Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Abstract Collagens and collagenous proteins undergo several post-translational modifications that are important for their structure and functions. Lysine hydroxylation produces hydroxylysines, which are important for collagen cross-link formation and provide attachment sites for galactose and glucosylgalactose. Glycosylated hydroxylysines are crucial for embryonic development and the assembly of certain collagen types. They may also facilitate interactions between collagen and adjacent molecules as well as control the diameter of collagen fibrils. Lysine hydroxylation is catalyzed by three lysyl hydroxylases (LH1, LH2 and LH3). In addition to lysyl hydroxylase activity, LH3 possesses collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. In this study, polyclonal antibodies against the lysyl hydroxylase isoforms were produced for protein level studies to localize the expression and understand the functions of the different isoenzymes. The results indicated ubiquitous expression during embryonic development compared to the more restricted, cell and tissue specific expression patterns observed in adult mouse tissues. Differences were seen also in the alternative splicing of LH2 during embryogenesis and between tissue types. Analyses of the subcellular localization revealed that LH3 is also present in extracellular space. Tissue and cell specific differences were noted in the distribution of LH3 between cellular compartments. Substrate analysis suggested an additional and novel role for LH3 as an enzyme catalyzing lysine modifications of collagenous proteins in the extracellular space. The importance of LH1 and LH2 has been highlighted in Ehlers-Danlos type VI and Bruck syndromes, respectively. In this study, the lysyl hydroxylase 3 gene was linked to a heritable disorder for the first time. Urinary screening revealed a patient that lacked a glucosylgalactosyl derivative of a pyridinium cross-link. The GGT activity levels measured from the patient’s serum and lymphoblastoid cells were also reduced, which suggested a defect in the lysyl hydroxylase 3 gene. Genetic analyses revealed two mutations, one in each allele of LH3 in this compound heterozygous patient. Recombinant mutant proteins showed defects in lysyl hydroxylase and collagen glycosyltransferase activities, respectively. In conclusion, it was shown that a defect in LH3 catalyzed modifications leads to a novel disorder, which shares features with many other connective tissue disorders.

Patients with connective tissue diseases (CTD) such as systemic lupus erythematosus (SLE) have an increased risk for infections. This study investigated the outcome and characteristics of CTD patients under intensive care unit (ICU) treatment for sepsis

La bilharziose reste un problème de santé majeur. L'équipe du Pr Dessein a montré que les infections élevées étaient déterminées par un locus majeur en 5q31 et que des polymorphismes dans un gène à ce locus,IL13, aggravent l'infection. Notre premier objectif était d'évaluer si des variants d'autres gènes de la voie de l'IL13 intervenaient dans le contrôle de l'infection. Nous avons observé une association entre le SNP rs324013, dans le promoteur de STAT6,et les niveaux d'infection à S. haematobium. Ce polymorphisme a un effet additif avec le polymorphisme IL13rs1800925. Ce SN modifie la fixation de facteurs nucléaires au niveau du promoteur de STAT6. L'équipe du Pr Dessein avait également montré que les fibres hépatiques avancées et sévères étaient déterminées par un autre locus majeur localisé en 6q23. Notre deuxième objectif fut d'évaluer dans le laboratoire du Pr Dessein et en étroite collaboration avec le laboratoire du Pr Li(Yueyang Institute of Parasitic disease)deux gènes candidats(IFNGR1 et CTGF) situés dans cette région chromosomique. Nous avons observé une association entre les deux polyporphismes(rs17066192 er rs673156)localisés dans le promoteur du gène. Nous avons observé une association entre les deux polymorphismes(rs17066192 et rs673156)localisés dans le promoteur du gène IFNGR1 et la fibrose hépatique: le génotype rs673156A/A et rs17066192C/C sont associés à un risque 7.3 fois et 1.5 fois plus élevé, respectivement, de fibrose avancée. Nous avons également montré que les variants rs9402373 et rs12526196 du gène CTGF sont indépendamment associés à la fibrose chez les fermiers et pêcheurs chinois infectés par S.japonicum. Sur la population chinoise d'étude, les risques relatifs associés aux polymorphismes rs9402373 et rs12526196 sont de 2.8 et 3 / Schistosomiasis remains one of the world’s most prevalent diseases. It comprises a group of chronic diseases caused by helminths of