, results were not satisfactory. This was due in part to limits imposed on the PrevailTM OA column method, which prevented the use of gradient elution. The Aminex HPX-87H+ column method outlined herein provides superior resolution for the nitric acid oxidation of D-glucose to D-glucaric acid, and in conclusion it is suggested that the Aminex HPX-87H+ column method be used.

This study used the Gordon-Schaefer model which was extended to Open Access (OA) model and Present Value Maximization (MPV) model to discuss the equilibrium levels for bigeye tuna in the Western and Central Pacific Ocean by purse seine fishery data. I compared the catches and the biomass with the two model¡¦s equilibrium value, and the result showed that the operating mode of bigeye tuna in the Western and Central Pacific Ocean tended to the MPV model, and the bigeye tuna resources were diminishing. In addition, the sensitivity analysis was used in order to understand the impact of various parameter changes on the two fisheries model¡¦s equilibrium value. In the OA model, the change of the price, the cost per unit effort and the catch coefficient would have greater impact on the equilibrium biomass. In the MPV model, the equilibrium biomass was sensitive to the change of the environmental capacity. Finally, in order to understand whether the management of the Western and Central Pacific Commission (WCPFC) was effective, I used simulation analysis in accordance with the catches restriction and effort restriction management strategies. The result showed that the catches restriction strategy which WCPFC established was still not proper enough. It must cut a large number of catches immediately and continue for some time enough to make the resource recover. The effort restriction strategy could make good effect on the bigeye tuna resource recovery.

Gait analysis is the process of collecting and analyzing quantitative information about walking patterns of the people. Gait analysis enables the clinicians to differentiate gait deviations objectively. Diagnostic decision making from gait data only requires high level of medical expertise of neuromusculoskeletal system trained for the purpose. An automated system is expected to decrease this requirement by a &lsquo / transformed knowledge&rsquo / of these experts. This study presents a clinical decision support system for the detecting and scoring of a knee disorder, namely, Osteoarthritis (OA). Data used for training and recognition is mainly obtained through Computerized Gait Analysis software. Sociodemographic and disease characteristics such as age, body mass index and pain level are also included in decision making. Subjects are allocated into four OA-severity categories, formed in accordance with the Kellgren-Lawrence scale: &ldquo / Normal&rdquo / , &ldquo / Mild&rdquo / , &ldquo / Moderate&rdquo / , and &ldquo / Severe&rdquo / . Different types of classifiers are combined to incorporate the different types of data and to make the best advantages of different classifiers for better accuracy. A decision tree is developed with Multilayer Perceptrons (MLP) at the leaves. This gives an opportunity to use neural networks to extract hidden (i.e., implicit) knowledge in gait measurements and use it back into the explicit form of the decision trees for reasoning. Individual feature selection is applied using the Mahalanobis Distance measure and most discriminatory features are used for each expert MLP. Significant knowledge about clinical recognition of the OA is derived by feature selection process. The final system is tested with test set and a success rate of about 80% is achieved on the average.

<p>  </p> <p>Osteoarthritis (OA) is prevalent in equine and can be career-ending for performance horses due to lameness limitations and decreased quality of life. OA is a progressive, multifactorial disease that compromises the synovial joints' normal function, resulting in subchondral bone and articular cartilage deterioration over time. OA is a complex disease that impacts the entire joint, wherein activation of the innate immune system has an essential role in the disease progression and the development of pain. The synovial membrane, or the synovium, is a crucial contributor to the inflammation of diseased joints, regardless of the intra-articular tissue type initially affected. Synoviocytes are a predominant cell type of the synovium and contribute to inflammation by releasing key mediators and degradative enzymes, such as interleukin (IL)-6, IL-1β, a disintegrin, and metalloproteinase (ADAM) domains, and matrix metalloproteinases (MMPs). The production of pro-inflammatory molecules sequentially influences the expression of degradative enzymes and cartilage destruction. Therefore, the pathophysiological processes within synovial joints afflicted by OA can be further understood by studying the characteristics of synoviocytes.</p> <p>We aimed to investigate the inflammatory component of OA in an <em>in vitro</em> model using a primary cell line of equine fibroblast-like synoviocytes (eqFLS) stimulated with tumor necrosis factor-alpha (TNF-α) to represent an initial inflammatory stimulus. Our studies have shown that stimulating eqFLS with TNF-α for 24 hours significantly increased the gene expression of pro-inflammatory biomarkers. Among several pro-inflammatory candidate genes assayed, only pro-inflammatory cytokine IL-6 gene expression could be detected reproducibly following stimulation with the TNF-α gene in eqFLS. We characterized the pro-inflammatory response of eqFLS and utilized this system to examine the impact of novel therapeutic compounds designed <em>in-silico</em> with the goal of reducing the inflammatory response of eqFLS. A piperazine-based compound (C3) and its derivative (02-09) were primarily designed to mimic the interactions of the growth factor pigment epithelium-derived factor (PEDF) with its receptor, the non-integrin laminin receptor 1 (LAMR1). Based on previous <em>in vitro</em> studies in the laboratory, C3 and 02-09 had been proposed to have a strong potential for inhibiting inflammation while reducing angiogenesis and chondrocyte hypertrophy. The efficacy of these two novel compounds on eqFLS was examined in the present work by assessing the gene expression levels of inflammatory biomarkers, including IL-6, IL-1β, IL-8, ADAMs, and MMPs relative to a control housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in various study designs. An <em>in-vitro</em> screen with the IL-1β promoter driving a reporter green fluorescent protein (GFP) was also designed to detect and track the inflammatory response of eqFLS by imaging following stimulation with or without (+/-) TNF-α relative to controls. This screen will be utilized in future studies to potentially identify more effective compounds in the LAMR1-interacting series. The current findings suggest that the novel compounds, especially 02-09, might exhibit an anti-inflammatory effect on eqFLS; therefore, it is a potential therapeutic agent in modulating inflammation during OA development. </p> <p><br></p>

The purpose of this research is to explore preservice teachers’ attitudes and beliefs towards mathematics, mental and written computations, and mental computation anxiety, to investigate their use of different mental computation strategies using different approaches (i.e., Direct Teaching (DT) and Open-Approach (OA)) among the three different groups, and to identify how the use of preservice teachers’ mental computation strategies affects their flexibility regarding mental computation. The participants were preservice teachers (PTS). Three classes were used for this study: two classes in a mathematics class (Course A) for experimental groups and one class for the control group. One class from professional education courses was selected. A mixed methods design was used, more specifically, the Mathematics Attitude Survey (MAS) was administrated before and after intervention to examine PTS’ attitudes towards mathematics, mental and written computation, and mental computation anxiety. In addition, to determine whether there is any statistically significant difference among the three groups, the one-way analysis of variance (ANOVA) was used. Then, the MAS was analyzed descriptively. Next, a pre-and post-Mental Computation Test (MCT) was given to investigate PTS’ mental computation knowledge in relation to whole numbers, integers, and rational numbers (i.e., fractions, decimals, and percentages). A one-way analysis of covariance (ANCOVA) was conducted to determine if there were significant differences in mental computation performance among the three groups (i.e., DT, OA, and Control) with different instructions. Further, before and after intervention, face-to-face interviews were given to both the experimental and control groups to identify how they arrived at their answers. During interviews, 38 interviewees in the pre-interviews and 36 in the post-interviews for all groups participated. The interview items were selected from the pre-and post-MCT problems. Three levels of problems (i.e., high, medium, and low difficulty) for each operation were selected. The results of the MAS showed that with respect to the attitudes towards mathematics, PTS were generally shown positive attitudes towards learning mathematics and were aware of the importance of learning mathematics; however, in reality, about half of them did not want to spend time on learning or studying mathematics. In terms of PTS’ attitudes towards mental and written computation, PTS were aware that learning mental computation is more useful in real life situations and provides benefits in their mathematics learning. However, they do not feel comfortable and safe when using mental computation because of their lack of confidence and teaching abilities. For the mental computation, PTS showed slightly higher anxiety levels from pre-to post-tests. The findings of Mental Computation Test (MCT) revealed that there was a statistically significant difference in post-MCT scores between the different instructional groups when adjusted for pre-MCT scores. In particular, PTS using Open-Approach (OA) performed better than the PTS in the group using Direct Teaching (DT). The PTS in the control group performed worst. Significant differences between pre-and post-MCT performance were found among the three groups in solving multiplication, fraction, and decimal operations. The results of interviews suggest that there was an association between each interviewee’s quintile level and their flexibility in the use of the mental computation strategies. Regarding the whole number operation strategies, the results revealed that the interviewees in the middle and upper quintiles in both DT and OA used more than two different strategies with higher accuracy and were more likely to use the strategies. Interviewees in the middle and upper quintiles for the DT and OA groups were more likely to use the strategies that reflect efficient number facts or number-sense (e.g., Adding by place, Decomposing, & Compensation). The mental image of the Traditional method was frequently observed in the OA group. In contrast, for the lower quintiles, alternative strategies were not provided for both groups. The interviewees in the control group offered the smallest range of strategies. For the integer and rational operations, the interviewees in the DT group showed strategies that focused more on conceptual understanding. Surprisingly, the interviewees in the OA group were more likely to apply teacher-taught methods, including the Traditional method. The control group was not able to provide any alternative strategies. Plans for future research are set forth to add to the body of knowledge that exists regarding mental computation.

