Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 210-235).

Inflammatory bowel disease causes structural and functional alterations in the enteric nervous system (ENS). Since the onset of intestinal inflammation involves the activation of resident immune cells as well as rapid influx of infiltrating cells, we proposed that changes in the ENS are a result of the release of toxic inflammatory factors. We hypothesized that early damage to the ENS in inflammation is caused by harmful levels of nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) found in immune cells. This was assessed in the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-model of colitis in rats. Large increases in infiltrating granulocytes, particularly neutrophils and blood-derived monocytes were found in the muscularis layers adjacent to the ENS. A rapid increase in iNOS immunoreactivity in the muscularis regions during early stages of inflammation (6 – 24 hr) was observed. Whether high NO levels generated by chemical donors could be toxic to neurons was tested in a co-culture model of myenteric neurons, smooth muscle and glia enzymatically isolated from neonatal rats. Exposure of co-cultures to NO for 48 hr resulted in significant, concentration dependent decrease in neuron survival. We then developed a model that permitted the direct study of immune cell interactions with myenteric neurons. Myenteric neurons were co-cultured with activated peritoneal immune cells that expressed iNOS and generated high NO levels (49 + 6.2µM) for 48 hr. This caused significant neuronal death, reducing neuron number by 19 + 5%, and disruption of axons. Pre-treatment of immune cells with a selective iNOS-inhibitor, L-NIL resulted in neuron numbers that were not significantly different from control (96 + 2%) suggesting that NO played a central role in mediating the damaging effects of immune cells. Lastly, when direct contact between immune cells and neurons was prevented in the previous experiment through use of trans-wells, unanticipated neurotrophic effects were observed. Increased axon outgrowth (282 + 57%) was detected in addition to loss of the neurotoxic effects in spite of similar experimental conditions. We concluded that proximity and contact plays an important role in determining the nature of immune cell mediated alterations in enteric neurons. / Thesis (Master, Physiology) -- Queen's University, 2009-11-30 10:09:38.384

Contamination of drinking water sources, such as groundwater, by pathogens (protozoa, bacteria and viruses) is of major concern globally. Due to their small size, mobility and high infectivity, enteric viruses have been a focus of groundwater research. However, the behaviour of enteric viruses in aquifer media is still poorly understood, which is partially attributable to the lack of reliable surrogates for these viruses. In the study reported in this thesis, a new type of surrogate was characterised and validated for its use in studying virus fate and transport in groundwater. The surrogates developed were composed of 70 nm carboxylated silica nanoparticles, labelled with dsDNA tags for sensitive detection, and coated with selected proteins to mimic the physico-chemical characteristics (size, charge, density) of two enteric viruses, human rotavirus and adenovirus, frequently found in faecal-contaminated groundwater. The selected enteric viruses and a commonly used virus surrogate, the MS2 bacteriophage, were purified and characterised in terms of size, surface charge, hydrophobicity and aggregation. For validation, the characteristics, the adsorption, degradation and transport of the surface-modified nanoparticles and the viruses were investigated in laboratory studies and compared. The characterisation of the viruses and particles revealed that the modified silica nanoparticles resemble the size and negative surface charge of the rotavirus and adenovirus. In general, the nanoparticles were found to be less hydrophobic than the enteric viruses, thus presumably less interactive with hydrophobic media. In contrast, the MS2 bacteriophage was smaller in size than the enteric viruses studied and considerably more hydrophobic implying stronger interactions with hydrophobic media. The surface-modified nanoparticles were found to be more stable and remained more monodispersed over time than the purified enteric viruses. In laboratory studies using simulated groundwater, the DNA-labelled nanoparticles were more stable over time than the rotavirus, the adenovirus or a plasmid DNA on its own. Interestingly, the study revealed that rotavirus was more persistent than the adenovirus over time in terms of degradation and aggregation, however, day light considerably enhanced rotavirus degradation. The adsorption studies revealed strong interactions between the enteric viruses and natural aquifer media (gravel and sand), whereas most of the surface-modified nanoparticles adsorbed weakly to these media. Only the casein-coated nanoparticles adsorbed strongly to the sand. The MS2 adsorbed to the gravel strongly, but weakly to the sand implying different interactions. The studies on virus and nanoparticle adsorption to hydrophobic-coated and non-modified Ottawa sand supported the results of characterisation. Column studies investigating the transport of the viruses and the nanoparticles in gravel and sand showed that even though gravel had high adsorption capacity in the adsorption tests, all viruses and nanoparticles travelled though the gravel columns with little retention, probably due to insufficient interaction time. This highlights the vulnerability of gravel aquifers to virus contamination. Experiments using sand columns showed great differences in the transport of the particles. Results suggested that the recovery of the DNA-labelled nanoparticles was similar to the recovery of the adenovirus, however, their transport pattern was different. The glycoprotein-, the protein A- and the AMBP-coated nanoparticles mimicked the transport pattern and low recovery of the rotavirus. In contrast, the streptavidin- and casein-coated nanoparticles were not recovered, emphasising the great importance of surface structure in particle transport. The results of this study demonstrated the usefulness of protein-coated silica nanoparticles as virus surrogates in groundwater studies. Surface-modified nanoparticles are able to mimic the surface characteristics of viruses. The glycoprotein-, protein A- and AMBP-coated particles were found to be suitable surrogates for rotavirus, whereas the DNA-labelled nanoparticles resembled adenovirus behaviour in hydrophilic media. Using particles with different material, size and protein-coating other pathogens can be modelled as well. Furthermore, these particles are expected to besafe to humans and the environment, thus can be used in a great variety of experiments in environmental research.