the Schistosoma genus. Schistosoma haematobium causes obstructive nephropathy that can be aggravated by urinary bacterial infections. S.japonicum and S.mansoni cause hepatic fibrosis associated with portal blood hypertension, which can be lethal. In previous studies, our laboratory had shown that worm burden in S.haematobium infections were aggravated by IL13 variants and that severe hepatic fibrosis (HF) was controlled by gene(s) located on 6q23. The present study is to further evaluate other IL-13 pathway genes (STAT6) in the control of infection in Malian farmers and to test candidate genes in the 6q23 region in hepatic fibrosis (HF) in S.japonicum infected Chinese fishermen and farmers. First we have developped an improved FTA® technology technique to perform SNP genotyping. This technique allows us to use saliva samples for genotyping SNPs. Subsequently, this improved FTA® technology was used in our study on HF.Our work on a Malian sample infected with S. haematobium indicated that a polymorphism (rs324013) in the promoter of STAT6 gene was associated with the control of S. haematobium infection levels and has an additive effect with IL13rs1800925, a polymorphism previously associated with infection in this same population. Both SNPs modify the binding of nuclear factors to the promoter regions of their respective genes. Thus, both SNPs may play a crucial role in controlling S. haematobium infection levels. In order to study HF in S.japonicum infections, we have participated actively in the study that recruited of a large sample of Chinese fishermen and farmers who had been exposed to the infection for most of their life. HF was evaluated by ultrasound and covariates that could affect HF were evaluated by interviews. Then, we tested two genes (IFNGR1, CTGF) of the 6q23 region that were good candidates for the control of HF on these samples. Both genes encode molecules that were shown in animal and human studies to have strong effect on extracellular matrix proteins deposition and turnover. We found that two polymorphisms (rs17066192 and rs673156) in IFNGR1 promoter were associated with HF: the rs673156A/A genotype was associated with a 7.3-fold increased risk of advanced HF; and rs17066192C/C genotype with a 1.5-fold increased risk of HF. These results must now be confirmed in another population sample. We also found that variants of CTGF rs9402373 and rs12526196 were independently associated with HF in Chinese fishermen and farmers, in Sudanese, and in Brazilians infected with either S. japonicum or S. mansoni. Our results provide additional evidence for a protective role of IL-13 in schistosome infections, and they also demonstrate that TGFβ / CTGF pathway plays a key role in HF and should be targeted by chemotherapy. Ongoing studies evaluate whether CTGF variants could be used in the prognosis of the HF caused by schistosomes and also by other infectious agents.

Background Pulmonary arterial hypertension (PAH) is a rare progressive disease with no known cure. Of various aetiologies, PAH in association with connective tissue disease (PAH-CTD) is the most rapidly progressive and difficult to treat. Management of PAH has evolved significantly in the past ten years since the introduction of oral therapies. Evidence for the efficacy of these agents outside randomised controlled trials is limited, but guidelines exist. Aim To measure the impact of first-line monotherapy with bosentan or sildenafil and the introduction of prescribing guidelines on outcomes in PAH-CTD. Methods Following a retrospective analysis of consecutive, incident, treatment-naive PAH-CTD cases identified by the ASPIRE registry, influences on outcome measures have been compared. First-line monotherapy episodes for 247 patients was analysed against four distinct endpoints: change in exercise capacity, WHO functional class, time on monotherapy and all-cause mortality. Results Treatment with bosentan or sildenafil resulted in clinical stability at 2 years for nearly 1/4 patients. No difference was identified between the groups in terms of either exercise capacity or WHO functional class. Sildenafil patients were found to remain on monotherapy longer than those prescribed bosentan. Patients prescribed sildenafil have improved survival over those treated with bosentan. Unexpected baseline differences in between groups may confound the results as the haemodynamics of the bosentan patients were more severe. Conclusions A significant number of patients with PAH-CTD remain clinically stable on monotherapy at 2 years. Both agents seem equally effective in this aggressive form of PAH. A novel endpoint “TOM” may be of value in future research assessing response to treatment.

