The historical notion of a microbial population has been of a clonal population of identical swimming planktonic cells in a laboratory flask. As the field has advanced, we have grown to appreciate the immense diversity in microbial behaviors, from their propensity to grow in dense surface-attached communities as a biofilm, to the consequences of social dilemmas between cells, to their ability to form spores able to survive nearly any environmental insult. However, the historically biased view of the clonal microbial population still persists – even when a rare phenotype is investigated, the focus simply shifts to that narrower focal population - and this bias can lead to some of the broader questions relating to the consequences of phenotypic diversity within populations to be overlooked. This work seeks to address this gap by investigating the evolutionary causes and consequences of phenotypic heterogeneity, with a focus on clinically relevant phenotypes. We first develop and experimentally validate a theoretical model describing the evolution of a microbial population faced with a trade-off between survival and fecundity phenotypes (e.g. biofilm and planktonic cells), which suggests that simultaneous investment in both types maximizes lineage fitness in heterogeneous environments. This model helps to inform the experimental studies in the following chapters. We find that biofilm-mediated phenotypic resistance to antibiotics is evolutionarily labile, and responsive to antibiotic dose and whether biofilm or planktonic cells are passaged. We also show that persistence in E. coli is age-independent, supporting the current hypothesis of stochastic metabolic fluctuations as the cause of this rare phenotype. Finally, we explore phenotypic variation across a library of natural isolates of P. aeruginosa, and find few organizing principles among key phenotypes related to virulence. Together these results suggest that phenotypic heterogeneity is a crucial component in the ecology and evolution of microbial populations, and directly affects pressing applied concerns such as the antibiotic resistance crisis.

This study examined the moderating role of motivational tendencies for social approach and avoidance behavior, as measured by anterior EEG asymmetry, on symptom expression. In particular, this study aimed to replicate and extend previous findings that measures of anterior EEG asymmetry provide an important marker of subgroups of HFA children that significantly differ from each other, and controls, on measures of social communication impairment. EEG data were collected across two occasions on 51 HFA and 44 non-HFA children. EEG asymmetry was computed for homologous electrode pairs (e.g., lnF4-lnF3). More positive scores were indicative of relative left frontal asymmetry. Data on social and behavioral functioning were collected via parent- and self-report. Results of this short-term longitudinal study revealed moderate test-retest reliability for midfrontal asymmetry, r (65) = .39, p < .01. Results supported previous research demonstrating the differential relation of EEG asymmetry to symptom impairment among HFA children, such that parents of LFA-HFA children reported lower levels of impairment than RFA-HFA children on the SCQ Total Score, F (3, 47) = 3.58, p = .065, and Social Interaction Domain, F (3, 47) = 4.59, p < .05. Results also indicated that parents of LFA-HFA children reported higher levels of general communicative competence on the CCC-2, GCC, F (3, 47) = 6.83, p = .01, but greater impairment in pragmatic communication when compared to RFA-HFA children, SIDC, F (3, 47) = 4.41, p < .05. Additional analyses indicated that RFA was associated with early and more confident recognition of atypical (and stereotypically autistic) development based on retrospective parent-report (ADI-R #86), while LFA was associated with early, but less unambiguously autistic impairment, X2 (51) = 3.75, p = .05. This study demonstrates that anterior EEG asymmetry subgroups are reliable and useful markers of phenotypic variability that are meaningfully related to the experience and expression of symptoms of core autism impairment.

