This work investigates the death and degradation of the second polar body of the nematode C. elegans in order to improve our understanding how pluripotent undifferentiated cells deal with dying cells. With the use of fluorescence microscopy this work demonstrates that both polar bodies loose membrane integrity early. The second polar body has contact to embryonic cells and gets internalized, dependent on the Rac1-ortholog CED-10. The polar body gets degraded via LC3-associated phagocytosis. While lysosome recruitment depends on RAB-7, LC3 does not improve lysosome recruitment but still accelerates polar body degradation. This work establishes the second polar body as a genetic model to study cell death and LC3-associated phagocytosis and has revealed further aspects of phagosome maturation and degradation. / Um besser zu verstehen, wie undifferenzierte pluripotente Zellen mit abgestorbenen Zellen umgehen, wird in dieser Dissertation die Phagozytose und der Abbau des 2. Polkörpers der weiblichen Meiose im Fadenwurm C. elegans untersucht. Mithilfe von fluorenzenzmikroskopischen Aufnahmen wird in dieser Arbeit gezeigt, dass beide Polkörper schon früh ihre Membranintegrität verlieren. Der 2. Polkörper, welcher direkten Kontakt zu embryonischen Zellen hat, wird daraufhin mithilfe des Rac1-Orthologs CED-10 phagozytiert. Es wird gezeigt, dass es sich bei dem Abbauprozess um LC3-assoziierte Phagozytose handelt. Die RAB-7 GTPase ist notwendig für die Rekrutierung von Lysosomen, während LC3 darauf keinen Einfluss hat, aber trotzdem den Abbau des Polkörpers beschleunigt. Mit dieser Arbeit konnte ein genetisches Modell für die Erforschung von Zelltod und der LC3-assoziierten Phagozytose entwickelt werden und weitere Aspekte der Phagosomreifung und des -abbaus aufgedeckt werden.

Quiescent adult stem cells are important for both tissue maintenance and for responding to stress. C. elegans provides an ideal context to dissect pathways involved in the maintenance of long-term cellular quiescence. In favorable environmental conditions, larvae develop continuously into reproductive adults. In adverse environmental conditions, larvae can undergo an alternative life history and enter dauer diapause, a long-lived state with characteristics that promote survival and dispersal. Animals that enter dauer can survive for months, many times the normal life span of an animal that develops continuously. Entry into dauer interrupts the development of the vulva and is associated with a reprogramming-like event that ensures that Vulval Precursor Cells (VPCs) remain multipotent and quiescent until conditions improve. In this work, I aim to understand how the pathways that regulate dauer entry dictate cellular outcomes that oppose VPC fate acquisition and cell division.VPC specification is initiated when an inductive EGF signal from the somatic gonad activates EGFR-Ras-ERK signaling in the nearest VPC, P6.p. Using an ERK activity biosensor, we found that EGFR signal transduction is activated in P6.p prior to dauer entry. However, during the molt into dauer, EGFR signal transduction itself is downregulated and ERK activity remains low in P6.p throughout dauer. To understand how the VPCs are maintained as multipotent precursors in dauer larvae, we investigated the level at which negative regulation of EGFR signaling occurs. We found that dauer VPCs are desensitized to both endogenous and ectopic expression of EGF despite the presence and correct localization of the EGFR. A constitutively active allele of Ras, but not the EGFR, was sufficient to increase ERK activity in the VPCs. This suggests that during dauer, regulation of EGFR signal transduction occurs at or above the level of Ras. We conclude that EGFR signaling is opposed within the VPCs themselves at the level of membrane associated events. Entry into dauer is regulated by Insulin (IIS), TGF-β and Nuclear Hormone Receptor (NHR) signaling pathways. To understand how these pathways might act to block VPC specification and cell division, we characterized mutants acting in either the IIS or NHR pathway that show inappropriate VPC developmental progression in dauer larvae. We found that the phosphatase DAF-18/PTEN, a modulator of IIS, is required to maintain VPC quiescence during dauer. We created an endogenously floxed daf-18 allele and used tissue-specific Cre recombinase drivers to determine the cellular focus of DAF-18/PTEN in regulating VPCs. Our data is consistent with DAF-18/PTEN acting nonautonomously to prevent VPC division and to maintain competence in dauer. DAF-16/FOXO, the major downstream effector of IIS, and DIN-1S/SHARP, which acts in NHR signaling, were previously implicated in regulation of the VPCs during dauer. We looked at null mutants over time in dauer life history and found that each transcription factor opposes VPC division during distinct stages in dauer development. While DIN-1S/SHARP appears to be required to maintain quiescence at the end of the L2d-dauer molt, DAF-16/FOXO is required to maintain quiescence in dauer itself. This suggests that regulation of the VPCs during dauer life history is dynamic and occurs in phases with each stage having distinct regulatory mechanisms, which is like what has been described for dauer exit. Our research provides insights into robust protective mechanisms that maintain multipotency and quiescence over long periods of time. While the pathways required to enact the dauer program are well defined, the downstream consequences of these pathways on individual or groups of cells are less understood. Future work will aim to link dauer regulating pathways to the downregulation of EGFR signaling in the VPCs.

