Studies on interactions between NKR-P1D and Clrb membrane receptors Interaction between murine NKR-P1D and Clrb receptors was originally described as a novel type of "MHC class-I independent missing-self recognition" and was shown to confer protection from killing by natural killer cells.[1] However, further study brought conflicting results suggesting that NKR-P1D does not binds Clrb strongly if it does at all.[2] In order to address the issues arising from these conflicting results, we have recombinantly expressed the extracellular domains of both receptors in E. coli cells and refolded the proteins in vitro. The quality of refolding was confirmed both by determining the disulphide bonding pattern using FTMS and measuring 1 H/15 N-HSQC spectra. By means of size exclusion chromatography and analytical ultracentrifuge we were unable to provide convincing results for the interaction itself. However, using SPR technique, a weak, specific, pH-dependent interaction was observed. Interaction between the proteins in solution was immobilized using chemical cross-linking technique. Three cross-linking reagents, EDC, DSG and DSS were used. The reaction mixture was separated by means of SDS-PAGE and protein bands corresponding to dimers were digested in gel. Using FT-MS we were able to find peptides from both...

In eukaryotic translation, eukaryotic initiation factors (eIFs) are at least as important as the ribosome itself. Some of these factors play different roles throughout the entire process to ensure proper assembly of the preinitiation complex on mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most important factor integrating signals from others and coordinating their functions on the ribosome is eIF3. In Saccharomyces cerevisiae, eIF3 is formed by five subunits. All these subunits contain structural motifs responsible for contact with ribosomal proteins and RNAs. In addition to these highly structured parts, the rest of eIF3 is unstructured and very flexible. Therefore, despite the recent progress thanks to the use of a cryo-electron microscopy, a precise structure and position of eIF3 on the 40S ribosomal subunit are still not known. Also, the presence of eIF3 on 80S during early elongation and its role in reinitiation and readthrough are not fully understood. In order to crack mysteries of yeast eIF3, we used x-ray crystallography, chemical cross- linking coupled to mass spectrometry, and various biochemical and genetic assays. We demonstrated that eIF3 is very compactly packed when free in solution. This...

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L'α-Synuclein (αSyn) fibrillaire, impliqué dans la maladie de Parkinson et d’autres synucleinopathies, peut se propager entre cellules de manière « prion-like » et cette propagation est liée à la progression de la maladie. Durant cette étude, nous nous sommes tournés vers les chaperons moléculaires impliqués dans l’agrégation de l’αSyn ou bien dans sa toxicité afin de trouver des candidats capables d’interférer avec la propagation. Nous avons ensuite testé l’effet des chaperons capables de se lier aux fibres d’αSyn sur l’internalisation des fibres d’αSyn par les cellules Neuro-2a. Nous démontrons que l’interaction avec l’αSyn agrégeant avec αB-crystallin (αBc) ou Carboxyl terminus of Hsc70-interacting protein (CHIP) a mené à la formation de fibres qui sont moins internalisées par les cellules. Enfin, en passant par une stratégie de pontage chimique optimisé couplé à la spectrométrie de masse, nous avons identifié les zones d’interaction entre l’αSyn fibrillaire et soit αBc, soit CHIP. Ces résidus issus des chaperons, se trouvant à proximité des fibres d’αSyn dans les complexes, pourraient être développés dans des mini-chaperons peptidiques, capables d’enrober la surface des fibres et ainsi de bloquer la liaison à la membrane et l’internalisation des fibres. De surcroît, des polypeptides issus des partenaires précédemment identifiés d’αSyn ont été testé pour leur liaison aux fibres et leur effet sur la propagation des fibres. / Fibrillar α-Synuclein (αSyn) is the molecular hallmark of Parkinson’s Disease and other synucleinopathies. Its prion-like propagation between cells is linked to disease progression. In this study, we looked to molecular chaperones previously implicated in αSyn fibrillation and/or toxicity to identify proteins capable of binding αSyn fibrillar aggregates in order to target their propagation. We further assessed the effect of the fibril-binding chaperones on internalization of αSyn fibrils by Neuro-2a cells. We demonstrate that the interaction of aggregating αSyn with αB-crystallin (αBc) or Carboxyl terminus of Hsc70-interacting protein (CHIP) led to the formation of fibrils that are less internalized by cells. Finally, using an optimized chemical cross-linking and mass spectrometry strategy, we identified the interaction areas between fibrillar αSyn and either αBc or CHIP. These chaperone residues, located proximally to αSyn fibrils, could be subsequently developed into peptidic mini chaperones, capable of coating the fibril surface and thereby blocking fibrillar cell binding and internalization. Furthermore, polypeptides derived from previously identified αSyn binding partners were tested for their binding to αSyn fibrils and subsequent effect on fibril propagation.

