No description available.

Afin de caractériser les mécanismes moléculaires sous-jacents au processus d’adhésion des bactéries commensales, nous avons utilisé Streptococcus salivarius comme modèle. Streptococcus salivarius est une bactérie pionière dans la colonisation des surfaces orales chez le nouveau né, et devient par la suite un composant majoritaire du microbiote oral de l'adulte avec un rôle écologique majeur. Nous avons développé une méthode pour identifier, par des tests de criblage phénotypique, les gènes impliqués dans l’adhésion de S. salivarius aux surfaces bactériennes ou de l’hôte. Notre approche a permis d’identifier un ensemble de gènes codant pour des protéines de surfaces, des glycosyltransférases, des transporteurs qui sont impliqués dans les phénomènes d’auto-agréation et / ou de co-agrégation avec d’autres espèces et / ou l’adhésion aux protéines de l’hôte.En particulier, nous avons montré que le système SecA2Y2, qui comprend des gènes codant pour des protéines dédiées à la glycosylation et l'export de protéines de surface riche en sérine (SRRPs), participe aux processus d’agrégation, de formation de biofilms, à l'adhésion in vitro aux protéines de l’hôte et in vivo à la colonisation du tractus digestif de souris. Alors que toutes les bactéries contenant un système similaire possèdent un substrat unique au système, une SRRP, le locus génétique secA2Y2 comprend trois SRRPs qui présentent des rôles complémentaires dans les phénotypes précédement cités. SrpB est spécifiquement impliquée dans la liaison aux cellules epitheliales, tandis que SrpC participe à l’adhésion aux protéines de la matrice extracellulaire et le mucus. De manière atypique, nous avons démontré que le processus de maturation des SRRPs est supporté par glycosyltransférases extra-cluster. Cette étude est le premier rapport indiquant la présence dans une bactérie de trois SRRPs, qui présentent des rôles complémentaires dans l'interaction bactéries-hôte. Bien que le système SecA2Y2 soit principalement associé à la virulence des bactéries pathogènes, il semble être clairement impliqué dans les caractères de commensalité de S. salivarius, tels que la colonisation de ses niches écologiques orales et intestinales. Ce travail offre de nouvelles perspectives sur les mécanismes de colonisation des bactéries commensales. / To characterize molecular mechanisms underlying adhesion of commensal bacteria, we used Streptococcus salivarius (SSAL) as a model. SSAL is among the most important pioneer colonizers of neonatal oral mucosal surfaces, and later becomes a predominant component of the human adult oral microbiota with pre-eminent ecological role. We developed a method to identified, through phenotypic screening assays, genes involved in SSAL adhesion to host or bacterial surfaces. In particular, we showed that the SecA2Y2 system, which comprises genes devoted to glycosylation and export of surface Serine Rich Repeat Proteins (SRRPs), participates to bacterial aggregation, biofilm formation, in vitro adhesion and colonization of mice. While all bacteria containing a similar system possess only one SRRP, the SSAL secA2Y2 locus comprises three SRRPs with complementary role in line with the previous phenotypes. Interestingly, SrpB is specifically involved in the binding to epithelial cells, while SrpC to the extracellular matrix and mucus proteins. We showed that these interactions require glycosylation of both bacterial SRPs and host surfaces. Surprisingly, we demonstrated that this essential process is shared by glycosyltransferases located in other genomic regions. This work is the first report showing the presence in a bacterium of three SRPs, which display complementary roles in bacterial-host interaction. While the SecA2Y2 system is mostly associated to virulence in pathogenic bacteria, it appears to be involved in the expression of commensal traits in SSAL, such as its colonization and its resilience to oral and intestinal niches. This work may offer new insights into the mechanisms of niche establishment (host, microbial communities) of commensal bacteria.

No description available.