Ehlers-Danlos Syndrome (EDS) comprises a spectrum of heritable connective tissue disorders with varying genetic origins and clinical manifestations such as soft tissue fragility and skin hyperextensibility. There are multiple EDS subtypes, the first few of which were defined by collagen mutations. Many new EDS variants have been discovered involving mutations that do not necessarily implicate collagen biosynthesis but do involve extracellular matrix (ECM) proteins. One of these proteins, Aortic Carboxypeptidase-Like Protein (ACLP), is a large secreted protein encoded by the AEBP1 (adipocyte enhancer binding protein 1) gene. Previous research has shown that ACLP plays a vital role in binding collagen via its discoidin domain and therefore regulates connective tissue assembly. Thus far, individuals from 7 different families have been identified with different EDS-causing ACLP mutations. Some mutations are ACLP null whereas other mutations lead to expressed mutant ACLP. One of these mutations is characterized by a single-nucleotide deletion that causes the insertion of 40 amino acids in the discoidin domain of ACLP. It is therefore denoted “ACLP-Ins40”. The goal of this research was to characterize the ACLP-Ins40 protein and investigate how mutations in ACLP disrupt ECM homeostasis and cause EDS. We initially sought to determine if the ACLP-Ins40 mutation would alter ACLP’s ability to bind collagen. To achieve this goal we generated expression vectors of full length human ACLP carrying the Ins40 mutation. By western blot, it was determined that ACLP-Ins40 was not secreted from fibroblasts and was retained intracellularly. We then hypothesized that the retention of ACLP-Ins40 in the secretory pathway would induce ER stress due to misfolding. 3T3 fibroblasts were co-transfected with the ACLP-Ins40 expression vector and an XBP1u-EGFP sensor of ER stress. Immunofluorescence imaging revealed that in comparison to WT, fibroblasts expressing ACLP-Ins40 experienced ER stress with significantly increased spliced XBP1. This may then cause cell death, the improper secretion of other important ECM proteins, or defective collagen scaffolding, all which could contribute to symptoms of EDS. These studies contribute to our current understanding of how mutations in the AEBP1 gene and alterations in the ACLP protein cause EDS. This connection provides a framework for future research and for targeted interventions to treat EDS. / 2021-06-17T00:00:00Z

A elasticidade do tecido conjuntivo desempenha uma função protetora agindo como uma mola tênsil durante o trabalho muscular, No entanto, existem poucos estudos sobre a distribuição das fibras do sistema elástico no coração. Considerando que: a) o estudo da distribuição destas fibras pode ajudar a compreender a mecânica cardíaca, e b) o rato tem sido usado como o melhor modelo animal para as disfunções cardiovasculares, o objetivo deste estudo é apresentar uma descrição sistemática da distribuição diferencial das fibras do sistema elástico do endocárdio, epicárdio e miocárdio ventricular. Cortes histológicos de ventrículo esquerdo obtido de ratos normais adultos foram estudados pela comparação do padrão ultraestrutural de cada um dos tipos fibrilares com coloração pela técnica da resorcina-fucsina com prévia oxidação, para microscopia de luz. As observações ultra-estruturais foram feitas em tecidos fixados com ácido tânico - glutaraldeído, que permite a identificação mais precisa das fibras oxitalânicas, elaunínicas e elásticas. Foi aplicado um sistema semiquantitativo de avaliação. A análise dos cortes histológicos corados pela Resorcina-fucsina com oxidação prévia mostrou um estrato de fibras elásticas em estreita associação ao endotélio no endocárdio e ao mesotélio no epicárdio. Quando observado ao microscópio eletrônico é possível identificar que nestas ambas localizações as fibras do sistema elástico estão arranjadas em dois estratos dispostos ortogonalmente. Ao nível ultraestrutural, notou-se a distribuição diferencial das fibras do sistema elástico nos três compartimentos do tecido conjuntivo associado ao miocárdio propriamente dito: epimísio, perimísio e endomísio. O epimísio apresenta fibroblastos, fibras colágenas grossas, fibras elaunínicas e elásticas entremeadas à substância amorfa. No perimísio, a microscopia eletrônica mostrou uma grande quantidade de