The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. Although mainly considered to be a protective mechanism, prolonged and severe ISR can result in cell death. The ISR is activated by diverse stress pathways converged on phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) that inhibits the guanine nucleotide exchange activity of its partner eIF2B and attenuates overall rates of protein synthesis. Numerous mutations in eIF2B are linked to a fatal neurodegenerative disease of vanishing white matter. A new chemical inhibitor of the ISR (ISRIB), a bis-O-arylglycolamide, can reverse the attenuation of mRNA translation by phosphorylated eIF2 protecting mice from prion-induced neurodegeneration and traumatic brain injury. The work presented in this dissertation describes identification of mammalian eIF2B as a cellular target of ISRIB by implementing biochemical, biophysical, structural and chemogenetic methods. The herein reported cryo-electron microscopy-based structure of eIF2B uncovers a novel allosteric site on the translation factor capturing the ISRIB-binding pocket at the interface between its β and δ regulatory subunits. The extensive CRISPR/ Cas9-based screen for ISRIB-resistant and analogue-sensitive phenotypes revealed residues on the eIF2B dimer interface important for ISRIB binding. Based on the results reported in this dissertation along with the similar findings of others the potential molecular basis of ISRIB action, and its implication for the regulation of eIF2B's activity is broadly discussed. The identification of the ISRIB binding pocket away from the known interaction sites between eIF2B and eIF2 is also put into the context of a possible molecular mechanism of eIF2B's guanine exchange inhibition by phosphorylated eIF2. The work described in this dissertation provides new insight into the translational regulation and points to the importance of fine-tuning the activity of translation factors by small chemical molecules.

mTOR is the central kinase in biochemical pathways that regulate cellular growth, protein synthesis and cell survival. Deregulation of mTOR signaling results in uncontrolled cell proliferation and hence is implicated in various cancers and autoimmune diseases. mTOR functions through two distinct signaling complexes, called mTORC1 and mTORC2. CCT is a cytosolic chaperonin that assists in folding of several protein substrates. In these studies, we have identified two components of the mTOR complexes, mLST8 and Raptor, as substrates of CCT. We have performed biochemical and signaling studies which indicate that CCT is involved in assembly and signaling of mTOR complexes by folding β-propeller domains of mLST8 and Raptor. We have also obtained high resolution structural information of the mLST8-CCT complex by cryo-EM and mass spectrometric cross-linking. Moreover, we have explored the role of PhLP1 as a co-chaperone for CCT in the assembly of mTOR complexes. Interestingly, we found that PhLP1 plays very different roles in the case of mLST8 and Raptor. While PhLP1 participate in assembly of mLST8 into mTOR complexes, it facilitates degradation of Raptor. These biochemical data, combined with structural information, can be used to design small molecules that modulate mTOR signaling by affecting the formation of intact mTOR complexes.

Single particle electron cryomicroscopy (cryo-EM) is a rapidly developing structural biology technique for the study of macromolecular protein complexes. Presently, cryo-EM fills an important niche by facilitating acquisition of 3-D structures of protein complexes not amenable to structure determination by other techniques. Expansion of cryo-EM beyond this niche requires continued improvement in the types of specimens that can be studied as well as the final resolutions achieved. Two studies were undertaken to address these issues. The first examined resolution limitations by quantifying the effect of beam-induced motion in images of beam-sensitive paraffin crystals. The second explored the possibility of using cryo-EM to study the interaction of small effector proteins with a large multi-protein complex, V-ATPase. The results of these studies exposed the fact that fundamental aspects of the imaging and specimen preparation processes remain poorly understood and must be addressed to facilitate future improvements in cryo-EM structure determination.

Cette thèse porte sur une étude expérimentale et théorique de surfaces micro-structurées de silicium, obtenues par traitement dans un plasma SF6/02 à des températures cryogéniques. La texturation qui résulte de ce traitement confère à ces surfaces des propriétés remarquables. L'une d'entre elles est la capacité de piéger et absorber la lumière, qui se traduit par une couleur noire de ces surfaces, d'où l'appellation Black Silicon. Cette propriété qu'on retrouve dans la gamme spectrale du visible et du proche infra-rouge, présente un intérêt particulier pour la conversion d'énergie solaire, aussi bien par voie photovoltaïque que par voie photo-thermique. L'étude que nous avons menée a toutefois porté sur une gamme spectrale plus large, s'étendant jusqu'aux Térahertz. A cet effet, différentes techniques de caractérisation spectrales ont été mises en œuvre. L'analyse des résultats a été effectuée également au moyen de simulations électromagnétiques. Des corrélations ont été mises en évidence entre les propriétés optiques et les caractéristiques morphologiques des surfaces micro-structurées. L'analyse d'images prises au microscope électronique a permis d'esquisser une théorie pour tenter d'expliquer le mécanisme de formation des microstructures de Black Silicon. Enfin, un microcomposant a été réalisé en vue de mettre en œuvre le premier volet applicatif de ce travail. Il s'agit d'un dispositif de conversion photo-thermique qui incorpore des thermo-résistances en platine sur une surface de Black Silicon réalisée sur une membrane thermiquement isolée

Sponge are primitive multi-cellular organisms. They are important sources of secondary metabolites. In the previous studies indicated that the sponges harbor stable symbiotic microbial consortia. The mechanisms for maintenance and transmission of microbial consortia to the next generations are still not fully understood. The sponge, Cinachyrella australinesis, was chosen to further investigate relationship of the symbiotic bacteria within to the host. Fluorescent in situ hybridization ¡]FISH¡^was employed with non-specific ¡]EUB338¡^and specific oligonucleotide probes for bacteria. The sponge was cryo-sectioned¡]1£gm¡^and hybridized with fluorescent probes. The distribution and ratios of the bacteria in the sponge agreed with those of previous studies indicating that the symbiotic bacteria of C. australiensis are stable and endosymbiotic in nature.