The life of microorganisms is characterized by two main tasks, rapid growth under conditions permitting growth and survival under stressful conditions. The environments, in which microorganisms dwell, vary in space and time. The microorganisms innovate diverse strategies to readily adapt to the regularly fluctuating environments. Phenotypic heterogeneity is one such strategy, where an isogenic population splits into subpopulations that respond differently under identical environments. Bacterial persistence is a prime example of such phenotypic heterogeneity, whereby a population survives under an antibiotic attack, by keeping a fraction of population in a drug tolerant state, the persister state. Specifically, persister cells grow more slowly than normal cells under growth conditions, but survive longer under stress conditions such as the antibiotic administrations. Bacterial persistence is identified experimentally by examining the population survival upon an antibiotic treatment and the population resuscitation in a growth medium. The underlying population dynamics is explained with a two state model for reversible phenotype switching in a cell within the population. We study this existing model with a new theoretical approach and present analytical expressions for the time scale observed in population growth and resuscitation, that can be easily used to extract underlying model parameters of bacterial persistence. In addition, we recapitulate previously known results on the evolution of such structured population under periodically fluctuating environment using our simple approximation method. Using our analysis, we determine model parameters for Staphylococcus aureus population under several antibiotics and interpret the outcome of cross-drug treatment. Next, we consider the expansion of a population exhibiting phenotype switching in a spatially structured environment consisting of two growth permitting patches separated by an antibiotic patch. The dynamic interplay of growth, death and migration of cells in different patches leads to distinct regimes in population propagation speed as a function of migration rate. We map out the region in parameter space of phenotype switching and migration rate to observe the condition under which persistence is beneficial. Furthermore, we present an extended model that allows mutation from the two phenotypic states to a resistant state. We find that the presence of persister cells may enhance the probability of resistant mutation in a population. Using this model, we explain the experimental results showing the emergence of antibiotic resistance in a Staphylococcus aureus population upon tobramycin treatment. In summary, we identify several roles of bacterial persistence, such as help in spatial expansion, development of multidrug tolerance and emergence of antibiotic resistance. Our study provides a theoretical perspective on the dynamics of bacterial persistence in different environmental conditions. These results can be utilized to design further experiments, and to develop novel strategies to eradicate persistent infections. / Das Leben von Mikroorganismen kann in zwei charakteristische Phasen unterteilt werde, schnelles Wachstum unter Wachstumsbedingungen und Überleben unter schwierigen Bedingungen. Die Bedingungen, in denen sich die Mikroorganismen aufhalten, verändern sich in Raum und Zeit. Um sich schnell an die ständig wechselnden Bedingungen anzupassen entwickeln die Mikroorganismen diverse Strategien. Phänotypische Heterogenität ist eine solche Strategie, bei der sich eine isogene Popolation in Untergruppen aufteilt, die unter identischen Bedingungen verschieden reagieren. Bakterielle Persistenz ist ein Paradebeispiel einer solchen phänotypischen Heterogenität. Hierbei überlebt eine Popolation die Behandlung mit einem Antibiotikum, indem sie einen Teil der Bevölkerung in einem, dem Antibiotikum gegenüber tolerant Zustand lässt, der sogenannte "persister Zustand". Persister-Zellen wachsen unter Wachstumsbedingungen langsamer als normale Zellen, jedoch überleben sie länger in Stress-Bedingungen, wie bei Antibiotikaapplikation. Bakterielle Persistenz wird experimentell erkannt indem man überprüft ob die Population eine Behandlung mit Antibiotika überlebt und sich in einem Wachstumsmedium reaktiviert. Die zugrunde liegende Popolationsdynamik kann mit einem Zwei-Zustands-Modell für reversibles Wechseln des Phänotyps einer Zelle in der Bevölkerung erklärt werden. Wir untersuchen das bestehende Modell mit einem neuen theoretischen Ansatz und präsentieren analytische Ausdrücke für die Zeitskalen die für das Bevölkerungswachstums und die Reaktivierung beobachtet werden. Diese können dann einfach benutzt werden um die Parameter des zugrunde liegenden bakteriellen Persistenz-Modells zu bestimmen. Darüber hinaus rekapitulieren wir bisher bekannten Ergebnisse über die Entwicklung solch strukturierter Bevölkerungen unter periodisch schwankenden Bedingungen mithilfe unseres einfachen Näherungsverfahrens. Mit unserer Analysemethode bestimmen wir Modellparameter für eine Staphylococcus aureus-Popolation unter dem Einfluss mehrerer Antibiotika und interpretieren die Ergebnisse der Behandlung mit zwei Antibiotika in Folge. Als nächstes betrachten wir die Ausbreitung einer Popolation mit Phänotypen-Wechsel in einer räumlich strukturierten Umgebung. Diese besteht aus zwei Bereichen, in denen Wachstum möglich ist und einem Bereich mit Antibiotikum der die beiden trennt. Das dynamische Zusammenspiel von Wachstum, Tod und Migration von Zellen in den verschiedenen Bereichen führt zu unterschiedlichen Regimen der Populationsausbreitungsgeschwindigkeit als Funktion der Migrationsrate. Wir bestimmen die Region im Parameterraum der Phänotyp Schalt-und Migrationsraten, in der die Bedingungen Persistenz begünstigen. Darüber hinaus präsentieren wir ein erweitertes Modell, das Mutation aus den beiden phänotypischen Zuständen zu einem resistenten Zustand erlaubt. Wir stellen fest, dass die Anwesenheit persistenter Zellen die Wahrscheinlichkeit von resistenten Mutationen in einer Population erhöht. Mit diesem Modell, erklären wir die experimentell beobachtete Entstehung von Antibiotika- Resistenz in einer Staphylococcus aureus Popolation infolge einer Tobramycin Behandlung. Wir finden also verschiedene Funktionen bakterieller Persistenz. Sie unterstützt die räumliche Ausbreitung der Bakterien, die Entwicklung von Toleranz gegenüber mehreren Medikamenten und Entwicklung von Resistenz gegenüber Antibiotika. Unsere Beschreibung liefert eine theoretische Betrachtungsweise der Dynamik bakterieller Persistenz bei verschiedenen Bedingungen. Die Resultate könnten als Grundlage neuer Experimente und der Entwicklung neuer Strategien zur Ausmerzung persistenter Infekte dienen.

