Potassium Starvation in Yeast: Mechanisms of Homeostasis Revealed by Mathematical Modeling

The intrinsic ability of cells to adapt to a wide range of environmental conditions is a fundamental process required for survival. Potassium is the most abundant cation in living cells and is required for essential cellular processes, including the regulation of cell volume, pH and protein synthesis. Yeast cells can grow from low micromolar to molar potassium concentrations and utilize sophisticated control mechanisms to keep the internal potassium concentration in a viable range. We developed a mathematical model for Saccharomyces cerevisiae to explore the complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis. By using a novel inference method (“the reverse tracking algorithm”) we predicted and then verified experimentally that the main regulators under conditions of potassium starvation are proton fluxes responding to changes of potassium concentrations. In contrast to the prevailing view, we show that regulation of the main potassium transport systems (Trk1,2 and Nha1) in the plasma membrane is not sufficient to achieve homeostasis

Use of physiological constraints to identify quantitative design principles for gene expression in yeast adaptation to heat shock

BACKGROUND: Understanding the relationship between gene expression changes, enzyme activity shifts, and the corresponding physiological adaptive response of organisms to environmental cues is crucial in explaining how cells cope with stress. For example, adaptation of yeast to heat shock involves a characteristic profile of changes to the expression levels of genes coding for enzymes of the glycolytic pathway and some of its branches. The experimental determination of changes in gene expression profiles provides a descriptive picture of the adaptive response to stress. However, it does not explain why a particular profile is selected for any given response. RESULTS: We used mathematical models and analysis of in silico gene expression profiles (GEPs) to understand how changes in gene expression correlate to an efficient response of yeast cells to heat shock. An exhaustive set of GEPs, matched with the corresponding set of enzyme activities, was simulated and analyzed. The effectiveness of each profile in the response to heat shock was evaluated according to relevant physiological and functional criteria. The small subset of GEPs that lead to effective physiological responses after heat shock was identified as the result of the tuning of several evolutionary criteria. The experimentally observed transcriptional changes in response to heat shock belong to this set and can be explained by quantitative design principles at the physiological level that ultimately constrain changes in gene expression. CONCLUSION: Our theoretical approach suggests a method for understanding the combined effect of changes in the expression of multiple genes on the activity of metabolic pathways, and consequently on the adaptation of cellular metabolism to heat shock. This method identifies quantitative design principles that facilitate understating the response of the cell to stress

A meta-analysis to determine factors associated with the severity of enteritis in Atlantic salmon (Salmo salar) fed soybean meal-based diets

A meta-analytic approach was used to determine factors associated with the severity of enteritis in distal intestine of Atlantic salmon fed soybean meal (SBM)-based diets. Dataset from 26 fish studies were extracted and standardized for use in the meta-analysis. After standardization, the data were analyzed with ordinal logistic regression model by comparing the SBM treatment(s) in each study with the neutral-reference treatment. The log-odds ratio of the proportional odds model and its standard error were extracted and analyzed using the random effects model to estimate the effect size of dietary SBM on enteritis using four semi-quantitative histological variables: reduction in mucosal fold height; disappearance of supranuclear vacuolization; inflammatory cell infiltration of lamina propria, and of submucosa. Both univariate and multivariate meta-regression were used to identify study factors with significant association to the severity of enteritis in Atlantic salmon. The results showed that fish production phase, feed type, SBM inclusion level, year of study and water temperature are significantly associated with the severity of enteritis in Atlantic salmon. Further meta-analysis of sub datasets according to production phase, revealed that fish reared in seawater were more prone to develop enteritis compared with fish reared in freshwater. The absence of positive relationship between SBM inclusion level and the severity of enteritis was probably due to difference in source, batch, processing, and level of anti-nutritional factors in the SBM used in the different studies combined in the meta-analysis. Subgroup analysis based on year of study revealed that the severity of enteritis in fish fed SBM-based diets has decreased over the years. Additional results revealed that fish fed SBM-based diet at low water temperature showed increased severity of enteritis, compared with fish raised in high water temperature. Linear and quadratic regressions conducted to explore possible impact of enteritis on fish performance, showed that the specific growth rate and thermal growth coefficient of fish decreased with increased severity of enteritis. However, this relationship depends on the fish production phase and the histological variables used for the regression analysis. The current study concluded that the severity of enteritis in Atlantic salmon fed SBM-based diets are significantly associated with fish production phase, feed type, SBM inclusion level, year of study and water temperature, but not the exposure time. Also, the study showed that increased severity of enteritis reduced specific growth rate and thermal growth coefficient of fish fed SBM-based diets.A meta-analysis to determine factors associated with the severity of enteritis in Atlantic salmon (Salmo salar) fed soybean meal-based dietspublishedVersio

