No description available.

No / ß2-microglobulin (ß2m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How ß2m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on ß2m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (¿N6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that ¿N6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of ß2m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type ß2m with that of ¿N6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.

Assembly of the extracellular matrix (ECM) protein fibronectin (FN) is a mechanical process that involves cell binding to FN through cell surface integrin receptors and application of tensional forces generated in the cell's contractile actin cytoskeleton. Deformation-induced exposure of cryptic sites, defined as buried molecular recognition sites, in FN has been proposed as a mechanism by which cell tension drives FN fibrillogenesis. The primary integrin attachment site on FN is the RGD loop in the 10FNIII domain. In this thesis, I set out to define the molecular biophysical mechanism by which cell tension application at the RGD site promotes unfolding and thereby induces FN-FN self-assembly leading to matrix fibril formation. Chapter 1 of this dissertation provides an overview of the current knowledge behind the biophysical and molecular basis of FN assembly in the ECM and its key role in development and disease. In Chapter 2, steered molecular dynamic simulations show that the 10FNIII domain under force applied through its N-terminus and RGD loop (N-to-RGD) unfolds to a preferred kinetic intermediate with solvent-exposed N-terminal hydrophobic residues in a manner different from past analyses in the literature where force through the N- and C- termini leads to multiple unfolding pathways. Use of single-molecule atomic force spectroscopy in Chapter 3 experimentally reveals that a mechanically stable intermediate of 10FNIII exposed by N-to-RGD pulling shows a length extension that agrees with the predicted kinetic intermediate. Results of biochemical and cellular studies using synthetic peptides with sequences from the 10FNIII intermediate show in Chapter 4 that the twenty-three amino acid sequence that spans the unraveled N-terminus of the predicted intermediate mediates FN multimerization and contains a minimal seven amino acid sequence we call the multimerization motif that is sufficient to induce FN-FN multimer assembly. Finally, Chapter 5 summarizes the new insights supported by this work regarding the role that mechanical forces applied at the cell binding site in 10FNIII plays in the physiological unfolding of FN with respect to FN fibrillogenesis and ECM assembly.

Collagen, the most abundant protein of connective tissues, in various forms has a wide applications due to their diverse biological and chemical properties. One of the forms are collagen hydrogels, which are considered very suitable material for applications in tissue engineering, because they are able to provide biodegradable scaffolds that its properties correspond with living tissues. These systems are used for example as scaffold for targeted drug delivery with controlled release, in combination with cells can be used for the regeneration and reconstruction of tissues and organs. Heating the aqueous solution of collagen leads to spontaneous self-assembly process to variously distributed fibrillar structures, which are at a later stage of fibrillogenesis prerequisite for creating a three-dimensional supporting network, which is the basic building block of the gel. The resulting properties of the hydrogel depend not only on its structure, but also on the conditions which cause self-assembly process. Hydrogels were performed at 37 ° C and physiological pH. Studied structural variable was the concentration of collagen. So far, for the research of self-assembly were used spectrometric methods, which only provide information about kinetics of morphogenesis. In this work to study the kinetics of collagen I self-assembly were used rheological methods, which additionally give information about viscoelastic properties of the resulting material. The obtained experimental data confirmed two-step process of collagen I fibrillogenesis consisting of nucleation and growth process. Rheological hydrogels collagen behaved as a nonlinear yield-pseudoplastic. An attempt was made to molecular interpretation of the results. Using two-parametric Avrami equation was determined the rate of self-assembly for each concentration of collagen and the value of Avrami exponent determining the shape of produced units. The prepared hydrogels were subjected to increasing shear stresses (strain amplitude, shear rate). Larger amplitudes leads to collapse of the hydrogel structure, which is able to again partially regenerated.