Mycobacterial populations are known for the heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions. Such population heterogeneity had been observed in TB patients, in animal models, and in in vitro cultures. Also, the physiological relevance of population heterogeneity under nutrient starvation has been studied. However, the physiological significance of population heterogeneity in oxidative and nitrite stress has not been addressed yet. Our laboratory had earlier shown that a subpopulation of mycobacterial mid-log phase cultures divide by highly deviated asymmetric division, generating short cells and normal-sized/long cells. This proportion has been found to be consistent and reproducible, and has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum, which is known to have high levels of oxidative stress. The highly deviated asymmetric cell division has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, the physiological significance of the population heterogeneity generated by the highly deviated asymmetric division remained to be addressed. Therefore, in the present study, we addressed the physiological significance of the generation of population heterogeneity in terms of cell size in Mycobacterium smegmatis and Mycobacterium tuberculosis. In this regard, we explored whether the minor subpopulation of short cells generated in the population has any relevance in the response of mycobacteria to oxidative and nitrite stress for survival. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on the phenotypic heterogeneity in diverse bacterial systems and the physiological significance of such heterogeneity. Subsequently, an account of the phenotypic heterogeneity reported in mycobacteria is given, with examples of its significance implicated for survival under nutrient stress. Then an account of various studies on the oxidative and nitrite stress response of mycobacteria and on the genes involved in those processes are given. Further, the present study is justified by stating that so far there has not been any study to find out the physiological relevance of phenotypic heterogeneity on oxidative and nitrite stress response in mycobacteria. Finally, the Introduction is concluded by stating that the present study investigates and reports for the first time the physiological significance of the minor subpopulation of short cells for survival under oxidative and nitrite stress conditions. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the separation of the short cells, their characterisation, stress response, and so on are given in great detail. The Chapter 3 forms the first data chapter that presents results on the nature of response of Mycobacterium smegmatis and Mycobacterium tuberculosis against oxidative and nitrite stress. Here the cell size natural distribution, in terms of short cells and normal-sized/long cells in the mid-log phase population, the fractionation and enrichment of these subpopulations, differential susceptibility of the cells in the fractions to the stress conditions, the enhanced survival of the population upon mixing of these cell populations at the natural proportion, and the decreased survival upon mixing them at unnatural proportion are presented. The differential survival of the short cells and normal-sized/long cells was studied at a variety of stress concentrations for oxidative (H2O2) and nitrite (acidified sodium nitrite, pH 5), cell densities and exposure time to show the robustness of the phenomenon. Enhanced survival upon extended exposure to stress also has been documented. Essentially the data in this chapter shows that although the different sized populations show differential stress susceptibility to the stress conditions, their combined presence at the proportion that naturally exists in the mid-log phase population enhances the survival of the population, at the cost of the highly susceptible short cells for the enhanced survival of the less susceptible normal-sized/long cells, kin selection and altruism. The Chapter concludes with a discussion on the results. The Chapter 4 delineates the mechanism of the altruistic phenomenon that results in the enhanced survival of the population at the sacrifice of the minor subpopulation of short cells. Here we present evidence that hydroxyl radical generated through Fenton reaction is responsible for the enhanced survival through the induction of the synthesis of catalase-peroxidase (KatG) for the degradation of H2O2. The free iron deficient short cells acquire more iron, which in turn becomes stoichiometrically detrimental to them due to the high levels of hydroxyl generation in the presence of H2O2. On the contrary, the free iron containing normal-sized/long cells do not acquire iron and hence the hydroxyl radical produced in the population becomes stoichiometrically beneficial to them. Thus, the deficiency of free iron which consequentially necessitates the short cells to acquire more iron becomes a maladaptive trait in the presence of H2O2 but gets co-opted in kin selection, for the survival of the normal-sized/long cells that form major proportion of the population – a phenomenon reminiscent of altruism. The Chapter concludes with a model depicting the entire phenomenon and a discussion on the results and their implications.