Comparative study of homologous structures in closely related species allows the identification of changes in gene regulatory mechanisms and their impact on the evolution of developmental processes. Nematodes, the invertebrate roundworms, are well suited for such studies, especially the Caenorhabditis briggsae and its famous cousin C. elegans. These two worms diverged from a common ancestor roughly 30 million years ago, yet appear morphologically almost identical. My Ph.D. thesis has focused on a set of nuclear factors in C. briggsae that negatively regulate cell proliferation to generate the hermaphrodite-specific mating and egg-laying organ, i.e., vulva. To this end, I have taken a two-pronged approach: one, developing resources to facilitate genetic and genomic studies in this species, and two, characterizing the roles of a novel class of genes and known repressors of vulval development. My work has uncovered substantial differences in the underlying genetic networks that regulate vulva formation in C. briggsae and C. elegans. / Thesis / Candidate in Philosophy

El sistema nervioso consiste en una red muy compleja de billones de neuronas que se comunican entre sí. Las sinapsis químicas son primordiales para la comunicación neuronal rápida y eficiente, y están mediadas por la liberación de neurotransmisores a la hendidura sináptica para unirse a sus receptores específicos ubicados en otras células o neuronas postsinápticas. Entre los receptores de neurotransmisores se encuentran los receptores Cys-loop, que pertenecen a la superfamilia de canales pentaméricos activados por ligando (pLGIC). Son proteínas integrales de membrana que convierten la señal química en una respuesta eléctrica al permitir el paso de iones de un lado a otro de la membrana. Estos receptores se constituyen por 3 regiones fundamentales, el dominio extracelular, donde se encuentra el sitio de unión del neurotransmisor o sitio ortostérico; el dominio transmembrana, que conforma el poro del canal y contiene los sitios de unión para diferentes moduladores alostéricos; y el dominio intracelular que posee sitios de modulación y aminoácidos determinantes de la conductancia del canal. En vertebrados, esta familia de receptores está formada por los receptores nicotínicos de acetilcolina y el receptor de serotonina tipo 3 (5-HT3), los cuales son permeables a cationes, y por los receptores de ácido γ-aminobutírico tipo A (GABAA) y receptores de glicina, permeables a aniones. El repertorio de receptores Cys-loop en invertebrados es más variado y extenso, incluyendo entre ellos un canal aniónico activado por serotonina, denominado MOD-1. Los receptores Cys-loop son ampliamente estudiados por ser blancos moleculares terapéuticos en una extensa variedad de patologías. Es por ello por lo que ampliar el conocimiento de la función y modulación de estos receptores permitirá generar mejores estrategias farmacológicas. En esta Tesis Doctoral se realizaron estudios de la función y modulación de los receptores Cys-loop activados por serotonina humano, 5-HT3, y de Caenorhabditis elegans, MOD-1, para la búsqueda de nuevos fármacos y la generación de estrategias de reposicionamiento de medicamentos de uso clínico. En el Capítulo 1 se estudió la funcionalidad molecular del receptor 5-HT3A humano. Se logró descifrar las diferencias entre la activación ortostérica y la activación y modulación alostérica por terpenoides, utilizando la valiosa técnica de patch-clamp. Se definieron las bases mecanísticas de la activación de 5-HT3A por los agonistas ortostéricos serotonina y triptamina, y de la activación alostérica por los terpenoides timol y carvacrol. Se demostró que triptamina es un agonista de muy baja eficacia y potencia. Mediante registros de whole-cell, se demostró que los terpenoides potencian las corrientes evocadas por serotonina, pero también poseen capacidad para evocar alostéricamente respuestas macroscópicas, con mayor potencia y eficacia que triptamina, aunque de forma más lenta que serotonina. Mediante registros de canal único, se evidenció que dichos compuestos activan al receptor 5-HT3 como agonistas muy eficaces dado que generan episodios de activación más prolongados que serotonina. Nuestro estudio mostró la primera caracterización