This thesis focuses on elucidating the electrical mechanisms underlying excitation of small intestinal and colonic smooth muscle initiated by interstitial cells of Cajal (ICC). All the ICC subtypes are involved in the orchestration, generation, and/or transmission of electrical signals to smooth muscle to pace gut motor patterns. Some ICC types have intrinsic activity leading to omnipresent rhythmic changes in smooth muscle excitability; others respond to stimuli, inducing pacemaker activity as required. Together they orchestrate motor patterns such as propulsion and segmentation, essential functions of the gut. To study ICC electrophysiology, I utilized patch clamping to record ion channel currents from single intestinal ICC and sharp microelectrodes to record colonic smooth muscle membrane potentials. I have made several discoveries contributing to our understanding of ICC electrophysiology. Firstly, my research increased our understanding of the properties of intrinsic pace-maker activity. I showed that maxi Cl– channels from small intestinal ICC make a significant contribution to slow wave depolarization triggered by intracellular calcium. Secondly, I showed that colonic intramuscular ICC (ICC-IM) selectively express KV7.5 channels, which are suppressed by cholinergic agonists, meaning that excitatory stimuli triggering acetylcholine release deactivate KV7.5 channels, leading to increased excitability. Thirdly, I have shown that the bile acid chenodeoxycholic acid and the nitric oxide donor sodium ni-troprusside both induce pacemaker activity, rhythmic transient depolarisations in mouse colonic muscle, which led to the hypothesis that nitrergic nerves are involved in generating inducible myenteric plexus ICC (ICC-MP) pacemaker activity. It is only when ICC are suitably stimulated by intracellular processes such as rhythmic Ca2+ transients or extracellular signalling from neurotransmitters or small molecules, that ICC produce membrane potential rhythmicity, required for generation of intrinsic slow waves, low-frequency rhythmic transient depolarisations and transmission of excitation into the muscle. / Thesis / Doctor of Philosophy (PhD) / The gut is essential for digestion and absorption of food. The gut has special cells called interstitial cells of Cajal (ICC), which control the contractions of the gut muscle. ICC are pacemaker cells, like those that pace heart beats. To pace gut muscle contractions, ICC generate electrical signals which cause the muscle to contract in an organized rhythmic manner, which promotes mixing or propulsion of gut contents, called motility. I used tiny electrodes to record electrical activity from ICC or gut muscle, to improve our understanding of how ICC pacemaker activity controls motility. My research characterised ion channels, which are microscopic protein pores that allow cells to make electrical currents, that enable generation of pacemaker signals by ICC. I also investigated activation of ICC electrical activity that causes propulsive colonic motility. This will hopefully lead to treatment improvements for patients with motility disorders in the future.

In aquatic environments Cu is both a vital nutrient and an important toxicant. Consequently fish require Cu as a micronutrient and can obtain this metal from either water or their diet. Inadequate intake of Cu is associated with reduced growth and development, while decreased growth rates, mortality and reduced swimming capacity have been reported in fish when Cu accumulates in excess of cellular needs. Characterization of Cu uptake is therefore critical in understanding the dynamics that govern toxicity and the risks associated with exposure to an aquatic contaminant. While mechanisms of waterborne uptake and toxicity are well understood, far less is known about gastrointestinal Cu uptake in fish. In vivo and in vitro techniques were therefore used in this study to investigate dietary Cu uptake in freshwater rainbow trout. The mid and posterior regions of the intestine emerged as important sites for Cu absorption in trout, while the role of the stomach and anterior intestine in Cu absorption requires further investigation. The intestinal uptake route was kinetically characterized as a low affinity absorption pathway as compared to the branchial route. Cu uptake appeared to occur via a hypoxia-resistant, carrier-mediated, saturable process which could be fueled by Cu(II)^2+ at concentrations typically found in the fluid phase of chyme in the trout intestine. Experimental manipulation of mucosal NaCl levels stimulated Cu uptake, Na2SO4 had an identical effect, implicating Na rather than the anion. These responses were unrelated to solvent drag, osmotic pressure or changes in TEP. The presence of excess luminal Ag and L-histidine stimulated Cu and Na uptake indicating that a portion of Cu transport was mediated by a Na-Cu co-transport system. Partial inhibition of Cu and Na uptake by phenamil and hypercapnia stimulated Na and Cu transport suggest Cu entry could also occur via the apical Na channel. The Na-dependent mechanism thus either involves more than one component or a unique Na-Cu co-transport mechanism with these combined characteristics mediates part of Cu uptake. Cu uptake was sensitive to pH and competed by Fe and Zn implicating DMT1 in the transport of Cu in the trout intestine. These factors had no effect on Na uptake, leading to the identification of a Na-independent mechanism for Cu uptake in the trout intestine. While the Na dependent nature of Cu uptake and Ag stimulated Cu transport argue against a role for Ctrl in this process, Cu transport characteristics identified in this study compare well with a recently identified Cu transporter in Ctr1 deficient mouse embryonic cells, indicating the existence of a similar transport mechanism in the trout intestine. / Thesis / Master of Science (MSc)

No description available.