Mosaicism occurs due to postzygotic genetic alterations during early embryonic development. The phenomenon is common, present in all humans, animals, and plants, and is associated with phenotypic variability and heterogeneity. Mosaic pathogenic gene variants result in a mosaic disease state, in which the individual can present with mild, generalized disease, a localized disease phenotype in specific organs and tissue regions, or full-blown clinical features which are indistinguishable from the heterozygous disease state. Multiple studies have described the prevalence and clinical correlations associated with low-level mosaicism for various genetic disorders, including several tumor suppressor gene (TSG) syndromes, which are well-known to display mosaicism. However, the extent of mosaicism research varies widely between TSG syndromes. Currently there is no comprehensive, up to date review covering multiple TSGs and focusing on mosaicism prevalence, diagnostic strategies and transmission risk. Here, in this literature review, I focus on 8 common tumor suppressor genes NF1, NF2, TSC1, TSC2, RB1, PTEN, VHL, and TP53; reporting the following disease aspects: • Role and function of each tumor suppressor gene, disease prevalence, inheritance pattern, penetrance/expressivity pattern, age of onset clinical features, organs affected, and benign or malignant tumors seen • Different types of mosaicism, including critical review of recent, representative publications for each tumor suppressor gene syndrome • Established criteria for clinical diagnosis of inherited versus mosaic disease, molecular diagnosis, and current methods of genetic analysis Then more extensively, this thesis discusses the most informative, representative original studies for each TSG and provides a summary which covers: • The number of mosaic patients analyzed and the spectrum of clinical features of the cohort they were sampled from • The spectrum of variant allele frequency (VAF), tissue types analyzed, and different analysis methods performed • Whether or not the mosaic patients met clinical criteria for diagnosis of inherited disease • The number of patients who were persistently classified as no mutation identified (NMI) after genetic analysis • Spectrum and type of mosaic mutational event(s) identified • Age of onset and age range of mosaic patients • Patient ascertainment and family history (sporadic or familial cases) and • Type of mosaicism seen Furthermore, it compares and discusses disease severity, possibility of malignancy, and genotype-phenotype correlations for each TSG. Ultimately, by juxtaposing these TSGs, this review aims to centralize existing knowledge about mosaicism and provide insight into how molecular techniques can be broadly applied for better diagnosis of mosaic disease. / 2022-11-10T00:00:00Z

Toxoplasma gondii bildet im Nerven- und Muskelgewebe seines Zwischenwirts persistente enzystierte Bradyzoiten, die Immunreaktionen und medizinischen Behandlungen entgehen. Der experimentelle Zugang zu reifen Zysten ist auf ex vivo Modelle beschränkt und die Bradyzoiten-Biologie unzureichend erforscht. Das Metabolom oder Wirtszell-Bradyzoiten-Interaktionen und Persistenzmechanismen sind mit aktuellen in vitro und in vivo Modellen schwer zu adressieren. Um dies zu ermöglichen, war es das Ziel dieser Arbeit ein in vitro Modell zur Generierung reifer T. gondii Zysten zu etablieren und zu charakterisieren. Dieses System wurde verwendet, um (1) das Metabolom von reifen enzystierten Bradyzoiten im Vergleich zu Tachyzoiten zu charakterisieren, (2) Wirtszell-Bradyzoiten-Interaktionen zu untersuchen und (3) einen Zellteilungsmarker für Bradyzoiten zu etablieren. Bradyzoiten wurden in ausdifferenzierten menschlichen Myotuben generiert und zeigten typische ultrastrukturelle Charakteristika und Antigenexpression. Die Bradyzoiten zeigten auch funktionelle Merkmale wie Resistenz gegen Pepsin, Temperatur und Antiparasitika, die von der Reifungszeit abhängig waren. Der metabolische