Background: Collagen cross-linking of the sclera is a promising approach to strengthen scleral rigidity and thus to inhibit eye growth in progressive myopia. Additionally, cross-linking might inhibit degrading processes in idiopathic melting or in ocular inflammatory diseases of the sclera. Different cross-linking treatments were tested to increase resistance to enzymatic degradation of the rabbit sclera. Methods: Scleral patches from rabbit eyes were cross-linked using paraformaldehyde, glutaraldehyde or riboflavin combined with UV-A-light or with blue light. The patches were incubated with collagenase I (MMP1) for various durations up to 24 h to elucidate differences in scleral resistance to enzymatic degradation. Degraded protein components in the supernatant were detected and quantified using measurements of Fluoraldehyde o-Phthaldialdehyde (OPA) fluorescence . Results: All cross-linking methods reduced the enzymatic degradation of rabbit scleral tissue by MMP1. Incubation with glutaraldehyde (1%) and paraformaldehyde (4%) caused nearly a complete inhibition of enzymatic degradation (down to 7% ± 2.8 of digested protein compared to control). Cross-linking with riboflavin/UV-A-light reduced the degradation by MMP1 to 62% ± 12.7 after 24 h. Cross-linking with riboflavin/blue light reduced the degradation by MMP1 to 77% ± 13.5 after 24 h. No significant differences could be detected comparing different light intensities, light exposure times or riboflavin concentrations. Conclusions: The application of all cross-linking methods increased the resistance of rabbit scleral tissue to MMP1-degradation. Especially, gentle cross-linking with riboflavin and UV-A or blue light might be a clinical approach in future.

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The use of degradable biomedical materials has during the past decades indeed modernized medical science, finding applications in e.g. tissue engineering and drug delivery. The key question is to adapt the material with respect to mechanical properties, surface characteristics and degradation profile to suit the specific application. Degradation products are generally considered non-toxic and they are excreted from the human body. However, large amounts of hydroxy acids may induce a pH decrease and a subsequent inflammatory response at the implantation site. In this study, macromolecular design and a combination of cross-linking and adjusted hydrophilicity are utilized as tools to control and tailor degradation rate and subsequent release of degradation products from biomedical polyester-ethers. A series of different homo- and copolymers of -caprolactone (CL) and 1,5-dioxepan-2-one (DXO) were synthesized and their hydrolytic degradation was monitored in phosphate buffer solution at pH 7.4 and 37 °C for up to 546 days. The various materials comprised linear DXO/CL triblock and multiblock copolymers, PCL linear homopolymer and porous structure, and random cross-linked homo- and copolymers of CL/DXO using 2,2’-bis-(ε-caprolactone-4-yl) propane (BCP) as a cross-linking agent. The results showed that macromolecular engineering and controlled hydrophilicity of cross-linked networks were useful implements for customizing the release rate of acidic degradation products in order to prevent the formation of local acidic environments and thereby reduce the risk of inflammatory responses in the body. / QC 20101109