The small intestine and yolk sac (YS) are important organs for nutrient absorption and innate immunity in chickens during the post-hatch or prehatch periods. These organs share a similar structure of epithelial cell-lined villi with tight junctions between adjacent cells. Probiotics have been reported to improve chicken growth performance and gut health including promotion of intestinal morphology. However, there are few studies that show the effect of probiotics on ontogeny of intestinal epithelial cells and antimicrobial peptides, or intestinal integrity in young healthy chicks. Heat stress during incubation was shown to increase mortality and decrease hatchability of chicks, while no studies have investigated the effect of heat stress on the integrity of the YS, which might be related to hatching performance. There were four studies conducted in this research: 1) a comparison of the effect of two probiotics on the ontogeny of small intestinal epithelial cells in young chicks; 2) the effect of two probiotics on mRNA abundance of tight junction proteins in the small intestine of young chicks; 3) the effect of high incubation temperature on mRNA abundance of tight junction proteins in the YS of broiler embryos; and 4) comparison of avian defense peptide mRNA abundance in the YS of broilers and layers. In study 1, Probiotics transiently decreased body weight gain (BWG) from day 2 to day 4, but did not affect body weight (BW) from day 2 to day 8, and small intestinal weight and intestinal morphology from day 2 to day 6. Probiotics did not affect marker gene expression of intestinal stem cells (Olfm4) and goblet cells (Muc2) in all small intestinal segments, but did increase expression of a marker gene of proliferating cells (Ki67), and decreased an antimicrobial peptide (liver-enriched antimicrobial peptide 2, LEAP2) in the jejunum at day 4. Probiotic 1 decreased PepT1, a marker of enterocytes in the duodenum at day 4. These results suggest that probiotics did not improve growth performance and intestinal morphology in young healthy chicks, but temporarily promoted intestinal epithelial cell proliferation and decreased LEAP2 antimicrobial peptide expression in the jejunum. In situ hybridization (ISH) showed that Ki67+ proliferating cells were mainly located in the crypt region and the blood vessels of villi. In study 2, Probiotic supplementation to newly hatched chicks for less than one week did not affect mRNA abundance of the tight junction proteins in the small intestine. Occludin (OCLN) mRNA, which was detected by ISH to be expressed in intestinal epithelial cells in both the villus and crypt regions, was greater in the duodenum of female chicks than males. In study 3, high incubation temperature starting from embryonic day 12 (E12) affected mRNA abundance of the tight junction proteins in the YS, including increased zonula occluden 1 (ZO1) at E13, increased junctional adhesion molecule A (JAMA) and heat shock protein 90 (HSP90) at E17, but decreased tight junction protein JAMA at E19 and OCLN at day of hatch (DOH). These results showed that the YS tight junction proteins were increased by short term heat exposure but decreased by long term heat exposure. In study 4, the expression of avian β defensin 10 (AvBD10), CATHs and toll-like receptors in the YS was examined. Toll-like receptors were highly expressed in the YS at early incubation stages (E7), while CATHs showed a peak expression from E9 to E13, which was similar to the expression pattern of AvBD10. CATHs and AvBD10 mRNA temporal expression patterns were similar in broilers and layers, while their expression levels were different. Layers, especially brown layers, had greater mRNA abundance for antimicrobial peptides such as AvBD10, CATH1, and CATH2 in the YS. These results demonstrate that the antimicrobial peptide temporal expression patterns in the YS are not affected by breed, but their expression levels are affected by breed. In summary, the small intestine and the YS are essential for nutrient uptake, innate immunity, and maintenance of integrity. The ontogeny of intestinal epithelial cells, such as proliferating cells can be modulated by probiotic supplementation. Similar to the small intestine, the YS can also express tight junction proteins, which can be affected by high incubation temperature. Antimicrobial peptide expression in the intestine of healthy young chicks is also transiently decreased by probiotic supplements. Avian defensin and cathelicidin expression patterns in the YS were not affected by breed. / Doctor of Philosophy / The small intestine and yolk sac are important organs for nutrient absorption in hatched chicks or embryonic chicks. These organs also serve as a barrier to prevent pathogens from entering the blood circulation. Intestinal epithelial cells along the villi renew rapidly by proliferation and differentiation. In this research, probiotics which are also known as direct fed microbials temporarily increased expression of the proliferating cell marker Ki67 in the jejunum of healthy young chicks, which suggests that probiotics promote intestinal epithelial cell proliferation. However, probiotics transiently decreased expression of an antimicrobial peptide, which may reduce immune protection in the gut. The yolk sac can also express tight junction proteins. The expression of tight junction proteins was affected by elevated incubation temperature in broiler embryos, which might be related to low hatchability of eggs exposed to heat stress. Avian defense peptides and pathogen recognition receptors were expressed in the YS, which implied that the yolk sac contained an innate immune function. The expression pattern of avian defense peptides was affected by breed (broilers and layers), while the expression level of avian defense peptides was greater in layers than broilers. In summary, the small intestine and the yolk sac are multifunctional organs. Their cell composition, structural integrity, and secretion of antimicrobial peptides can be affected by environmental factors, such as probiotic supplementation or high incubation temperature.