Alzheimer’s Disease (AD) is a neurodegenerative disease marked by progressive neuronal cell death, leading to dementia. AD is the most common disease that results in dementia and largely affects the elderly, with five million people in the United States diagnosed with the disease as of 2015 and approximately 35 million people worldwide. Diseases resulting in dementia cost the US healthcare system an estimated $172 billion in 2010 and that cost is expected to increase as the population ages and as diagnostic techniques improve so that more people are treated (Holtzman, 2011). The disease was first reported by psychiatrist Alois Alzheimer at the onset of the 20th century, when one of his patients “suffered memory loss, disorientation, hallucinations and delusions and died at the age of 55,” then was found to have severe brain atrophy post-mortem (Cipriani, Dolciotti, Picchi, & Bonuccelli, 2011). There are palliative treatments available that marginally slow disease progression but there is currently no cure for the disease (Awasthi, Singh, Pandey, & Dwivedi, 2016). More research is needed to develop effective therapeutic strategies to combat the disease. Currently, AD cytotoxicity is believed to be caused by increased amyloid β (Aβ) peptide plaque deposition in the brain, as described by the amyloid cascade hypothesis (Barage & Sonawane, 2015). The current understanding is that oligomers of Aβ peptide lead to neuronal death through multiple mechanisms, most notably hyper-phosphorylation of the tau protein. Having a better understanding of the structural changes in the fibrillization process of Aβ will provide a broader insight into mechanisms of cell death and open new possibilities for pharmacological treatments, which is what this research intends to provide.

No description available.

Recentemente, descobrimos que coelhos, Nova Zelândia, imunizados com colágeno tipo V humano mais adjuvante de Freund apresentavam fibrose e vasculite de órgãos normalmente afetados na esclerose sistêmica. Deste modo, nós estudamos o processo de fibrilogênese para identificar possíveis fatores envolvidos na alteração do remodelamento observado neste modelo de esclerodermia. Adicionalmente, fizemos uma comparação preliminar com pele humana obtida de pacientes com esclerodermia (n=3). Coelhos fêmeas, Nova Zelândia (n=14), foram imunizados subcutaneamente com duas doses de 1mg de colágeno V mais adjuvante completo de Freund, com intervalo de 30 dias, seguido de duas imunizações em adjuvante incompleto de Freund, via intramuscular, com intervalo de 15 dias. Os animais do grupo controle (n- 14) foram inoculados somente com adjuvante completo e incompleto de Freund, nas mesmas condições dos imunizados. Foram realizadas análises histológicas das peles dos animais e pacientes através da coloração com tricrômico de Masson e imunofluorescêcia, a fim de detectar fibras de colágeno e interação dos colágenos I, III e V. A análise da pele dos animais demonstrou depósito precoce de fibras de colágeno na derme após 7 dias da sensibilização, com aumento destes depósitos após 75 e 120 dias respectivamente. Depósito de colágeno na pele e atrofia de anexos foram mais intensos nos animais sacrificados em 120 dias e se correlacionaram com a quantidade aumentada de colágeno. Surpreendentemente, o colágeno V foi expresso em animais e pacientes, formando fibras densas e atípicas na derme. Sugerimos que esta expressão anômala de colágeno V, morfologicamente diferente, possa justificar o remodelamento observado na placa esclerodérmica / Recently, we discovered that New Zealand rabbits immunized with human type V collagen plus Freund`s adjuvant presented fibrosis and vasculitis of organs usually affected in systemic sclerosis. In this way, we studied the fibrillogenesis process regarding to identify any possible factor involved in altered remodeling observed in this scleroderma-like model. Additionally, we done a very preliminary comparison with human skins obtained from scleroderma patients (N=3). Female New Zealand rabbits (N=14) were immunized subcutaneously with two doses of 1mg collagen V plus complete Freunds adjuvant at a 30 days interval, followed by two additional intramuscular booster immunizations in incomplete Freunds adjuvant at a 15-day interval. Animals from control group (N=14), were only inoculated with complete and incomplete Freunds adjuvant given at same conditions of collagen type V group. Histological analysis of skins from animals and patients were done by Massons trichrome staining, and immunofluorescence method used to detect collagen fibers and interactions of types I, III and V collagen in the remodeling process. The analysis of animal skins showed precocious collagen fibril deposits in the dermis after 7 days of immunization and increase of this process in 75 and 120 days. Skin collagen deposit and atrophy of annexes were progressively more intense in late sacrificed animals and correlated with increased amount of collagen deposition. Surprisingly, type V collagen was over expressed both in animals and patients, forming dense and atypical collagen fibers in the dermis. We suggest that this anomalous expression of morphologically different type V collagen could justify the remodeling observed in scleroderma plaque

