Yes / Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The Cterminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region. / MRC New Investigator Research Grant: G0900936; BBSRC grants: BB/L006952/1 and BB/L006510/1; BBSRC grant: BB/N006267/1; Wellcome Trust Investigator Award in Science: 204957/Z/16/Z; BBSRC grant: BB/J014567/1

Droplet evaporation is ubiquitous to multitude of applications such as microfluidics, surface patterning and ink-jet printing. In many of the process like food processing tiny concentrations of suspended particles may alter the behavior of an evaporating droplet remarkably, leading to partially viscous and partially elastic dynamical characteristics. This, in turn, may lead to some striking mechanical instabilities, such as buckling and rupture. In this thesis, we provide a comprehensive physical description of the vaporization, self-assembly, agglomeration and buckling kinetics of sessile nanofluid droplet pinned on a hydrophobic substrate in various configurations. We have deciphered five distinct regimes of droplet lifecycle. Regime I-III consists of evaporation induced preferential agglomeration that leads to the formation of unique dome shaped inhomogeneous shell with stratified varying density liquid core. Regime IV involves capillary pressure initiated shell buckling and stress induced shell rupture. Regime V marks rupture induced cavity inception and growth. We provide a regime map explaining the droplet morphology and buckling characteristics for droplets evaporating on various substrates. Specifically, we find that final droplet volume and radius of curvature at buckling onset are universal functions of particle concentration. Furthermore, flow characteristics inside the heated and unheated droplets are investigated and found to be driven by the buoyancy effects. Velocity magnitudes are observed to increase by an order at higher temperatures with self-similar flow profiles. With an increase in the surface temperature, droplets exhibit buckling from multiple sites over a larger sector in the top half of the droplet. In addition, irrespective of the initial nanoparticle concentration and substrate temperature, hydrophobicity and roughness, growth of daughter cavity (subsequent to buckling) inside the droplet is found to be controlled by the solvent evaporation rate from the droplet periphery. The results are of great significance to a plethora of applications like DNA deposition and nanofabrication. In the next part of the thesis, we deploy the droplet in a rectangular channel. The rich physics governing the universality in the underlying dynamics remains grossly elusive. Here, we bring out hitherto unexplored universal features of the evaporation dynamics of a sessile droplet entrapped in a 3D confined fluidic environment. Increment in channel length delays the completion of the evaporation process and leads to unique spatio-temporal evaporation flux and internal flow. We show, through extensive set of experiments and theoretical formulations, that the evaporationtimescale for such a droplet can be represented by a unique function of the initial conditions. Moreover, using same theoretical considerations, we are able to trace and universally merge the volume evolution history of the droplets along with evaporation lifetimes, irrespective of the extent of confinement. These results are explained in the light of increase in vapor concentration inside the channel due to greater accumulation of water vapor on account of increased channel length. We have formulated a theoretical framework which introduces two key parameters namely an enhanced concentration of the vapor field in the vicinity of the confined droplet and a corresponding accumulation lengthscale over which the accumulated vapor relaxes to the ambient concentration. Lastly, we report the effect of confinement on particle agglomeration and buckling dynamics. Compared to unconfined scenario, we report non-intuitive suppression of rupturing beyond a critical confinement. We attribute this to confinement-induced dramatic alteration in the evaporating flux, leading to distinctive spatio-temporal characteristics of the internal flow leading to preferential particle transport and subsequent morphological transitions. We present a regime map quantifying buckling & non-buckling pathways. These results may turn out to be of profound importance towards achieving desired morphological features of a colloidal droplet, by aptly tuning the confinement space, initial particle concentration, as well as the initial droplet volume. These findings may have implications in designing functionalized droplet evaporation devices for emerging engineering and biomedical applications.