a nivel de canal único de la activación de 5-HT3A humano por ligandos alostéricos, de creciente interés como herramientas terapéuticas. En el Capítulo 2 se halló un nuevo blanco promisorio para la terapia antihelmíntica, estudiando ampliamente al receptor MOD-1 de C. elegans. Este nematodo es un buen modelo parasitario ya que comparte las características funcionales y farmacológicas con los nematodos parasitarios. Además C. elegans es sensible a la mayoría de los fármacos antiparasitarios que están dirigidos a los receptores Cys-loop de los parásitos. Dado que MOD-1 está ausente en vertebrados, se encuentra limitado al filo, y está presente en nematodos parasíticos, emerge como un blanco farmacológico promisorio. En este capítulo se revelaron las diferencias en la selectividad agonista y en los sitios ago-PAM entre MOD-1 y el receptor de serotonina humano 5-HT3. Se encontró que la triptamina, un agonista muy parcial de 5-HT3A, es un agonista eficaz de MOD-1, y se demostró además que posee una acción antihelmíntica. Por otro lado, se realizó un testeo de una variedad de compuestos en búsqueda de nuevos moduladores de MOD- 1. Se demostró, mediante estudios electrofisiológicos, que muscimol y piperazina (PZE) son antagonistas no competitivos de MOD-1. Mediante ensayos de comportamiento en gusanos se confirmó el efecto de PZE sobre MOD-1, hallando de esta manera un nuevo blanco molecular para este fármaco de uso antiparasitario. Estos estudios ampliaron el conocimiento de receptores Cys-loop de nematodos para el descubrimiento de nuevos compuestos con actividad nematicida. Por lo tanto, se concluye que, derivados de triptamina y PZE podrían ser explorados como nuevos compuestos con potencial actividad antiparasitaria. En el Capítulo 3, se comenzó a plantear estrategias para reposicionar medicamentos de uso clínico, para lograr superar las desventajas de generar un fármaco de novo, acortando los tiempos y recursos necesarios para realizar los estudios farmacológicos, famacocinéticos y de toxicidad requeridos para la producción de nuevos fármacos dirigidos a tratar patologías en las que los receptores estudiados en esta Tesis están involucrados. Con tal fin, se exploró el efecto de fármacos de uso comercial derivados de triptamina y PZE, debido a lo postulado en el capítulo 2, y se encontró que sumatriptan, un fármaco comercializado para tratar la migraña, sería un buen candidato para reposicionar para la terapia antiparasitaria. Además, PZE podría reutilizarse como modulador alostérico negativo de los receptores 5-HT3A humano abordando patologías en las que este receptor está involucrado, tales como náuseas y vómitos relacionados a la quimioterapia, radioterapia y anestesia, así como para mitigar los síntomas del síndrome de intestino irritable y para patologías del sistema nervioso como depresión, ansiedad y esquizofrenia, entre otras en las que este receptor está involucrado. En resumen, aprovechando la potencialidad que brindan los registros de canal único y de corrientes macroscópicas en el estudio de las bases moleculares del funcionamiento y modulación de estos receptores Cys-loop, se logró hallar un nuevo blanco antiparasitario, descifrar por primera vez las diferencias de activación ortostérica y alostérica del receptor 5-HT3A humano por timol y carvacrol, y generar estrategias de reposicionamiento de fármacos de uso clínico mediante la modulación alostérica de estos receptores tanto en C. elegans como en humano. Estos estudios amplían el conocimiento de la función y modulación de los receptores Cys-loop, generando una base para abordar patologías que involucran a estos receptores y para el desarrollo de nuevos y más selectivos fármacos. / The nervous system consists of a highly complex network of billions of neurons that communicate with each other. Chemical synapses are key to the rapid and efficient neuronal communication and are mediated by the release of neurotransmitters into the synaptic cleft to bind to their specific receptors located either on other cells or postsynaptic neurons. Among the neurotransmitter receptors are the Cys-loop receptors, which belong to the superfamily of ligand-gated pentameric channels (pLGIC). They are integral membrane proteins that convert