The incubation period is crucial for development and overall quality of a chick. The selection for fast growing broilers has allowed the birds to reach market weight at a faster rate making the incubation period a larger portion of a broiler's life. A faster growth rate can lead to the release of more metabolic heat inside of the egg toward the second half of incubation because the embryo shifts to a homeothermic state. More heat being released into the incubator can cause the incubation temperature to rise if the incubator is not electronically regulated or cannot be ventilated properly due to malfunction. A high incubation temperature can impact the hatchability, growth, and development of the chick. This thesis provides a more in-depth analysis of the effects of high incubation temperature (37.5°C versus 39.5°C) on the developing small intestine and yolk sac, which provide the chick with nutrients posthatch and during embryogenesis. Studying these organs and mechanisms occurring during this time could potentially indicate why chicks from eggs subjected to a higher incubation temperature are not developing and growing properly. Chicks from eggs incubated at a higher temperature had lower body weights, lower hatchability and lower villus height in the duodenum, jejunum, and ileum. There were also differences seen in the depth of the crypt, which is the site for stem cells. Chicks from eggs incubated at a higher temperature had a lower crypt depth in the duodenum and jejunum. There was no difference in the expression of the intestinal stem cell marker olfactomedin 4 (Olfm4) and mucin 2, which is secreted by goblet cells and forms mucus. In the yolk sac, heat shock proteins (HSP) 70 and 90 were elevated at embryonic day 15, and HSP90 still remained elevated at embryonic day 17. Chicks from eggs incubated at a higher temperature showed greater expression of peptide transporter 1 and avian beta-defensin 10 mRNA at embryonic day 13. Even though small intestinal morphology was impacted early posthatch and expression of genes in the yolk sac were elevated at embryonic day 13, there does not seem to be a long-lasting effect on the development of the small intestine or the yolk sac. It is still important to study the impact of the incubation environment to understand the development and growth of the chicks and how different incubation factors can impact the overall hatchability and health of the chick. / Master of Science / The incubation period is crucial for development and overall quality of a chick. The selection for fast growing broilers has allowed the birds to reach market weight at a faster rate making the incubation period a larger portion of a broiler’s life. A faster growth rate can lead to the release of more metabolic heat inside of the egg toward the second half of incubation because the embryo shifts to a homeothermic state. More heat being released into the incubator can cause the incubation temperature to rise if the incubator is not electronically regulated or cannot be ventilated properly due to malfunction. A high incubation temperature can impact the hatchability, growth, and development of the chick. This thesis provides a more in-depth analysis of the effects of high incubation temperature (37.5°C versus 39.5°C) on the developing small intestine and yolk sac, which provide the chick with nutrients posthatch and during embryogenesis. Studying these organs and mechanisms occurring during this time could potentially indicate why chicks from eggs subjected to a higher incubation temperature are not developing and growing properly. Chicks from eggs incubated at a higher temperature had lower body weights, lower hatchability and lower villus height in the duodenum, jejunum, and ileum. There were also differences seen in the depth of the crypt, which is the site for stem cells. Chicks from eggs incubated at a higher temperature had a lower crypt depth in the duodenum and jejunum. There was no difference in the expression of the intestinal stem cell marker olfactomedin 4 (Olfm4) and mucin 2, which is secreted by goblet cells and forms mucus. In the yolk sac, heat shock proteins (HSP) 70 and 90 were elevated at embryonic day 15, and HSP90 still remained elevated at embryonic day 17. Chicks from eggs incubated at a higher temperature showed greater expression of peptide transporter 1 and avian beta-defensin 10 mRNA at embryonic day 13. Even though small intestinal morphology was impacted early posthatch and expression of genes in the yolk sac were elevated at embryonic day 13, there does not seem to be a long-lasting effect on the development of the small intestine or the yolk sac. It is still important to study the impact of the incubation environment to understand the development and growth of the chicks and how different incubation factors can impact the overall hatchability and health of the chick.

