Resource conditions in nature can fluctuate markedly and how organisms adapt to survive in these conditions is of great interest in the fields of evolutionary biology and ecology. Experimental evolution using microbes has been shown to be effective in answering general evolutionary questions. Using this technique, I studied the bacterium Escherichia coli adapting to fluctuating environments to investigate the evolution of growth traits and the dynamics of adaptation. My results have provided general insight into bacterial adaptation which may allow for better prediction of growth trait evolution in a range of conditions. (1) I have shown that evolution in both predictable and unpredictable environments resulted in the evolution of a reduced lag phase, an increased growth rate and a higher maximum population size. My results suggest that bacteria do not adapt to conditions by anticipating the timing of the resource renewal. (2) I found that a trade-off exists for evolved populations between a reduced lag phase and a higher mortality rate in all environments, and propose this as an explanation as to why some bacteria retain a lag phase. (3) I show that the dynamics of adaptation do not differ between populations adapted to conditions which involved varying periods of time in stationary phase between transfers. There seem to be different mutations for different traits, with mutations to the lag reducing first, followed by growth rate, and finally population size. These findings highlight the dynamics of growth trait evolution in environments in which a complex interplay exists between reproducing and growing faster than competitors, and being able to survive in starvation conditions.

La microbiologia predictiva és una de les parts més importants de la microbiologia dels aliments. En el creixement d'un cultiu bacterià es poden observar quatre fases: latència, exponencial, estacionària i mort. La fase de latència té un interès específic en microbiologia predictiva; al llarg de dècades ha estat abordada des de dues perspectives diferents: a nivell cel·lular i intracel·lular (escala microscòpica), i a nivell de població (escala macroscòpica). La primera estudia els processos que tenen lloc a l'interior dels bacteris durant la seva adaptació a les noves condicions del medi, com els canvis en l'expressió gènica i en el metabolisme. La segona descriu l'evolució de la població bacteriana per mitjà de models matemàtics continus i d'experiments que avaluen variables relacionades amb la densitat cel·lular. L'objectiu d'aquest treball és millorar la comprensió de la fase de latència dels cultius bacterians i dels fenòmens intrínsecs a la mateixa. Aquest objectiu s'ha abordat amb la metodologia Individual-based Modelling (IbM) amb el simulador INDISIM (INDividual DIScrete SIMulation), que ha calgut optimitzar. La IbM introdueix una perspectiva mecanicista a través de la modelització de les cèl·lules com a unitats bàsiques. Les simulacions IbM permeten estudiar el creixement d'entre 1 i 106 bacteris, així com els fenòmens que emergeixen de la interacció entre ells. Aquests fenòmens pertanyen al que anomenem escala mesoscòpica. Aquesta perspectiva és imprescindible per entendre l'efecte en la població dels processos d'adaptació individuals. Per tant, la metodologia IbM és un pont entre els individus i la població o, el que és el mateix, entre els models a escala microscòpica i a escala macroscòpica.En primer lloc hem estudiat dos dels diversos mecanismes que poden causar la fase de latència: inòculs amb massa mitjana petita, i canvis de medi.S'ha verificat també la relació de la durada de la latència amb variables com la temperatura o la grandària de l'inòcul. En aquest treball s'ha identificat la distribució de biomassa del cultiu com una variable cabdal per analitzar l'evolució del cultiu durant el cicle de creixement. S'han definit les funcions matemàtiques que anomenem distàncies per avaluar quantitativament l'evolució d'aquesta distribució.Hem abordat, també, la fase de latència des d'un punt de vista teòric. L'evolució de la velocitat de creixement al llarg del cicle ha permès distingir dues etapes en la fase de latència que anomenem inicial i de transició. L'etapa de transició s'ha descrit per mitjà d'un model matemàtic continu validat amb simulacions INDISIM. S'ha constatat que la fase de latència ha de ser vista com un procés dinàmic, i no com un simple període de temps descrit per un paràmetre. Les funcions distància també s'han utilitzat per avaluar les propietats del creixement balancejat.Alguns dels resultats de les simulacions amb INDISIM s'han corroborat experimentalment per mitjà de citometria de flux. S'ha comprovat, al llarg de les diverses fases del creixement, el comportament de la distribució de biomassa previst per simulació, així com l'evolució de les funcions distància. La coincidència entre els resultats experimentals i els de simulació no és trivial, ja que el sistema estudiat és molt complex. Per tant, aquests resultats permeten comprovar la bondat de la metodologia INDISIM.Finalment, hem avançat en l'optimització d'eines per parametritzar IbMs, un pas essencial per poder utilitzar les simulacions INDISIM de manera quantitativa. S'han adaptat i assajat els mètodes grid search, NMTA i NEWUOA. Aquest darrer mètode ha donat els millors resultats en termes de temps, mantenint una bona precisió en els valors òptims dels paràmetres. Per concloure, podem afirmar que INDISIM ha estat validat com una bona eina per abordar l'estudi dels estats transitoris com la fase de latència. / Predictive food microbiology has become an important specific field in microbiology. Bacterial growth of a batch culture may show up to four phases: lag, exponential, stationary and death. The bacterial lag phase, which is of specific interest in the framework of predictive food microbiology, has generally been tackled with two generic approaches: at a cellular and intracellular level, which we call the microscopic scale, and at a population level, which we call the macroscopic scale. Studies at the microscopic level tackle the processes that take place inside the bacterium during its adaptation to the new conditions