<p>Osteoarthritis (OA) is one of the most prevalent diseases in the United States and also in the world. Cartilage plays a vital role in articular joints and its structural integrity and mechanical properties are diminished by OA. Post traumatic osteoarthritis (PTOA) is a prevalent type of OA and occurs after a significant joint injury. Currently, no treatments are available to prevent or delay the progression of any form of OA.</p><p>Collagen crosslinking improves the material properties of cartilage and has been proposed as a potential treatment for OA. The wear resistance of cartilage that had been crosslinked with CASPc, a light activated crosslinking agent, was tested. Results suggested that photo-initiated crosslinking improves the wear characteristics of cartilage.</p><p>Another treatment for PTOA is through biological intervention. Preliminary data from our lab showed that metformin rescues the chondrocyte response to injurious impact overloading in the initial 24 hours following impact. However, whether this treatment maintained cartilage integrity for an extended duration had not been investigated. Material properties of cartilage were analyzed with an indentation test at different time points post-impact to determine the functional effect of cartilage injury and metformin treatment. Changes in the composition of the cartilage were investigated through biochemical techniques.</p><p>Having an in vivo model for PTOA is key for testing any new therapeutic intervention. In this study a model was developed to deliver a consistent impact load to the posterior aspect of medial condyle of a rabbit knee. A drop tower was designed for impacting the rabbit knee, and load and acceleration were measured during the impact. A k-wire was passed through the condyles in the medial-lateral direction under the impact site to secure the condyle during the impact. Whether the impact parameters were affected by the location of the k-wire was evaluated. The location of the k-wire was varied in the anterior/posterior and proximal/distal directions in a knee joint of cadaveric rabbits and impact parameters were recorded. Multiple linear regression showed a correlation between the location of the k-wire and peak stress, loading rate, impact duration and work. Moreover a correlation was found between the damage induced to the cartilage and loading rate, impact duration and peak stress. This study indicated that k-wire location is critical to prevent fracture of the subchondral bone.</p><p>A pilot study was designed to investigate the in-vivo effect of the metformin treatment on PTOA. Impacted knee joints in rabbits were treated with intraarticular metformin or were untreated controls. At 12 weeks post-injury, the progression of OA in the rabbit knees was quantified by histology, and OA severity was assessed using OA Research Society International (OARSI) scoring. Although the number of animals in the study were limited, intraarticular metformin appeared to prevent the development of PTOA in the impacted rabbit knees.</p>

La douleur est une réponse normale qui nous permet de réagir en réponse à un trauma ou une situation qui pourrait potentiellement causer du tort à notre corps. Cette expérience désagréable n’est pas seulement physiologique, mais elle est aussi psychologique, émotionnelle et socio-culturelle. C’est ce qui la rend aussi complexe et subjective. Lorsque les mécanismes de modulation de la douleur ne fonctionnent plus normalement, ces douleurs peuvent devenir chroniques et être très handicapantes pour les patients. Dans cette étude, nous nous sommes plus particulièrement focalisés sur certaines maladies musculosquelettiques présentant de la douleur chronique telles que l’encéphalomyélite myalgique (EM), la fibromyalgie (FM) et l’arthrose ou l’ostéoarthrite (OA). L’EM et la FM sont deux maladies complexes et hétérogènes qui ont plusieurs symptômes qui se chevauchent. L’EM se caractérise généralement par de la fatigue chronique qui n’est pas soulagée par le repos et par des malaises après-effort (PEM). Alors que la FM se caractérise davantage par de la douleur chronique musculaire et articulaire. Puis, l’OA est une maladie débilitante où les patients ont une dégradation progressive du cartilage