The identification of antimicrobial compounds that inhibit multidrug-resistant (MDR) bacteria continues to be a significant area of research to combat the public health threat posed by MDRs. Pseudomonas aeruginosa (PA) is a Gram-negative, MDR bacterium found within both the environment and healthcare settings. Our laboratory has observed another Gram-negative bacterium, Proteus species, exerts an interesting polymicrobial interaction with Pseudomonas aeruginosa by compromising the quorum sensing (QS) factor, pyocyanin. Production of pyocyanin by Pseudomonas is a main method the bacterium uses for communication and coordination of virulence. In this study, we examined Proteus mirabilis (PM) and Proteus vulgaris (PV13) effectiveness to compromise pyocyanin production in Pseudomonas aeruginosa. Through the use of pyocyanin isolation and extraction techniques, data was gathered for Pseudomonas aeruginosa’ s molecular interaction with the two Proteus bacteria described. Further observations were made on microbial interaction between Pseudomonas aeruginosa and Proteus mirabilis through measuring the rate of metabolic activity, twitching motility rate, and observing differences in biofilm formation. Using the data obtained from this research, we hope to identify new methods of controlling Pseudomonas virulence and infection by inhibiting its ability to communicate and coordinate in polymicrobial infections.

The identification of antimicrobial compounds that inhibit multidrug-resistant (MDR) bacteria continues to be a significant area of research to combat the public health threat posed by MDRs. Pseudomonas aeruginosa is a Gram-negative, MDR bacterium found within both the environment and healthcare settings. Our laboratory has observed another Gram-negative bacterium, Proteus species, exerts an interesting polymicrobial interaction with Pseudomonas aeruginosa by potentially compromising the quorum sensing (QS) factor, pyocyanin, of Pseudomonas aeruginosa. Production of pyocyanin by Pseudomonas is the main method the bacterium uses for communication and coordination of virulence. In this study, we examined the effectiveness of Proteus mirabilis and Proteus vulgaris to compromise pyocyanin production in Pseudomonas aeruginosa. Through the use of pyocyanin isolation and extraction techniques, data was gathered for Pseudomonas aeruginosa’ s molecular interaction with the two Proteus species of bacteria. Further observations were made on microbial interaction between Pseudomonas aeruginosa and Proteus mirabilis through measuring the rate of metabolic activity, twitching motility rate, and observing differences in biofilm formation. Using the data obtained from this research, we hope to identify new methods of controlling Pseudomonas virulence and infection by inhibiting its ability to communicate and coordinate in polymicrobial infections

<p>Although there are many examples of microbial biogeography, few microbes have been studied at high taxonomic resolution over large spatial scales. As a result, the environmental and ecological processes that drive niche partitioning, diversity, composition, and activity of microbial taxa are often poorly understood. To address this gap, I examine the most abundant phytoplankton in the global ocean, Prochlorococcus sp., a marine cyanobacterium. Using amplicon libraries of the Prochlorococcus internal transcribed spacer (ITS) region and 23S rRNA gene as markers, I demonstrate several key differences between the two major high light (HL) clades of Prochlorococcus. First, by examining ITS amplicon libraries at high taxonomic resolution it is revealed that “sub-ecotype” clades have unique, cohesive responses to environmental variables and distinct biogeographies, suggesting that presently defined ecotypes can be further partitioned into ecologically meaningful units. Whereas unique combinations of environmental traits drive the distribution of the HL-I sub-ecotype clades, the HL-II sub-ecotype clades appear ecologically coherent. Second, using 23S rRNA and rDNA libraries I show that activity (rRNA) and abundance (rDNA) are highly correlated for Prochlorococcus across all sites and operational taxonomic units (OTUs) in the surface ocean, demonstrating a tight coupling between activity and abundance. Finally, I investigate the associations between Prochlorococcus and the rest of the microbial community in the North Pacific and find region-specific trends in both strength and sign. Associations with other microbes are strongest for HL-I in the temperate region and strongest for HL-II in the sub-tropical gyre. This dissertation clarifies the relative importance of the environment, geography, community, and taxonomy in terms of their role in creating complex assemblages of Prochlorococcus and helps improve our understanding of how marine microbial communities are assembled in situ.</p> / Dissertation

