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Background: The composition of the vaginal microbiota (VMB) in the female genital tract (FGT) can impact the vaginal epithelium and protect against or increase risk of sexually transmitted viral infections. The VMB grows as a biofilm, a complex structure formed by bacteria for increased survival. When the VMB consists of a diverse bacterial community it correlates with pathogenic effects that lead to adverse health conditions and an increased risk of HIV infection. When the VMB contains Lactobacillus species, beneficial health effects and decreased susceptibility to infection are observed. The aim of this project is to optimize an in vitro model of biofilm formation for different bacteria associated with the VMB, identify the effects that biofilm has on vaginal epithelial cells and test biofilm treatment strategies. We hypothesize that a Lactobacillus biofilm will enhance barrier function and decrease cytotoxicity of vaginal epithelial cells whereas dysbiotic biofilm will decrease barrier function and induce cytotoxicity. We also hypothesize that various conditions, such as presence of estradiol and eubiotic short-chain fatty acids, will stimulate Lactobacillus biofilm growth and suppress dysbiotic biofilm growth in a vaginal epithelial cell model. Methods: For optimization of the biofilm model, VK2/E6E7 cells were grown in air-liquid interface (ALI) or liquid-liquid interface (LLI) cultures in presence or absence of L. crispatus, L. iners, G. vaginalis or P. bivia bacteria. Biofilm formation was assessed using FilmTracerTM SYPRO® Ruby biofilm matrix protein stain. Hormone effects were tested by adding estradiol (10-9 M) and progesterone (10-7 M) to culture media. Short-chain fatty acid (SCFA) effects were tested by adding lactic acid, acetic acid, succinic acid and butyric acid in varying concentrations to culture media. Enzyme effects were tested by adding sialidase to Vk2 cells before bacteria inoculation. Results: A novel in vitro model of biofilm formation on vaginal epithelial cells was created. Vk2 cells in ALI and LLI cultures remained viable in anaerobic conditions and showed mucin-1 production in aerobic and anaerobic conditions. Matrix protein staining provided a means to accurately visualize and quantify biofilm formation in this model. L. crispatus and L. iners biofilm growth maintained vaginal epithelial barrier integrity without cytotoxicity. G. vaginalis and P. bivia biofilm growth significantly reduced barrier integrity (p=0.0166, p=0.0115) and increased cytotoxicity (p=0.0024, p<0.0001). Estradiol significantly increased the growth of L. crispatus biofilm in the co-culture system (p<0.0001). Progesterone significantly increased G. vaginalis biofilm growth in the Vk2 cell co-culture (p=0.006). L. crispatus biofilm formation in the estradiol condition, G. vaginalis biofilm formation in the progesterone condition and P. bivia biofilm growth in the normal media condition were significantly decreased in the presence of sialidase (p<0.0001, p=0.0001, p=0.0380). Conclusion: A novel in vitro model of biofilm formation on a vaginal epithelial cell line that can be used to visualize and quantify biofilm growth was generated. This model was used to test various strategies for biofilm enhancement or dissociation. Estradiol enhanced beneficial Lactobacillus biofilm growth, while progesterone enhanced dysbiotic biofilm growth. Mucin- digesting enzyme sialidase was effective at dissociating all biofilms. This model can be used in the future to test different strategies of dysbiotic biofilm dissociation and enhancement of Lactobacillus biofilm in order to investigate treatments for Bacterial Vaginosis (BV) and reduce susceptibility to HIV transmission in women. / Thesis / Master of Science (MSc)

<p>Hydrogen sulfide (H₂S) is an important modulator of many aspects of digestive function, both in health and disease. Colonic tissue H₂S synthesis increases markedly during injury and inflammation and contributes to resolution. Some of the bacteria residing in the colon also produce H₂S. The extent to which bacterial H₂S synthesis contributes to what is measured as colonic H₂S synthesis is not clear. When comparing conventional and germ-free mice we found no differences in colonic H₂S synthesis. Furthermore, we found that colonic H₂S synthesis is markedly increased when colonic tissue is inflamed, and, in proportion to the extent of inflammation, however fecal H₂S synthesis does not change. Finally, rats fed a B vitamin-deficient diet for 6 weeks exhibited significantly diminished colonic H₂S synthesis, but fecal H₂S synthesis was not different from that of rats on the control diet. Our results demonstrate that H₂S production by colonic bacteria does not contribute significantly to what we measure as colonic tissue H₂S production.