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Connective tissue extracellular matrix (ECM) consists of an interwoven network of contiguous collagen fibers that inform cell activity, direct biological function, and guide tissue homeostasis throughout life. Recently, ECM analogs have emerged as a unique ex vivo culture platform for studying healthy and diseased tissues and in the latter, enabling the screening for and development of therapeutic regimen. Unfortunately, current commonly used platforms, such as tissue-culture polystyrene (TCPS) or the basement membrane matrix, Matrigel, fail to fully recapitulate the physical and biochemical properties of the ECM. Tissue-culture polystyrene is significantly stiffer than typical ECM tissues and lacks the composition and 3-dimensional architecture that is critical for ECM function. Improving upon TCPS’s shortcomings, Matrigel retains a natural ECM structure and is comprised of native biopolymers. However, it is derived from mouse sarcomas, and thus, has significant batch-to-batch variability and often contains growth factors at non-physiologic concentrations. Moreover, despite being biopolymer based, Matrigel has relatively low amounts of type I collagen and high levels of type IV collagen, and as such, compositionally does not match the predominantly type I collagen matrix intrinsic to connective tissues. Thus, it is clear that new and improved models of the ECM are needed for in vitro culture. In pursuit of developing a highly biomimetic ECM analog, the objectives of this work were three-fold— first, to fabricate collagen-based ECM analogs with nanoscale mimicry, second, to systematically optimize crosslinking protocols in order to produce a stable substrate with continuous fibrous architecture, and third, to evaluate the substrate’s biocompatibility and utility as a platform for studying biomineralization. It was hypothesized that an architecturally and chemically relevant fibrous substrate could be prepared from gelatin and provide an optimal ex vivo platform for cell culture and new therapy screening and development. Thus, the ECM analog will be collagen-like, biocompatible, consist of continuous fibers, demonstrate both viscoelastic and elastic behavior, exhibit relevant mechanical properties, and remain stable for at least 14 days at cell culture conditions. To this end, first, a “green” electrospinning method was developed for preparing fibrous meshes from gelatin, which avoids typical electrospinning solvents that present significant health risks and barriers to large scale production. Next, crosslinking methods were developed using the reactive dialdehyde, glutaraldehyde (GTA), and the naturally derived enzyme, transglutaminase (TGase). These methods stabilized the meshes for over 28 days under cell culture conditions without disrupting its biomimetic architectures and chemical properties. In addition, a third approach to mesh fabrication using gelatin methacryloyl (gelMA) was developed to overcome the shortcomings of GTA and TGase crosslinking. With gelMA, the number of crosslinking sites were customized and, by taking advantage of its ability to undergo free radical polymerization, stable fibrous meshes were prepared with reproducible architecture, chemistry, and tunable mechanical properties. Following fabrication, the biocompatibility of the meshes was evaluated through macrophage, stem cell, and differentiated cell cultures. During culture, the macrophages maintained a naïve, non-polarized state, indicating they were not triggered towards an inflammatory response by the meshes. In addition, fibrochondrocytes, a cell critical for maintaining the collagen-based matrices where ligaments attach to bone, remained viable and maintained phenotypic expression on the meshes, as evident by their enhanced proteoglycan and collagen production relative to TCPS cultures. After demonstrating biocompatibility, the gelatin platform was coupled with a synthetic matrix vesicle (SMV) system and successfully acted as a mineralization platform in the presence of human osteoblast-like cells. Additionally, the platform supported mesenchymal stem cell expansion and mineralization when cultured with an alkaline phosphate conjugated SMV. In this work, three unique methods were developed for preparing ECM analogs. These efforts led to the production of a collagen-like mesh with nano- and micro-scale cues, fibrous continuity with little batch-to-batch variability, and proven stability in both dry and wet conditions. Importantly, these meshes did not instigate any inflammatory responses and supported fibrochondrocyte, osteoblast, and stem cell culture. Furthermore, the mesh successfully functioned as a template for biomineralization using both human osteoblast-like cells and stem cells. Collectively these findings demonstrate the potential of a collagen-like ECM analog with physiological relevance for ex vivo cell culture studies; and furthermore, its potential as a high-fidelity platform for studying cell-mediated biomineralization, cell-matrix interactions, and developing new therapeutic approaches for the treatment of connective tissue disorders.

A text is a linguistic structure that is more than a random collection of sentences. A text is cohesive (Halliday &amp / Hasan, 1976) and coherent (Mann &amp / Thompson, 1987, 1988). Mainly ignored in the field of linguistics until recently, the text and the discourse structure have been inquired from various points of view (Asher, 1993 / Asher &amp / Lascarides, 1998 / Grosz &amp / Sidner, 1986 / Mann &amp / Thompson, 1987, 1988 / Webber, 2004). D-LTAG is a discourse grammar work that extends a lexicalized sentence level grammar LTAG (Joshi, 1987) to low-level discourse (Webber, 2004 / Webber &amp / Joshi, 1998). In this framework, discourse connectives such as coordinating conjunctions, subordinating conjunctions, parallel connectives and discourse adverbials are predicates of discourse structure that take text spans that can be interpreted as abstract objects (Asher, 1993). Turkish has a flexible word order in comparison to languages like English. In English, the discourse adverbials are noted for their ability to occupy positions unavailable to other discourse connectives. In Turkish, word order of other discourse connectives, coordinators and subordinators are not expected to be as restricted. This thesis examines the connective position, argument order and the information structure of five Turkish discourse connectives in their eleven uses. The analyses show that the examined features of discourse connectives are related to the syntactic group the connective belongs to. Discourse connectives of the same syntactic groups exploit similar connective position and argument order possibilities, and they tend to be included in similar information units.

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.

The thesis aims to examine Incels discourses concerning their collective identity on the online forum Incels.is. Further investigation discovered that Incels produce and reproduce their collective identity through discourses. Incels are men in “involuntary celibacy” who gather online to share their frustration and dissatisfaction against society, and especially women. The data was collected through netnography and encompasses fifteen threads from the forum Incels.is. The material was analyzed through critical discourse analysis and the theories of collective identity and connective action. The theories uncover Incels collective identity and discourses created via language. The study shows that Incels create a collective identity through sharing specific characteristics, sharing emotional experiences, positioning themselves against the rest of society, sharing life experiences, sharing ideological perceptions concerning societal constructs, giving and receiving advice, as well as using slang and memes. Discourses were further related to each of these findings. Such discourses include how Incels create a “we” against “them”. This study addresses a research gap concerning Incels and identity and has thus contributed to the research tradition of radical online groups and identity. Many countries have identified Incels as a concern due to their violent tendencies and permeating misogynistic views. Hence, research on Incels is of societal significance.

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