the chemical signal into an electrical response by allowing ions to pass from one side of the membrane to the other. These receptors are made up of 3 fundamental regions: the extracellular domain, where the neurotransmitter binding site or orthosteric site is located; the transmembrane domain, which forms the channel pore and contains the binding sites for different allosteric modulators; and the intracellular domain which contains modulation sites and amino acids that determine channel conductance. In vertebrates, this family of receptors consists of the nicotinic acetylcholine receptors and the serotonin receptor type 3 (5- HT3), both of which are permeable to cations, and the receptors for γ-aminobutyric acid type A (GABAA) and glycine receptors, permeable to anions. The repertoire of Cys-loop receptors in invertebrates is more varied and extensive, including among them, a serotonin-activated anion channel called MOD-1. Cys-loop receptors have been extensively studied as they are therapeutic molecular targets in a wide variety of pathologies. Expanding our knowledge on the function and modulation of these receptors will therefore contribute to generating better pharmacological strategies for the treatment of these disorders. In this Ph. D. thesis studies were carried out on the function and modulation of the Cys-loop receptors activated by human serotonin, 5-HT3, and of Caenorhabditis elegans, MOD-1, for the search for new drugs and the generation of repositioning strategies for drugs for clinical use. In Chapter 1 the molecular functionality of the human 5-HT3A receptor was studied. It was possible to decipher the differences between orthosteric activation and allosteric activation and modulation by terpenoids, using the valuable patch-clamp technique. The mechanistic bases of 5-HT3A activation by the orthosteric agonists serotonin and tryptamine, and allosteric activation by the terpenoids thymol and carvacrol, were defined. It is revealed that tryptamine is an agonist with very low efficacy and potency. Using whole-cell recordings, they show that terpenoids not only potentiate the currents evoked by serotonin but also have the ability to allosterically evoke macroscopic responses, with greater power and efficiency than tryptamine, although more slowly than serotonin. Using single channel recordings, these compounds were shown to activate the 5-HT3A receptor as highly effective agonists, since they generate longer episodes of activation than serotonin. Our study showed the first characterization at the single channel level of the activation of human 5-HT3A by allosteric ligands, of increasing interest as therapeutic tools. In Chapter 2, a promising new target for anthelmintic therapy was found by extensively studying the MOD-1 receptor of C. elegans. This nematode is a good parasitic model since it shares functional and pharmacological characteristics with parasitic nematodes. In addition, C. elegans is sensitive to most antiparasitic drugs that are directed at the Cys-loop receptors of the parasites. Since MOD-1 is absent in vertebrates, limited to the phylum, and present in parasitic nematodes, it emerges as a promising drug target. In this chapter we revealed the differences in agonist selectivity and ago-PAM sites between MOD-1 and the human serotonin 5-HT3 receptor. Tryptamine, a very partial agonist of 5-HT3A, was found to be an effective agonist of MOD-1, and was further shown to have an anthelmintic action. On the other hand, we carried out a test of a variety of compounds in search of new modulators of MOD-1. We demonstrated by means of electrophysiological studies that muscimol and piperazine (PZE) are non- competitive antagonists of MOD-1. Also, through behavioral tests in worms, we confirmed the effect of PZE on MOD-1, thus finding a new molecular target for this drug for antiparasitic use. These studies have considerably expanded the knowledge of nematode Cys-loop receptors for the discovery of new compounds with nematicidal activity. We conclude that tryptamine and PZE derivatives could be explored as new compounds with potential antiparasitic activity. In Chapter 3, we began to propose strategies to reposition drugs for clinical use in order to overcome the disadvantages