The current period of human history is considered to be the plastics age due to its versatile characteristics, especially the lightweight, durability and low production cost. Plastics can be manufactured to suit multifarious functions, for example, for personal care products, food/drink storage and medical purposes. Thus, the use of plastics is unavoidable now, finally contributing to the severe pollution worldwide. In 2018 alone, the global plastics production amount has exceeded 359 million tons, around 10% of which ultimately become waste persisting in the environment. When plastic wastes exposed to the sun's radiation, climate change and mechanic abrasion, degradation and fragmentation may occur. Once the size of the fragmentation products is less than 5 mm, they are commonly defined as microplastics (MPs) by the National Oceanographic and Atmospheric Administration. Currently, microplastics have been regarded as the most pervasive environmental pollution problems, not only because of their physical hazards but also due to their interactions with other pollutants in the environment. Pollution can be attributed by the release of additives from MPs, as well as the MPs with adsorbed toxic contaminants. Moreover, MPs additives together with adsorbed chemicals can be easily uptaken by animals, which may cause further propagated effects on the ambient ecosystem. Through the bioaccumulation and biomagnification effect, MPs can even be accumulated in the organisms from different trophic levels and cause serious impacts on aquatic ecology and human health. Despite growing number of evidences that have confirmed the presence and consequential effects of microplastics, researches on microplastic pollution are still lacking. Investigations on occurrence, determination and environmental fate of MPs in aquatic systems are clearly needed. Therefore, the major objective of this study is to elucidate the distribution of MPs in natural environment, to develop novel determination methods to characterize the micro-(nano-)plastics (MNPs), and to study the interactions of MPs with other contaminants in different conditions, as well as their consequential fate in different matrices (e.g. freshwater, cold-blooded intestine, and warm-blooded intestine). The spatial-temporal distribution of the MPs along the Maozhou River was investigated for both the surface water and sediments from 17 sites. Results showed that MPs were widely and unevenly distributed along the river. The MP abundances in dry season ranged from 4.0 ± 1.0 to 25.5 ± 3.5 items·L-1 in water and 35 ± 15 to 560 ± 70 item·kg-1 in sediments, which were relatively higher than those observed in wet season (water: 3.5 ± 1.0 to 10.5 ± 2.5 items·L-1; sediments: 25 ± 5 to 360 ± 90 item·kg-1; p value < 0.05). The dominant types of MPs were identified as: polyethylene (PE, water: 45.0%, sediments: 42.0%), polypropylene (PP, water and sediments: 12.5%), polystyrene (PS, water: 34.5%; sediments 14.5%) and polyvinyl chloride (PVC, water: 2.0%; sediments: 15%). Moreover, metals such as Al, Si, Ca were discovered on the rough surface of the MPs, indicating the interactions between the MPs and the aquatic environment. After obtaining the occurrence of the MPs in the aquatic systems, we proposed an accurate method for MNPs identification and quantification with the employment of the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). By optimizing the conditions (e.g. the laser energy, matrix, analyte, cationization agent and their ratio), the peaks of PS and polyethylene terephthalate (PET) were successfully identified. A quantitative correlation was built between the normalized signal intensity and ln[polymer concentration], with a correlation coefficient above 0.96 for low-molecular-weight (LM-) polymers and 0.98 for high-molecular-weight (HM-) polymers. Furthermore, two types of environmental MPs samples were prepared, including the particles of an aviation cup as the fresh plastics and the aged MPs extracted from river sediment. By using MALDI-TOF MS, the PS-related micro-(nano-)plastics (in both aviation cup and sediment) consisted of C8H8 and C16H16O oligomers, while the PET-related MNPs (only found in sediment) were identified with compositions of C10H8O4 and C12H12O4. The contents of PS and PET MNPs in sediment were quantified as 8.56 ± 0.04 and 28.71 ± 0.20 mg·kg-1, respectively. Also, the interaction between MPs and bisphenols was investigated. PVC was selected as the representative target because it is comparatively easy to decompose into MPs with the release of additives, especially the bisphenols. The released bisphenols may then be readsorbed by the PVC MPs and cause consequential pollution to the ecosystem. To elaborate on the interactions mechanism, a systematic study was carried out to determine the adsorption mechanisms of five bisphenol analogues (BPA, BPS, BPF, BPB, and BPAF) on PVC MPs. The equilibrium adsorption numbers of the bisphenols on PVC MPs are 0.19 ± 0.02 mg/g (BPA), 0.15 ± 0.01 mg/g (BPS), 0.16 ± 0.01 mg/g (BPF), 0.22 ± 0.01 (BPB), 0.24 ± 0.02 mg/g (BPAF), respectively. Intraparticle diffusion modeling (kinetics) divided the adsorption process into three stages: external mass transport, intraparticle diffusion and dynamic equilibrium. The isotherm results showed a better fit of the adsorption to the Freundlich model. Furthermore, the adsorption mechanisms of the five bisphenol analogues were explored intensively, with respect to hydrophobic interaction, electrostatic force and noncovalent bonds. Besides the adsorption process, the transfer and release behaviors of contaminated MPs are of critically importance in the exploration of their role as culprits and/or vectors for the aforementioned toxicity. Therefore, experiments were performed to examine desorption behaviors and cytotoxicity performance of contaminated MPs in aquatic surroundings and intestinal environment after ingestion by organisms (cold-/warm-blooded). The kinetic study showed that the rate of desorption for bisphenols could be enhanced threefold under simulated warm intestinal conditions. The Freundlich isotherms indicated multiple-layer desorption of the bisphenols on the heterogeneous surfaces of PVC MPs. Hysteresis was detected in the adsorption/desorption of bisphenols in a water environment, but no adsorption/desorption hysteresis was observed in the simulated intestinal conditions of warm-blooded organisms. Due to the enhanced bioaccessibility, the desorption results implied that the environmental risk of contaminated PVC MPs might be significantly increased after ingestion at a high bisphenols dosage. Although with different IC50, the five bisphenols released under the intestinal conditions of warm-blooded organisms can cause higher proliferation reduction in fish and human cell lines than the bisphenols released in water. In summary, this study elucidated the spatial-temporal distribution behaviors of MPs, developed effective determination methods for MNPs revealed the interactions mechanisms of MPs with other contaminants, and explored their consequential fate in different environments. The obtained results are helpful of better understanding on the land-based input of MPs from the intensively affected inland waters, realizing the role of microplastics as both source and carrier for emerging organic pollutants, and providing a novel alternative for MPs determination in future studies.