microfibrilas associadas a fibras elásticas, elaunínicas e mesmo fibras colagênicas. Freqüentemente, neste compartimento, as microfibrilas estão compactadas formando feixes que correspondem ao padrão ultra-estrutural das fibras oxitalânicas. O endomísio é rico em fibras oxitalânicas associadas à lâmina basal dos cardiomiócitos. Uma rede microfilamentar conecta os elementos do endomísio entre si. Ainda que as implicações funcionais sejam especulativas, a distribuição diferencial das fibras do sistema elástico nos compartimentos da parede ventricular sugere que diferenças na elasticidade conferem versatilidade biomecânica ao tecido como um todo. A análise ultra-estrutural mostra que as fibras oxitalânicas (menos elásticas) se co-localizam com fibras colagênicas finas no endomísio e perimísio, enquanto que a presença de fibrilas colágenas mais grossas coincide com fibras elásticas e elaunínicas (com mais elasticidade) no endo- e epicárdio. Estas observações sugerem que o sistema elástico, em co-evolução com o sistema colagênico, contribui para acomodar a diversidade funcional / The connective tissue elasticity has a protective function acting as a tensile spring during muscular work. Nevertheless, few is known about the distribution of the elastic system fibers in the heart. Considering that a) the study of the distribution of these fibers may help understand the cardiac mechanics, and b) rat models are used to study cardiac dysfunctions, our aim is to study the distribution of elastic system fibers in the ventricular endocardium, epicardium and myocardium of normal rats. Histological tissue sections of left ventricle (obtained from adult rats) were studied by comparing the typical ultrastructural picture of each of the fiber types with Resorcinfuchsin staining technique for light microscopy. The ultrastructural observation was made in tissues fixed with tannic acid-glutaraldehyde, which provided a reliable means to identify the elastic system fibers, as oxytalan, elaunin and elastic fibers. A semiquantitative evaluation was performed. The analysis of the histological sections stained by Resorcin-fuchsin technique after oxidation shows a stratum of elastic system fibers in close association to the endothelium in the endocardium and to the mesothelium in the epicardium. When observed at the transmission electron microscope, it was possible to see that in both locations the elastic system fibers are arranged in two orthogonally disposed layers. At the ultrastructural level, the epimysium presents fibroblasts, thick collagen, elaunin and elastic fibers interspersed in the amorphous substance. In the perimysium, the electron microscope disclosed a great amount of microfibrils, surrounding all fibrilar components: elastic fibers, elaunin fibers and even collagen fibers. Frequently, at this location, the microfibrils are closely packed, forming bundles devoid of elastin that correspond to the ultrasctructural picture of the oxytalan fibers. The endomysium is rich in oxytalan fibers in a close association with the basal lamina of the myocytes. A microfibrilar network interconnects the endomysium elements each other. In spite of the functional implications being speculative, the differential distribution of the elastic system fibers in the compartments of the ventricular wall suggests that the differences in elasticity provide biomechanical versatility to the intire system. Ultrastructural analysis shows that oxytalan fibres and thin collagen fibrils are co-localized in endomysium and perimysium, whereas, the presence of thicker collagen fibrils coincides with elaunin and elastic fibers in endo- and epicardium. These specific co-localizations suggest that elastic system, in co-evolution with collagen, has contributed to accommodating functional diversity