Nucleobindins are a class of secreted, multi-domain Ca2+ binding proteins that interact with nucleic acids. Two nucleobindins, nucleobindin-1 (NUCB1) and nucleobindin-2 (NUCB2) have been identified so far. In 2006, nesfatin-1, an 82 amino acid peptide encoded in NUCB2 was discovered. Nesfatin-1 is an anorexigenic and insulinotropic peptide found abundantly in hypothalamus, pancreas and stomach. Meal responsive insulin secretion is regulated by glucagon like peptide-1 (GLP-1), glucose dependent insulinotropic polypeptide (GIP), peptide YY (PYY) and cholecystokinin (CCK) secreted by intestinal mucosal cells. Since both nesfatin-1 and intestinal hormones modulate insulin secretion, nesfatin-1 could regulate intestinal hormones to elicit its insulinotropic action. Nucleobindin-1 primarily regulates Ca2+ homeostasis. Like NUCB2, NUCB1 is also present in the pancreas, stomach, intestine and pituitary. NUCB2 has a high similarity (62% in humans) to NUCB1. Both proteins also retain their prohormone convertase cleavage sites. However, no information exists on whether NUCB1 encodes bioactive peptides. The fact that NUCB1 is a secreted protein suggests an endocrine function for NUCB1 and/or its encoded peptide. This research hypothesizes that nesfatin-1 is enterotropic, and NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP). Nesfatin-1 protein expression was found in STC-1 cells and it co-localized GLP-1, GIP, CCK and PYY in mouse enteroendocrine cells. Treatment of STC-1 cells with nesfatin-1 stimulated GLP-1, GIP, CCK mRNA expression and protein secretion, while opposite effects were found for PYY. In silico analysis of the NUCB1 amino acid sequence found a 77 amino acid NLP. Mouse pancreatic islets and MIN6 cells express NUCB1 mRNA and protein. NUCB1 was co-localized with insulin in mouse pancreatic islets. While treatment of cells with synthetic NLP increased preproinsulin mRNA expression and secretion, a scrambled peptide based on NLP was ineffective, indicating that the specific amino acid sequence is crucial for its insulinotropic action. Overall, the data presented supports the hypotheses. The studies reaffirm NUCB2 expression in intestine and provide the first set of evidence for nesfatin-1 regulation of enteric hormones. It also found a novel NUCB1 encoded insulinotropic NLP that could elicit other functions of nesfatin-1.

The enteric nervous system (ENS) runs within the intestinal wall and is responsible for initiating and enacting several reflexes and motor patterns, including peristalsis and the complex interdigestive motor programs, known as migrating motor complexes (MMCs). The ENS consists of several neuron types including intrinsic sensory neurons, interneurons and motor neurons. A great deal is known about the anatomy, pharmacology and electrophysiology of the ENS, yet there is almost no understanding of how enteric neural circuits perform the functions that they do and how they switch from one function to another. The ENS contains intrinsic sensory neurons (ISNs) that connect to every neuron type in the ENS, including making recurrent connections amongst themselves. Thus, they are likely to play a key role, not just in sensory transduction, but in coordination of reflexes and motor patterns. This thesis has explored how these functions are performed by developing and analysing mathematical and computer models of the network of ISNs. (For complete abstract open document)

Thesis (M.S.)--Rutgers University, 2006. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references (p. 89-93).