Tesis por compendio / Aunque el uso de altos niveles de fuentes de proteína vegetal en piensos para doradas de engorde se ha alcanzado con éxito en cuanto al crecimiento, estas dietas todavía están asociadas a efectos negativos en la eficiencia nutricional y en la capacidad inmunitaria. El intestino es el órgano donde se produce la primera interacción entre el pez, los nutrientes y las bacterias del medio, y desarrolla un papel crucial en la digestión de los nutrientes y la respuesta infamatoria e inmune. Esta tesis doctoral se centra en el impacto de distintas dietas con altos niveles de proteína vegetal, y especialmente, en la evaluación del estatus intestinal de las doradas de engorde alimentadas con altos niveles de sustitución de la harina de pescado durante un periodo largo de tiempo. Los cambios observados en el intestino se caracterizaron mediante el uso de distintas estrategias, como el análisis de la digestibilidad y la retención de amino ácidos, de la excreción de amonio, de la actividad de enzimas digestivos, de los cambios histológico o de la expresión de genes relacionados con la función y el mantenimiento de la arquitectura intestinal, así como técnicas ómicas para el análisis del proteoma y de la microbiota intestinal. Se ensayaron distintos niveles de sustitución de harina de pescado, pero el impacto de las dietas con una sustitución completa, bien complementada con subproductos de origen marino o suplementada con aminoácidos libres sintéticos, recibió mayor atención. La sustitución completa de la harina de pescado provocó una reducción, aunque ligera, del crecimiento y de la eficiencia digestiva y nutritiva de la dorada de engorde, aunque el impacto sobre el crecimiento era mayor cuando los peces eran alimentados desde la época de juveniles con estas dietas. La digestibilidad y el nivel de síntesis de proteína se vio alterada, aunque no se observaron diferencias significativas en la actividad enzimática digestiva. No obstante, el impacto de las fuentes vegetales cuando no había fuentes de proteína marina en la dieta era especialmente crítico para la supervivencia de los peces. En el intestino de estos peces solo se observaron diferencias menores relacionadas con la inflamación a nivel histológico, pero también se observó una disminución en la expresión génica de genes involucrados en la inflamación y la respuesta inmune. El análisis de la microbiota intestinal reveló cambios significativos en la composición de su composición, especialmente en el intestino posterior, sugiriendo una posible falta de capacidad de regular la respuesta inmune y de modular la colonización de bacterias patógenas tras un largo periodo de alimentación con esta dieta. Por otro lado, el análisis del proteoma de la mucosa intestinal también mostró un claro impacto sobre distintos procesos biológicos relacionados con el mantenimiento del homeostasis intestinal y de la integridad epitelial. Por el contrario, no se observó un impacto de la sustitución de la harina de pescado a nivel de expresión génica o del proteoma cuando se incorporaba a la dieta una fuente de proteína marina complementaria, aunque sí que se observaron algunos signos menores de inflamación. Por último, se desarrolló un sistema ex vivo para estudiar la respuesta inflamatoria e inmune de la mucosa intestinal a la presencia de distintas bacterias, y se realizó un ensayo preliminar en dorada para evaluar el efecto de la dieta sobre esta respuesta. En resumen, en este trabajo se ha realizado una evaluación extensa y detallada de los efectos a nivel intestinal de la inclusión de altos niveles de proteína vegetal en la dieta para doradas de engorde. Los resultados indican que las alteraciones en la capacidad inmune, la homeostasis y la microbiota intestinal aparecían solo cuando la proteína procedía exclusivamente de fuentes vegetales, y podrían explicar la mayor mortalidad registrada con esta dieta. / Malgrat que la utilització d'alts nivells de proteïna vegetal en pinsos per a dorades en la fase d'engreixament s'ha aconseguit amb èxit en quan al creixement, aquestes dietes encara s'associen amb freqüència amb efectes negatius en l'eficiència nutricional i la capacitat immunitària. L'intestí és l'òrgan on es produeix la primera interacció entre el peix, els nutrients de la dieta i les bactèries de l'ambient, i juga un paper fonamental en la digestió dels nutrients i en la resposta inflamatòria i immune. Aquesta tesi doctoral es centra en l'impacte de diferents dietes experimentals amb un alt nivell de proteïna vegetal, i especialment, en l'avaluació de l'estat de l'intestí de les dorades d'engreixament alimentades durant un llarg període amb alts nivells de substitució de farina de peix. Els distints canvis observats a nivell intestinal es van descriure mitjançant l'ús de distintes estratègies, com l'anàlisi de la digestibilitat i la retenció dels aminoàcids, de l'excreció d'amoni i de l'activitat enzimàtica, dels canvis histològic o de l'expressió de gens relacionats amb la funció i el manteniment de l'estructura intestinal, així com tècniques òmiques per a l'anàlisi del proteoma i de la microbiota intestinal. Es van assatjar diferents nivells de substitució de farina de peix, però l'impacte de les dietes amb substitució completa, bé complementada amb subproductes d'origen marí o suplementada amb aminoàcids lliures sintètics, va rebre major atenció. La substitució completa de la farina de peix va tenir un efecte lleugerament negatiu sobre el creixement i l'eficiència digestiva i nutritiva de la dorada d'engreixament, encara que l'impacte era major quan els peixos eren alimentats des de la fase de juvenils amb aquesta dieta. La digestibilitat i el nivell de síntesis de proteïna es va veure alterada, encara que no s'observaren diferències significatives en l'activitat dels enzims digestius. No obstant, l'impacte de les fonts vegetals quan s'eliminaven per complet les fonts de