O coração é formado por tecido muscular especializado e por um esqueleto de tecido conjuntivo que sustenta e dá inserção a musculatura. Este tecido conjuntivo é formado predominantemente por fibras colágenas tipo I, tipo III e fibras elásticas, as quais influenciam diretamente as propriedades estruturais e funcionais do miocárdio. Algumas doenças podem provocar alterações qualitativas e quantitativas no colágeno comprometendo a funcionalidade do órgão. Diante da inexistência de informações sobre os efeitos diretos do diabetes na estrutura atrial e a concentração de pesquisas em torno dos ventrículos, especialmente o esquerdo, que está ligado à circulação sistema, a proposta deste trabalho foi analisar as alterações morfológicas quantitativas e qualitativas do colágeno nas câmaras atriais comparando as diferenças entre os grupos de cães sadios e diabéticos. Além disto, esta pesquisa verificou a distribuição de outros constituintes do átrio como fibras elásticas e colágenas do tipo III. Foram utilizados corações de cães sem raça definida, machos e fêmeas, sadios e diabéticos. As estruturas foram examinadas por microscopia óptica, imunofluorescência e microscopia eletrônica de varredura. A taxa de colágeno nos átrios de cães diabéticos foi maior do que nos átrios de cães sadios. Isso indica que há uma mudança estrutural nos átrios de animais com doença metabólica, a partir da verificação de uma proteína estrutural importante como o colágeno. / The heart is composed of specialized muscle tissue and a skeleton of connective tissue that supports and gives muscle insertion. This tissue connective is formed predominantly of collagen type I, type III and elastic fibers, which directly influence the structural and functional properties of the myocardium. Some diseases can cause qualitative and quantitative changes in collagen compromising the functionality of the organ. Due to the lack of information on the direct effects of diabetes on atrial structure and the concentration of researches about the ventricles, especially the left, which is connected to the macro circulation system, the purpose of this study was to analyse the morphological quantitative and qualitative collagen in the chambers atrial comparing the differences between the groups of healthy and diabetic dogs. Moreover, this study found the distribution of other constituents like elastic and collagen type III fibers. We used hearts of mongreal dogs, males and females, healthy and diabetics. The structures were examined by light microscopy, immunofluorescence and scanning electron microscopy. The rate of collagen in the atria of diabetic dogs was greater than in the healthy dogs. This indicates that there is a structural change in the atria of animals with metabolic disease, from the finding of a major structural protein like collagen.

As a good and competent surgical simulator, it should provide surgeons with visual, tactile and behavioral illusion of reality. In literature, methods for object deformation range from non-physically based models to physically based models. Early works of non-physically based models focused on pure geometrical models that were originally employed in computer-aided design. These methods could be used to produce vivid deformable effects in computer animation. However, the soft tissue simulation in surgical applications requires more realistic models based on physical properties of human tissues. As a result, the mass-spring model and the finite element model have become the most popular representations for deformable organs in surgical simulation. Our research focuses on the real-time soft tissue deformable model based on the finite element method for surgical application. / Extended from the hybrid condensed finite element model, an interactive hybrid condensed model with hardware acceleration by the graphics processing unit (GPU) is proposed. Two methods are developed in order to map the data onto the GPU in accordance with the application data structure. The performance of the primary calculation task in the solver is enhanced. Furthermore, an improved scheme is presented to conduct the newly applied forces induced by dragging or poking operations in the non-operational region. / In the thesis, new approaches to establish a physically based model for soft tissue deformation and cutting in virtual-reality-based simulators are proposed. A deformable model, called the hybrid condensed finite element model, based on the volumetric finite element method is presented. By this method, three-dimensional organs can be represented as tetrahedral meshes, divided into two regions: the operational region and the non-operational one. Different methods treat the regions with different properties in order to balance the computational time and the level of the simulation realism. The condensation technique is applied to only involve the calculation of the surface nodes in the non-operational