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Lai Kit Ching Jan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 121-141). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.vi / List of abbreviations --- p.vii / List of figures --- p.ix / Table of contents --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- "The structure, function and regulation of growth hormone" --- p.1 / Chapter 1.2 --- Historical perspective of growth hormone secretagogues --- p.5 / Chapter 1.3 --- Ghrelin and growth hormone secretagogue receptor --- p.7 / Chapter 1.4 --- Calcium signalling of growth hormone secretagogues --- p.12 / Chapter 1.5 --- Pancreas and functions of exocrine pancreas --- p.15 / Chapter 1.6 --- Regulation of exocrine pancreatic function --- p.22 / Chapter 1.7 --- Functions of ghrelin in pancreas --- p.32 / Chapter 1.8 --- Aims of study --- p.34 / Chapter Chapter 2 --- Materials and methods --- p.35 / Chapter 2.1 --- Experimental animal and cell line models --- p.35 / Chapter 2.1.1 --- Rat model --- p.35 / Chapter 2.1.2 --- Isolation of pancreatic lobules and acinar cells --- p.35 / Chapter 2.1.3 --- Omeprazole-induced gastric acid inhibition --- p.36 / Chapter 2.1.4 --- Cerulein-induced acute pancreatitis --- p.37 / Chapter 2.1.5 --- Starvation rat model --- p.37 / Chapter 2.1.6 --- AR42J cell line --- p.38 / Chapter 2.2 --- Reverse transcriptase-polymerase chain reaction --- p.39 / Chapter 2.2.1 --- Total RNA extraction and quantification --- p.39 / Chapter 2.2.2 --- Reverse transcription --- p.40 / Chapter 2.2.3 --- Polymerase chain reaction --- p.40 / Chapter 2.2.4 --- Gel electrophoresis --- p.41 / Chapter 2.2.5 --- Optimization of semi-quantitative RT-PCR --- p.42 / Chapter 2.3 --- Western blot analysis --- p.43 / Chapter 2.3.1 --- Extraction of total protein and quantification --- p.43 / Chapter 2.3.2 --- Sodium Dodecyl-sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.43 / Chapter 2.3.3 --- Tricine Sodium Dodecyl-sulphate Polyacrylamide Gel Electrophoresis (Tricine-SDS-PAGE) --- p.44 / Chapter 2.3.4 --- Electroblotting and immunodetection of proteins --- p.45 / Chapter 2.4 --- Immunocytochemistry --- p.47 / Chapter 2.4.1 --- Preparation of isolated acinar cells for paraffin sections --- p.47 / Chapter 2.4.2 --- Preparation of AR42J cells slices --- p.47 / Chapter 2.4.3 --- Immunofluorescent double staining --- p.47 / Chapter 2.5 --- Functional studies of digestive enzyme secretion from pancreatic isolated acini and lobules --- p.49 / Chapter 2.5.1 --- Incubation of pancreatic isolated acini and lobules with different peptides --- p.49 / Chapter 2.5.2 --- Quantification of protein content --- p.50 / Chapter 2.5.3 --- Measurement of a-amylase secretion by α-amylase assay --- p.50 / Chapter 2.6 --- Spectrofluorimetric measurement --- p.52 / Chapter 2.6.1 --- Incubation of AR42J cells with Fluo-4/AM --- p.52 / Chapter 2.6.2 --- Pretreatment of antagonist or blockers and Ca2+-free treatment --- p.52 / Chapter 2.6.3 --- Calcium mobilization assay --- p.53 / Chapter 2.7 --- Statistics and data analysis --- p.54

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Bontebok, Damaliscus pygargus pygargus, formerly classified as D. dorcas dorcas, are territorial animals with interdigital glands between the toes of the forelegs. Males regularly defecate on dung heaps, on which they often lie, to communicate with other members of their species. They also communicate by means of visual displays, scent marking and occasionally with scraping or pawing of dung heaps. It is assumed that scent marking with the interdigital secretion serves to define territories frequented by these antelope. These glands secrete a complex mixture of volatile and non-volatile compounds and the volatile compounds in the secretion serve as a chemical signal for other bontebok. It has been suggested that the interdigital secretion is not produced in its final composition by the interdigital gland alone, but that microbial activity is responsible for many of the compounds present in the secretion. In general, many