such as the changes in genetic expression and in metabolism. Studies at the macroscopic scale deal with the description of a population growth cycle by means of mathematical continuous modelling and experimental measurements of the variables related to cell density evolution.In this work we aimed to improve the understanding of the lag phase in bacterial cultures and the intrinsic phenomena behind it. This has been carried out from the perspective of Individual-based Modelling (IbM) with the simulator INDISIM (INDividual DIScrete SIMulation), which has been specifically improved for this purpose. IbM introduces a mechanistic approach by modelling the cell as an individual unit. IbM simulations deal with 1 to 106 cells, and allow specific study of the phenomena that emerge from the interaction among cells. These phenomena belong to the mesoscopic level.Mesoscopic approaches are essential if we are to understand the effects of cellular adaptations at an individual level in the evolution of a population.Thus, they are a bridge between individuals and population, or, to put it another way, between models at a microscopic scale and models at a macroscopic scale.First, we studied separately two of the several mechanisms that may cause a lag phase: the lag caused by the initial low mean mass of the inoculum, and the lag caused by a change in the nutrient source. The relationship among lag duration and several variables such as temperature and inoculum size were also checked. This analysis allowed identification of the biomass distribution as a very important variable to follow the evolution of the culture during the growth cycle. A mathematical tool was defined in order to assess its evolution during the different phases of growth: the distance functions.A theoretical approach to the culture lag phase through the dynamics of the growth rate allowed us to split this phase into two stages: initial and transition. A continuous mathematical model was built in order to shape the transition stage, and it was checked with INDISIM simulations. It was seen that the lag phase must be defined as a dynamic process rather than as a simple period of time. The distance functions were also used to discuss the balanced growth conditions.Some of the reported INDISIM simulation results were subjected to experimental corroboration by means of flow cytometry, which allow the assessment of size distributions of a culture through time. The dynamics of biomass distribution given by INDISIM simulations were checked, as well as the distance function evolution during the different phases of growth. The coincidence between simulations and experiments is not trivial: the system under study is complex; therefore, the coincidence in the dynamics of the different modelled parameters is a validation of both the model and the simulation methodology.Finally, we have made progress in IbM parameter estimation methods, which is essential to improve quantitative processing of INDISIM simulations.Classic grid search, NMTA and NEWUOA methods were adapted and tested, the latter providing better results with regard to time spent, which maintains satisfactory precision in the parameter estimation results.Above all, the validity of INDISIM as a useful tool to tackle transient processes such as the bacterial lag phase has been amply demonstrated.

abstract: The discovery that mechanical forces regulate microbial virulence, stress responses and gene expression was made using log phase cultures of Salmonella Typhimurium (S. Typhimurium) grown under low fluid shear (LFS) conditions relevant to those encountered in the intestine. However, there has been limited characterization of LFS on other growth phases. To advance the growth-phase dependent understanding of the effect of LFS on S. Typhimurium pathogenicity, this dissertation characterized the effect of LFS on the transcriptomic and phenotypic responses in both stationary and lag phase cultures. In response to LFS, stationary phase cultures exhibited alterations in gene expression associated with metabolism, transport, secretion and stress responses (acid, bile salts, oxidative, and thermal stressors), motility, and colonization of intestinal epithelium (adherence, invasion and intracellular survival). Many of these characteristics are known to be regulated by the stationary phase general stress response regulator, RNA polymerase sigma factor S (RpoS), when S. Typhimurium is grown under conventional conditions. Surprisingly, the stationary phase phenotypic LFS stress response to acid and bile salts, colonization of human intestinal epithelial cells, and swimming motility was not dependent on RpoS. Lag phase cultures exhibited intriguing differences in their LFS regulated transcriptomic and phenotypic profiles as compared to stationary phase cultures, including LFS-dependent regulation of gene expression, adherence to intestinal epithelial cells, and high thermal stress. Furthermore, the addition of cell-free conditioned supernatants derived from either stationary phase LFS or Control cultures modulated the gene expression of lag phase cultures in a manner that differed from either growth phase, however, these supernatants did not modulate the phenotypic responses of lag phase cultures. Collectively, these results demonstrated that S. Typhimurium can sense and respond to LFS as early as lag phase, albeit in a limited fashion, and that the lag phase transcriptomic and phenotypic responses differ from those in stationary phase, which hold important implications for the lifecycle of this pathogen during the infection process. / Dissertation/Thesis / Transcriptomic Data / Doctoral Dissertation Microbiology 2020

No / ß2-Microglobulin (ß2m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven ß-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E¿)), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native ß2m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59¿71 (peptide E) and 59¿79 (peptide E¿) of intact ß2m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59¿71 may be important in the self-association of partially folded ß2m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.