A crescente resistência dos micro-organismos patogênicos aos fármacos já existentes gera intensa demanda por novos agentes terapêuticos. Em contrapartida, a eficiência na descoberta de compostos com novas estruturas químicas diminuiu nos últimos anos. Tendo em vista a vasta biodiversidade existente de micro-organismos, a redução da eficiência na descoberta de novos produtos naturais não indica que todos compostos existentes já foram descritos, mas sim que as metodologias para isolamento dos mesmos devem ser aperfeiçoadas e diversificadas, e novos nichos devem ser explorados. Esta tese compreende três capítulos, que trazem abordagens que podem ser utilizadas na busca por produtos naturais. O capítulo 1 aborda a aplicação de conhecimentos de biologia molecular na pesquisa de produtos naturais, através da metagenômica. O capítulo 2 aborda o conceito de química ecológica para estimular o metabolismo secundário, através da utilização de interações microbianas. O capítulo 3 aborda o uso de genome mining para entender a capacidade metabólica de uma linhagem bacteriana, bem como o uso de variações nos parâmetros da cultura para alterar o metabolismo desta. Metagenômica: a triagem anti-parasitária das bibliotecas metagenômicas detectou clones bioativos contra Leishmania major. As análises químicas e biológicas das culturas destes clones não permitiram a identificação dos compostos responsáveis pelas atividades observadas. Esta abordagem apresenta grandes desafios técnicos e tem passado por ajustes envolvendo sequenciamento e bioinformática para aumentar a taxa de sucesso em seu uso. Interações microbianas: esta metodologia mostrou-se promissora para a busca por compostos bioativos, tendo em vista que diversos pares exibiram nova atividade antibiótica, corroborando a hipótese que interações microbianas podem levar à expressão diferenciada do metabolismo secundário. Foi escolhida para caracterização química e biológica a interação entre uma actinobactéria rara (Krasilnikovia sp. T082) e uma actinobactéria endossimbionte de besouro (Streptomyces sp. SPB78). Esta interação é robusta e estimula a biossíntese de um antibiótico polar capaz de inibir o crescimento de uma bactéria multirresistente. Diversas técnicas foram testadas para o isolamento do composto indutor e do antibiótico induzido. Este processo foi desafiador devido ao caráter polar de ambos compostos e pelo fato da atividade antibiótica ser instável. Foi demonstrado que o antibiótico induzido é capaz de inibir o crescimento do micro-organismo indutor, sugerindo importância ecológica deste composto. A utilização desta abordagem metodológica permitiu a identificação de uma linhagem pertencente a um gênero de actinobactéria rara que nunca teve seu metabolismo secundário estudado (Krasilnikovia). Isto foi realizado através da investigação de seu genoma e também através do isolamento de compostos produzidos por esta linhagem. Esta linhagem demonstrou potencial para a produção de metabólitos secundários, apresentando pelo menos 21 potenciais clusters gênicos biossintéticos detectados pelo antiSMASH em seu genoma. Esta linhagem foi cultivada em dois meios de cultura (ISP2 e TSB) e diferentes metodologias de extração foram empregadas. O metabolismo secundário desta linhagem é expresso de maneira diferente de acordo com a metodologia de cultivo, evidenciando a importância da variação da composição do meio de cultura para o acesso da real capacidade metabólica de microorganismos. Foram identificadas algumas dicetopiperazinas, que são conhecidas por seu amplo espectro de atividades biológicas, e três compostos pertencentes a uma classe de peptídeos nãoribossomais não usuais com atividade antibiótica, que possuem estrutura química complexa e incomum para produtos naturais / Increasing drug resistance among microbial pathogens is a public health threat; therefore, new antibiotics are needed. On the other hand, the rate of discovering new compounds has diminished. Considering the wide biodiversity of microorganisms, reduced efficiency on the discovery of new natural products does not indicate that all existing compounds have been described, but that methods for isolation should be improved and diversified and new niches should be explored. This thesis comprises three chapters that demonstrate approaches that can be used in the search for natural products. Chapter 1 demonstrates the application of molecular biology tools on the search for natural products through metagenomics. Chapter 2 discusses the concept of chemical ecology for the stimulation of secondary metabolism by the use of microbial interactions. Chapter 3 discusses the use of genome mining to understand the metabolic capacity of a bacterial strain as well as the use of different culture parameters to alter bacterial