</p> <p>In another study, the contributions of three enzymatic pathways to colonic H₂S synthesis were determined in tissues taken from healthy rats and rats with colitis.<strong> </strong>The ability of colonic tissue to inactivate H₂S was also determined. The majority of increased H₂S synthesis, in both healthy and inflamed tissue, was derived via a pyroxidal-5’-phosphate-independent pathway. Ulcerated mucosal tissue accounted for the greatest levels of H₂S synthesis, and the extent of granulocyte infiltration into the tissue did not appear to be a significant determinant of the levels of H₂S production. Inactivation of H₂S by colonic tissue occurred rapidly, but was significantly reduced in ulcerated colonic tissue from rats with colitis. Damage to colonic tissue appears to be the major stimulus for enhanced H₂S synthesis. Together, the increased production and decreased inactivation of H₂S may contribute to promoting resolution of inflammation and repair of damaged colonic tissue.</p> / Master of Science (MSc)

Gut microbiota has been shown to regulate the growth and development of the central and enteric nervous systems (CNS and ENS) after birth. There is ample evidence to suggest that intestinal bacteria can influence behavior of the host through both immune and immune-independent mechanisms. Gut-microbiota-regulated CNS structural changes are focused in the limbic system, at centres associated with memory, social and emotional behaviour. Several studies using germ-free (GF) and specific pathogen free (SPF) mice demonstrated microbial influence on behaviour development accompanied by neurochemical changes in the brain. Higher exploratory and lower anxiety-like behavior was found in GF mice compared to SPF mice with lower central expression of neurotrophins, such as nerve growth factor and BDNF. The mechanisms by which the microbiota influences behavior are unknown but could be immune-mediated, neural, or humoral in origin. In this study I investigated the role of immune system maturation on mouse behaviour after bacterial colonization. I showed that mono-colonization of GF mice with E. coli normalizes behaviour similar to colonization with complex microbiota (SPF and ASF) and the continuous presence of bacteria is not required to maintain this normal behaviour. I also showed that innate immunity through the MyD88/Ticam pathway is crucial for the development of normal behaviour and that multiple innate immunity and neuronal genes are involved in this process. Together these results suggest that bacterial colonization primes and matures the innate immunity and development of the central nervous system ultimately leading to normal behaviour. I believe that bacterial colonization at birth is not only important for the epithelial barrier function, gut homeostasis, and immune functions, but also for the development of normal behaviour. Altered immune priming during the postnatal period due to abnormal microbial colonization may have wider implications when considering psychiatric disorders in humans. / Thesis / Doctor of Philosophy (PhD)

Exercise is known to induce changes in the gut, typically referred to as the ‘forgotten organ’, and changes in gut microbiota can also occur with exercise possibly imparting systemic benefits. The question remains whether or not microbiota from an exercised animal can independently affect skeletal muscle morphology. Our first objective was to examine whether an endurance exercise program could modify the microbiota in donor mice. Second, we aimed to elucidate if such an endurance-trained microbiota could be transferred to germ-free mice via fecal inoculation. Finally, we sought to determine how the morphology and functional characteristics of skeletal muscle were influenced as a result of fecal inoculation. We hypothesized that germ-free mice recipients inoculated with the microbiota from endurance trained donors would undergo morphological changes in muscle fibre type composition and physiological changes in skeletal muscle function associated with a more oxidative phenotype. Eight-week-old male C57BL/6NCrl donor mice (n = 20) were randomized into two groups: one group completed an endurance exercise training protocol on a treadmill machine 3x/week for 11 weeks (n = 10) while one group remained cage-bound (n = 