A novel image analysis approach to characterise the effects of dietary components on intestinal morphology and immune system in Atlantic salmon

The intestinal tract of salmonids provides a dynamic interface that not only mediates nutrient uptake but also functions as the first line of defence against ingested pathogens. Exposure of the immune system to beneficial microorganisms and different dietary immunostimulants via the intestine has been shown to prime the immune system and help in the development of immune competence. Furthermore, the morphology and function of teleostean intestines are known to respond to feed components and to ingested and resident bacterial communities. Histological appraisal is still generally considered to be the gold standard for sensitive assessment of the effects of such dietary modulation. The aim of the present study was to improve understanding of salmonid intestinal function, structure and dynamics and to use the knowledge gained to develop a model for analysis, which would allow intestinal health to be assessed with respect to different intestinal communities and feed components. Virtual histology, the process of assessing digital images of histological slides, is gaining momentum as an approach to supplement traditional histological evaluation methodologies and at the same time, image analysis of digitised histological sections provides a practical means for quantifiable assessment of structural and functional changes in tissues, being both objective and reproducible. This project focused on the development of a rapid, practical analytical methodology based on advanced image analysis, that was able to measure and characterise a range of features of the intestinal histology of Atlantic salmon in a quantitative manner. In the first research chapter, the development of a novel histological assessment system based upon advanced image analysis was described, this being developed with the help of a soybean feed model known to induce enteropathy in Atlantic salmon. This tool targeted the evaluation of the extent of morphological changes occurring in the distal intestine of Atlantic salmon following dietary modulation. The final analytical methodology arrived at, could be conducted with minimal user-interaction, allowing rapid and objective assessment of 12 continuous variables per histological frame analysed. The processing time required for each histological frame was roughly 20-25 min, which greatly improved the efficiency of conducting such a quantitative assessment with respect to the time taken for a subjective semi-quantitative alternative approach. Significant agreement between the fully automated and the manual morphometric image segmentation was achieved, however, the strength of this quantitative approach was enhanced by the employment of interactive procedures, which enabled the operator / observer to rectify preceding automated segmentation steps, and account for the specimen’s variations. Results indicated that image analysis provided a viable alternative to a pathologist’s manual scoring, being more practical and time-efficient. In the second research chapter, feeding Atlantic salmon a high inclusion level of unrefined SBM (25 %) produced an inflammatory response in the distal intestine as previously described by other authors. The model feed trial successfully generated differentiable states, although these were not, for the most part, systemically differentiable through the majority of standard immunological procedures used, being only detectable morphologically. Quantitation of morphometric parameters associated with histological sections using the newly developed image analysis tool successfully allowed identification of major morphological changes. Image analysis was thus shown to provide a powerful tool for describing the histomorphological structure of Atlantic salmon distal intestine. In turn, the semi-automated image analysis methods were able to distinguish normal intestinal mucosa from those affected by enteritis. While individual parameters were less discriminatory, use of multivariate techniques allowed better discrimination of states and is likely to prove the most productive approach in further studies. Work described in the third research chapter sought to validate the semi-automated image analysis system to establish that it was measuring the parameters it was purported to be measuring, and to provide reassurance that it could reliably measure pre-determined features. This study, using the same sections for semi-quantitative and quantitative analyses, demonstrated that the quantitative indices performed well when compared to analogous semi-quantitative descriptive parameters of assessment for enteritis prognosis. The excellent reproducibility and accuracy performance levels indicated that the image analysis system was a useful and reliable morphometric method for the quantification of SB-induced enteritis in salmon. Other characteristics such as rapidity, simplicity and adaptability favour this method for image analysis, and are particularly useful where less experienced interpreters are performing the analysis. The work described in the fourth research chapter characterised changes in the morphology of the intestinal epithelial cells occurring as a result of dietary modulation and aspects of inflammatory infiltration, using a selected panel of enzyme and IHC markers. To accomplish this, image analysis techniques were used to evaluate and systematically optimise a quantitative immunolabelling assessment protocol. Digital computer-assisted quantification of labelling for cell proliferation and regeneration; programmed cell death or apoptosis; EGCs and t-cell like infiltrates; mobilisation of stress-related protein regenerative processes and facilitation of nutrient uptake and ion transport provided encouraging results. Through the description of the intestinal cellular responses at a molecular level, such IHC expression profiling further characterised the inflammatory reaction generated by the enteropathic diet. In addition, a number of potential diagnostic parameters were described for fish intestinal health e.g. the relative levels of antigenicity and the spatial distribution of antigens in tissues. Work described in the final research chapter focused on detailed characterisation of intestinal MCs / EGCs in order to try to elucidate their functional role in the intestinal immune responses. Through an understanding of their distribution, composition and ultrastructure, the intention was to better characterise these cells and their functional properties. The general morphology, histochemical characteristics and tissue distribution of these cells were explored in detail using histochemical, IHC and immunogold staining / labelling, visualised using light, confocal and TEM microscopy. Despite these extensive investigations, their physiological function and the content of their granules still remain somewhat obscure, although a role as immunodulatory cells reacting to various exogeneous signals through a finely regulated process and comparable to that causing the degranulation of mammalian MCs is suggested. The histochemical staining properties demonstrated for salmonid MCs / EGCs seem to resemble those of mammalian mucosal mast cells, with both acidophilic and basophilic components in their granules, and a granule content containing neuromodulator / neurotransmitter-peptides such as serotonin, met-enkephalin and substance-p. Consequently, distinguishable bio-chromogenic markers have been identified that are of utility in generating a discriminatory profile for image analysis of such cells