Self-assembly of biomolecules into beta-sheet structures can be applied in the creation of nano-materials with novel electrical, optical, catalytical, or/and mechanical characteristics. This work was directed towards the construction of nano-derivatives based on amyloid fibrils forming proteins (Abeta40 peptide, a-Synuclein (a-Syn), equine lysozyme (EL)). Such nanostructures can be used to produce nanoscale functional systems. Herein, different mutant and hybrid proteins, which were able to form fibrillar structures, were constructed and the properties of fibrils were investigated. Designed cysteine mutants of Abeta40 and a-Syn can be modified through thiol group of cysteine. Herein, for the first time, it was demonstrated that a-Syncys141 fibrils could be modified with biotin and gold nanoparticles with neutravidin molecules. Hybrid proteins of Abeta40 or a-Syn and other non-amyloid proteins were designed on purpose to obtain fibrils with active functional non-amyloid proteins. Under appropriate conditions, these proteins aggregated into beta-sheet structures. Hybrid protein of streptavidin and Abeta40 formed a net-like fibrillar structure, and streptavidin was active. For the first time it was described the production of recombinant EL in E. coli. Moreover, active EL can form fibrils, which are similar to the fibrils formed by native EL. Constructed novel hybrids and mutants that are able to form amyloid fibrils, can be applied for the creation of functionalized nanodevices... [to full text] / Savitvarkės biomolekulės, gebančios formuoti beta-klosčių struktūras, gali būti pritaikomos nanomedžiagų su naujomis elektrinėmis, optinėmis, katalitinėmis ir/ar mechaninėmis savybėmis, kūrimui. Šiame darbe buvo siekiama kurti nanodarinius, grįstus amiloidines fibriles formuojančiais baltymais (Abeta40 peptidas, a-sinukleinas (a-Syn), kumelės pieno lizocimas (EL)), kurie būtų pagrindas nano dydžio funkcinių sistemų gamybai. Šiam tikslui buvo sukonstruoti mutantiniai ir hibridiniai baltymai bei tiriamos jų fibrilinių struktūrų savybės. Sukurti Abeta40 ir a-Syn cisteino mutantai, kurie gali būti modifikuojami per cisteino tiolinę grupę. Pirmą kartą buvo pademonstruotas a-Syncys141 baltymo fibrilių modifikavimas biotinu ir aukso nanodalelėmis su neutravidinu. Sukonstruoti hibridiniai baltymai, kurie sudaryti iš Abeta40 ar a-Syn bei GDH, streptavidino ir hidrofobino. Buvo tikimasi, kad tokie baltymai formuos fibrilines struktūras, o funkciniai baltymai bus aktyvūs. Esant atitinkamoms sąlygoms, šie baltymai agregavo suformuodami skirtingos morfologijos beta-klostines struktūras. Streptavidino ir Abeta40 hibridinis baltymas formavo fibrilinę struktūrą – tinklą, o streptavidinas buvo aktyvus. Šiame darbe pirmą kartą aprašoma rekombinantinio EL gamyba E. coli ląstelėse. Aktyvus EL gali formuoti panašias į natyvaus EL fibrilines struktūras. Sukonstruoti nauji hibridiniai ir mutantiniai baltymai, gebantys formuoti amiloidines fibriles, yra geras pagrindas, kuriant funkcionalizuotus... [toliau žr. visą tekstą]

Savitvarkės biomolekulės, gebančios formuoti beta klosčių struktūras, gali būti pritaikomos nanomedžiagų su naujomis elektrinėmis, optinėmis, katalitinėmis ir/ar mechaninėmis savybėmis, kūrimui. Šiame darbe buvo siekiama kurti nanodarinius, grįstus amiloidines fibriles formuojančiais baltymais (Abeta40 peptidas, a-sinukleinas (a-Syn), kumelės pieno lizocimas (EL)), kurie būtų pagrindas nano dydžio funkcinių sistemų gamybai. Šiam tikslui buvo sukonstruoti mutantiniai ir hibridiniai baltymai bei tiriamos jų fibrilinių struktūrų savybės. Sukurti Abeta40 ir a-Syn cisteino mutantai, kurie gali būti modifikuojami per cisteino tiolinę grupę. Pirmą kartą buvo pademonstruotas a-Syncys141 baltymo fibrilių modifikavimas biotinu ir aukso nanodalelėmis su neutravidinu. Sukonstruoti hibridiniai baltymai, kurie sudaryti iš Abeta40 ar a-Syn bei GDH, streptavidino ir hidrofobino. Buvo tikimasi, kad tokie baltymai formuos fibrilines struktūras, o funkciniai baltymai bus aktyvūs. Esant atitinkamoms sąlygoms, šie baltymai