10). Ten-week-old male (n = 7) and female (n = 9) germ-free mice were colonized with the cecal microbiota of the donor mice in that, equal numbers of germ-free mice were inoculated with exercised-microbiota as sedentary-microbiota. Glucose metabolism and performance measures were evaluated in the donors as well as the recipients post-inoculation. Muscle tissue was extracted for immunohistochemistry and mitochondrial assays. During the intra-peritoneal glucose tolerance test (IPGTT), significant differences in blood glucose were found at 30min between exercise-inoculated and sedentary-inoculated (23.4 ± 2.2; 29.0 ± 1.9 mmol/L, p<0.05).and change in blood glucose relative to baseline (12.04 ± 2.4; 18.3 ± 1.9 mmol/L, p<0.01). There were significant sex-based differences in the blood glucose response in inoculated animals such that there were significant differences in blood glucose between the exercise-inoculated females and sedentary-inoculated females at 15mins (28.4 ± 2.4; 30.6 ± 1.1 mmol/L, p<0.05) and 30mins (24.7 ± 3.6; 29.9 ± 2.4 mmol/L, p<0.01), however no differences between exercise-inoculated males and sedentary-inoculated males. In addition, there were significant differences in the change in blood glucose relative to baseline between the exercise-inoculated females and sedentary-inoculated females at 15mins (12.3 ± 1.9; 20.6 ± 0.8 mmol/L, p<0.01) and 30mins (10.2 ± 2.6; 19.9 ± 2.1 mmol/L, p<0.001). This novel characterization of a link between gut microbiota and skeletal muscle suggests a transmissible capacity of microbiota to impart properties of ‘healthy’ muscle into compromised populations. / Thesis / Master of Science (MSc) / The gut microbiome or microbiota describes the composition of the human gut – remarkably, over 100 trillion bacterial cells live in symbiosis with the cells of the human body. Research from the past decade has elucidated the salient nature of the human gut microbiome on immunity, metabolic homeostasis, and overall health and disease. Transformative research in the field has demonstrated the ability to transfer these bacterial colonies from one individual to another and elicit change, such as altering body mass and adiposity, respective to their donor. The interaction between gut microbiota and other organ systems i.e. brain, liver, adipose tissue has been the focus of several recent investigations, suggesting that lifestyle changes such as diet and exercise can influence communication between the gut and various other organs and contribute to changes in function. Skeletal muscle is the largest muscle in the human body accounting for 40% of total mass and although the main role of skeletal muscle is locomotion and postural stabilization, it is integral for the regulation of blood glucose as well as a reservoir for other macronutrients. Acute and chronic physical exercise cause a myriad of adaptive responses throughout the human body including in skeletal muscle and the gut. Therefore, the existence and influence of a gut-muscle link or ‘axis’ on human health cannot be ignored. What is unclear exactly, is if exercise-induced adaptations in the gut of an individual can be transferred to elicit change in the gut of a recipient and further induce adaptations at the level of the skeletal muscle.

Research has shown that our gut microbiota confers many beneficial functions, including aiding the development of the immune system, metabolism, modulating stress reactivity and behaviour. The diverse population of the gut microbiota has been shown to be heterogeneous between individuals, with host genetic factors emerging as a contributor to gut microbiota composition. Recent work suggests that microRNA may act as a mediator of communication between the host and resident gut microbiota. Here we explore host genetic differences in gut microbiota composition and fecal miRNA profiles in two inbred mouse strains BALB/C and C57BL/6, in relation to gastrointestinal homeostasis. Furthermore, we evaluate the role of host genetics in response to perturbation of the gut microbiota using broad-spectrum antibiotic treatment. Distinct differences in the gut microbiota composition evaluated by fecal 16s rRNA gene sequencing between BALB/c and C57BL/6 mice were found with notable significant differences in genera Prevotella, Alistipes, Akkermansia and Ruminococcus. Significant host genetic differences were also observed in fecal miRNA profiles evaluated using the nCounter Nanostring platform. A BLASTn analysis was used to identify conserved fecal miRNA target regions in bacterial metagenomes, which identified numerous