Kan fisk vokse på trær? Næringsverdi og funksjonelle egenskaper av gjær i fôret til Atlantisk laks (Salmo salar)

Yeasts are gaining attention as alternative ingredients in fish feeds. The nutritional and health potentials of non-saccharomyces yeasts in fish are scarce in literature. Three non-saccharomyces yeasts; Cyberlindnera jadinii (CJ), Blastobotrys adeninivorans (BA) and Wickerhamomyces anomalus (WA) are the focus of this thesis. The objective of the current thesis was to investigate the nutritional values and health effects of the three selected yeasts in the diets of Atlantic salmon (Salmo salar) with focus on growth performance, intestinal health, gut microbiota, and immune responses of fish. The three yeasts were produced in-house using a growth medium containing a blend of enzymatic hydrolysates of pre-treated spruce wood (Picea abies) and chicken by-products. After harvesting, the selected yeasts were processed by direct heat-inactivation with spray-drying (ICJ, IBA and IWA) or autolyzed at 50 ºC for 16 h, followed by spray-drying (ACJ, ABA and AWA). The present thesis comprises of seven papers. Paper I used a desk study approach to review the state-of-the art on the use of yeasts in fish feeds and identified gaps in literature regarding the use of yeasts as aquafeed ingredients. Yeasts are efficient converter of low-value non-food biomass into high-value resources. Yeasts showed comparatively similar amino acids with fishmeal (FM) and soybean meal (SBM), except for methionine, lysine, arginine, and phenylalanine which need to be supplemented when used in fish feeds. Genetic modification and/or nutrient digestibility through exogenous enzyme supplementation and the use of cost-effective down-stream processing (DSP) are possible strategies to increase the nutritive values of yeasts in fish. Additional investment in large-scale production at competitive price is needed for yeasts to be considered as feasible replacement for FM and SBM in fish feeds. Paper II investigated the impacts of yeast species and processing on performance, immune response and gut health of Atlantic salmon fry fed SBM-based diet in freshwater. In a 37-day feeding experiment, the fish were fed one of the nine experimental diets: a FM-based diet, a challenging diet with 40% SBM and six other diets containing 40% SBM and 5% each of ICJ, ACJ, IBA, ABA, IWA and AWA yeast products. An additional control containing 40% SBM and 5% of a reference inactivated C. jadinii (ICU), known for its ability to counteract SBM-induced enteritis (SBMIE) was used in this experiment. C. jadinii and W. anomalus yeasts showed the most promising effects on gut health based on widening of lamina propria and immune response parameters. The AWA was effective in ameliorating SBMIE in fish, while only limited effects were observed for other yeasts products. The ability of yeasts to counteract SBMIE is linked to the activation of immune responses in fish. The results also revealed that the amounts, length, adhesion, and accessibility of cell wall components could be important for the ameliorating effects of yeasts on SBMIE in fish. Paper III assessed the effects of yeasts species and processing on systemic immune response of Atlantic salmon fry fed SBM-based diet in freshwater and demonstrated whether spleen can be used as a target organ to characterize immunomodulatory effects of functional ingredients in fish. The production of yeasts, experiment diets and fish experimental protocol were fully described in Paper II. Four experimental diets (FM, SBM, ICJ, and ACJ) were used in Paper III. The immunomodulatory effects of the diets were analyzed in the spleen of fish after 37 days of feeding, using a transcriptomic evaluation by RNA sequencing and protein expression of specific immunological markers through indirect ELISA. The results showed that SBM induced a down-regulation of pathways associated with ion binding and transport, along with an increase at the protein levels of pro-inflammatory cytokines TNFα and IFNγ. The inclusion of ACJ in the diet was able to control the inflammatory profile caused by SBM through activation of biological pathways related to endocytosis, along