The use of cell transplantation to treat enzyme deficiency disorders is limited by the immune response targeted against foreign tissue or the use of life-long immunosuppressants. Hiding cells from the immune system in an encapsulation device is promising. Cells encapsulated within an anionic calcium alginate hydrogel bead are protected through a semi-permeable membrane formed by polycation, poly-L-lysine (PLL). A final layer of alginate is added to hide the cationic PLL surface but this has proved to be difficult creating capsules which are prone to fibrotic overgrowth, blocking exchange of nutrients, waste and therapeutic enzymes through the capsule. For long term applications these capsules need to be both biocompatible and mechanically robust. This thesis aims to address the biocompatibility issue of high cationic surface charge by synthesizing polycations of reduced charge using N-(3- aminopropyl)methacrylamide hydrochloride (APM) and N-(2- hydroxypropyl)methacrylamide (HPM) and study the associated mechanical properties of the capsules using micropipette aspiration. Micropipette aspiration was applied and validated for alginate based capsules (gel and liquid core) to quantify stiffness. Varying ratios of APM were used to control the overall charge of the polycations formed while HPM was incorporated as a neutral, hydrophilic, nonfouling comonomer. The molecular weight (MW) was controlled by using reversible addition-fragmentation chain transfer (RAFT) polymerization. The biocompatibility of these polymers was tested by cell adhesion and proliferation of 3T3 fibroblasts onto APM/HPM copolymer functionalized surfaces and by solution toxicity against C2C12 myoblasts. The ability for the APM/HPM copolymers to bind to alginate and form capsules was also assessed, along with the integrity and stiffness of the capsule membrane with or without additional covalent cross-linking by reactive polyanion, poly(methacrylic acid-co-2-vinyl-4,4- dimethylazlactone) (PMV60). Thermo-responsive block copolymers of N-isopropylacrylamide (NIPAM) and 2- hydroxyethylacrylamide (HEA) were also synthesized as potential drug delivery nanoparticles, showing control over micelle morphology with varying NIPAM to HEA ratios. / Thesis / Doctor of Science (PhD) / The treatment of enzyme deficiency disorders by cell transplantation is limited by the immune attack of foreign tissue in absence of immunosuppressants. Cells protected in an encapsulation device has shown promise. Poly-L-lysine, a widely used membrane material in these protective capsules, binds to the anionic gel entrapping living cells because it is highly cationic. The high cationic charge is difficult to hide causing the immune system to build tissue around the capsule, preventing the encapsulated cells from exchanging nutrients and therapeutic enzymes. This thesis aims to replace poly-L-lysine by synthesizing a series of more biocompatible materials of decreasing cationic charge. These materials were studied for the ability to support tissue growth and form stable capsules. The membrane strength was measured using an aspiration method validated for these types of capsules. Reducing the cationic charge of the materials increased the biocompatibility of the capsule membrane but also made for weaker membranes.

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<p>The rotator cuff is a group of four muscles and tendons in the shoulder that function to lift and rotate the arm. Rotator cuff tendon tears are increasingly common: more than 545,000 rotator cuff surgeries occur annually in the US. However, treatment is often complicated by disorganized collagen matrix formed via fibrosis and results in high re-tear rates. Tendon tissue engineering seeks to solve the problem using biomaterials to promote neo-tendon formation to augment repair or regenerate tendon. However, while current biomaterials provide the opportunity to improve tendon healing, they frequently still exhibit fibrosis in preclinical studies. Therefore, a critical need exists to understand the mechanisms of aligned collagen formation when designing biomaterials for tendon tissue engineering. Matrix architecture and transient receptor potential cation channel subfamily V member 4 (TRPV4) regulate aligned collagen formation during tenogenesis in vitro, but the mechanism remains to be determined. Recently, TRPV4 stimulation was found to induce nuclear localization and activation of transcriptional co-activators Yes-associated protein (YAP). YAP expression is upregulated during tendon development, a process characterized by aligned collagen formation, and in response to physiological mechanical stimulation, suggesting it could play an important role in tendon. The objective of this work is to improve tissue engineering strategies and progress toward making a device that regenerate tendon after injury. Aim 1 incorporates tendon-derived matrix into synthetic polymer scaffolds to add biological signaling cues to induce tenogenesis. Aim 2 uses a 2D photolithography system (microphotopatterning) to optimize architectural and structural cues to promote stem cell differentiation toward tenogenic, chondrogenic, and osteogenic lineages. Aim 3 investigates dynamic tensile loading protocols to promote collagen matrix synthesis and improve engineered tendon mechanical function. Aim 4 investigates the role of TRPV4 and YAP in collagen alignment during engineered tendon development.</p>