metabolism. The anti-parasitic metagenomic screening on metagenomic libraries detected bioactive clones against L. major. Chemical and biological analysis of cultures of these clones did not permit identifying the compounds responsible for the observed activities. This approach faces diverse technical challenges and is currently being improved by the use of sequencing and bioinformatics analysis in order to increase its hit rate. Microbial interactions: this approach has shown to be promising in the search for bioactive compounds, considering that several pairs exhibited new antibiotic activity, supporting the hypothesis that microbial interactions can stimulate differential expression of secondary metabolism. It was chosen for chemical and biological characterization the interaction between a rare actinobacteria (Krasilnikovia sp. T082), which belongs to a genus that its secondary metabolism has not yet been studied in the literature, and an endosymbiont actinobacteria of beetle (Streptomyces sp. SPB78). This interaction is robust and stimulates the biosynthesis of a polar antibiotic capable of inhibiting the growth of multi-resistant bacteria. Several techniques have been tested for the isolation process of the inducer compound and the induced antibiotic. This process has been particularly challenging due to the polar character of both compounds, and because the antibiotic activity is unstable. It has been shown that the induced antibiotic is capable of inhibiting the growth of the inducer microorganism, suggesting an ecological importance of this compound. The use of this co-culture approach led to the identification of a strain belonging to a rare actinobacteria genus whose secondary metabolism has never been studied before (Krasilnikovia). This was accomplished through sequencing and analysis of its genome and also by isolating compounds produced by this strain (chapter 3). This strain has shown great potential for the production of secondary metabolites, exhibiting at least 21 biosynthetic gene clusters detected by antiSMASH in its genome, and only four of them showed high similarity to any known gene cluster. This strain was cultured in two different culture media (ISP2 and TSB), and different methods of extraction were used. The secondary metabolism of this strain is expressed differently according to the method of cultivation, showing the importance of variation in the composition of the culture medium to access the actual metabolic capacity of microorganisms. Some diketopiperazine were isolated, which are known for their wide spectrum of biological activities, and also three compounds belonging to a class of non-ribosomal peptides known for their high bioactivity, which have complex and unusual chemical structures for natural products

Burkholderia seminalis tem sido encontrada tanto em interações patogênicas, quanto não patogênicas. O gene wcbE codifica uma glicosiltransferase e pertence ao cluster wcb, que está relacionado à síntese de cápsula. O objetivo deste trabalho foi investigar o papel do gene wcbE e da temperatura nas interações microbianas de B. seminalis TC3.4.2R3. A produção de biofilme, EPS e compostos antifúngicos foi maior a 28 ºC. Por outro lado, a motilidade, virulência e respostas ao estresse foram maiores a 37 ºC. wcbE produziu menos biofilme que WT e foi atenuada em G. mellonella a 37 ºC, destacando a importância da glicosiltransferase na patogênese. Além disso, wcbE perdeu a habilidade de inibir fungos fitopatogênicos. Embora B. seminalis seja um membro do Bcc, é eficiente contra patógenos clínicos e ambientais, indicando que esta linhagem pode ter interações múltiplas no ambiente. A temperatura e o gene de glicosiltransferase desempenharam um papel crucial nas interações ambientais de B. seminalis TC3.4.2R3. / Burkholderia seminalis has been found in both pathogenic and nonpathogenic interactions. The wcbE gene encodes a glycosyltransferase and belongs to the wcb cluster, which is related to capsule synthesis. The aim of this work was to investigate the role of the wcbE gene and temperature in the microbial interactions of B. seminalis TC3.4.2R3. The production of biofilm, EPS and antifungal compounds was higher at 28 ºC. On the other hand, the motility, virulence and stress responses were higher at 37 ° C. wcbE produced less biofilm than WT and was attenuated in G. mellonella at 37 ° C, highlighting the importance of glycosyltransferase in the pathogenesis. Furthermore, wcbE lost the ability to inhibit phytopathogenic fungi. Although B. seminalis is a member of Bcc, it is effective against clinical and environmental pathogens, indicating that this strain may have multiple interactions in the environment. The temperature and the glycosyltransferase gene played a crucial role in the environmental interactions of B. seminalis TC3.4.2R3.