with increased protein expression of IL-10 and decreased level of TNFα. The functionality of yeasts in improving gut health of fish is dependent on the yeast species and DSP used after harvesting the yeasts. The results also showed that spleen was a good target organ to characterize the immunomodulatory effects of functional ingredients in Atlantic salmon. Paper IV investigated the effects of yeast species and processing on nutrient digestibility of yeasts in Atlantic salmon. The production and processing of yeasts used in Paper IV were as described in Paper II. Seven experimental diets were used in paper. The control feeds consisted of 100% reference diet (REF) and six other diets comprising of 70% REF diet and 30% each of the yeast products (ICJ, ACJ, IBA, ABA, IWA and AWA). The protein and amino acids of the three yeast species were moderately digested in Atlantic salmon. Autolysis slightly increased protein digestibility of C. jadinii and W. anomalus in Atlantic salmon, but not B. adeninivorans. The results revealed that cell wall porosity as demonstrated by nitrogen solubility had larger impact on nutrient digestibility of yeasts than cell wall thickness. The nutrient digestibility of yeasts in Atlantic salmon is dependent on the yeast species and DSP used after harvesting the yeasts. Based on the results of Papers II, III and IV, a second batch of C. jadinii and W. anomalus yeast were produced to understand the response of Atlantic salmon reared in seawater to dietary yeasts. Therefore, Paper V evaluated the effects of yeasts species and processing on intestinal health and transcriptomic profile from DI and spleen tissue of Atlantic salmon fed SBM-based diet in seawater. The yeasts were produced and processed following the procedure described in Paper II. The ICJ, ACJ, IWA and AWA yeasts products were used in this paper. Six diets were formulated, one based on FM, a challenging diet containing 30% SBM and four other diets containing 30% SBM and 10% each of the yeast products (ICJ, ACJ, IWA and AWA). The inclusion of ICJ and ACJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α positive cells in the lamina propria of fish fed SBM diets. The ICJ and ACJ yeasts controlled the inflammatory profile through upregulation of pathways connected to wound healing and taurine metabolism. The IWA and AWA yeasts controlled the inflammatory profile in fish fed SBM through down-regulation of pathways associated with toll-like receptor signaling, C-lectin receptor and signal transduction. This paper strengthened our earlier observations (Papers II and III) that C. jadinii and W. anomalus are promising novel ingredients with health beneficial effects in terms of controlling distal intestine inflammation associated by feeding plant based diets to Atlantic salmon. Paper VI investigated the effects of yeast species and processing on gut microbiota of fish. The yeast production and processing, experimental diets and fish experimental protocol were as described in Paper V. After 42 days of feeding, six fish from each tank were randomly selected to collect digesta samples from the DI for 16S rRNA sequencing. Water samples (from the source and rearing tanks) and feed samples were also collected for the sequencing analysis. The microbiota of fish fed SBM diet differed from those fed FM diet. The microbiota composition, richness and diversity were similar in fish fed ICJ, IWA and SBM diets. Fish fed ACJ increased relative abundance of Pediococcus, and mucin O-glycan degradation pathway, while fish fed AWA diet increased relative abundance of Bacillaceae compared with fish fed the other diets. Despite the significant modulation of intestinal microbiota of fish fed the autolyzed yeasts (ACJ and AWA), the histological and transcriptomic results revealed that the autolyzed yeasts did not improve gut health of fish beyond the level observed for the inactivated yeasts (ICJ and IWA) (Paper V). These results suggest that the ameliorating effects of yeasts on SBMIE is connected to their ability to stimulate immune responses in Atlantic salmon (Papers II, III and V), rather than through modulation of intestinal microbiota (Paper VI).publishedVersio