No description available.

In recent years, there has been an increasing demand for newer, more accurate, technologies that can detect and identify biomolecules or biological entities related to health, agriculture or the environment. With the discovery of new properties of nucleic acids beyond the storage and transfer of genetic information, a new class of nucleic acid-based biosensors is emerging, using DNA and RNA as target recognition elements with the advantage of being simpler and more cost-effective compared to antibodies-based biosensor. Two sequences, TrG14MC and TrG10SC, with evidence to suggest that they are capable of inhibit the metalloenzyme CIP, were isolated from a selection conducted by Dr. Razvan Nutiu. Here we study the inhibitory properties of these two aptamer candidates and measure the IC50 value, determined as 94 nM for TrG14MC and 83 nM for TrG10SC. Different bivalent constructs, designed to increase the inhibitory effect of the isolated sequences, are studied showing a pronounce influence of the linker length improving the inhibitory effect over CIP. Modulating the interaction of the isolated sequences and the CIP is of key importance in order to develop a successful biosensor. Therefore, we try to recover CIP from the inhibition effect by using antisense sequences complementary to different segments of the construct. The maximum recovery, 75%, was achieved by an antisense sequence fully complemented to the inhibitory bivalent construct. We also study here the use of a linker in the bivalent construct that forms a secondary hairpin structure, and the effect of linearizing that structure with an antisense sequence complementary to the linker. This resulted in as 12% of the inhibitory effect. The purpose of this investigation was to establish the first steps toward the development of a new class of biosensors capable of disinhibiting CIP upon the recognition of a specific target, taking advantage of the suggested CIP-inhibitory properties of the isolated sequences TrG14MC and TrG10SC. / Thesis / Master of Applied Science (MASc)

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The number of tissue eosinophils were counted and phospholiphase B activity was assayed in the intestines of nonsensitized and sensitized and sensitized mice infected with Trichinella spiralis.