Fingerabdruck von Bradyzoiten wurde mit Tachyzoiten mit Hilfe einer ungezielten HILIC-UHPLC / MS-basierten Metabolomik-Plattform verglichen. Während die Hemmung der Aconitase durch Natriumfluoracetat letal in Tachyzoiten wirkte, tolerierten Bradyzoiten eine längere Hemmung des Enzyms. Stabile isotopenmetabolische Markierung und pharmakologische Modulation des Wirt Lipidstoffwechsels wiesen auf eine entscheidende Rolle der Wirts Carnitinester für den Fettsäureimport und die Entgiftung der antimikrobiellen Linolsäure hin. Um einen Zellteilungsmarker auf Einzelzellebene zu entwickeln, wurden Klick-Chemie nachweisbare Nukleosidanaloga auf Toxizität in beiden Stadien und ihr jeweiliges Inkorporationsprofil untersucht. Drei der Analoga wurden ohne toxische Wirkungen in die DNA von beiden Stadien eingebaut. / Toxoplasma gondii forms persistent encysted bradyzoites inside neuronal and muscle tissue of its intermediate host, which resist immune responses and medical treatments. Experimental access to mature tissue cysts is limited to ex vivo models and the biology of bradyzoites remains understudied. Aspects like the metabolome or bradyzoite-host-interactions and mechanisms of persistence are difficult to address using current in vitro and in vivo models. To overcome these restrictions, the aim of this thesis was the establishment and characterization of an in vitro model for the generation of matured T. gondii tissue cysts. This system then should be used to (1) characterize the metabolome of matured encysted bradyzoites in comparison with tachyzoites, (2) interrogate bradyzoite-host-interactions and (3) establish a cell division marker for bradyzoites that allows studying bradyzoite heterogeneity. Encysted bradyzoites were grown in terminally differentiated human myotubes and showed typical ultrastructural hallmarks and antigen expression. These bradyzoites contained functional hallmarks like resistance to pepsin, temperature and commonly used antiparasitics that were dependent on maturation time. The metabolic fingerprint of bradyzoites was compared to tachyzoites using an untargeted HILIC-UHPLC / MS based metabolomics platform. While tachyzoites succumbed to inhibition of their aconitase by sodium fluoroacetate, bradyzoites tolerated prolonged inhibition of this enzyme. Further, stable isotope-metabolic labeling and pharmacological modulation of host lipid metabolism indicated a critical role of host carnitine esters for fatty acid import and for the detoxification of antimicrobial linoleic acid. To develop a single cell-resolved cell division marker, we screened click chemistry-detectable nucleoside analogues for toxicity on both stages and their respective incorporation profile. Three compounds labelled both bradyzoite and tachyzoite nuclei without toxic effects.

Les émulsions fluorées de Pickering sont étudiées et mises au point dans la technologie demicrofluidique en gouttes pour des applications d’études sur des cellules adhérentes isolées.Les principaux résultats de ce projet sont : l’établissement d’un lien entre la couverture desurface des nanoparticules et la fluidité de l’émulsion de Pickering ; l’établissement deslignes directrices pour la stabilisation des gouttes avec un débit de production élevé et unminimum de déchets de particules ; et la mise en oeuvre d’une plateforme technologiquecomplète pour l’étude des cellules RPE, pour mesurer leur hétérogénéité phénotypique auniveau de la cellule individuelle. / Fluorinated Pickering emulsions are studied and engineered within droplet-based microfluidicstechnology for adherent-cell studies applications. The main findings of this projectinclude: linking the nanoparticles surface coverage to the bulk flowability of the Pickeringemulsion; deriving guidelines for droplet stabilization with high production throughput andminimal particle waste; and implementing the full technological platform for the study ofRPE cells, while unraveling their phenotypic heterogeneity at the single cell level.