of generating a new drug, shortening the time and resources necessary to perform the pharmacological, pharmacokinetic, and toxicity studies required for the production of new drugs aimed at treating pathologies in which the receptors studied in this Ph. D. thesis are involved. To this end, the effect of drugs in commercial use derived from tryptamine and PZE was explored based on the results collected in Chapter 2. It was found that sumatriptan, a drug marketed to treat migraine, appears to be a good candidate to reposition for antiparasitic therapy. In addition, PZE could be reused as a negative allosteric modulator of human 5-HT3A receptors, addressing pathologies in which this receptor is involved, such as nausea and vomiting related to chemotherapy, radiotherapy, and anesthesia, as well as to alleviate the symptoms of irritable bowel syndrome and for pathologies of the nervous system, such as depression, anxiety and schizophrenia, among others in which this receptor is involved. Summing up, taking advantage of the potential offered by single channel recordings and macroscopic currents in the study of the molecular bases of the functioning and modulation of the Cys-loop receptors explored in this Ph. D. thesis, it was possible to: i) find a new antiparasitic target, ii) to decipher for the first time the differences of orthosteric and allosteric activation of the human 5-HT3A receptor by thymol and carvacrol, and iii) to generate drug repositioning strategies for clinical use through allosteric modulation of these receptors in both C. elegans and humans. These studies broaden the knowledge of the function and modulation of Cys- loop receptors, generating a basis for addressing pathologies that involve these receptors as well as for developing new and more selective drugs.

Nervous systems are made of a large diversity of neuron types, that are characterized by specific molecular markers, their unique morphology and connectivity patterns, or their electrophysiological profiles. Those terminal differentiation features are specified by combination of transcription factors, defining genetic programs that controls neuronal identity. Some transcription factors act as "master regulators" or "terminal selectors", binding directly and continuously to many effector genes of a specific neuron type. Neuronal identity is specified by the code that the combinations of those transcription factors define. It is hard to assess to which extent those mechanisms shape more complex nervous systems, while C. elegans has a small number of well- defined neuron classes that make it more experimentally tractable. Thanks to CRISPR tagged alleles, a novel multicolor C. elegans reporter transgene strain and recent scRNA-Seq datasets, it has been possible to determine the full expression pattern of the gene family that is most likely to control neuronal identity, the highly conserved homeodomain transcription factors. From this, it is now possible to study their functional effects on neuronal identity on the full complement of C. elegans neuron types. I show that previously understudied homeodomains can indeed act as terminal selectors. I find that multiple types of neurons that had no previous known master regulators can be attributed a homeodomain regulator. I focus on the SIX homeodomain subfamily and identify a new type of subordinate transcription factor, "subtype terminal selectors". I find that such a "subtype terminal selector" is sufficient and necessary to specify a subset of features separating two very closely related neuron types, under the control of a shared terminal selector that control both shared and subtype specific features in those classes. Homeodomains remain front and center in neuronal identity control and could plausibly contribute to the specification of every single neuron class in C. elegans. Moreover, they have very conserved roles starting even in primitive nervous systems and likely played a major contribution to the evolution of cellular diversity in the nervous system. With my subtype terminal selector examples, I show possible mechanisms through which an ancestrally shared neuron type could progressively diverge towards two distinct neuron types.

No description available.