region while the fully calculation of the volumetric deformation is processed in the operational part. This model guarantees the smooth simulation of cutting operation with the exact cutting path when users manipulate a virtual scalpel. Moreover, we discuss the relevant aspects on what affect the efficiency of implementing the finite element method, as well as the issues considered for choosing the effective solving method to our problem. Three numerical methods have been examined in our model. / Surgical simulator, which benefits from virtual reality techniques, presents a realistic and feasible approach to train inexperienced surgeons within a safe environment. It plays more and more important role in medical field and also changes the world of surgical training. Especially, the minimally invasive microsurgery, which offers patients various attractive advantages over the traditional surgery, has been widely used in otolaryngology, gastroenterology, gynecology and neurology in the last two decades. / Through the combination of these approaches, a physically based model which allows users to freely perform the soft tissue cutting and detecting, such as poking or dragging operations, with soft tissue deformation is achieved in real-time. / Wu Wen. / "August 2006." / Adviser: Pheng Ann Heng. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1745. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 112-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Biocompatibilidade é uma das principais propriedades que um cimento retrobturador deve possuir, para não provocar no hospedeiro reações tóxica e imunopatológica. Desta forma, a sucessão de experimentos que comprovem tal propriedade, é fundamental para o uso clínico desse material. O objetivo deste estudo foi comparar a reação tecidual frente ao MTA Branco Angelus®, MTA Bio Ângelus® e Sealepox RP. Para isso, foram utilizados 54 ratos Wistar albinos, que foram divididos em 3 grupos iguais. Os materiais foram implantados no tecido subcutâneo do dorso desses animais, dentro de tubos de polietileno. Como controle, todos os tubos tiveram uma de suas extremidades seladas com guta-percha. Os períodos experimentais analisados foram de 15, 30 e 60 dias após o procedimento cirúrgico, quando, então, os animais foram mortos. As amostras coletadas passaram pelo processamento histotécnico e foram feitos cortes de 5µm de espessura e coloração com hematoxilina e eosina. Os dados obtidos após a análise morfométrica foram submetidos à análise de variância a dois critérios (ANOVA) e teste de Kruskal-Wallis para comparação; a significância foi para p<0,05. Os resultados demonstraram que em todos os grupos e períodos houve um padrão de comportamento da reação inflamatória, ou seja, aos 15 dias havia uma densidade de volume de células inflamatórias maior, que foi decrescendo com o passar do tempo. Não houve diferença estatisticamente significante entre os materiais com relação a densidade de volume de células inflamatórias observadas. Com base na metodologia empregada e levando em conta suas limitações, pode-se concluir que todos os materiais testados são biocompatíveis. / Biocompatibility is one of the most important properties that a root-end filling material might have. Thus, experiments that prove this characteristic are very important. The purpose of this study was to compare the connective tissue reaction caused by MTA White Angelus, MTA Bio and Sealepox RP. Fifty-four Wistar albino rats were divided in three equal groups. The materials were implanted in subcutaneous connective tissue of the animals, into polyethylene tubes. As control group, gutta-percha was placed in one of the tubes border. The rats were sacrificed after 15, 30 and 60 days. Histologic samples were sectioned in 5µm thickness and stained with hematoxylin and eosin. ANOVA and Kruskal-Wallis tests were used for statistical analysis (p<0,05). The results showed that in all groups there was a standard result on the inflammatory infiltrate observed. The 15 days period showed a more severe response, which has been diminishing over time. There was no statistically significant difference between the materials with respect to the volume density of inflammatory cells observed. Based on the methodology used and taking into account its limitations, it can be concluded that all materials are biocompatible.