bacterial gene targets. Of those miRNA targets that were conserved in our dataset, 14 significant correlations were found between fecal miRNA and predicted taxa relative abundance. Treatment with broad-spectrum antibiotics for a period of 2 weeks resulted in BALB/c mice exhibiting a decrease in barrier permeability while C56BL/6 barrier permeability remained unchanged, demonstrating a host-specific physiological response to antibiotics at the gastrointestinal barrier. Differential response to antibiotics was also observed in the expression of barrier regulating genes in both host strains. Individual taxa were found to respond differentially by host strain, with Parabacteroides and Bacteroides associating with changes in barrier function. Together these findings suggest that host genetics play a role in determining the host-microbe relationship in both healthy homeostatic conditions and altered microbial conditions. / Thesis / Master of Science (MSc)

The human gut and brain have a bidirectional communication that has shown to play a pivotal role in our health and disease. Literature has shown that microbiota composition and diversity can be influenced by both genetic and environmental factors, contributing to shaping an individual’s microbial composition. The current work includes analysis of the microbiome of several mouse models to better understand how gene-environmental interactions during early development can influence the composition of microbiota within the gut. Here, male and female mice from several strains (C57BL/6, Balb/C, FVB, CD1) and genetically modified mice including T-cell receptor knock out mice (TCRβ-/-δ-/-) and Fragile-X-mice (FMR1-KO) were exposed to early life stressors including lipopolysaccharide (LPS) injection on postnatal day 3 (P3) and/or overnight maternal separation on P9. Fecal samples were collected at P24 and microbiota composition was determined by amplifying the 16s rRNA gene variable 3(v3) region and sequenced using the MiSeq Illumina platform. DADA2, was used to analyze this data in R software. Among the group, strain was found to be significant among alpha and beta diversity metrics while sex and stress were found to contribute to within strain variation, which demonstrated that both genetic and environmental factors are important in shaping an individual’s microbial composition. Secondly, we also explored the role of gut microbiota on the development of the immune system in TCRβ-/-δ-/- and C57BL/6 mice. Mice that lack T-lymphocytes were found to have a lower alpha diversity, as well as separated from their wild-type controls by beta diversity. Several bacterial taxa were found to be influenced by the immune system, demonstrating a bidirectional communication between the gut and T-cells. Lastly, the influence of litter, an environmental factor on microbial composition was explored within inbred mouse strains, C57BL/6 and Balb/C. Litter was found to influence alpha diversity, in which litters among C57BL/6 exhibited the greatest variation in such diversity. Beta diversity was also found to be influenced by litter, as related litters were found to cluster together. Differences in bacterial taxa between the inbred strains were observed and a subset of those taxa were found to be influenced by litter. Hierarchical clustering and co-occurrence analysis revealed different clusters of co-occurring taxa between both strains. These findings demonstrate that environmental factors can contribute to influence the composition of microbiota. / Thesis / Master of Science (MSc)

Individuals living with depression and anxiety show systemic increases of the bacterial endotoxin lipopolysaccharide (LPS), which induces an inflammatory cascade, resulting in negative effects across the gut-brain axis (GBA). LPS administration in mice has previously been used as a rodent model of depression/anxiety. Flaxseed (FS) contains key bioactives, including an omega-3 fatty-acid, dietary fibre, and a poly-phenolic compound which all may attenuate the effects of LPS through modulation of the GBA. The objectives of this thesis were to examine the effects of LPS on the GBA in C57Bl/6 mice and to determine if dietary supplementation with FS and/or FS oil (FO) provided protection against the LPS challenge. The LPS-induced negative effects across the GBA were partially attenuated by dietary supplementation with FS, but not FO, through changes in microbiota composition/function and systemic-/neuro-inflammation. Therefore, the potential benefits of FS are independent of the oil or are synergistic of all bioactives.