proteïna marina de la dieta era especialment crític en la supervivència dels peixos. En l'intestí d'aquests peixos sols s'observaren xicotets indicis d'inflamació a nivell histològic, però també es va observar una disminució l'expressió de gens involucrats amb el procés inflamatori i la resposta immune. L'estudi de la microbiota intestinal va revelar canvis significatius en la composició, especialment a l'intestí posterior, suggerint una possible falta de capacitat de regular la resposta immunitària i de modular la colonització per part de patògens després d'un llarg període d'alimentació amb aquesta dieta. D'altra banda, l'anàlisi del proteoma de la mucosa intestinal també va mostrar un impacte clar sobre diferents processos biològics relacionats amb el manteniment de l'homeòstasi intestinal i de la integritat de l'epiteli. Per contra, no es van observar un impacte de la substitució de la farina de peix a nivell d'expressió gènica o proteoma quan s'incloïa a la dieta una font complementària de proteïna d'origen marí, encara que sí que s'observaven alguns signes d'inflamació. Per últim, es va desenvolupar un sistema ex vivo per avaluar la resposta inflamatòria i immune de la mucosa intestinal davant la presència de diferents bactèries, i es va realitzar un assaig preliminar per determinar l'efecte de la dieta sobre aquesta resposta. En resum, en aquest treball s'ha realitzat una avaluació extensa i detallada dels efectes a nivell intestinal de la inclusió d'alts nivells de fonts de proteïna vegetal a les dieta per a les dorades d'engreixament. Els resultats indiquen que les alteracions en la capacitat immunitària, l'homeòstasi i la microbiota intestinal eren observades solament quan la proteïna era exclusivament obtinguda de fonts vegetals, i podrien explicar la major mortalitat observada amb aquesta dieta. / Although the inclusion of plant protein sources at high levels in aquafeeds for on-growing gilthead seabream has been successfully achieved on gilthead seabream in terms of growth, these diets are still associated to detrimental effects in feed efficiency and immune capacity. The intestine is the organ where takes place the first interaction of the host with dietary antigen or environmental bacteria, and plays a major role in the digestion of nutrients and the inflammatory and the immune response. The present PhD thesis focus on the impact of classical formulated high plant protein diets on fish performance, but especially, on evaluation of the intestinal status in on-growing fish long-term fed with high levels of fishmeal replacement. Changes at intestinal level were characterized by using different approaches, including protein and amino acid digestibility and retention and ammonia excretion, digestive enzyme activity, histology, expression of genes related with inflammation, immunity, structure and digestion, but also using whole tissue-level techniques for the analysis of the impact on proteome and gut microbiota. Different levels of fishmeal replacement were assayed, although the impact of diets with total replacement, complemented by inclusion of alternative marine by-products or supplemented by free amino acids, received greater attention. Total fish replacement produced a negative but minor impact on the growth and nutritive and digestive performance of on-growing gilthead seabream. Nevertheless, when fish were fed from juvenile stage with plant protein based diets, a higher negative impact in growth terms was noticed. Digestibility and metabolic use of amino acids was altered, but no differences were observed in the digestive enzyme activities. Nonetheless, feeding fish with total dietary fishmeal replacement by plant protein without any marine protein source was especially critical for survival rate. In these fish, gut histological assessment only revealed minor alterations related with an inflammatory response, but gene expression assay showed a down-regulation of several genes involved in the inflammatory and immune response. Moreover, a drastic change in the microbiota composition was observed, especially at the hindgut, revealing a possible lack of capacity to regulate a defensive response and to face with pathogen colonisation after a long-term coupling with these diet. Likewise, gut mucosa proteome analysis also suggests an impact on biological processes related with the maintenance of gut homeostasis and the epithelial integrity. In contrast, total fishmeal replacement did not induce alterations at transcript or proteomic level when diet was complemented with marine ingredients, although some minor inflammatory signs were reported. On the other hand, an ex vivo system to study the inflammatory and immune response of the gut mucosa to the presence of different bacteria was developed, and a preliminary assay evaluating the impact of the diet on this response was performed. To sum up, present works represents a wide assessment at intestinal level of the effects of including plant protein sources at high levels in aqua feeds for on-growing gilthead seabream. Results indicate that alterations in the immune capacity, the gut homeostasis and the microbiota were observed when protein was exclusively provided by plant sources, and could explain the higher mortality reported with this diet. / Estruch Cucarella, G. (2018). ASSESSMENT OF THE LONG-TERM IMPACT OF HIGH PLANT PROTEIN DIETS ON THE INTESTINAL STATUS OF THE ON-GROWING GILTHEAD SEA BREAM (Sparus aurata, L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/113063 / Compendio

Yes / Background: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. Objectives: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. Methods: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC–tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. Results: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). Conclusions: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy.