Recentemente, descobrimos que coelhos, Nova Zelândia, imunizados com colágeno tipo V humano mais adjuvante de Freund apresentavam fibrose e vasculite de órgãos normalmente afetados na esclerose sistêmica. Deste modo, nós estudamos o processo de fibrilogênese para identificar possíveis fatores envolvidos na alteração do remodelamento observado neste modelo de esclerodermia. Adicionalmente, fizemos uma comparação preliminar com pele humana obtida de pacientes com esclerodermia (n=3). Coelhos fêmeas, Nova Zelândia (n=14), foram imunizados subcutaneamente com duas doses de 1mg de colágeno V mais adjuvante completo de Freund, com intervalo de 30 dias, seguido de duas imunizações em adjuvante incompleto de Freund, via intramuscular, com intervalo de 15 dias. Os animais do grupo controle (n- 14) foram inoculados somente com adjuvante completo e incompleto de Freund, nas mesmas condições dos imunizados. Foram realizadas análises histológicas das peles dos animais e pacientes através da coloração com tricrômico de Masson e imunofluorescêcia, a fim de detectar fibras de colágeno e interação dos colágenos I, III e V. A análise da pele dos animais demonstrou depósito precoce de fibras de colágeno na derme após 7 dias da sensibilização, com aumento destes depósitos após 75 e 120 dias respectivamente. Depósito de colágeno na pele e atrofia de anexos foram mais intensos nos animais sacrificados em 120 dias e se correlacionaram com a quantidade aumentada de colágeno. Surpreendentemente, o colágeno V foi expresso em animais e pacientes, formando fibras densas e atípicas na derme. Sugerimos que esta expressão anômala de colágeno V, morfologicamente diferente, possa justificar o remodelamento observado na placa esclerodérmica / Recently, we discovered that New Zealand rabbits immunized with human type V collagen plus Freund`s adjuvant presented fibrosis and vasculitis of organs usually affected in systemic sclerosis. In this way, we studied the fibrillogenesis process regarding to identify any possible factor involved in altered remodeling observed in this scleroderma-like model. Additionally, we done a very preliminary comparison with human skins obtained from scleroderma patients (N=3). Female New Zealand rabbits (N=14) were immunized subcutaneously with two doses of 1mg collagen V plus complete Freunds adjuvant at a 30 days interval, followed by two additional intramuscular booster immunizations in incomplete Freunds adjuvant at a 15-day interval. Animals from control group (N=14), were only inoculated with complete and incomplete Freunds adjuvant given at same conditions of collagen type V group. Histological analysis of skins from animals and patients were done by Massons trichrome staining, and immunofluorescence method used to detect collagen fibers and interactions of types I, III and V collagen in the remodeling process. The analysis of animal skins showed precocious collagen fibril deposits in the dermis after 7 days of immunization and increase of this process in 75 and 120 days. Skin collagen deposit and atrophy of annexes were progressively more intense in late sacrificed animals and correlated with increased amount of collagen deposition. Surprisingly, type V collagen was over expressed both in animals and patients, forming dense and atypical collagen fibers in the dermis. We suggest that this anomalous expression of morphologically different type V collagen could justify the remodeling observed in scleroderma plaque

Polymerní biomateriály jsou jedním ze současných populárních témat vzhledem k možnosti potenciální aplikace v tkáňovém inženýrství a řízeného dávkování léčiv v organismech. Kolagen je jako jeden z nejčastěji se vyskytujících proteinů zvláště zajímavý díky svým rozmanitým vlastnostem bez imunoreakce organismu příjemce. Tato práce je zaměřena na samouspořádávací procesy, kinetiku, obecné zákonitosti řídící proces samouspořádání a mechanické vlastnosti kolagenních roztoků. Dále je zkoumán efekt hydroxyapatitových nanočástic na samouspořádávání kolagenu a mechanické vlastnosti výsledných nanokompozitních hydrogelů. Jsou objasněny možné mechanismy interakcí mezi kolagenem I a hydroxyapatitem spolu s popisem vývoje struktury a vlastností na různých úrovních struktury. Byly měřeny a molekulárně interpretovány závislosti viskoelastických veličin na smykové rychlosti spolu s viskoelastickým chováním. Dále byla studována struktura kolagenních scaffoldů a určen vliv HAP a síťování. Závěrem byly diskutovány výsledky v souvislosti s jejich aplikovatelností v tkáňovém inženýrství chrupavek tvrdých tkání a v regenerativní medicíně.