As part of the ongoing loss of global biodiversity, amphibian populations are experiencing declines and extinctions. A primary factor in these declines is the skin disease chytridiomycosis, which is caused by the fungus Batrachochytrium dendrobatidis (Bd). Recent research suggests that the amphibian skin microbiota has anti-Bd activity and may be an important factor in host disease resistance. However, little is known about the basic ecology of this host-microbe symbiosis, such as how much variation there is in microbial symbionts among host species and populations, and the nature of symbiont transmission, culturability, and function. My dissertation research addressed these basic questions in microbial ecology, as well as used a novel system to examine the long-standing ecological theory of community structure-function relationships. First, host-specificity, population-level variation and potential environmental transmission of the microbiota were examined by conducting a field survey of bacterial communities from bullfrogs, newts, pond water, and pond substrate at a single pond, and newts from multiple ponds. There was variation among amphibian host species and populations in their skin symbionts, and, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment. Second, the culturability of amphibian skin bacteria was assessed by directly comparing culture-dependent and -independent bacterial sequences from the same individuals. Although less than 7% of the amphibian skin microbes were captured using R2A medium, most of the dominant bacteria were represented in our cultures, and similar patterns of diversity among four amphibian species were captured with both approaches. Third, the relationship between microbial community structure and function and selective forces shaping structure and function were examined in bullfrogs by tracking microbial community structure and function following experimental manipulation of the skin microbiota and pathogen exposure. Results of this study demonstrated that Bd is a selective force on cutaneous bacterial community structure and function, and suggest that beneficial states of bacterial structure and function may serve to limit infection and negative fitness consequences of Bd exposure. Using a combination of observational and experimental approaches, my dissertation contributes to understanding structure-function relationships of these complex symbiotic communities of vertebrates. / Ph. D.

Tesis por compendio / [EN] The importance of the microbiota for host health and disease is now widely appreciated. Microbial communities that inhabit the human body have been proposed as an essential aspect of the hypothesis focused on the developmental origin of health and disease (DOHaD), suggesting that altered microbial exposure during infancy could play a role in the risk of some immune-based diseases, such as obesity, allergy development or T2 diabetes mellitus. Indeed, associational studies have related perinatal factors that could disrupt neonatal microbial colonization, such as antibiotic intake, Csection and formula feeding, to these diseases later in life. Some authors have suggested that the interaction between the initial microbiome and the developing immune system could be crucial for a correct maturation of the latter. However, the exact molecular mechanisms that regulate the host−microbiome interconnection during this period are still unknown. Furthermore, despite the importance of maternal microbiota on initial neonatal microbial seeding, data concerning the effect of these perinatal factors on maternal microbiota are still scarce. Objectives: The general aim of the thesis was to to ascertain the impact of perinatal factors, such as mode and place of delivery, antibiotic use, maternal diet and gestational age on maternal-neonatal microbiota composition and their implications on health outcomes. Subjects: Participants in this study were part of a larger longitudinal cohort study conducted in the Mediterranean area, between 2015 and 2019. Mothers were enrolled in the Hospital Universitario y Politécnico La Fe, Hospital Clinico Universitario de Valencia, primary health-care units from Valencia and CEIC-Parc de Salut MAR. In this thesis, faecal samples at birth, 7 and 31 days were used as well as the saliva, amniotic fluid and placenta samples from a subset of participants. Methods: Next-generation sequencing targeting the V3−V4 region of the 16S rRNA gene was performed to assess the microbiota composition at each time point. Enzymelinked immunosorbent assay and Luminex assay were used for the determination of the molecular hormonal, metabolic and immune status of the participants and/or cellular models. Quantitative polymerase chain reaction (PCR) and reverse transcription PCR were also performed in a quantitative measurement of microbiota composition and for the assessment of gene expression in