EPR-based structural and functional characterization of the C-terminal domain of the osmoregulated glycine betaine transporter BetP from Corynebacterium glutamicum

Als Gram-positives Bodenbakterium ist Corynebacterium glutamicum regelmäßig diversen Stresssituationen ausgesetzt, die das Überleben der Zelle beeinträchtigen können. Neben wechselnden pH- und Temperatur-Werten, werden die Zellen auch mit Osmolaritätschwankungen des umgebenden Mediums konfrontiert. Es konnte gezeigt werden, dass das sekundäre Glycinbetain Aufnahmesystem BetP aus diesem Bakterium in der Lage ist, den unter hyperosmotischen Bedingungen intrazellulär erhöhten Kaliumgehalt als spezifischen Stimulus autonom zu detektieren (Osmo-/Chemosensor) und seine Aktivität daraufhin an das Ausmaß des Stresses anzupassen (Osmoregulator). Weiterführende Untersuchungen zeigten, dass Änderungen in der Struktur und/oder der relativen Orientierung der C-terminalen Domäne des carrier einen starken Einfluss auf die Stimulusdetektion und/oder die nachgeschaltete Signaltransduktion haben. Da die molekularen Mechanismen solcher sensorischen Eigenschaften von Transportproteinen bislang nur unzureichend untersucht und verstanden sind, wurde in der vorliegenden Arbeit eine Kombination aus ortspezifischer Spinmarkierung (site-directed spin labelling, SDSL) und Elektronenspinresonanzspektroskopie (ESR) angewendet, um die molekulare Dynamik während des Aktivierungsprozesses von BetP zu untersuchen. Im Fokus lagen dabei zum einen die tertiäre Struktur des Transporters als auch die Aufklärung von strukturellen Änderungen der C-Domäne sowie deren mögliche Interaktion mit angrenzenden Protein- und/oder Lipid-Bereichen im aktivierten Protein. Strategisch eingeführte Cystein-Reste zu Beginn (545), in der Mitte (572) und nahe dem Ende (589) der C-Domäne wurden auf ihre jeweilige Aktivitätsregulation hin überprüft. Darüber hinaus wurden die Markierungs- und Rekonstitutionsschritte für jede Mutante optimiert, um eine hohe Ausbeute an spinmarkiertem, gereinigtem Material für die EPR-basierten Analysen zu erhalten. Es konnte eine hochgradige Absorption von BetP durch den direkten Kontakt zu den Bio-Beads (bis zu 90%) während des herkömmlichen Rekonstitutions-Assay identifiziert werden. Daraufhin wurde eine neue Rekonstitutionsmethode für den verlustfreien Einbau von spinmarkiertem BetP Protein in E. coli-Liposomen etabliert, deren Effizienz um etwa 60% höher lag als mit der herkömmlichen Methode. Die SDSL-ESR-Studien zeigten zum einen, dass der Einbau von solubilisiertem BetP in E. coli-Liposomen einen ausgeprägten Einfluss auf die Konformation und/oder räumliche Orientierung der C-Domäne hat. Eine hyperosmotisch induzierte Aktivierung des Transportproteins zeigte zudem eine erhöhte spinlabel-Mobilität am Ende der C-Domäne auf, die auf strukturelle Änderungen während des Aktivierungsprozesses hinwies. Das Ausmaß der Mobilisierung war dabei maßgeblich von der Art (Ionenstärke, Osmolalität) des benutzten Osmolytes abhängig. Die vorläufigen Ergebnisse der Abstandsmessungen von Einzel- und