Con il primo lavoro è stato identificato un tratto fenotipico di un ceppo di L.crispatus associato alla capacità di persistere e colonizzare il colon dell’ospite e di modificarene la composizione microbica, tale L.crispatus M247 è in grado di modificare, nell’epitelio del colon, il livello di espressione dei TLR2 dei TLR4 sia in vitro che in vivo. Con il secondo studio si identifica un meccanismo antinfiammatorio, prima sconosciuto, indotto da un ceppo probiotico che coinvolge l’attivazione di PPAR-γ e fornisce una nuova visuale sui meccanismi molecolari coinvolti nel dialogo tra epitelio intestinale e microbiota simbionte. / The colonic microbiota is a major modulator of the mucosal immune system; therefore, its manipulation through supplementation with probiotics may significantly affect the host’s immune responses. Since different probiotics seem to exert various effects in vivo, we tested the relevance of the autoaggregation phenotype on the intestinal persistence of lactobacilli and their ability to modulate the host’s innate immune responses. After 14 days of diet supplementation, the aggregating strain Lactobacillus crispatus M247 but not aggregation-deficient isogenic mutant MU5 was recovered from the feces and colonic mucosa of mice. This observation was confirmed by strain-specific PCR amplification and by Lactobacillus-specific denaturing gradient gel electrophoresis analysis. Indeed, L. crispatus M247 increased Toll-like receptor 2 (TLR2) mRNA levels, while it reduced TLR4 mRNA and protein levels in the colonic mucosa, whereas MU5 was ineffective. In colonic epithelial cells (CMT-93 cells) L. crispatus M247 but not MU5 induced time-dependent extracellular signal-regulated kinase-1 (ERK1) tyrosine phosphorylation and TLR modulation, which were abolished in the presence of PD98059 (an ERK1 inhibitor). To assess the functional relevance of probiotic-induced TLR modulation, we determined the consequences of L. crispatus preexposure on TLR4 (lipopolysaccharide [LPS]) and TLR2 [Pam3Cys-Ser-(Lys)4] ligand-mediated effects in intestinal epithelial cells. Preexposure to L. crispatus M247 blunted LPS-induced interleukin-6 (IL-6) release and inhibition of CMT-93 migration over a wound edge, whereas it enhanced TLR2-mediated IL-10 up-regulation. In summary, the aggregation phenotype is required for L. crispatus persistence in the colon and for modulation of TLR2/TLR4 expression through an ERK-dependent pathway. We speculate that the aggregation phenotype in L. crispatus M247 is required to temper epithelial cell responsiveness to bacterial endotoxins, which thus affects the evolution of intestinal inflammatory processes. Accumulating evidence indicates that the peroxisome proliferator activated receptor (PPAR)- is a major player in maintaining intestinal mucosa homeostasis, but whether PPAR- is directly involved in probiotic-mediated effects and the molecular events involved in its activation are not known. Methods: We investigated the role of PPAR- in the immunomodulatory effects of Lactobacillus crispatus M247 on intestinal epithelial cells (IEC) and the role of probiotic-derived H2O2 on PPAR- activity. Results: L crispatus M247 supplementation in mice significantly increased PPAR- levels and transcriptional activity in the colonic mucosa. L crispatus M247 induced PPAR- nuclear translocation and enhanced transcriptional activity in epithelial (CMT-93) cells, as demonstrated by the increased luciferase activity of a PPAR- –responsive element, PPAR- – responsive gene up-regulation, and reduced activity of an nuclear factor- B–responsive element. Pharmacologic PPAR- inhibition or silencing by small interfering RNA cancelled the L crispatus M247–mediated effects in CMT-93 cells. Because Lactobacillus strains producing little H2O2 failed to activate PPAR- , we investigated the role of L crispatus M247– derived H2O2 in PPAR- activation. L crispatus M247 induced a transient rise in intracellular H2O2 and PPAR- transcriptional activity was cancelled by antioxidant or H2O2 scavenger. Toll-like receptor (TLR)-2 was not required for PPAR- up-regulation mediated by L crispatus M247 in mice, although the protective effects of L crispatus M247 on dextran sodium sulfate-induced colitis were less pronounced in TLR-2 / mice. Conclusions: L crispatus M247 uses H2O2 as a signal transducing molecule to induce PPAR- activation in IEC, directly modulating epithelial cell responsiveness to inflammatory stimuli.