agregavo suformuodami skirtingos morfologijos beta klostines struktūras. Streptavidino ir Abeta40 hibridinis baltymas formavo fibrilinę struktūrą – tinklą, o streptavidinas buvo aktyvus. Šiame darbe pirmą kartą aprašoma rekombinantinio EL gamyba E. coli ląstelėse. Aktyvus EL gali formuoti panašias į natyvaus EL fibrilines struktūras. Sukonstruoti nauji hibridiniai ir mutantiniai baltymai, gebantys formuoti amiloidines fibriles, yra geras pagrindas, kuriant funkcionalizuotus... [toliau žr. visą tekstą] / Self-assembly of biomolecules into beta-sheet structures can be applied in the creation of nano-materials with novel electrical, optical, catalytical, or/and mechanical characteristics. This work was directed towards the construction of nano-derivatives based on amyloid fibrils forming proteins (Abeta40 peptide, a-Synuclein (a-Syn), equine lysozyme (EL)). Such nanostructures can be used to produce nanoscale functional systems. Herein, different mutant and hybrid proteins, which were able to form fibrillar structures, were constructed and the properties of fibrils were investigated. Designed cysteine mutants of Abeta40 and a-Syn can be modified through thiol group of cysteine. Herein, for the first time, it was demonstrated that a-Syncys141 fibrils could be modified with biotin and gold nanoparticles with neutravidin molecules. Hybrid proteins of Abeta40 or a-Syn and other non-amyloid proteins were designed on purpose to obtain fibrils with active functional non-amyloid proteins. Under appropriate conditions, these proteins aggregated into beta-sheet structures. Hybrid protein of streptavidin and Abeta40 formed a net-like fibrillar structure, and streptavidin was active. For the first time it was described the production of recombinant EL in E. coli. Moreover, active EL can form fibrils, which are similar to the fibrils formed by native EL. Constructed novel hybrids and mutants that are able to form amyloid fibrils, can be applied for the creation of functionalized nanodevices... [to full text]

α-synuclein is a small presynaptic protein whose misfolding and aggregation are considered drivers of the neurological disorders Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies and related synucleopathies. α-synuclein exists in a dynamic state that changes from an α-helical conformation when bound to liposomes to natively unfolded in solution, the majority being in the latter state. The disease process by which native healthy α-synuclein undergoes a change in conformation to form β-sheet oligomers and fibrils is still unresolved. The fibrillation process has been widely studied by several different techniques and the structure of the fibrils has been determined by NMR, scanning transmission electron microscopy and X-ray diffraction. The early stages of aggregation into β-sheet rich oligomers, despite having been widely studied, has proven difficult to follow due to the heterogeneity of the species formed and the unpredictability of the process. The goal of the work reported here was to design and develop a novel, reproducible and quantitative assay to study the early stages of α-synuclein aggregation and to establish a platform for discovery of novel compounds that inhibit this process. These compounds could then be taken as a starting point for the development of new drugs for the treatment of synucleopathies. The assay developed herein has been designed, established and demonstrated to be suitable for the screening of α-synuclein aggregation inhibitors. The assay quantitatively measures aggregation using α- synuclein site-specifically labelled with green and red fluorescent dyes. Proteins labelled with the green dye are bound to microbeads. α-synuclein labelled with the red dye aggregates on the bead-linked green α-synuclein. The first part of the thesis describes the development of the tools required for the assay. α-synuclein single cysteine mutants were produced to introduce a specific attachment point to the protein. Single isomer carboxytetramethylrhodamine was synthesised in