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

INTRODUCTION: Foods enter the oral cavity and mix with saliva. Some foods are not well tolerated, for instance, gluten proteins in individuals suffering from celiac disease (CD). Celiac disease is a chronic immune-mediated inflammation of the duodenum, triggered by gliadin component of gluten contained in wheat, barley and rye. In previous studies we showed that oral Rothia bacteria can degrade gliadin in vitro. The objective of this study was to gain more insights into the role of Rothia bacteria and subtilisin-A enzyme on gliadin digestion in vivo, with the ultimate goal to find new therapeutic options for CD. MATERIALS AND METHODS: Part I: Rothia bacterial proteins were analyzed for enzyme activity and subjected to LC-ESI-MS/MS. For in vivo, mice chow was prepared with and without R. aeria. Gliadin epitope abolishment was assessed in the mice stomach contents (n=9/group) by ELISA. Part II: Subtilisin-A was dissolved in various solutions, temperatures and incubation time to assess enzyme activity by using enzyme substrate (Suc-AAPF-pNA). Part III: PEGylation of subtilisin-A (Sub-A) was performed by mixing Sub-A with methoxy-polyethylene glycol (mPEG) and further encapsulated by polylactic-glycolic acid (PLGA). The activity of the modified enzyme to detoxify the immunogenic gliadin epitopes was evaluated at pH3.0. RESULTS: Part I: R. aeria gliadin-degrading enzyme was found to be a member of the subtilisin family. In vivo, gliadins immunogenic epitopes were reduced by 32.6%. Part II: Sub-A dissolved at pH1.5 showed a band of 27kDa, while it only showed bands below 10kDa when dissolved at pH7.0, suggesting auto-proteolysis. The enzyme activity was completely lost at temperatures exceeding 60°C and also reduced 4-fold after 6hr incubation at 37°C. Part III: PEGylation protected Sub-A from autolysis. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gliadin immunogenic epitopes were decreased by 60% in the stomach of the mice fed with chow containing Sub-A-mPEG-PLGA. CONCLUSION: The results provide proof for the contribution of oral Rothia bacteria to gliadin digestion and pharmaceutical modification can protect Sub-A from auto-digestion as well as from acidic insults, thus rendering the usefulness of coated subtilisins as a digestive aid for gluten degradation. / 2019-01-13T00:00:00Z