Microorganisms live in complex communities and interact either through secreting soluble molecules or by delivering effectors in a contact dependent manner. Microbial interactions range from cooperative to competitive. Contact-dependent growth inhibition (CDI), discovered in Escherichia coli EC93, is becoming increasingly studied, as this mode of interaction seems to be widespread among proteobacteria. CDI is mediated by cdiBAI genes which encode for a two-partner secretion system; i.e. CdiB is an outer membrane protein that transports CdiA to the surface of the cell. CdiA can interact with a specific receptor on a target cell and deliver a toxin localized in its C-terminal domain to the target cell. CdiI is a small immunity protein that neutralizes the toxic effect of CdiA toxin. Recently, evidence from our research group has shown that E. coli EC93 harbours two cdi loci. The first cdi locus has been extensively studied but the role of second locus remained unknown. In this study we wanted to elucidate the activity and the role of second E. coli EC93 cdi locus in intra-strain bacterial interactions. Bacterial competitions of E. coli EC93 wild type versus E. coli EC93 targets that had deletions for one or both cdi loci showed that the second locus is indeed active in inhibiting the targets, albeit to a lesser extent than the first. The toxic activity of the second cdi-locus was neutralized specifically by the second immunity protein. The expression of both these systems is higher under carbon starvation conditions than in nutrient rich conditions. Unfortunately, we could not elucidate the mechanism of toxicity for the second cdi locus toxin. Taken together, our results show that E. coli EC93 actively uses both of its cdi loci during bacterial interactions and that these systems are more active during stressful conditions.

La disponibilité en eau exerce un contrôle majeur sur les cycles des nutriments terrestres, à travers ses impacts sur le fonctionnement des plantes et des microorganismes du sol. Les changements de magnitude et de fréquence des épisodes de pluie (c’est-à-dire les régimes de précipitations) prédits par les modèles et associés au changement climatique vont ainsi avoir des conséquences importantes sur le fonctionnement des écosystèmes. Les écosystèmes arides et semi-arides sont particulièrement vulnérables à des changements de régime de précipitations, car ils sont déjà contraints par la disponibilité en eau. Cependant, des systèmes plus tempérés peuvent aussi être soumis à des périodes sèches qui peuvent affecter le fonctionnement plante-sol. Dans la présente thèse, les effets d’un historique de régimes de précipitations contrastés ont été étudiés dans des systèmes sol seul et plante-sol, afin de déterminer dans quelle mesure plusieurs semaines de régime hydrique peuvent moduler la réponse des écosystèmes à une réhumectation lors d’un événement pluvieux important. Premièrement, nous avons évalué les effets de régimes de précipitations contrastés dans des mésocosmes de sol seul, sur les communautés bactériennes et fongiques actives et inactives dans le sol, 2 et 5 jours après réhumectation. Nous avons employé une approche de 18O-SIP (stable isotope probing), en réhumectant le sol avec H218O puis en utilisant la métagénomique ciblée sur les bactéries et champignons du sol. Deuxièmement, nous avons mis en place deux expériences séparées en mésocosmes plante-sol avec couvert de blé. La première expérience sol-plante s’est intéressée à la profondeur de sol. Nous avons évalué les effets de régimes de précipitations contrastés sur le flux de C depuis les plantes vers les microorganismes du sol ainsi que la la réponse des microorganismes à différentes profondeurs de sol (de 0 à 35 cm) en utilisant des approches de traceur isotopiques stables (13C-CO2) et 18O-SIP, respectivement. La deuxième expérience plante-sol a évalué les effets de régimes de précipitations contrastés sur la dynamique temporelle (durant 29h) de la réponse du système plante-sol à la réhumectation. En outre, deux niveaux de fertilisation azotée ont permis de déterminer l’éventuelle modulation de la réponse par la disponibilité en N dans le sol. La réponse des communautés bactériennes et fongiques potentiellement actives dans le sol a été évaluée par métagénomique ciblée. La réponse de cycles biogéochimiques a été évaluée à l’aide de traceurs isotopiques stables (13C-CO2 et 15N- NO3-) pour quantifier le flux de C des plantes vers les microorganismes du sol et déterminer la compétition plantes-microorganismes du sol au cours du temps après réhumectation.Nos résultats ont montré un contrôle du régime de précipitation sur la morphologie et physiologie des plantes, les communautés microbiennes du sol ainsi