Following their endosymbiotic uptake, plastids undergo profound changes to genome content and to their associated biochemistry. I have investigated how evolutionary transitions in plastid genomes may impact on biochemical pathways associated with plastid gene expression, focusing on the highly unusual plastids found in one group of eukaryotes, the alveolates. The principal photosynthetic alveolate lineage is the dinoflagellate algae. Most dinoflagellate species harbour unusual plastids derived from red algae. The genome of this plastid has been fragmented into small, plasmid-like elements termed “minicircles”. Transcripts of this genome receive a 3’ poly(U) tail and, in some species, undergo extensive sequence editing. Some dinoflagellates have replaced their original plastids with others, in a process termed “serial endosymbiosis”. The major non-photosynthetic alveolates are the apicomplexans, which include the malaria parasite Plasmodium. Apicomplexans are descended from free-living algae and possess a vestigial plastid, which originated through the same endosymbiosis as the ancestral red dinoflagellate plastid. This plastid has lost all genes involved in photosynthesis and does not possess a poly(U) tail addition pathway. I have investigated the consequences of the fragmentation of the red algal dinoflagellate plastid genome on plastid transcription. I have characterised non-coding transcripts in plastids of the dinoflagellate Amphidinium carterae, including the first evidence for antisense transcripts in an algal plastid. Antisense transcripts in dinoflagellate plastids do not receive poly(U) tails, suggesting that poly(U) tail addition may play a role in strand discrimination during transcript processing. I have additionally characterised transcript processing in dinoflagellate plastids that were acquired through serial endosymbiosis. I have shown that poly(U) tail addition and editing occur in the haptophyte-derived serial endosymbionts of the fucoxanthin-containing dinoflagellates Karenia mikimotoi and Karlodinium veneficum. This is the first evidence that plastids acquired through serial endosymbiosis may be supported by pathways retained from previous symbioses. Transcript editing constrains the phenotypic consequences of divergent mutations in fucoxanthin plastid genomes, whereas poly(U) tail addition plays a central role in recognising and processing translationally functional fucoxanthin plastid mRNAs. I have additionally shown that certain genes within fucoxanthin plastids are located on minicircles. This demonstrates convergent evolution in the organisation of the fucoxanthin and red algal dinoflagellate plastid genomes since their endosymbiotic acquisition. Finally, I have investigated transcript processing in the algae Chromera velia and Vitrella brassicaformis. These species are closely related to apicomplexans but are still photosynthetic and apply poly(U) tails to plastid transcripts, as with dinoflagellates. I have shown that poly(U) tails in these species are preferentially associated with translationally functional mRNAs of photosynthesis genes. This is the first plastid transcript processing pathway documented to target a specific functional gene category. Poly(U) tail addition may direct transcript cleavage and allow photosynthesis gene transcripts to accumulate to high levels. The loss of this pathway from ancestors of apicomplexans may have contributed to their transition from photosynthesis to parasitism.

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Cell adhesion is the first step of cell response to materials and the extracellular matrix (ECM), and is essential to all cell behaviours such as cell proliferation, differentiation, migration and apoptosis for anchor-dependent cells. Therefore, studies of cell attachment have important implications to control and study cell behaviours. During many developed techniques for cell attachment, the manipulation of surface chemistry is a very important method to control initial cell attachment. To control cell adhesion on a two-dimensional surface is a simple model to study cell behaviours, and is a fundamental topic for cell biology, tissue engineering, and the development of biosensors. From the engineering point of view, the preparation of a material with controllable surface chemistry can help studies of cell behaviours and help scientists understand how surface features and chemistry influence cell behaviours. During the fabrication, the challenge is to create a surface with heterogeneous surface properties in the micro scale and subsequently to guide cell initial adhesion. In order to control cell adhesion in a spatial and temporal manner, a photochemical method to control surface chemistry was employed to control the surface property for cell adhesion in this project. Two photocleavable derivatives of the nitrobenzyl group were tried on two types of surfaces: a model self-assembled monolayer (SAM) with alkanethiol-gold surface and biodegradable chitosan. Reactive functional groups on two different surfaces can be inactivated by covalent binding with these photocleavable molecules, and light can be further introduced into the system as a stimulus to recover their reactivity. By simply applying a photomask with diffe

Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

As a first project, collision-induced dissociation experiments were carried out using electrospray ionisation mass spectrometry on gas phase complexes involving different poly(methylmetacrylate) oligomers with three amino acids: glycine, leucine, and phenylalanine. After acquiring breakdown diagrams, RRKM modeling was used to fit the experimental data in order to obtain the 0 K activation energy and the entropy of activation. These thermodynamic data were then used to understand the competing dissociation channels observed (except for gas phase complexes involving glycine that had only one dissociation channel). Molecular dynamics simulated annealing calculations were carried on the gas phase complexes to understand further the energetic and entropic effects involved as well as the 3D conformation of these complexes. Valuable insight information was found on the 3D conformations, on a qualitative level. Using rotational constants and vibrational harmonic frequencies, it was possible to evaluate the entropy variation between the experimentally observed competing channels. Reasonable agreement was found between the experimental and theoretical variations of entropies. Finally, the proton affinity of poly(methylmetacrylate) oligomers is being discussed. Even though no absolute values for the proton affinity were found, the experimental and computational results help to understand the variation that accompanies the oligomers length. The second project presents the development an efficient and reproducible screening method for identifying low molecular weight compounds that bind to amyloid beta peptides (Abeta) peptides using electrospray ionization mass spectrometry (ESI-MS). Low molecular weight (LMW) compounds capable of interacting with soluble Abeta may be able to modulate/inhibit the Abeta aggregation process and serve as potential disease-modifying agents for Alzheimer’s disease. The present approach was used to rank the binding affinity of a library of compounds to Abeta1-40 peptide. The results obtained show that low molecular weight compounds bind similarly to Abeta1-42, Abeta1-40, as well as Abeta1-28 peptides and they underline the critical role of Abeta peptide charge motif in binding at physiological pH. Finally, some elements of structure-activity relationship (SAR) involved in the binding affinity of homotaurine to soluble Abeta peptides are discussed. As a third project, the gas phase binding of small molecules to the Abeta1-40 peptide generated by electrospray ionization has been explored with collision-induced dissociation mass spectrometry and kinetic rate theory. This project presents a simple procedure used to theoretically model the experimental breakdown diagrams for the Abeta1-40 peptide complexed with a series of aminosulfonate small molecules, namely homotaurine, 3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO), 3-(1,3,4,9-tetrahydro-2H-beta-carbolin-2-yl) propane-1-sulfonic acid, 3-(1,3,4,9-tetrahydro-2H-beta-carbolin-2-yl)butane-1-sulfonic acid, and 3-(cyclohexylamino) propane-1-sulfonic acid. An alternative method employing an extrapolation procedure for the microcanonical rate constant, k(E), is also discussed.

Suite à un accident, les lésions du ligament croisé antérieur sont de plus en plus fréquentes devenant un problème de santé publique. En réponse à la demande de solutions alternatives aux traitements chirurgicaux actuels, nous avons développé un renfort ligamentaire en biomatériaux dégradables. Pour répondre à l'exigence de la régénération ligamentaire, ce dernier doit être suffisamment résistant pour répondre aux contraintes physiologiques du genou, et doit se dégrader tout en permettant la régénération d'un nouveau ligament. Nous avons synthétisé de nouveaux copolymères bloc à base de PLA et de poloxamère ou poloxamine qui ont été filés puis tricotés pour concevoir un textile tubulaire. Ce textile tubulaire possède des caractéristiques mécaniques intéressantes pour le remplacement d'un ligament. En parallèle, a été développée une matrice poreuse à trois dimensions en collagène et glycoaminoglycanes. Cette matrice permet de favoriser l'adhésion et la prolifération cellulaire. Le renfort tricoté associé à la matrice de collagène a été implanté in vivo durant 3 mois. A 3 mois, parfaitement intégrée, la structure composite permet la formation d'un néo-tissu tout en perdant progressivement ses propriétés mécaniques. / Following accidents, anterior cruciate ligament (ACL) damages are increasingly becoming a common public health problem. To comply with the demand for alternatives to current surgical treatments, we have developed ligament reinforcement with degradable biomaterials. To meet the requirement of ligament regeneration, ligament reinforcement must be strong enough to support knee physiological strains, and must degrade while allowing the new ligament regeneration. Novel block copolymers PLA- and poloxamer or poloxamine based have been synthesized which were then spun for designing a tubular knitted fabric. The tubular fabric has interesting mechanical characteristics for ligament replacement. In parallel, a collagen and glycosaminoglycans porous three-dimensional matrix has been developed. This matrix is able to promote cell adhesion and proliferation. The knitted reinforcement associated with the collagen matrix has been implanted in vivo for 3 months. Fully integrated, the composite structure allows néo-tissue formation while gradually losing its mechanical properties after 3 months.

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