Doppelcystein-Mutanten bestätigten einen oligomeren Zustand (z.B. Trimer) von solubilisiertem und in E. coli-Liposomen eigebautem BetP. Darüber hinaus konnte gezeigt werden, dass der zusätzliche Einbau eines Prolins in einer deregulierten Dreifachmutante BetP-S545C/Y550P/S589C einen Einfluss auf die Struktur der C-terminalen Domäne im nicht aktiven Zustand des Proteins hat. Die in der vorliegenden Arbeit gewonnenen Daten konnten in einem aktuellen Topologie-Modell kombiniert werden, das die möglichen Konformationen und die Dynamik der C-terminalen Domäne während des Aktivierungsprozesses von BetP zusammenfasst

Mathematical modelling of ion regulations in fungi

Intracellular ion concentration and cation transporter activities are important determinants of many fundamental physiological parameters, including cell turgor, plasma membrane potential and intracellular pH. In order to maintain these parameters within physiological ranges despite external perturbations, cells regulate their transporter activities through both post-translational modifications and gene regulation. In this thesis, the ion regulations in two model fungal species, Saccharomyces cerevisiae and Aspergillus nidulans, are investigated. We use a mathematical modelling approach to gain a quantitative understanding of the impact of collective transporter activities on the intracellular cation concentrations and the cellular adaptation processes. This thesis is mainly composed of two parts: 1) A biophysical and mathematical model is built for the cation transporters and their regulatory proteins to describe the temporal changes of cell volume, intracellular pH and cation concentrations during hyper-osmotic stress, ionic stress and alkaline pH stress in S. cerevisiae. 2) Four models are built for the activation of the alkaline pH responsive transcription factor, PacC, in A. nidulans, based on competing hypotheses. The integrated model in the first part shows that calcineurin activation in response to stress conditions results in a rapid and transient decrease of membrane potential, which we speculate is an important strategy for the cell to respond to unknown external ionic perturbations. The model also confirms the importance of Hog1p phosphorylation on Nha1p and Tok1p for immediate adaptation to salt stress and predicted that activated Hog1p down-regulates Tok1p activity. In alkaline stress conditions, the induction of Ena1p expression results in increased membrane potential. This model provides a theoretical framework for the study of ion homeostasis in stress conditions, the understanding of drug effects, such as FK506. And since the membrane potential is an important determinant of drug uptake, the model is well suited for the drug development. In the second part of the thesis, results from those competing models for PacC activation show significant difference for the pacC904 mutant strain and suggests that further experiments on this strain would be able to uncover the role of the intermediate form, PacC53, plays in the activation process