placental tissue and cellular models, respectively. The culture of different cell lines, including intestinal epithelial cells (Caco-2), mucusproducer cells (LSTH-17) and monocyte-like cells (THP-1), were used for the in vitro assays. Results and conclusion: Maternal microbiota at delivery was influenced by several perinatal factors, including immune status of amniotic cavity, diet during gestation and delivery mode. Maternal consumption of fat was positively related to Firmicutes spp. while carbohydrates, fibre and polyunsaturated fatty acids (PUFA) intakes were associated with higher relative abundance of Bacteroidetes and Proteobacteria levels. Some of these relations were also reflected in the neonatal microbiota at delivery. Indeed, children born by C-section and from mothers with higher fat intakes showed higher body mass index (BMI) index z-scores than those born from mothers with higher fibre consumption. Two bacterial profiles were found during the study. The first one was composed of health-related bacteria with short-chain fatty acids (SCFA) production activity, such as Roseburia, Faecalibacterium, Blautia, Lachnospira or Bacteroides genera. This group was associated with a distinct amniotic cytokine profile characterized by higher concentrations of IL-2, IL-5, IL-17 and TNF-α. Indeed, salivary cortisol concentration also showed positive relations with some of these genera independently of the delivery mode. The other group was characterized by Finegoldia, Peptoniphilus, Anaeroaoccus, Porphyromonas and Campylobacter genera. Those were associated with another amniotic cytokine profile dominated by IL-4, IL-13, IL-18, and IL-10 cytokines and more highly represented in mothers who had undergone a Csection. Some of these genera were negatively associated with salivary cortisol concentration. Thus, our results suggested that amniotic fluid immune status and cortisol concentration were related to maternal microbiota at delivery with implications also in neonatal microbiota. Furthermore, we described the effect of mode and place of birth in the microbial colonization evolution during the first month of life, showing that neonates born by Csection had a distinct microbial pattern and higher BMI index z-scores than vaginalborn infants, both at home and hospital. We proposed an in vitro molecular mechanism by which the differences observed in C-section-born neonates in terms of microbiota composition would promote a shift in immune system maturation through the lack of immune stimulation observed in these samples compared with those from vaginal-born infants, especially those from the home birth group. Thus, disruptions in the colonization process during the first month of life could have long-lasting consequences through an alteration of immune system development during this period. / [ES] El gran impacto del microbioma humano sobre la salud del huésped es ampliamente conocido. Se cree que las comunidades microbianas que habitan el cuerpo humano serían un aspecto fundamental en la hipótesis promulgada sobre el posible origen de la enfermedad durante el desarrollo (DOHad) sugiriendo que alteraciones en la exposición microbiana durante la infancia podrían estar implicadas en el mayor riesgo de algunas enfermedades de base inmunológica como la obesidad, la alergia o la diabetes mellitus de tipo II. Además, se han encontrado relaciones significativas entre este tipo de enfermedades con algunos factores perinatales que se han descrito como disruptores del proceso de colonización, como la cesárea, el uso de antibióticos o la alimentación mediante leche de formula. Algunos autores sugieren que la interacción entre la microbiota inicial y el sistema inmunológico que está desarrollándose sería crucial para una correcta maduración de este. Sin embargo, los mecanismos exactos que mediarían en esta relación huésped-microbiota durante este periodo todavía se desconocen. Además, a pesar de la importancia de la microbiota materna para el inicio del proceso de colonización, todavía es escasa la información sobre como los diferentes factores perinatales afectan a la microbiota materna. Objetivos: El objetivo general de esta tesis fue definir el impacto de los diferentes factores perinatales como el tipo de parto, el uso de antibióticos, la dieta materna o la edad gestacional en la composición de la microbiota materno-neonatal y sus posibles implicaciones para la salud. Participantes: Los participantes del presente análisis son parte de un estudio longitudinal de cohorte desarrollado en el área mediterránea entre 2015 y 2019. Las madres fueron reclutadas en el Hospital Universitario y Politécnico de la Fe, el Hospital Clínico Universitario de Valencia, centros de atención primaria de la ciudad de Valencia y el CEIC-Parc de Salut MAR. En el presente trabajo se utilizaron las muestras fecales recogidas al parto, a los 7 y 31 días, así como las muestras