Résumé : Saccharomyces cerevisiae, utilisée depuis des millénaires pour la fermentation du vin du fait de son endurance et de ses qualités inégalables, est de nos jours largement utilisée pour inoculer les mouts de raisin. Néanmoins, lors de l'inoculation, les souches oenologiques doivent faire face à des stress spécifiques qui peuvent compromettre le début de la fermentation. L’objectif de ce travail est d'élucider les bases métaboliques et moléculaires de la résistance multi-stress pendant la phase de latence en conditions oenologiques. Nous avons tout d'abord caractérisé un ensemble de levures oenologiques en mettant l'accent sur des facteurs de stress caractéristiques des vins rouges et des vins blancs. La température et le stress osmotique affectent fortement cette phase pour toutes les souches, alors que le SO2, les lipides et la thiamine ont un effet souche-dépendant. Ces données ont servi de base à deux approches parallèles. Une approche d'évolution expérimentale a permis, en appliquant des pressions sélectives caractéristiques de la phase de latence, de sélectionner des souches évoluées présentant une phase de latence plus courte. Plusieurs mutations de novo potentiellement impliquées dans le phénotype évolué ont été identifiées par séquençage de leur génome. En parallèle, une approche QTL combinant des croisements inter-souches, une étape de propagation industrielle et séchage des descendants, et la sélection de cellules bourgeonnantes par FACS a été développée. Ces deux stratégies ont permis d’identifier plusieurs variants alléliques impliqués dans la paroi cellulaire, le transport du glucose, le cycle cellulaire et la résistance au stress, jouant un rôle potentiellement important pendant la phase de latence. L’ensemble de ces résultats apporte de nouvelles connaissances sur la diversité et les bases génétiques de l'adaptation des levures à la phase de latence oenologique et offre un cadre d’amélioration des propriétés des souches. De plus, nous avons montré que K. marxianus a un potentiel pour des cultures mixtes et des contributions aromatiques positives en conditions oenologiques, ouvrant de nouvelles possibilités pour des études ultérieures.Titre : Résistance au stress lors de la phase de latence en fermentation oenologique et développement de levures optimiséesMots clés : Fermentation oenologique, levure, phase de latence, résistance multi-stress, QTL, évolution adaptative, K. marxianus / Abstract: Saccharomyces cerevisiae has been used for millennia to perform wine fermentation due to its endurance and unmatched qualities and is nowadays widely used as wine yeast starter. Nevertheless, at the moment of inoculation, wine yeasts must cope with specific stress factors that can compromise the fermentation start. The objective of this work was to elucidate the metabolic and molecular bases of multi-stress resistance during wine fermentation lag phase. We first characterized a set of commercialized wine yeast strains by focusing on stress factors typically found at this stage in red wines and in white wines. Temperature and osmotic stress had a drastic impact in lag phase for all strains whereas SO2, low lipids and thiamine had a more strain dependent effect. Based on these data, we developed two parallel approaches. Using an evolutionary engineering approach where selective pressures typically present in lag phase were applied, we obtained evolved strains with a shorter lag phase in winemaking conditions. Whole genome sequencing allowed to identify several de novo mutations potentially involved in the evolved phenotype. In parallel, a QTL mapping approach was conducted, combining an intercross strategy, industrial propagation and drying of the progeny populations and selection of the first budding cells by FACS. Both strategies allowed the identification of several allelic variants involved in cell wall, glucose transport, cell cycle and stress resistance, as important in lag phase phenotype. Overall, these results provide a deeper knowledge of the diversity and the genetic bases of yeast adaptation to wine fermentation lag phase and a framework for improving yeast lag phase. Additionally, we showed that K. marxianus has potential for mixed cultures and positive aromatic contributions under oenological conditions, opening new possibilities for further studies.Title: Stress resistance during the lag phase of wine fermentation and development of optimized yeastsKeywords: Wine fermentation, yeast, lag phase, multi-stress resistance, QTL, adaptive evolution, K. marxianus