articulaire causant de la douleur chronique qui est davantage localisée au niveau des joints articulaires atteints. Actuellement, il n’existe pas de biomarqueurs idéaux pour mesurer le niveau de douleur pour ces maladies dont l’étiologie reste incertaine. Toutefois, une étude récente a démontré une diminution de l’expression du gène MFAP3 au niveau des cellules mononuclées du sang périphérique (PBMC) chez des individus présentant des états de douleur élevée à la suite d’un syndrome post-traumatique. Ce gène code pour une protéine appelée protéine associée aux microfibrilles 3 (MFAP3), qui participent dans plusieurs processus biologiques dont l’assemblage des microfibrilles, l'élastinogenèse et l'homéostasie tissulaire, et pourrait aussi être une protéine clé impliquée dans l’inhibition de la douleur. L’objectif de cette étude est de mieux comprendre le rôle de la protéine MFAP3 et son implication dans la douleur dans ces différentes pathologies. L’expression du gène MFAP3 a été mesurée dans les PBMC de patients atteints d’EM, de FM, d’OA et de sujets sains (HC) et a été corrélée aux niveaux de douleur des patients. Une recherche in silico nous a permis d’identifier des récepteurs membranaires impliqués dans la douleur et pouvant interagir physiquement avec la protéine MFAP3 dont le récepteur HTR3A (le récepteur à sérotonine 3A). La spectroscopie cellulaire diélectrique a été utilisée pour valider cette interaction dans des cellules Jurkat (lymphocytes T humains immortalisés) dans des conditions standards. Il a été observé que la protéine MFAP3 inhibait la réponse induite par la stimulation de ce récepteur. Cette recherche pourrait éventuellement conduire au développement de nouvelles thérapies pour traiter la douleur associée à ces maladies musculosquelettiques. / Pain is a normal response that allows us to react in response to trauma or in situations that are dangerous or could potentially harm our body. This unpleasant experience is not only physiological, but it is also psychological, emotional, and socio-cultural. This is what makes it so complex and subjective. When pain modulation mechanisms no longer function normally, pain can become chronic and be very disabling for patients. In this study, we particularly focused on some musculoskeletal diseases presenting chronic pain such as myalgic encephalomyelitis (ME), fibromyalgia (FM) and osteoarthritis (OA). ME and FM are two complex and heterogeneous diseases that exhibit several overlapping symptoms. ME is characterized by a chronic fatigue that is not relieved by rest and post-exertional malaise (PEM), whereas FM is rather characterized by more chronic muscle and joint pain. In regard to OA, this debilitating disease leads to a progressive degradation of articular cartilage joints resulting in chronic pain which is more localized in the affected joints. Currently, there are no ideal biomarkers to measure pain level for these diseases and their etiology remains unclear. However, a recent study demonstrated a decrease in MFAP3 gene expression in peripheral blood mononuclear cells (PBMC) in individuals presenting high pain states following a post-traumatic syndrome. This gene encodes a protein called Microfibril-associated protein 3 (MFAP3), which participates in several biological processes including microfibril assembly, elastinogenesis and tissue homeostasis, and could also be a key protein involved in pain inhibition. The objectives of this study were to better understand the role of the MFAP3 protein and its involvement in pain in these different pathologies. MFAP3 gene expression was measured in PBMCs from patients with ME, FM, OA and healthy subjects (HC) and correlated with their pain levels. In silico research allowed us to identify membrane receptors involved in pain that can physically interact with the MFAP3 protein, including the HTR3A receptor (Serotonin receptor 3A). Cellular dielectric spectroscopy was used to validate this interaction in Jurkat cells (immortalized human T lymphocytes) under standard conditions. It was observed that MFAP3 inhibits the response induced by this receptor stimulation. This research could eventually lead to the development of new therapies to treat pain associated with these musculoskeletal diseases.