que sur le cycle de l’azote du sol dans nos systèmes. En particulier, des régimes de précipitations peu fréquentes (cycles de périodes sèches longues suivies de périodes de pluie plus importantes) se sont traduits par une augmentation des potentiels de transformation de l’azote dans le sol et une réduction des stocks d’azote minéral dans le sol. Ceci a façonné l’environnement de la réponse de nos systèmes à la réhumectation, que nous avons évaluée en déterminant les dynamiques du C (couplage plantes-microbes et émissions de CO2 du sol), de l’azote du sol (compétition plantes-microorganismes du sol pour le N et émissions de N2O) et de la composition des communautés microbiennes du sol (bactéries et champignons actifs et potentiellement actifs) après réhumectation (...). / Water availability governs terrestrial nutrient cycles by impacting the functioning of both plants and of soil microorganisms. The predicted changes in precipitation patterns (i.e. the magnitude and frequency of precipitation events) associated with climate change, will thus likely have important consequences on ecosystem functioning. Dry and seasonally dry ecosystems are particularly vulnerable to changes in precipitation patterns, as they are already constrained to a large extent by water availability. However, more mesic systems may also experience dry periods that may impact plant-soil functions. In this thesis, experiments in soil-only systems and plant-soil systems were used to gain insight into how the legacy effects of several weeks of exposure to contrasted precipitation patterns set the scene for the rewetting response of the system. First, in an experiment using soil-only mesocosms, we evaluated the effects of contrasting precipitation regimes on the actively growing as well as the inactive bacterial and fungal communities 2 and 5 days after rewetting, using an 18O-SIP (stable isotope probing) approach by applying H218O followed by metagenomics targeting soil bacteria and fungi. Second, we performed two separate and complementary experiments using plant-soil mesocosms with wheat plant cover. The first plant-soil experiment focused on soil depth. It determined the effects of contrasting precipitation patterns on the flux of C from plants to microbes and the microbial response to rewetting at different soil depths, using a heavy isotope tracer approach (13C-CO2) and 18O-SIP with metagenomics respectively. The second plant-soil experiment evaluated the effects of a history of contrasting precipitation patterns on the dynamics of the rewetting response of the plant-soil system over time (over 29 hours post-rewetting). In addition, two levels of N inputs allowed to determine how N availability modulated plant-soil responses. The response of the potentially active soil bacterial and fungal communities to rewetting was assessed using targeted metagenomics. The responses of biogeochemical cycles were evaluated using heavy isotope tracers (13C-CO2 and 15N-NO3-) to quantify C flux from plants to soil microorganisms and plant-microbial competition for N over time post-rewetting.We found that precipitation patterns shaped plant morphology and physiology, microbial community composition as well as soil N cycling in our systems, which set contrasting scenes for the rewetting responses in our systems. In particular, infrequent precipitation patterns (cycles of longer dry periods followed by larger magnitude rain events) resulted in increased microbial N transformation potentials and smaller inorganic N pools. The rewetting responses were determined by evaluating C dynamics (plant-microbial coupling and soil CO2 efflux rate), N dynamics (plant-microbial competition for N and soil N2O efflux rate) and microbial dynamics (composition of active and potentially active bacterial and fungal communities after rewetting). First, we found that plant-microbial coupling (i.e the microbial assimilation of C from fresh photosynthate) may be reduced under more infrequent precipitation patterns, especially near the soil surface, and under conditions of low N availability. Our findings also suggest that whilst in soil-only systems, dead microbial cells appear to be a major source fuelling soil CO2 efflux pulse upon rewetting, in plant-soil systems root respiration plays an important role in the magnitude of the CO2 efflux upon rewetting. Second, concerning soil N dynamics, we found, in concurrence with previous studies, that soil microorganisms were the stronger competitor for N over short time scales, likely due to their overall fast response rates and high affinity for substrate, whilst plants outcompeted soil microbes for soil N assimilation, over longer time scales likely taking advantage of the fast microbial turnover (...).