La matrice extracellulaire est composée d’environ 300 protéines et protéoglycanes qui constituent le matrisome et de 800 protéines associées (Naba et al., 2012a) et glycosaminoglycanes. C’est un protéome sous-exploré qui est modifié dans de nombreuses pathologies. Les fragments bioactifs issus de la matrice extracellulaire (matricryptines) sont capables de réguler des processus physiopathologiques et notamment l’angiogenèse et les pathologies cérébrales (Ricard-Blum and Salza, 2014). Environ 90 % des patients atteints de la maladie d’Alzheimer (MA) ont une angiopathie amyloïde cérébrale. L’angiogenèse contribue au déroulement de la MA. Nous nous sommes intéressés à l’endostatine (ES), une matricryptine du collagène XVIII qui possède des activités anti-angiogéniques, anti-tumorales et est également présente dans les plaques amyloïdes chez les patients atteints de la MA. Elle est libérée par les neurones et est capable de former des fibrilles amyloïdes in vitro (Kranenburg et al., 2003). Elle pourrait donc avoir une implication dans la MA. Nous avons montré que l'ES est présente dans le liquide céphalorachidien et que le rapport de sa concentration à celle des marqueurs classiques de la MA permet d’améliorer le diagnostic des patients atteint de démence fronto-temporale (DFT) et de discriminer les patients atteints de MA de ceux atteint de DFT et de pathologie nonMA/nonDFT. Nous avons établi les répertoires d’interactions extracellulaire du peptide -amyloïde (1-42) sous formes monomérique, oligomérique, fibrillaire ou agrégée et montré que l’oligomérisation et la fibrillogenèse augmentent la capacité d’interaction du peptide -amyloïde. Nous avons établi le réseau d’interaction global de l’endostatine par résonance plasmonique de surface en mode imagerie et identifiés 21 nouveaux partenaires de cette matricryptine. Nous avons plus particulièrement caractérisé son interaction avec la Procollagen C-Proteinase Enhancer-1, une protéine dont nous avons montré qu’elle donne naissance à une matricryptine anti-angiogénique. Nous avons enfin construit les réseaux d’interactions extracellulaires spécifiques de l’angiogenèse et de la maladie d’Alzheimer et des processus amyloïdes pour identifier les protéines connectant ces deux processus qui sont des cibles thérapeutiques potentielles. Ces réseaux d’interactions ont été créés à l’aide de 239 interactions que nous avons identifiées expérimentalement et des interactions décrites dans la littérature. Ces données seront à terme disponibles dans la base de données spécifique des interactions extracellulaires créée au laboratoire, MatrixDB, dans la nouvelle version à laquelle nous avons contribué. / The extracellular matrix include approximately 300 proteins and proteoglycans which constitute the matrisome and 800 associated proteins (Naba et al., 2012a) and glycosaminoglycans. It is an under-explored proteome which is modified in many diseases. Extracellular matrix bioactives fragments (matricryptins) are able to regulate physiopathological process like angiogenesis and cerebral disorders (Ricard-Blum and Salza, 2014). About 90 % of patients with Alzheimer's disease (AD) have cerebral amyloid angiopathy. Angiogenesis contributes to the development of AD. We are studying endostatin (ES), a matricryptin of collagen XVIII which has anti-angiogenic and anti-tumoral activities and is also present in amyloid plaques in AD patients. ES is released by neurons and is able to form amyloid fibrils in vitro (Kranenburg et al., 2003). This anti-angiogenic matricryptin could therefore be involved in AD. We have shown that ES is present in the cerebrospinal fluid of AD patients and the ratio of its concentrations to conventional markers of AD improves the diagnosis of patients with frontotemporal dementia (FTD) and discriminate AD patients from those suffering from FTD and pathology noAD/noDFT. We have established the extracellular interactions repertoires of the -amyloid peptide (1-42) in monomeric, oligomeric, fibrillar or aggregated forms and showed that the oligomerization and fibrillogenesis increase the interaction capacity of the -amyloid peptide. We have established the global interaction network of endostatin by surface plasmon resonance imaging and identified 21 new partners of this matricryptin. Specifically, we characterized its interaction with the Procollagen C-Proteinase Enhancer-1, a protein which gives rise to an anti-angiogenic matricryptin. We finally built networks of specific extracellular interactions of angiogenesis and of Alzheimer's disease and amyloid process to identify proteins connecting these two processes that are potential therapeutic targets. These interaction networks have been built using 239 interactions including those we have identified experimentally and those described in the literature. This data will be available in the database specific of extracellular interactions created in the laboratory, MatrixDB, in the new version of which we contributed.