Systems neuroscience has entered in the past 20 years into an era that one might call "large scale systems neuroscience". From tuning curves and single neuron recordings there has been a conceptual shift towards a more holistic understanding of how the neural circuits work and as a result how their representations produce neural tunings. With the introduction of a plethora of datasets in various scales, modalities, animals, and systems; we as a community have witnessed invaluable insights that can be gained from the collective view of a neural circuit which was not possible with small scale experimentation. The concurrency of the advances in neural recordings such as the production of wide field imaging technologies and neuropixels with the developments in statistical machine learning and specifically deep learning has brought system neuroscience one step closer to data science. With this abundance of data, the need for developing computational models has become crucial. We need to make sense of the data, and thus we need to build models that are constrained up to the acceptable amount of biological detail and probe those models in search of neural mechanisms. This thesis consists of sections covering a wide range of ideas from computer vision, statistics, machine learning, and dynamical systems. But all of these ideas share a common purpose, which is to help automate neuroscientific experimentation process in different levels. In chapters 1, 2, and 3, I develop tools that automate the process of extracting useful information from raw neuroscience data in the model organism C. elegans. The goal of this is to avoid manual labor and pave the way for high throughput data collection aiming at better quantification of variability across the population of worms. Due to its high level of structural and functional stereotypy, and its relative simplicity, the nematode C. elegans has been an attractive model organism for systems and developmental research. With 383 neurons in males and 302 neurons in hermaphrodites, the positions and function of neurons is remarkably conserved across individuals. Furthermore, C. elegans remains the only organism for which a complete cellular, lineage, and anatomical map of the entire nervous system has been described for both sexes. Here, I describe the analysis pipeline that we developed for the recently proposed NeuroPAL technique in C. elegans. Our proposed pipeline consists of atlas building (chapter 1), registration, segmentation, neural tracking (chapter 2), and signal extraction (chapter 3). I emphasize that categorizing the analysis techniques as a pipeline consisting of the above steps is general and can be applied to virtually every single animal model and emerging imaging modality. I use the language of probabilistic generative modeling and graphical models to communicate the ideas in a rigorous form, therefore some familiarity with those concepts could help the reader navigate through the chapters of this thesis more easily. In chapters 4 and 5 I build models that aim to automate hypothesis testing and causal interrogation of neural circuits. The notion of functional connectivity (FC) has been instrumental in our understanding of how information propagates in a neural circuit. However, an important limitation is that current techniques do not dissociate between causal connections and purely functional connections with no mechanistic correspondence. I start chapter 4 by introducing causal inference as a unifying language for the following chapters. In chapter 4 I define the notion of interventional connectivity (IC) as a way to summarize the effect of stimulation in a neural circuit providing a more mechanistic description of the information flow. I then investigate which functional connectivity metrics are best predictive of IC in simulations and real data. Following this framework, I discuss how stimulations and interventions can be used to improve fitting and generalization properties of time series models. Building on the literature of model identification and active causal discovery I develop a switching time series model and a method for finding stimulation patterns that help the model to generalize to the vicinity of the observed neural trajectories. Finally in chapter 5 I develop a new FC metric that separates the transferred information from one variable to the other into unique and synergistic sources. In all projects, I have abstracted out concepts that are specific to the datasets at hand and developed the methods in the most general form. This makes the presented methods applicable to a broad range of datasets, potentially leading to new findings. In addition, all projects are accompanied with extensible and documented code packages, allowing theorists to repurpose the modules for novel applications and experimentalists to run analysis on their datasets efficiently and scalably. In summary my main contribution in this thesis are the following: 1) Building the first atlases of hermaphrodite and male C. elegans and developing a generic statistical framework for constructing atlases for a broad range of datasets. 2) Developing a semi-automated analysis pipeline for neural registration, segmentation, and tracking in C. elegans. 3) Extending the framework of non-negative matrix factorization to datasets with deformable motion and developing algorithms for joint tracking and signal demixing from videos of semi-immobilized C. elegans. 4) Defining the notion of interventional connectivity (IC) as a way to summarize the effect of stimulation in a neural circuit and investigating which functional connectivity metrics are best predictive of IC in simulations and real data. 5) Developing a switching time series model and a method for finding stimulation patterns that help the model to generalize to the vicinity of the observed neural trajectories. 6) Developing a new functional connectivity metric that separates the transferred information from one variable to the other into unique and synergistic sources. 7) Implementing extensible, well documented, open source code packages for each of the above contributions.