No description available.

Single particle electron cryomicroscopy (cryo-EM) is a rapidly developing structural biology technique for the study of macromolecular protein complexes. Presently, cryo-EM fills an important niche by facilitating acquisition of 3-D structures of protein complexes not amenable to structure determination by other techniques. Expansion of cryo-EM beyond this niche requires continued improvement in the types of specimens that can be studied as well as the final resolutions achieved. Two studies were undertaken to address these issues. The first examined resolution limitations by quantifying the effect of beam-induced motion in images of beam-sensitive paraffin crystals. The second explored the possibility of using cryo-EM to study the interaction of small effector proteins with a large multi-protein complex, V-ATPase. The results of these studies exposed the fact that fundamental aspects of the imaging and specimen preparation processes remain poorly understood and must be addressed to facilitate future improvements in cryo-EM structure determination.

The mitochondrial F-ATPases make about 90% of cellular ATP. They are multi-protein assemblies with a membrane extrinsic catalytic domain attached to a membrane embedded sector. They operate by a mechanical rotary mechanism powered by an electro-chemical gradient, generated across the inner mitochondrial membrane by respiration. A detailed molecular description has been provided by X-ray crystallographic studies and "single molecule" observations of the mechanism of the F1 catalytic domain. Details are known also of the architecture of the peripheral stalk of part of the stator and the membrane embedded region of the rotor. However, knowledge of the detailed structure of the rest of the membrane domain, and the detailed mechanism of generation of rotation is lacking. Recently, studies of the intact mitochondrial F-ATPases, determined by cryo-electron microscopy (cryo-em), have provided structural information at intermediate levels of resolution. Whilst these structures have given insights into the mechanism of generation of rotation, the information required for a molecular understanding of this mechanism is still lacking. Moreover, the locations and roles of six supernumerary membrane subunits are unclear. Some of them are likely to be involved in the formation of dimers of the enzyme which line the edges of mitochondrial cristae. Therefore, in this thesis, a procedure is described for the purification of dimers of the bovine and yeast F-ATPases. The structure of the bovine dimer has been determined by cryo-em at a resolution of ca. 6.9 Angstrom. This structure confirms features concerning the trans-membrane spans of the a-, A6L- and b-subunits observed in the monomeric complex. In addition, the single trans-membrane a-helix of the f-subunit has been located, and the subunit appears to mediate dimer formation. The structure of A6L has been extended, and the a-helices of subunits e- and g- have been located. Another novel feature has been assigned to the DAPIT subunit, and may provide links between dimers in forming larger oligomers. Further improvement in the resolution of the structure is hampered by the extreme conformational heterogeneity of the F-ATPase. To this end, the simpler Fo membrane domain has been isolated and characterized initially by electron microscopy in negative stain.

Este trabalho apresenta o estudo e caracterização de dois complexos moleculares, hRXRálfadeltaAB e hemocianina de Acanthoscurria gomesiana, através de técnicas estruturais e biofísicas. O uso da técnica de crio-microscopia eletrônica para o estudo da hemocianina de Acanthoscurria gomesiana, resultou em um modelo estrutural com resolução de 14 angstron- pelo métodode Fourier Shell Correlation com critério de 1/2 bit. Neste limite de resolução, já é possível observar detalhes estruturais que o mostram como sendo comptível com outros modelos de hemocianinas. Com relação ao estudo de hRXRalfadeltaAB, mostrou-se, através das técnicas de cromatografia analítica de exclusão por tamanho, eletroforese de gel de poliacrilamida e SAXS, que a proteína pode se apresentar no estado dimérico em solução, mesmo na ausência do seu ligante, 9-cis-RA. Também foi estudado a associação de hRXRalfadeltaAB a elementos responsivos: DR1, DR4, F2 e PAL. Suas constantes de dissociação foram calculadas através da técnica de espectroscopia por anisotropia de fluorescência. Os resultados obtidos mostram maior afinidade por DR1 e DR2 e indicam uma origem entrópica para o processo de associação / This work describes characterization of two biomolecular complexes: hRXR deltaAB and a hemocyanin from Acanthoscurria gomesiana using structural and biophysical techniques. Application of cryo-electron microscopy to studies of a hemocyanin from Acanthoscurria gomesiana resulted in its structural model to 14Å resolution, which was calculated by Fourier Shell Correlation with cut-off of 1/2 bit. At this resolution limit one can observe structural details of the complex which are compatible with other hemocyanin models. With respect to hRXR deltaAB, we showed using analytic size exclusion chromatography, SDS PAGE and SAXS, that the protein is dimeric in solution even at the absence of its ligand, 9-cis-RA. hRXR deltaAB binding to the responsive elements of DNA, DR1, DR4, F2 and PAL was investigated and the binding constants to these responsive elements have been determined using fluorescence anisotropy technique. Our results show higher affinity of the receptor to DR1 and DR4 and indicate entropic mechanism of DNA binding

No description available.