de saliva, líquido amniótico y placenta de un grupo reducido de participantes. Métodos: Se utilizaron técnicas de secuenciación de nueva generación dirigidas a la región V3-V4 del gen 16S rRNA para la determinación de la composición de la microbiota de cada muestra a los tiempo estudiados. Además, el ensayo por inmunoadsorción ligado a enzimas y la técnica Luminex fueron usadas para la determinación de moléculas relacionadas con el estado hormonal, metabólico e inmune de las muestras y/o de los ensayos celulares. La PCR cuantitativa y la de transcripción reversa fueron usadas para realizar una medida cuantitativa de la composición microbiana y de la expresión de algunos genes seleccionados en las muestras de placenta y modelos celulares, respectivamente. EL cultivo de diferentes líneas celulares, como líneas epiteliales (Caco-2), productoras de moco (LSTH-17) y líneas similares a macrófagos (THP-1) fueron utilizadas en los ensayos in vitro. Resultados y conclusión: La microbiota materna al parto estuvo influenciada por diversos factores perinatales, incluyendo el estado inmunológico de la cavidad amniótica, la dieta durante el embarazo y el tipo de parto. El consumo materno de grasa estuvo positivamente relacionado con Fimicutes spp. Mientras que el consumo de carbohidratos, fibra y ácidos grasos poliinsaturados fueron asociados con mayores abundancias relativas de los filos Bacteroidetes y Proteobacteria. Algunas de estas relaciones fueron observadas también en la microbiota neonatal. Además, los niños nacidos por cesárea y de madres con altos consumos de grasa mostraron mayores IMC (índice de masa corporal) z-scores que aquellos que nacieron de madres consumidoras de fibra. Dos patrones microbianos fueron encontrados a lo largo de todos los análisis. El primero de ellos estaba compuesto por especies asociadas a la salud y productoras de SCFA como los géneros Roseburia, Faecalibacterium, Blautia, Lachnospira o Bacteroides. Este grupo estuvo asociado a un perfil de citoquinas caracterizado por IL2, IL-5, IL-17 y TNF-α. Los niveles de cortisol en saliva estuvieron positivamente correlacionados con algunos de estos géneros independientemente del tipo de parto. El otro patrón microbiano estuvo caracterizado por los géneros Finegoldia, Peptoniphilus, Anaeroaoccus, Porphyromonas y Campylobacter. Estos estuvieron relacionados con un perfil de citoquinas en la cavidad amniótica dominados por la presencia de IL-4, IL-13, IL-18 y IL-10 y con el parto por cesárea. Además, algunos de estos géneros estuvieron negativamente correlacionados con los niveles de cortisol en saliva. Así, nuestros resultados sugieren que el ambiente inmunológico de la cavidad uterina y la concentración de cortisol estuvieron relacionados con la composición de la microbiota materna al parto con implicaciones para la microbiota neonatal. Además, describimos el efecto del tipo de parto y el lugar en el proceso de colonización durante el primer mes de vida mostrando que los niños nacidos por cesárea tienen un perfil diferencial de microbiota y mayores IMC z-scores que los niños nacidos de forma vaginal, tanto en el hospital como en casa. En nuestra investigación, hemos propuesto un mecanismo molecular por el cual las diferencias en la composición microbiana de los niños nacidos por cesárea podrían provocar una alteración en la maduración del sistema inmunológico mediante la falta de imnunoestimulación observada estas muestras comparadas con aquellas obtenidas de los niños nacidos por parto vaginal, especialmente de aquellos nacidos en casa. Así, las alteraciones en el proceso de colonización durante el primer mes de vida podrían tener consecuencias a largo plazo en la salud infantil debido a una alteración del desarrollo del sistema inmunológico durante este periodo. / [CA] El gran impacte del microbioma humà sobre la salut humana del hoste és amplament reconegut. Es creu que les comunitats microbianes que habiten el cos humà serien un aspecte fonamental en la hipòtesi promulgada sobre el possible origen de la malaltia durant el desenvolupament (coneguda com DOHad) suggerint que alteracions en l’exposició microbiana al llarg de la infància podrien estar implicades en el augment del risc a patir algunes malalties amb base immunològica com són l’obesitat, l’al·lèrgia o la diabetis mellitus tipus II. Així mateix, s’han trobat relacions significatives entre aquest tipus de malalties amb alguns factors perinatals que s’han descrit com disruptors del procés de colonització, com la cesària, l’ús d’antibiòtics o l’alimentació mitjançant llet de fórmula. Alguns autors suggereixen que la interacció entre la microbiota inicial i el sistema immunològic que estaria desenvolupant-se seria crucial per a una correcta maduració d’aquest últim. No obstant això, els mecanismes exactes que mediten en aquesta relació hoste-microbiota durant aquest període encara es desconeixen. A pesar de la