O presente trabalho teve como objetivo determinar a prevalÃncia de enteroparasitos em hortaliÃas, mais especificamente a alface (Lactuca sativa) de hortas comerciais e de uso coletivo no municÃpio de Fortaleza-CE e associÃ-la aos fatores ambientais, higiÃnico-sanitÃrios e sÃcio-econÃmicos locais. Inicialmente foram cadastradas, atravÃs de um censo, 165 hortas em Fortaleza, dentre as quais 57 foram escolhidas aleatoriamente para o estudo. Cinco unidades de alface (Lacuta sativa) foram obtidas por horta, totalizando uma amostra de 285 hortaliÃas. Considerou-se como unidade amostral, 01 pà ou touceira, independente do peso ou tamanho, colhidas ao acaso. O exame parasitolÃgico das unidades foi realizado atravÃs do mÃtodo de sedimentaÃÃo espontÃnea em Ãgua e o de centrÃfugo-flutuaÃÃo em sulfato de zinco empregados ao lavado das hortaliÃas e um questionÃrio foi utilizado para obtenÃÃo dos dados epidemiolÃgicos. Em 100% das hortas estudadas, uma ou mais amostras estavam contaminadas com enteroparasitos. Foi encontrada uma positividade em 73,3% das amostras de alface examinadas, sendo diagnosticados ovos e larvas de helmintos e cistos de protozoÃrios, dentre estes: Strongyloides sp (66,7%), AncilostomÃdeos (17,5%), Ascaris lumbricoides (3,2%), Entamoeba sp, Trichuris sp, Isospora sp, e Iodameba butschilii (1,1%); Taenia sp e Toxocara sp (0,7%); e Shistosoma mansoni (0,4%). Dentre as amostras parasitadas, 54,4% apresentaram uma espÃcie de parasito, enquanto que 18,9% apresentaram duas ou trÃs. A prevalÃncia de Strongyloides sp apresentou significÃncia estatÃstica (p<0,05) apenas para idade e renda dos trabalhadores das hortas. Os demais fatores como uso de Ãgua nÃo tratada na irrigaÃÃo, proximidades com coleÃÃes hÃdricas e poluentes, solos com declives acentuados, uso de adubo orgÃnico sem nenhum tipo de tratamento, presenÃa de vetores e animais domÃsticos e inexistÃncia de local adequado para os trabalhadores das hortas evacuarem, apesar de poderem ter contribuÃdo para a elevada prevalÃncia observada, nÃo atingiram significÃncia estatÃstica. Tal fato pode ser explicado pela homogeneidade das condiÃÃes ecolÃgicas que foram precÃrias na maioria das hortas. / The present study has the goal to determine the prevalence of enteric parasites on vegetables, more specifically the lettuce (Lacuta sativa) from commercial or collective vegetable gardens of the city of Fortaleza, located in Northeastern Brazil, and to associate the findings with the environmental factors, the hygienic-sanitation conditions and the socio-economic situations locally. Initially, 165 vegetable gardens were identified spread over the city, of which, 57 were randomly chosen to have 5 unit samples examined, reaching a total of 285 vegetable units analyzed from each vegetable garden from July/99 to May/00. A unit sample was considered the whole plant independent of the size or weight. The parasite search was done through the spontaneous sedimentation in water and the centrifugal-flotation on zinc sulfate used to wash the units. A questioner was used to obtain the epidemiological data. In 100% of the vegetable gardens, at least one unit was contaminated. The following parasites were found on 73.3% of lettuces: Strongyloides sp (66,7%), ancylostomideous (17.5%), Ascaris lumbricoides (3.2%), Entamoeba sp, Trichuris sp, Isospora sp, Iodameba butschi (1.1%); Taenia sp, Toxocara sp (0.7%) and Shistosoma mansoni (0.4%). Among the positive samples, 54.4% had one single species and 18.9% had 2 or 3 different parasites identified. The Strongyloides prevalence presented statistical significance (p<0.05) for the absence of vegetables cleaning before exit and for workerâs age, and income. The other risk factors such as untreated irrigation water, proximity to pools and pollutes, accentuated ground sloping, the usage of untreated organic fertilizer, the presence of flying vectors and domestic animals, and the inexistence of inappropriate place for fecal evacuation by the workers, reached no statistic significance, despite the possible contribution to the elevated numbers that were found; that was probably related to the homogeneous ecologic conditions observed in the majority of the kitchen gardens studied.

Hajj, the annual Muslim pilgrimage to Mecca, Saudi Arabia is a unique mass gathering event that attracts approximately 3 million pilgrims from around the globe. This diverse pilgrim population coupled with the nature of the performed activities raise major public health concerns in the host country with potential global implications. Although gastroenteritis and diarrhea are common among the pilgrims performing Hajj, the microbial etiologies of these infections are still unknown. We used molecular and antigenic approaches to identify the main pathogens associated with Hajj diarrhea. 544 fecal samples from pilgrims suffering from diarrhea whilst performing Hajj during three consecutive seasons (2011-2013) and 99 control samples from 2011 were screened for 16 pathogens that include bacterial, parasitic and viral etiologies that are commonly associated with diarrheal infections. At least one of the screened pathogens could be detected in 42% (n=228) of the samples from the diarrheal cases. Bacteria were the main agents detected in 83% (n=189) of the positive samples, followed by viral and parasitic agents detected in 6% (n=14) and 5% (n=12) respectively. We have also standardized a 16S-based metagenomic approach to identify the gut microbiome in diarrheal cases and non-diarrheal controls in 76 samples. Also, we have standardized a shotgun metagenomics protocol for the direct characterization (diagnosis) of enteric pathogens without cultivation. This approach was used successfully to identify viral (adenovirus) and bacterial causes of Enterotoxigenic E. coli diarrhea from Hajj samples. The findings in this study fill in clear gaps in our knowledge of the etiologies associated with diarrheal infections during Hajj. Foodborne bacteria were the major contributors to Hajj-diarrheal infections. This was coupled with the increased incidences of antimicrobial resistance loci associated with the identified bacteria. These findings would help the public health policy makers to develop and introduce appropriate public health measures to improve the food safety during Hajj.