The type IV pilus is a motility organelle found in a range of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The principle structural protein of the pilus is the major pilin, PilA, while a set of low abundance “minor pilins” are proposed to constitute the pilus tip. The minor pilins, FimU and PilVWXE, along with the non-pilin protein PilY1, prime assembly of surface-exposed pili. The fimU-pilVWXY1E operon is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is an adhesin and mechanosensor that, along with PilW and PilX, triggers virulence upon surface attachment. Here, we aimed to uncover the mechanism for PilWXY1-mediated virulence. We hypothesized that loss of PilWXY1 would relieve feedback inhibition on FimS-AlgR, resulting in increased transcription of the minor pilin operon and dysregulation of virulence factors in the AlgR regulon. Caenorhabditis elegans slow killing assays revealed that pilW, pilX, and pilY1 mutants had reduced virulence relative to a pilA mutant, implying a role in virulence independent of pilus assembly. FimS-AlgR were required for the increased promoter activity of the minor pilin operon upon loss of pilV, pilW, pilX, or pilY1. Overexpression or hyperactivation of AlgR by point mutation led to reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on FimS-AlgR expression. We propose that PilWXY1 inhibit their own expression at the level of FimS-AlgR, such that loss of pilW, pilX, or pilY1 leads to FimS-mediated activation of AlgR, and reduced expression of acute-phase virulence factors. Accumulation of mutations in the minor pilin operon may represent an evolutionary strategy for P. aeruginosa populations in chronic lung infections, as loss of PilWXY1 would upregulate the expression of AlgR-dependent virulence factors – such as alginate – characteristic of such infections. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes dangerous infections, including lung infections in cystic fibrosis patients. The bacteria use many strategies to infect their hosts, one of which involves a grappling hook-like fibre called the type IV pilus. There are many components involved in assembly and function of the pilus, including five proteins called “minor pilins” and a larger protein called PilY1 that may help the pilus detect surface attachment. We used a roundworm infection model to show that loss of PilY1 and specific minor pilins leads to delayed killing, while loss of other pilus proteins has no effect on worm survival. This effect was due to increased activation of a regulatory system called FimS-AlgR that inhibits expression of other factors used by this bacterium to infect its hosts. By studying how P. aeruginosa causes infection, we can design better strategies to disarm it and reduce the severity of infections.

Polymicrobial interactions are an important, yet understudied area of research. Candida albicans is the most common human fungal pathogen. The bacterial genus, Enterobacter, is a source of nosocomial acquired infections and increased drug resistance. Our lab has previously discovered that Enterobacter preferentially adheres to C. albicans hyphae. From an E. cloacae transposon library screen, six candidates displayed reduction in C. albicans attachment. These candidates were identified genetically and characterized for involvement in attachment to C. albicans. A fluorescent plasmid was introduced into E. cloacae to measure and observe adherence to C. albicans in planktonic and biofilm growth. In vivo experiments using Caenorhabditis elegans showed no significant differences in microbial burden or nematode survivability exposed to Candida and Enterobacter. Candida-Enterobacter co-infections were observed microscopically within C. elegans. This study highlights the complex dynamics of C. albicans-E. cloacae interactions, underscoring the importance of understanding polymicrobial relationships in research and clinical settings.

Fisetin, a flavanol with anti-inflammatory, anti-cancer, and anti-aging properties, has shown promise for reducing fat accumulation in tissue culture and animal models. This plant sourced compound has limited studies supporting its effects on fat accumulation. Therefore, this study was completed to determine fisetin’s role in fat reduction along with its mechanism of action using Caenorhabditis elegans. C. elegans is a small roundworm with roughly 65% of its genes being conserved in humans related to disease. In this study, 100 and 200 µM fisetin has shown to reduce fat accumulation in wild-type worms. Body size, locomotion, and pumping rate were assessed in wild-type worms to determine if fisetin modified worm size, speed, and feed behavior, respectively. Mutant strains were tested to elucidate a potential pathway, of which tub-1 knockout mutants failed to reduce fat accumulation after fisetin treatment, suggesting this gene’s involvement. Gene expression of tub-1 was not altered by fisetin treatment, suggesting potential post-transcriptional regulation of fisetin. This study serves as an introduction to fisetin’s fat reducing effects via a tub-1 dependent mechanism.