large scale for the label. Two different trifunctional tags that allow both the fluorescent labelling of the protein and the addition of a group for bead attachment in a single step were synthesised. Optimisation of the attachment of the functionalised proteins to beads of differing materials was accomplished enabling further development of the bead-based aggregation assay. With all tools established, the second part of the work comprised the development of the bead-based α-synuclein aggregation assay. Solid supports made of two different materials, TentaGel and Agarose, with two different types of bead surface attachment chemistry for α-synuclein were investigated, Ni-NTA on bead with His6-tag on the target or dibenzylcyclooctyne on bead and azide conjugation for the target. Only the combination of Ni-NTA agarose beads linking to His6-tag functionalised α-synuclein was found to be suitable for quantitative measurement of the aggregation process. Using 20 % EtOH, α-synuclein on-bead aggregation was reproducible within a 5 h time-frame with a linear dependence of aggregation rate as function of protein concentration on-bead. The third part of the thesis describes the research into novel starting points for the discovery of inhibitors of α-synuclein aggregation. In the peptides field, the most active peptides in the literature were selected and synthesised for study under the same conditions to find the most active ones. The most active peptide could be modified with non-natural amino acids to increase affinity and stability. While peptides and peptidomimetics would be applied in mechanistic studies, small molecular inhibitors of aggregation might represent lead compounds. One known inhibitor of α-synuclein aggregation was selected, NPT200-5, and an on-bead synthesis was developed so a diversity library could be generated around its four different building blocks. Finally the peptides, the NPT200-5 amide derivative and some known small molecule inhibitors of α-synuclein aggregation, such as curcumin, baicalein and EGCG amongst others, were screened on the bead-based α-synuclein aggregation assay. Strong inhibitory effects of curcumin and baicalein demonstrated the efficacy of the newly developed assay. In summary, the tools for the development of a novel micro-bead-based α-synuclein aggregation assay have been successfully produced. A novel bead-based α-synuclein early stage aggregation assay has been developed and optimised. Validation of this new technique was achieved with known small molecules inhibitors of α-synuclein aggregation.

Advances in recent nanoscience technologies have generated a new compilation of biocompatible, fluorescent nanoparticles derived from semiconductor quantum dots (QDs). QDs are extremely small in size and possess very large surface areas, which gives them unique physical properties and applications distinct from those of bulk systems. When exposed to biological fluid, these QDs may become coated with proteins and other biomolecules given their dynamic nature. These protein-QD systems may affect or enhance the changes in protein structure and stability, leading to the destruction of biological function. It is believed that these QDs can act as nucleation centers and subsequently promote protein fibril formation. Protein fibrillation is closely associated with many fatal human diseases, including neurodegenerative diseases and a variety of systemic amyloidoses. This topic of protein-QD interaction brings about many key issues and concerns, especially with respect to the potential risks to human health and the environment. Herein, the behavioral effects of dihydrolipoic acid (DHLA)-capped CdSe/ZnS (core/shell) QDs in hen egg-white lysozyme (HEWL) and human serum albumin (HSA) protein systems were systematically analyzed. This study gives rise to a better understanding of the potentially useful application of these protein-QD systems in nanobiotechnology and nanomedicine as a bioimaging tool and/or as a reference for controlled biological self-assembly processes.