Thesis advisor: Babak Momeni / Microbial communities are complex networks comprised of multiple species that are facilitating and inhibiting one another (as well as themselves). Currently, we lack an understanding of what mechanisms drive coexistence within these communities. We aimed to remedy this by studying the dynamics of coexisting communities, focusing on the complexity of their interaction networks, the impact of spatial dynamics, and the interplay of facilitating and inhibiting interactions. These limitations in our understanding prevent the furtherment of designing intentional communities for bioremediation, maintenance of healthy microbiota, and other functional communities. To better understand these microbial dynamics, we chose to address the problem from two fronts: computational modeling and exploring dynamics of cocultures. Through our 1-D model, spatial structure fostering more coexistence – especially when facilitation is present. For the coexistence assays, we determined that contact-dependent growth inhibition is a density dependent mechanism, and the use of a Tn-Seq mutant library to predict species interactions is possible, but needs further optimization to reconcile density dependent effects of interactions. / Thesis (MS) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Babak Momeni / Diseases of the upper respiratory tract encompass a plethora of complex multifaceted etiologies ranging from acute viral and bacterial infections to chronic diseases of the lung and nasal cavity. Due to this inherent complexity, typical treatments often fail in the face of recalcitrant infections and/or severe forms of chronic disease, including asthma. Thus, in order to provide improved standard of care, the mechanisms at play in hard-to-treat etiologies must be better understood. More recently, research has demonstrated a significant association between microbiota and many URT diseases. Previous work has also identified species capable of directly inhibiting standard treatments used to control asthma exacerbations. Despite an exhaustive collection of data characterizing microbiota composition in states of both health and disease, our knowledge of what microbiota profiles are observed in what specific disease etiologies is severely lacking. Yet, gaining these insights is crucial for the translation of such data into application. In this thesis I sought to: 1) identify gut microbiota profiles associated with severe and treatment resistant forms of childhood asthma, and 2) formulate a predictive model to facilitate the restructuring of microbiota for desired therapeutic outcomes. To identify gut microbiota and metabolites enriched in severe and treatment resistant childhood asthma, I looked to an ongoing longitudinal human study on vitamin D and childhood asthma. In this study, I find several fecal bacterial taxa and metabolites associated with more severe (i.e., higher wheeze proportion) and treatment resistant asthma in children at age 3 years. Specifically, several Veillonella species were enriched in children with higher wheeze proportion and in children that responded poorly to inhaled corticosteroid treatment (ICS) (i.e., non-responders). Haemophilus parainfluenzae, a species previously identified as enriched in the airway of adults with ICS-resistant asthma, was also uniquely enriched in children considered ICS non-responders in this study. Several metabolic pathways were also distinctly enriched: histidine metabolism was enriched in children with higher wheeze proportion while sphingolipid metabolism was enriched in ICS non-responders. Both metabolic pathways have been previously identified in association with asthma, further corroborating their role in this disease. Yet, this study is the first to identify these taxa and metabolites in children with preexisting and treatment resistant asthma. In the pursuit of improved treatment outcomes for recalcitrant URT diseases, recent efforts have turned towards microbiota-based therapies. While such treatments have proven successful in the treatment of gastrointestinal infections, these methods have not yet been extended to other conditions. Considering this, I ask whether a predictive model describing microbial interactions can facilitate the restructuring of microbiota for desired therapeutic outcomes. For this, I use a community of nasal microbiota to determine when a simply Lotka-Volterra-like (LV) model is a suitable representation for microbial interactions. I then utilize our LV-like model to examine whether environmental fluctuations have a major influence on community assembly and composition. For this, I looked specifically at pH fluctuations. In this study, I found that LV-like models are most suitable for describing community dynamics in complex low nutrient conditions. I also identified simple in vitro experiments that can reliably predict the suitability of a LV-like model for describing outcomes of a two-species community. When our LV-like model was applied to an in silico community of nasal species to determine the impact of environmental fluctuations, I find that nasal communities are generally robust against pH fluctuations and that, in this condition, facilitative interactions are a stabilizing force, and thus, selected for in in silico enrichment experiments. Overall, this thesis further corroborates the association of microbiota with URT diseases and treatment outcomes while also providing unique insight into their association with specific etiologies in childhood asthma. This thesis also provides a framework for developing models able to facilitate the development of future microbiota-based therapies while also determining how, and when, environmental factors impact community assembly and composition. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.