Ackumuleringen av plast och mikroplast i miljön är idag en globalt aktuell fråga. Forskning kring mikroplast och dess påverkan på miljö och biota har ökat kraftigt det senaste decenniet. En av de största utsläppskällorna till mikroplast är slitage från vägtrafiken, speciellt från däck, men mikroplast kan även komma från vägmarkeringar. Vägmarkeringar innehåller plastpolymerer (som bindemedel), fyllnadsmedel, pigment och tillsatsämnen (additiv), och ofta ingår även glaspärlor. Vissa ämnen kan ge en negativ påverkan på människa och miljö. I dagsläget saknas det data för att kunna kartlägga i vilken omfattning vägmarkeringsslitage bidrar till spridning av mikroplaster samt för att bedöma om och hur det skulle kunna påverka miljön och vattenlevande organismer. I detta examensarbete har toxicitetstest av lakvatten från vägmarkeringsprodukter utförts med algen Raphidocelis subcapitata för att studera om de är toxiska samt om det finns någon skillnad mellan olika vägmarkeringsprodukter. Partiklar från fyra olika typer av vägmarkeringsprodukter (prefabricerad termoplast, varmapplicerad termoplast, vattenbaserad akrylatfärg och 2-komponentsakrylatfärg) genererades och lakades var för sig med skakning i avjoniserat vatten under 24 timmar. Lakvattnen utan partiklar användes sedan till algtillväxthämningstest. Algerna exponerades under 72 timmar för lakvatten i olika koncentrationer, där den högsta motsvarade 100 g vägmarkeringspartiklar per liter, och algtillväxt mättes. Effektmåtten var maximal tillväxthastighet och lag-fas och dessutom analyserades korrelationen mellan dem som ett mått för anpassningseffektivitet. De fyra lakvattnen påverkade lag-fas och/eller tillväxthastighet i olika omfattning och medförde antingen inhibering eller stimulering av algtillväxten. Lakvattnet från den vattenbaserade akrylatfärgen påvisade störst inhibering av tillväxt och var även det mest toxiska eftersom ingen tillväxtanpassning skedde. För övriga lakvatten påvisades olika grad av anpassningseffektivitet av alger vid exponering. Dock krävs mer ingående statistisk utvärdering för att åtskilja de två lakvatten som hade minst påverkan, dvs. från den prefabricerade och den varmapplicerade termoplasten.  Detta är enbart en första studie. Upprepade tester med fler vägmarkeringsprodukter och fler tester med andra organismer, såsom bakterier, kärlväxter, kräftdjur och fisk, krävs för att bättre kunna utreda om och hur olika vägmarkeringar kan påverka biota. För ytterligare studier skulle det vara intressant att utvärdera vilka ämnen i vägmarkeringar som kan påverka miljön och om dessa skulle kunna ersättas med andra, mindre giftiga ämnen. / The accumulation of plastic and microplastics in the environment is a current global issue. Research on microplastics and its impact on the environment and biota has increased rapidly in the last decade. One of the largest sources of microplastics is wear from road traffic, especially tires, but they can also originate from road markings. Road markings contain plastic polymers (as a binder), fillers, pigments, and additives, and often also glass beads. Some substances may have a negative impact on humans and the environment. At present, there are no data to map the extent to which wear from road markings contributes to the emissions of microplastics, or to assess the effect they might cause on the environment and aquatic organisms. In this thesis, toxicity tests on leachates from road marking products were conducted on the alga Raphidocelis subcapitata to study the effect on the algae and if it differs between the different products.  Particles from four types of road marking products (prefabricated thermoplastic, hot-applied thermoplastic, acrylic water-based paint, and 2-component acrylic paint) were generated and leached separately by shaking in deionized water for 24 hours.  The leachates without particles were used in the standard algal growth inhibition tests. The algae were exposed for 72 hours to the leachates in different concentrations, with the highest corresponding to 100 g road marking particles per liter, and algal growth was monitored. Endpoints used were maximum growth rate and lag phase and, in addition, their correlation was analyzed as an indicator for adaptation capacity. The four leachates affected lag phase and/or growth rate to a varying extent and caused inhibition or stimulation of algae growth. Exposure to leachate from the water-based acrylic paint showed most inhibition of growth and was also the most toxic as no growth adaptation was observed. For the other tests, different rates of adaptation efficiency of the algae growth were found. However, more detailed statistical evaluation is needed to differentiate between the two leachates that had the least effect, i.e. the prefabricated and the hot-applied thermoplastic. This is only a first study. More tests with more road marking products and with other organisms, such as bacteria, vascular plants, crustaceans, and fish, are required to better understand if and how different road markings could affect biota. Additionally, an evaluation of what substances in road marking materials may cause effects and whether these can be replaced by other, less toxic substances, would be of interest.