Yarrowia lipolytica a la particularité de pousser sur de nombreux substrats hydrophobes et de les métaboliser via la β-oxydation ou de les stocker dans des corps lipidiques. Le premier contact entre la cellule et les substrats hydrophobes se déroule sur la surface cellulaire. A cette étape, la composition de la paroi cellulaire en protéines, en β-glucane et en chitine pourrait jouer un rôle important sur l’adhésion et la prise en charge des substrats hydrophobes. Dans le cadre de cette thèse, nous avons étudié l’effet d’une source de carbone hydrophobe (par rapport au glucose) et de la composition du milieu de culture sur la structure pariétale et la production de mélanine chez Y. lipolytica. Les résultats de nos analyses biochimiques et moléculaires ont montré que l’oléate de méthyle (un composé utilisé comme modèle de source de carbone hydrophobe) a induit des modifications au niveau de la paroi cellulaire et que ces modifications ont conduit ou non à l’adhésion de gouttelettes lipidiques à la surface cellulaire ; à une modification du contenu des composés pariétaux ; à la résistance des cellules à des composés toxiques ayant pour cible la paroi cellulaire ; et à la modification des profils d’expression de 27 gènes analysés codant des protéines de paroi cellulaire. L’identité des gènes surexprimés suggère la participation de leurs protéines dans l’intégrité de la paroi cellulaire, dans l’adhésion des gouttelettes lipidiques et dans la réponse au stress par l’oléate de méthyle et/ou le manque d’azote. Nos résultats ont aussi montré que la présence d’oléate de méthyle, huile de ricin et ricinoléate de méthyle a induit la production de DHN-mélanine chez une souche génétiquement modifiée de Y. lipolytica (MTLY40-2p) dont la β-oxydation est affectée / Yarrowia lipolytica can grow on many hydrophobic substrates and metabolize them via the β-oxidation pathway or store them into lipid bodies. The first contact between the cell and the hydrophobic substrates is by the cell wall. In this step, cell-wall proteins (CWP), β-glucane and chitin of the cell wall could play an important role for the adhesion and uptake of hydrophobic substrates. The aim of this work was to study the effect of a hydrophobic carbon source (compared with glucose) and the culture medium composition on the cell wall composition and the melanin production by Y. lipolytica. The results of our biochemical and molecular analysis showed that the presence of methyl oleate and the nutrients composition of the culture media have induced some modifications at the cell wall level. These modifications were linked with the adhesion or not of the lipid droplets to the cell surface; with a modified content of the cell wall components; with a resistance to compounds that are toxic for the cell wall, and with the modification of the expression patterns of 27 CWP genes analyzed in this work. The identity of the over-expressed CWP genes suggests the participation of their proteins in the cell wall integrity, in the adhesion of lipid droplets and in the stress response to methyl oleate and/or the nitrogen starvation. Our results have also shown that the presence of the lipids methyl oleate, castor oil and methyl ricinoleate induced the production of DHN-melanin by a mutant strain in which the β-oxidation is affected / Yarrowia lipolytica tiene la capacidad de crecer sobre numerosos substratos hidrofóbicos y demetabolizarlos a través de la β-oxidación o de almacenarlos en cuerpos lipídicos. El primercontacto entre la célula y los substratos hidrofóbicos se lleva a cabo mediante la pared celular.En esta etapa, la composición en proteínas, β-glucano y quitina de la pared celular podríajugar un papel importante en la adhesión y la toma de substratos hidrofóbicos. En el presentetrabajo se estudió el efecto de una fuente de carbono hidrofóbica (en comparación conglucosa) y la composición del medio de cultivo sobre la composición de la pared celular y laproducción de melanina en Y. lipolytica.Los resultados de nuestros análisis bioquímicos y moleculares mostraron que el oleato demetilo (un compuesto utilizado como fuente de carbono hidrofóbica) indujo modificacionesen la pared celular y estas modificaciones condujeron al aumento o la disminución de laadhesión de gotas lipídicas a la superficie celular; a la modificación del contenido de loscompuestos de la pared celular; a la resistencia de las células a compuestos tóxicos que tienencomo blanco la pared celular; y a la modificación del perfil de expresión de 27 genes quecodifican para proteínas putativas de la pared celular. La expresión de algunos de estos genesse indujo desde las primeras horas de cultivo en presencia de oleato de metilo. La identidad delos genes sobre-expresados sugiere la participación de sus proteínas en mecanismos como laintegridad de la pared celular, la adhesión de las gotas lipídicas a la superficie celular y larespuesta a estrés inducido por oleato de metilo y/o por la falta de nitrógeno en el medio.Nuestros resultados también mostraron que la presencia de oleato de metilo, de aceite dericino y de ricinoleato de metilo en el medio de cultivo, indujo la producción de un pigmentocafé en una cepa mutante de Y. lipolytica (MTLY40-2p) afectada en la β-oxidación. Ademásobservamos que el perfil espectroscópico la pared celular de la mutante MTYL40-2p, semodifica en función del substrato hidrofóbico presente en el medio de cultivo. Nuestrosresultados sugieren que el pigmento café es DHN-melanina y que la ausencia de peptona en elmedio de cultivo afecta la biosíntesis de esta melanina. A partir de un análisis in silicoproponemos los genes putativos para la biosíntesis de DHN-melanina en Y. lipolytica