The research field of conjugated polymers has grown due to the optical and electronic properties of the material, useful in applications such as solar cells and printed electronics, but also in biosensors and for interactions with biomolecules. In this thesis conjugated polymers have been used in two related topics; to detect conformational changes in proteins and to assemble the polymers with biomolecules into nanowires. Within biosensing, conjugated polymers have been used for detection of a wide range of biological events, such as DNA hybridization or enzymatic activity, utilizing both electronic and optical changes in the polymer. Here the focus has been to use the polymers as optical probes to discriminate between native and misfolded protein, as well as to follow the misfolding processes in vitro. The understanding and detection of protein misfolding, for example amyloid fibril formation, is a topic of growing importance. The misfolding process is strongly associated with several devastating diseases such as Alzheimer’s disease, Parkinson’s disease and Bovine Spongiform Encephalopathy (BSE). We have developed detection schemes for discrimination between proteins in the native or amyloid fibril state based on luminescent polythiophene derivatives. Through a synthesis strategy based on polymerization of trimer blocks rather than of monomers, polythiophene derivatives with higher optical signal specificity for amyloid-like fibrils were obtained. Self-assembly of nanowires containing conjugated polymers is a route to generate structures of unique opto-electrical characteristics without the need for tedious topdown processes. Biomolecules can have nanowire geometries of extraordinary aspect ratio and functionalities. The DNA molecule is the most well known and exploited of these. In this thesis work the more stable amyloid fibril has been used as a template to organize conjugated polymers. Luminescent, semi-conducting, conjugated polymers have been incorporated in and assembled onto amyloid fibrils. Using luminescence quenching we have demonstrated that the conjugated material can retain the electro-activity after the incorporation process. Furthermore, the amyloid fibril/conjugated polymer hybrid structures can be organized on surfaces by the means of molecular combing and soft lithography. In the process of generating self-assembled biomolecular nanowires functionalized with conjugated polymers, we have shown a new synthesis strategy for a water-soluble highly conducting polythiophene derivative. This material, PEDOT-S, has shown affinity for amyloid fibrils, but can also be very useful in conventional opto-electronic polymer-based devices.

In this thesis we present computer modelling studies that were implemented to investigate protein behavior in various environments causing their folding, unfolding and aggregation. Applications related to two important proteins - insulin and apolipoprotein C-II (ApoC-II) are presented. The use of atomistic simulation methodologies based on empirical force fields has enhanced our understanding of many physical processes governing protein structure and dynamics. However, the force fields used in classical modelling studies are often designed for a particular class of proteins and rely on continuous improvement and validation by comparison of simulations with experimental data. In Chapter 4 we present a comprehensive comparison of five popular force fields for simulation of insulin. The effect of each force field on the conformational evolution and structural properties of the protein is analysed in detail and compared with available experimental data. A fundamental phenomenon in nature is the ability of proteins to fold ab initio to their functional native conformation, also known as their biologically active state. Due to the heterogeneity and dimensionality of the systems involved, it is necessary to employ methodologies capable of accelerating rare events, specifically, configurational changes that involve the crossing of large free energy barriers. In Chapter 5, using the recently developed method BE-META we were able to identify the structural transitions and possible folding pathways of insulin. Another interesting phenomenon is the misfolding of proteins causing their aggregation, that may lead to formation of either amorphous compounds or structures of elongated-unbranched morphology known as amyloid fibrils. The deposition of amyloid fibrils in the human body may cause many debilitating diseases such as Alzheimer's and variant Creutzfeldt-Jakob diseases, thus making this field of research important and urgent. The human plasma protein apoC-II serves important roles in lipid transport, and it has been shown to form amyloid-like aggregates in solution. We have performed computational studies to investigate the effect of mutations, such as Met oxidation and the residue substitutions to hydrophobic Val and hydrophilic Gln, on dynamics of apoC-II(60-70) peptide. The conformation features relevant to the amyloidogenic propensities of the peptide were identified and presented in Chapter 6. The involvement of lipids at the various stages of development of amyloid diseases is becoming more evident in recent research efforts. In particular, micellar and sub-micellar concentrations have showed to have different effect on fibril growth and kinetics of native apoC-II and derived peptides. In Chapter 7 we investigated the influences of phospholipids at various concentrations on the structure of apoC-II(60-70) using MD and umbrella sampling methods. The molecular mechanisms of lipid effects on the peptide conformation and dynamics were identified. In Chapter 8 preliminary results on the structural stability of pre-formed oligomeric composites of apoC-II(60-70) peptide of different sizes and arrangements were also presented. The effects of mutation (oxidised Met, Met60Val and Met60Gln) on the most stable cluster was also investigated. To conclude, several ideas for continuation of research in the protein folding and aggregation field are discussed in the Future Work section of this thesis.