Mechanics, shape, and programmability in soft matter systems: From fluid membranes to spring and droplet networks

This thesis analyzes three different soft matter systems---membranes, polymers, and droplets---to answer questions about shape, mechanics, and programmability. For membranes, my collaborators and I have developed a theoretical model of endocytosis in yeast. Endocytosis is the process by which a cell membrane deforms to surround extracellular material to draw it into the cell. Endocytosis in yeast involves clathrin, actin, and Bar proteins. Our model breaks up the process into three stages: (i) initiation, where clathrin interacts with the cell membrane via adaptor proteins, (ii) elongation, where the membrane is then further deformed by polymerizing actin filaments, followed by (iii) pinch-off. Our results suggest that the pinch-off mechanism may be assisted by a pearling-like instability. In addition, we potentially rule out two of the three competing models for the organization of the actin filament network during the elongation stage. For polymers, the actin cytoskeleton network at the leading edge of the cell becomes anisotropic with filament alignment favoring the direction of motion of the cell. To begin to capture the mechanics of this anisotropic filament network, my collaborators and I have constructed an effective medium (mean field) theory of an anisotropic, disordered spring network. We find that increasing the anisotropy increases the filament density required for a nonzero shear modulus (rigidity). We also conduct numerical simulations and find good agreement with the effective medium theory. We then extend our analysis to include the mechanics of coupled disordered spring networks to study force transmission between the actin cytoskeletal network and DNA via the lamin filament network and potentially begin to establish a microscopic basis for the mechanical regulation of transcription via the actin cytoskeleton. For droplets, we study numerically a collection of aqueous droplets joined by single lipid bilayers to form a cohesive, tissue-like material. The droplets in these droplet networks can be programmed with different osmolarity gradients. These osmolarity gradients generate internal stresses via local flows and the network then folds into designed structures. In other words, global change is driven by local osmolarity gradients. Using molecular dynamics simulations, we study the formation of shapes ranging from rings to spirals to tetrahedra and determining the optimal range of parameters for such structures. By adding an osmotic interaction with a dynamic environment, a folding-unfolding process can also be realized. This latter result is a step towards osmotic robotics

Cellular stress characterisation in S. cerevisiae budding yeast using optical microscopy

The term "cellular stress" covers various environmental and metabolic events that threaten cell survival. In response, eukaryotic cells can adapt their metabolism to acute changes in their surrounding environment to maintain cellular homeostasis and ensure survival. The cell is an enclosed system with macromolecules and functional compartments diffusing in a liquid-like environment, the cytoplasm. The position in time and space of these elements influences every aspect of cell biology, from molecular interactions and enzymatic activities to the process of cell division itself. Cellular stress episodes can interfere with the timing of these physiological processes: perturbations modify the cytoplasm volume and composition (e.g., accumulation of damaged proteins), including changes of intracellular physical properties such as macromolecular crowding influencing diffusion and spatio-temporal dynamics of the whole system. Consequently, cellular stress dynamics have been of strong interest to physicists and biologists. In continuity with previous research in the field, this project aimed to explore these aspects of cellular physiology and gain new insight into cellular stress responses and crowding dynamics in the budding yeast Saccharomyces cerevisiae, a eukaryotic model sensitive to environmental stresses. This thesis sets out to investigate the influence of hyperosmotic shock, glucose availability, and cell growth on macromolecular crowding. I present a methodology developed to identify local regions of crowding in yeast cells using a previously generated Förster Resonance Energy Transfer Technology (FRET) crowding biosensor called CrGE. I describe experimental and analysis procedures to quantify crowding at subcellular levels and have developed new strains controlling the expression of fluorescently tagged cytoplasmic aggregates. To identify fluorescent clusters on cellular models, single molecule characterisations for stoichiometry and diffusion tracks in vivo were performed using bespoke Slimfield microscopy. Cellular sub-compartments were visualised and tracked over time using confocal microscopy, giving insight into polarised inheritance events in the budding yeast