[ES] La dieta es la principal vía de exposición a mercurio (Hg) para la mayoría de la población. Las principales formas de Hg en los alimentos son el metilmercurio (MeHg) y el Hg inorgánico divalente [Hg(II)]. Aunque el intestino es la principal puerta de entrada del tóxico en la circulación sistémica, los estudios sobre su toxicidad a nivel intestinal son escasos. El objetivo de la presente tesis ha sido evaluar la toxicidad y los mecanismos de acción del Hg(II) y el MeHg a nivel intestinal. Adicionalmente, se ha ensayado la eficacia de cepas de bacterias ácido- lácticas (BAL) como estrategia de reducción de esta toxicidad. Para los estudios in vitro se ha desarrollado un modelo celular en el que se han combinado enterocitos, células mucosecretoras y macrófagos en un sistema bicameral. Las células se han expuesto a Hg(II) y MeHg (0.1-1 mg/L) durante 10 días. Los datos han evidenciado que ambas especies generan un aumento de la respuesta inflamatoria, incrementando la liberación de la citoquina IL-8 e IL-1β, y una respuesta pro-oxidante, con un aumento de las especies reactivas de oxígeno/nitrógeno (ROS/RNS) y una sobreexpresión de proteínas de estrés (HSP70, HSP90 y MT2A). Esta respuesta ha sido más notable en macrófagos, indicando que posiblemente sean las células inmunitarias las que gobiernen la respuesta al tóxico. La situación de estrés va acompañada de una alteración de la expresión de la proteína de las uniones estrechas ZO-1 y una modificación de la morfología de la monocapa. Además, la exposición genera un aumento de la secreción de mucus y una sobreexpresión de su principal mucina, principalmente a MeHg. Los mecanismos de esta hipersecreción podrían estar relacionados con la ruta IL4/IL13/STAT6. Todos estos efectos sobre la monocapa epitelial conllevan un aumento de la permeabilidad y una reducción de la capacidad de regeneración. En los ensayos in vivo se han expuesto ratones BALB/c a Hg(II) y MeHg (1- 10 mg/L) durante 4 meses a través del agua de bebida. Los datos obtenidos han confirmado lo observado in vitro. La exposición a ambas especies (especialmente a 5 y 10 mg/L) induce un proceso inflamatorio en el colon, con un aumento de las citoquinas TNF-α e IL1-β y la presencia de infiltrados de neutrófilos. Paralelamente, la exposición genera estrés oxidativo con un incremento de las ROS/RNS y los peróxidos lipídicos. Se ha confirmado in vivo la participación en esta respuesta de algunas rutas apuntadas in vitro, en concreto p38 MAPK y JNK. Además, también se evidencian alteraciones en la expresión de proteínas de las uniones estrechas y de la mucina MUC2 en el colon, acompañada de una hiperplasia de las células mucosecretoras, especialmente en los tratamientos con MeHg. En estos animales se observa, además, un aumento de la expresión de IL13 e IL4, lo que apunta a la participación de la ruta IL4/IL13/STAT6, tal y como indicaban los ensayos in vitro. Además, estos ensayos in vivo muestran un efecto de ambas especies sobre el metabolismo de la microbiota intestinal con una reducción de los contenidos luminales de los ácidos grasos de cadena corta (SCFA), aunque los cambios detectados en la composición de la microbiota son mínimos. Finalmente, los animales tratados presentan un aumento de la permeabilidad. Todos los datos obtenidos in vitro e in vivo ponen de manifiesto que una exposición continuada a Hg(II) y MeHg genera una disrupción de la barrera intestinal. Los ensayos in vitro realizados para determinar la eficacia de las cepas de BAL de origen murino LE1 y LE2 como estrategia de protección se han realizado coexponiendo el modelo celular a Hg(II) o MeHg (1 mg/L) junto con las cepas durante 7 días. Los datos obtenidos han mostrado un efecto protector de ambas cepas, con una reducción de la respuesta inflamatoria, el estrés y los efectos sobre las uniones estrechas y la producción de mucus. Asimismo, la presencia de ambas bacterias restaura parcialmente la permeabilidad y la capacidad de regeneración de las monocapas. Teniendo en cuenta que en este estudio las BAL están inactivadas térmicamente, esta protección se asocia principalmente a su capacidad de quelación del Hg, aunque no hay que descartar que algún componente estructural de la pared bacteriana pueda ejercer otro tipo de efecto beneficioso. Los ensayos in vivo para confirmar el efecto protector de las BAL se han realizado en ratones BALB/c expuestos durante 2 meses a MeHg (5 mg/L) a través del agua de bebida. Las bacterias, en este caso viables, se han administrado por sonda gástrica diariamente. Los datos obtenidos muestran que, aunque las dos cepas reducen por igual los contenidos colónicos de MeHg, la reducción de los efectos tóxicos es mucho más notable con la cepa LE1. Se observan reducciones de los contenidos de mediadores de inflamación y estrés en el colon, hay una recuperación de la expresión de proteínas constituyentes de las uniones estrechas y del mucus. Se reestablecen los niveles luminales de los metabolitos de