Le cadre de cette étude est le coût énergétique lié à la production des Microfibrilles de Cellulose (MFC) qui est aujourd’hui un facteur limitant à son développement à l’échelle industrielle. Le but de cette étude est de caractériser les interactions cellulose/hémicellulose au sein de ces systèmes.Des MFC provenant de différentes pâtes à papier chimiques ont été caractérisées par RMN du solide afin d’obtenir des informations à l’échelle moléculaire. Suite à l’optimisation d’un protocole expérimental, les hémicelluloses contenues dans les MFC issues de pâte kraft de bouleau ont ensuite été extraites avec un rendement de 60% et sont composés uniquement d’un homopolymère de xylan de DP 75.La turbidimétrie a été utilisée pour qualifier la qualité des suspensions, dont il a été montré qu’elle dépend fortement du procédé de mise en pâte et du séchage. Des corrélations positives ont été établies entre l’état de dispersion et les propriétés mécaniques de feuilles de papier additionnées de microfibrilles. L’analyse RMN de modèles biomimétiques reconstitués a confirmé le changement de conformation du xylan lorsqu’il est adsorbé sur la cellulose et les mesures de surface spécifique ont montré que seule la couche de xylan en contact avec la cellulose était concernée par ce changement.Les interactions cellulose/xylane ont été étudiées par RMN du solide et par dynamique moléculaire atomistique (MD). Les simulations MD ont montré que le xylan s’adsorbe parallèlement aux chaines de cellulose. Des mesures d'interaction sur ce système ont conduit à une mesure d'énergie de 9kJ/résidu de xylose.Des tests de mesure d’adhésion ont également été réalisés à partir d’un modèle trois couches constitué de xylan entre deux films de cellulose et une forte adhésion a pu être observée.L’utilisation de xylanase comme prétraitement est proposé pour améliorer la production des MFC. / The study was motivated by the necessity to reduce the high energy costs of Micro-Fibrillated Cellulose (MFC) production, which is a limiting factor for its industrial development and aimed at understanding the cellulose/hemicelluloses interaction within this system. MFC resulting from different chemical pulps were characterized by solid-state NMR spectroscopy to get information on the hemicelluloses content and molecular conformation. By optimizing an extraction protocol, more than 60% of the residual hemicelluloses were extracted from birch kraft MFC and characterized as a high purity homopolymer of β-1,4 linked xylan of DP 75.Turbidimetry was used to qualify the quality of the suspensions, which strongly depended on the pulping and drying history. Positive correlations between the state of dispersion, specific surface and mechanical properties of MFC-reinforced handsheets were evidenced.Cellulose/xylan interactions were investigated using solid-state NMR and atomistic molecular dynamics (MD) simulation. NMR spectra confirmed that xylan in contact with cellulose altered its conformation, from the three-fold helix to a presumable cellulose-like two-fold one. In combination with specific surface area measurements, the conformational change was shown to happen only for the first layer of xylan adsorbed in direct interaction with the cellulose surface. MD simulations showed that adsorbed xylan tends to align parallel to the cellulose chain direction fully extended. Interaction energy between xylan chain and cellulose surface estimated with MD was 9kJ/xylose. Then a three-layers system made of xylan between two cellulose films were built to perform adhesion tests that showed strong adhesion between xylan and cellulose surfaces. Xylanase was proposed as a pulp pretreatment for MFC production.