Les études cliniques et in vitro suggèrent que la sclérose de l’os sous-chondral due aux ostéoblastes (Ob) anormaux est impliquée dans la progression de l’ostéoarthrose (OA). Les Ob OA humains isolés à partir d’os sous-chondral sclérosé montrent un phénotype altéré, un niveau réduit de signalisation Wnt/β-caténine canonique et une minéralisation in vitro réduite. Il existe également deux voies non-canoniques, Wnt/PKC et Wnt/PCP qui ont étés décrites dans la littérature. Cependant, il n’existe aucune étude qui traite de ces deux voies dans les Ob OA. Ces voies sont activées après qu’un ligand Wnt non-canonique tel que Wnt-5a se lie à un récepteur Wnt couplé à des corécepteurs de la voie non-canonique. Ceci enclenche, respectivement pour la voie Wnt/PKC-Ca2+ et Wnt/PCP, la phosphorylation de PKC (p-PKC) et la phosphorylation de JNK (p-JNK) et agit sur les cibles en aval. Nous avons voulu déterminer s’il était possible de constater des altérations dans les voies Wnt non-canoniques dans les Ob OA. Nous avons préparé des cultures primaires d’ostéoblastes sous-chondral humains à partir de plateaux tibiaux de patients OA subissant une arthroplastie totale du genou, ainsi qu’à partir de plateaux tibiaux recueillis à l’autopsie de patients « normaux ». L’expression des gènes impliqués dans les voies Wnt/PKC et Wnt/PCP a été évaluée par RT-qPCR et la production par Western Blot des protéines, ainsi que celle de p-PKC et p-JNK et que l’activité des facteurs NFAT et AP-1 utilisés par ces deux voies. L’activité phosphatase alcaline (ALPase) et la quantité d’ostéocalcine (OC) ont étés évaluées respectivement à l’aide d’hydrolyse de substrat et d’ELISA. Le niveau de minéralisation a été évalué par la coloration au rouge Alizarine. Nos résultats montrent que l’expression et la production de Wnt-5a étaient augmentées dans les Ob OA comparées aux Ob N et LGR5 était significativement plus élevée. De plus, l’expression de LGR5 est directement régulée via la stimulation ou la diminution de Wnt-5a, à la fois au niveau de l’ARNm et des protéines. Par ailleurs, Wnt-5a a stimulé la phosphorylation de JNK et de PKC ainsi que l’activité NFAT et AP-1. Les niveaux de minéralisation ainsi que d’activité ALPase et de sécrétion d’OC ont aussi été affectés par les changements du niveau de Wnt-5a. Ces résultats suggèrent que Wnt-5a, qui est augmentée dans les OA Ob, peut stimuler les voies Wnt non-canoniques et affecter le phénotype et la minéralisation des OA Ob humains. / Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts (Ob) is involved in the progression and/or onset of osteoarthritis (OA). Human Ob isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin signaling pathway (cWnt), and a reduced mineralization in vitro. Besides the cWnt pathway, at least two non-canonical signaling pathways, the Wnt/PKC and Wnt/PCP pathway have been described. These pathways are activated when a non-canonical Wnt ligand like Wnt-5a binds to a Wnt receptor coupled with non-canonical co-receptors. This activates, respectively for Wnt/PKC-Ca2+ and Wnt/PCP, the phosphorylation of PKC (pPKC) and the phosphorylation of JNK (pJNK) and their effect on downstream targets. However, there are no reports of either pathway in OA Ob. Here, we studied if alterations of the non-canonical pathways could be observed in OA Ob. We prepared primary human subchondral Ob using the tibial plateaus of OA patients undergoing total knee arthroplasty, or from tibial plateaus of normal individuals at autopsy. The expression of genes involved in Wnt/PKC and Wnt/PCP was evaluated by RT-qPCR and their protein production by Western blot analysis, in addition to p-PKC and p-PCP and NFAT and AP-1 activity with luciferase. Alkaline phosphatase activity and osteocalcin levels were evaluated respectively by substrate hydrolysis and ELISA respectively, and mineralization levels were evaluated with Alizarin red staining. OA Ob showed an increased alkaline phosphatase activity and osteocalcin release. The expression of Wnt5a was increased in OA Ob compared to normal. The expression of LGR5 was significantly increased in these cells. Moreover, the expression and production of LGR5 is directly modulated via the stimulation or inhibition of Wnt5a. However, Wnt5a did not stimulate the expression of LGR4. Wnt5a increased the phosphorylation of PKC and JNK as well as NFAT and AP-1 activity. Mineralization levels as well as alkaline phosphatase activity and osteocalcin secretion levels were also linked with changes in Wnt-5a levels. These data indicate that Wnt5a, which is increased in OA Ob, can directly stimulate the Wnt/PKC and Wnt/PCP pathways and this can affect the phenotype and mineralization observed in human OA Ob.