la microbiota y se restaura parcialmente la permeabilidad intestinal. Los resultados muestran que, además del proceso de quelación, la cepa LE1 activa la ruta de señalización Nrf2/Keap1/ARE y la producción de la citoquina anti-inflamatoria IL10. Los datos obtenidos in vitro e in vivo, apuntan a que la cepa LE1 podría ser una buena alternativa para reducir la toxicidad intestinal del Hg; reducción que puede tener también repercusiones beneficiosas a nivel sistémico. / [CA] La dieta és la principal via d'exposició a mercuri (Hg) per a la majoria de la població. Les principals formes de Hg en els aliments són el metilmercuri (MeHg) i el Hg inorgànic divalent [Hg(II)]. Encara que l'intestí és la principal porta d'entrada del tòxic a la circulació sistèmica, els estudis sobre la seua toxicitat a nivell intestinal són escassos. L'objectiu de la present tesi ha sigut avaluar la toxicitat i els mecanismes d'acció del Hg(II) i el MeHg a nivell intestinal. Addicionalment, s'ha assajat l'eficàcia de soques de bacteris àcid-làctics (BAL) com a estratègia de reducció d'aquesta toxicitat. Per als estudis in vitro s'ha desenvolupat un model cel·lular en el qual s'han combinat enteròcits, cèl·lules mucosecretores i macròfags en un sistema bicameral. Les cèl·lules s'han exposat des de l'inici a Hg(II) i MeHg (0.1-1 mg/L) durant 10 dies. Les dades han evidenciat que totes dues espècies generen un augment de la resposta inflamatòria, incrementant l'alliberament de la citocina IL- 8 i IL-1β, i una resposta pro-oxidant, amb un augment de les espècies reactives d'oxigen/nitrogen (ROS/RNS) i una sobreexpressió de proteïnes d’estrès (HSP70, HSP90 i MT2A). Aquesta resposta ha sigut més notable en macròfags, indicant que possiblement són les cèl·lules immunitàries les que governen la resposta al tòxic. La situació d’estrès va acompanyada d'una alteració de l'expressió de la proteïna de les unions estretes ZO-1 i una modificació de la morfologia de la monocapa. A més, l'exposició, principalment a MeHg, genera un augment de la secreció de mucus i una sobreexpressió de la seua principal mucina. Els mecanismes d'aquesta hipersecreció podrien estar relacionats amb la ruta IL4/IL13/STAT6. Tots aquests efectes sobre la monocapa epitelial comporten un augment de la permeabilitat i una reducció de la capacitat de regeneració. En els assajos in vivo s'han exposat ratolins BALB/c a Hg(II) i MeHg (1-10 mg/L) durant 4 mesos a través de l'aigua de beguda. Les dades obtingudes han confirmat l'observat in vitro. L'exposició a totes dues espècies (especialment a 5 i 10 mg/L) indueix un procés inflamatori en el còlon, amb un augment de les citocines TNF-α i IL1-β i la presència d'infiltrats de neutròfils. Paral·lelament, l'exposició genera estrès oxidatiu amb un increment de les ROS/RNS i els peròxids lipídics. S'ha confirmat in vivo la participació en aquesta resposta d'algunes rutes apuntades in vitro, en concret p38 MAPK i JNK. A més, també s'evidencien alteracions en l'expressió de proteïnes de les unions i de la mucina MUC2 en el còlon, acompanyada d'una hiperplàsia de les cèl·lules mucosecretores, especialment en els tractaments amb MeHg. En aquests animals hi ha, a més, un augment de l'expressió d'IL13 i IL4, la qual cosa apunta a la participació de la ruta IL4/IL13/STAT6, tal com indicaven els assajos in vitro. A més, aquests assajos in vivo mostren un efecte de les espècies de Hg sobre el metabolisme de la microbiota intestinal, amb una reducció dels continguts luminals dels àcids grassos de cadena curta (SCFA), encara que els canvis en la composició de microbiota intestinal son minims. Finalment, els animals tractats presenten un augment de la permeabilitat. Totes les dades obtingudes in vitro i in vivo posen de manifest que una exposició continuada a Hg(II) i MeHg genera una disrupció de la barrera intestinal. Els assajos in vitro realitzats per a determinar l'eficàcia de les soques de BAL d'origen murí LE1 i LE2 com a estratègia de protecció s'han realitzat coexposant el model cel·lular combinat a Hg(II) o MeHg (1 mg/L) juntament amb les soques durant 7 dies. Les dades obtingudes han mostrat un efecte protector de totes dues soques, amb una reducció de la resposta inflamatòria, l'estrès i els efectes sobre les unions estretes i la producció de mucus. Així mateix, la presència dels bacteris restaura parcialment la permeabilitat i la capacitat de regeneració de les monocapes. Tenint en compte que en aquest estudi les BAL estan inactivades tèrmicament, aquesta protecció s'associa principalment a la seua capacitat de quelació del Hg, encara que no cal descartar que algun component estructural de la paret bacteriana puga exercir un altre tipus d'efecte beneficiós. Els assajos in vivo per a confirmar l'efecte protector de les BAL s'han realitzat en ratolins BALB/c exposats durant 2 mesos a MeHg (5 mg/L) a través de l'aigua de beguda. Els bacteris, en aquest cas viables, s'han administrat per sonda gàstrica diàriament. Les dades obtingudes