The objectives of this study were first, to investigate the effect of stress and ascorbic acid (AA) supplement during stress on intestinal microflora of broilers and layers, and secondly, to determine nitric oxide (NO) production in intestinal tract of broilers during stress, when supplemented with L-arginine, and NO production in cecal bacteria. The intestinal microflora from broilers and layers were analyzed for bacterial populations during stress and when supplemented with ascorbic acid. In both studies, stress response was induced by adrenocorticotropic hormone (ACTH) via a mini osmotic pump for 7 days, and intestinal samples were collected before and after stress response was induced. During stress, there were no significant effects on intestinal bacterial populations, but changes in intestinal microflora were found in stressed layers and broilers. When AA was supplemented during stress, both short-term and long-term, the microbial population was changed. Cecal NO production during stress, cecal bacterial NO production, and large intestinal NO production when L-arginine was supplemented in broilers were determined as nitrite using Griess reagents. The stress response was induced as in the previous studies. The cecal pouches were collected at day 7 after ACTH insertion. Nitric oxide production by the ceca of broilers during stress was decreased. The cecal bacterial NO production was determined in vitro. The cecal bacteria that produced NO were identified as Lactobacillus fermentum, and Clostridium butyricum. Supplementing with L-arginine, Nù -nitro-L- arginine methyl ester (L-NAME), and sodium nitrate did not affect bacterial NO production on MRS agar incubated anaerobically, but sodium nitrate did affect bacterial NO production on tryptic soy and anaerobic agar incubated aerobically and anaerobically, respectively. L-arginine was supplemented in broiler diet to determine the effect on intestinal NO production and microbial populations. Supplemented with L-arginine affected cecal NO production, but did not affect large intestinal NO production or microbial populations. The positive correlation coefficient between NO contents and bacterial populations was only observed in the large intestine when L-arginine was supplemented in the diet.

No description available.

No description available.

The collection of microbes that inhabits the human gastrointestinal tract is known as intestinal microbiota, and an enormous body of work has shown that their activities contribute to health and disease. Ulcerative colitis (UC), which is a type of inflammatory bowel disease, is considered to arise due to a disruption in the balance between the immune system and microbiota. However, there is little consensus on the mechanism of action and microbes involved in the disease manifestation. In this work, I applied culture-enriched metagenomics (CEMG) to characterize the dynamics of gut microbiota in healthy individuals and UC patients. I showed that CEMG provides a higher resolution to study these microbial communities, and we used this approach to understand microbial colonization after fecal microbiota transplantation (FMT) therapy in UC patient. I showed that sequencing approaches alone did not reveal consistent engraftment across FMT responders. Using CEMG and a collection of bacterial whole-genome sequences, I showed patient-specific microbial strain transfer and a signature of commonly engrafted genes only in patients who responded to FMT. In this work, I also investigated the dynamics of a highly abundant bacteriophage, crAssphage, in an FMT donor and implemented a new method to detect bacteriophage engraftment post-FMT using SNP analysis. Finally, it has been suggested that antibiotic treatment before FMT may increase the efficacy of FMT. However, in this work, I show that while antibiotics alter the microbiome, there was no difference in the composition of the microbiome of antibiotic vs placebo group post-FMT. This is consistent with the randomized controlled trial results that shows pretreatment with antibiotics does not improve FMT outcome. Together, this work demonstrate the importance of in-depth microbiome analysis applied to culture-dependent and -independent sequencing to characterize microbial changes post-FMT. / Dissertation / Doctor of Philosophy (PhD) / Many bacteria reside in the human gut, and they are essential in our health and in disease. It is evident that these bacteria are associated with inflammatory bowel disease, but we do not yet know how and what bacteria are involved in this disease. In this work, I describe a method to study these bacteria from stool that relies on growing them and investigating their DNA. I showed that our approach helped us recover a greater diversity of these bacteria and their genetic content in healthy individuals and patients with inflammatory bowel disease compared to methods that use only DNA based approaches. Using this method, we could better understand why some patients responded to a treatment consisting of transferring stool content from healthy donor to patient. I also investigated a group of viruses that infect bacteria and implemented a new computational method based on DNA sequencing to test whether these viruses transfer to the patient after receiving the fecal therapy. We also found that antibiotic treatment before fecal therapy in patients with inflammatory bowel disease does not improve the patient’s recovery.