importància de la microbiota materna en l’inici del procés de colonització, encara és escassa la informació sobre els diferents factors perinatals que afecten a la microbiota materna. Objectius: L’objectiu general d’aquesta tesi fou definir l’impacte dels diferents factors perinatals com el tipus de part, l’ús d’antibiòtics, la dieta materna o l’edat gestacional en la composició de la microbiota materno-neoantal i les possibles implicacions sobre la salut. Participants: Els participants del present anàlisi són part d’un estudi longitudinal de cohort de l’àrea mediterrània entre el 2015 i 2019. Les mares van ser reclutades en l’Hospital Universitari i Politècnic La Fe, l’Hospital Clínic Universitari de València, centres d’atenció primària de la ciutat de València i el CEIC-Pac de Salut Mar de Catalunya. En el present treball s’utilitzaren les mostres fecals recollides al part, als 7 i als 31 dies, així com les mostres de saliva, líquid amniòtic i placenta. Mètodes: S’utilitzaren tècniques de seqüenciació de nova generació dirigides a la regió V3-V4 del gen 16S rRNA per a la determinació de la composició de la microbiota de cada mostra als temps estudiats. Així mateix, l’assaig per immune adsorció lligat a enzims i la tècnica Luminex foren utilitzades per a la determinació de molècules relacionades amb l’estat hormonal, metabòlic e immunològic de les mostres i/o els assajos cel·lulars. La PCR quantitativa i la de transcripció en mode revers foren utilitzades per a realitzar una mesura quantitativa de la composició de la microbiota i de l’expressió d’alguns gens seleccionats en les mostres de placenta i models cel·lulars, respectivament. El cultiu de diferents tipus cel·lulars, com tipus epitelials (Caco-2), productors de moc (LSTH-17) i línies similars a macròfags (THP-1) foren utilitzades en els assajos in vitro. Resultats i conclusió: La microbiota materna en el moment del part va estar influenciada per diversos factors perinatals, incloent l’estat immunològic de la cavitat amniòtica, la dieta durant l’embaràs i el tipus de part. El consum matern de greix es va vore associat amb Fimicutes spp. Pel contrari, el consum de carbohidrats, fibra i àcids grassos poliinsaturades es van veure relacionats amb majors abundàncies relatives dels phylum Bacteroidetes i Proteobacteria. Algunes d’aquestes relacions foren també observades en la microbiota neonatal. Tan mateix, els nounats nascuts per cesària i de mares consumidores de greixos van mostrar majors índexs de massa corporal (IMC) zscores que els nonats nascuts de mares consumidores de fibra. Dos patrons microbians es trobaren al llarg de totes les anàlisis. La primera d’elles va estar composta per espècies associades amb la salut i productes d’àcids grassos de cadena curta (SCFA), com són els gèneres Rosburia, Faecalibacterium, Blautia, Lachnospira o Bacteroides. Aquest grup va estar associat a un perfil de citocines caracteritzat per interleucines (IL)-2, IL-5, IL-17 i TNF-alpha. Els nivells de cortisol en saliva al part estigueren positivament correlacionats amb alguns d’aquests grups , independentment del tipus de part. L’altre patró microbià estava caracteritzat per els gèneres Finegoldia, Peptoniphilus, Anaeroccocus, Porphyromonas i Campylobacter. Aquests grups foren relacionats amb el part per cesària i un perfil de citoquines en la cavitat amniòtica dominat per la presència de IL-4, IL-13, IL.18 i IL-10. A més, alguns d’aquests gèneres estigueren negativament relacionats amb els nivells de cortisol en saliva en el moment del part. Així, els nostres resultats suggereixen que l’ambient immunològic de la cavitat uterina i la concentració de cortisol tenien una relació amb la composició de la microbiota materna al part amb implicacions per a la microbiota neonatal. Tan mateix, descrivim l’efecte del tipus de part i el lloc de naixement (hospital vs. casa) en el procés de colonització durant el primer mes de vida, mostrant que els nonats nascuts per cesària tenien un perfil diferencial de microbiota i majors índexs de massa corporal (IMC) z-scores que aquells nonats nascuts de forma vaginal, tant a l’hospital com a casa. En la nostra investigació, hem proposat un mecanisme molecular pel qual les diferències en la composició microbiana podrien provocar una alteració en la maduració del sistema immunològic mitjançant una manca de inmuno-estimulació, observada en les mostres obtingudes dels nounats nascuts per cesària, especialment si es compara amb aquells nascuts a casa. Així, les alteracions en el procés de colonització, durant el primer mes de vida podrien tenir conseqüències a llarg termini en la salut infant debut a alteracions en el desenvolupament del sistema immunològic durant el període. / Selma Royo, M. (2020). Impact of perinatal factors on the maternal- neonatal microbiota and influence on health outcomes [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153159 / Compendio