en aquest assaig mostren que, encara que els dues soques redueixen per igual els continguts colònics de MeHg, la reducció dels efectes tòxics és molt més notable amb la soca LE1. S'observen reduccions dels continguts dels mediadors d'inflamació i estrès en el còlon, hi ha una recuperació de l'expressió de proteïnes constituents de les unions estretes i el mucus. Es restableixen els nivells luminals dels metabòlits de la microbiota i es restaura parcialment la permeabilitat intestinal. Els resultats mostren que a més del procés de quelació, la soca LE1 activa la ruta de senyalització Nrf2/Keap1/ARE i la producció de la citocina anti-inflamatòria IL10. Les dades obtingudes in vitro i in vivo apunten al fet que la soca LE1 podria ser una bona alternativa per a reduir la toxicitat intestinal del Hg; reducció que pot tindre també repercussions beneficioses a nivell sistèmic. / [EN] A cell model for in vitro studies in which enterocytes, mucosecretory cells and macrophages have been combined in a bicameral system has been developed. Cells have been exposed to Hg for 10 days. The data have shown that both species induce an inflammatory response and a pro-oxidant response, with an increase in reactive oxygen/nitrogen species and an overexpression of stress proteins. The situation of stress is accompanied by alterations in the expression of the tight junction protein ZO-1 and changes in the morphology of the intestinal monolayer. In addition, exposure, especially to MeHg, generates an increase in mucus secretion and an overexpression of its main mucin. The mechanisms of this hypersecretion could be related to the IL4/IL13/STAT6 pathway. All these effects on the epithelial monolayer lead to and increased permeability and a reduced regeneration capacity. In vivo assays have been performed in BALB/c mice exposed to Hg (1-10 mg/L) for 4 months through drinking water. The data obtained have confirmed the previous results observed in vitro. Exposure to both species induces an inflammatory process in the colon and generates oxidative stress with an increase in ROS/RNS and lipid peroxides. The participation in this response of some signaling pathways targeted in vitro has been confirmed in vivo. In addition, alterations in the expression of tight junction proteins and the mucin MUC2 in the colon are also evidenced, accompanied by a hyperplasia of the mucosecretory cells, especially in MeHg treatments. In these animals, there is also an increase in the expression of IL-13 and IL-4 cytokines, which points to the participation of the IL4/IL13/STAT6 pathway. In addition, these in vivo assays show an effect of both species on the metabolism of the intestinal microbiota, with a reduction in the luminal contents of short-chain fatty acids, although changes in the intestinal microbiota composition are minimal. Finally, the treated animals showed an increase in permeability. The in vitro studies carried out to determine the efficacy of the BAL strains of murine origin LE1 and LE2 as a strategy of protection have been performed by co-exposing the combined cell model to Hg (1 mg/L) together with the strains for 7 days. The data obtained have shown a protective effect of both strains, with a reduction in the inflammatory response, oxidative stress and the effects on tight junctions and mucus production. In the same way, the presence of both bacteria partially restores the permeability and the regenerative capacity of the monolayers. Bearing in mind that in this study the LABs are heat-inactivated, this protection is mainly associated with their ability to bind Hg, although it cannot be ruled out that some structural components of the bacterial wall may exert another type of beneficial effect. In vivo assays carry out to confirm the protective effect of LAB have been performed in BALB/c mice exposed for 2 months to MeHg (5 mg/L) through drinking water. The strains of LAB have been administered daily by gavage. Data show that, the reduction of the toxic effects is much more pronounced when LE1 strain is administrated. Reductions in the contents of inflammatory and stress mediators in the colon are observed, together with a recovery in the expression of proteins of the tight junctions and the mucus layer. The luminal levels of microbiota metabolites are restored, and intestinal permeability is partially reestablished. The results show that in addition to the chelation process, LE1 strain activates the Nrf2/Keap1/ARE signaling pathway and the production of the anti-inflammatory cytokine IL10. The data obtained in vitro and in vivo suggest that LE1 strain could be a good strategy to reduce Hg intestinal toxicity; reduction that can also have beneficial repercussions at a systemic level. / Rodríguez Viso, P. (2023). Estrategias para mitigar la toxicidad asociada a la exposición crónica al mercurio a través de la dieta [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192865

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