El fluoruro se considera importante para la salud debido a su efecto beneficioso en la prevención de la caries dental y en el desarrollo óseo en la población infantil. Sin embargo, una ingesta excesiva produce determinadas patologías, como fluorosis dental y esquelética, incluso reducciones de las capacidades cognitivas en la población infantil. El objetivo de esta tesis doctoral es la evaluación de los contenidos de fluoruro en los alimentos, su bioaccesibilidad y su absorción por el epitelio intestinal, así como los efectos que este elemento puede ejercer a su paso por el tracto intestinal. Se ha desarrollado una metodología rápida para el análisis del fluoruro presente en alimentos basada en una digestión ácida asistida por microondas y posterior detección mediante potenciometría. Esta metodología presenta ventajas respecto a la fusión alcalina, método más empleado para el análisis de fluoruro. La aplicación de esta técnica a muestras de un estudio de Dieta Total de la Comunidad Valenciana ha generado datos sobre concentraciones e ingestas de este elemento y ha puesto de manifiesto que los productos pesqueros y el arroz son los alimentos que realizan un mayor aporte del elemento. Esta situación varía en poblaciones que presentan problemas de contaminación en las aguas de consumo. Para estas comunidades, se ha evidenciado que las pastas y leguminosas aportan cantidades muy importantes de fluoruro a la dieta, similares a las de los productos pesqueros. Esto es consecuencia del proceso de cocinado con aguas con altos contenidos de fluoruro. Para una aproximación más realista a la estimación del riesgo se ha analizado la cantidad de fluoruro solubilizada desde los alimentos durante la digestión gastrointestinal (bioaccesibilidad), evidenciándose que este parámetro oscila entre 38-103%. La consideración de la bioaccesibilidad reduce la exposición al elemento respecto a los cálculos en base a los contenidos del producto. Adicionalmente, se ha puesto de manifiesto que la solubilización gastrointestinal de fluoruro está especialmente influenciada por el pH de la etapa gástrica y la concentración de sales biliares de la etapa intestinal, así como por la presencia de cationes en el proceso digestivo. El estudio de los mecanismos de absorción intestinal de fluoruro han indicado que la vía paracelular es la mayoritaria en la absorción y secreción de este compuesto. Asimismo, se ha evidenciado la participación de transportadores transcelulares dependientes de pH, incluso la intervención de mecanismos implicados en el transporte de cloruros. La elevada contribución de la ruta paracelular ha puesto de manifiesto que el empleo de células Caco-2, modelo ampliamente utilizado para los estudios de biodisponibilidad, puede no ser la aproximación más idónea para el fluoruro. De hecho el uso de co-cultivos celulares de Caco-2 con HT29-MTX, ha mostrado resultados de permeabilidad más próximos a los reportados in vivo. Adicionalmente, se ha evidenciado que la exposición a fluoruro afecta al epitelio intestinal, alterando los procesos de diferenciación celular y permeabilidad y generando una respuesta proinflamatoria. Los resultados obtenidos en la presente tesis contribuyen a incrementar el escaso conocimiento sobre la presencia de fluoruro en los alimentos y los procesos que permiten su llegada a la circulación sistémica. / Rocha Barrasa, R. (2013). Fluoruro en alimentos: contenidos, bioaccesibilidad y absorción por el epitelio intestinal [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/27667