compounds can be attributed to the by-products of microbial hydrolysis of triglycerides, a common characteristic of sebum. It is well documented that micro-organisms inhabit the deep recesses of sebaceous glands and the presence of micro-organisms has been found to be consistent in all antelope exocrine glandular areas. This study involved the chemical characterisation of the volatile metabolites produced in vitro by micro-organisms from the interdigital cavity of the bontebok. Various comparative studies were made, one of which was comparison of the metabolites produced by the individual microbial species as well as the total community of bacteria incubated in different media. A comparison of the compounds identified in the interdigital secretion and the metabolites produced by the micro-organisms in the different media was also made. The volatile metabolite extracts of the individual bacterial species and of the total community were chemically characterised by low-resolution gas chromatography-mass spectroscopy. Classes of compounds identified from the volatile metabolite extracts include: • Acids - Aliphatic (saturated and unsaturated) • Alcohols - Aliphatic (saturated and unsaturated) • Aldehydes - Aliphatic (saturated and unsaturated) • Aromatic compounds • Ketones - Aliphatic (saturated and unsaturated) • Pyrazines • Dimethyldisulphide • Squalene and cholesterol Several qualitative differences were found between the compounds identified in the volatile metabolite extracts of the micro-organisms when incubated in tryptic soy broth (TSB) and minimal salt medium (MSM). In particular, when the microbes were incubated in TSB medium a number of pyrazines were found that were not present when utilising MSM as a medium. Additional qualitative differences were found between the compounds identified in the metabolite extracts of the individual bacterial species and the total community of bacteria, when incubated in both TSB and MSM media. A comparison of the interdigital secretion and the metabolite extracts of the microbial communities incubated in TSB and MSM revealed that many compounds produced in MSM corresponded to the compounds identified in the interdigital secretion. These corresponding compounds were found to be saturated and unsaturated acids, aldehydes and squalene. Furthermore, there was only one corresponding compound in the case of TSB as medium. / AFRIKAANSE OPSOMMING: Die bontebok, Damaliscus pygargus pygargus, voorheen geklassifiseer as D. dorcas dorcas, is 'n territoriale dier met interdigitale kliere tussen die kloutjies van die voorpote. Ramme ontlas gereeld op mishope, waarop hulle dikwels lê, om met ander lede van die spesie te kommunikeer. Hulle kommunikeer ook deur middel van visuele seine, reukmerking en soms deur mishope met die voorpote te kap of te skraap. Reukmerking met die interdigitale afskeiding dien klaarblyklik om gebiede wat deur hierdie diere bewoon word, af te baken. Die interdigitale kliere skei 'n komplekse mengsel van vlugtige en nie-vlugtige verbindings af en die vlugtige verbindings dien as chemiese sein vir ander bontebokke. Die vermoede bestaan dat die interdigitale klier nie alleen verantwoordelik is vir die finale samestelling van die interdigitale afskeiding nie, maar dat mikrobiese aktiwiteit bydra tot die produksie van baie van die verbindings wat in die afskeiding aanwesig is. Sekere verbindings kan in die algemeen toegeskryf word aan die vorming van die neweprodukte van mikrobiese hidrolise van trigliseriede, 'n algemene eienskap van sebum. Dit is bekend dat die diep holtes van vetkliere 'n goeie teelaarde is vir mikroorganismes en daar is gevind dat mikroorganismes feitlik deurgaans voorkom in alle anteloop eksokriene klierareas. Hierdie studie behels die chemiese karakterisering van die vlugtige metaboliete wat in vitro deur mikroorganismes van die interdigitale klierholte van die bontebok geproduseer word. Verskeie vergelykende studies is uitgevoer waarvan een die vergelyking was van die metaboliete wat deur die individuele mikrobiese spesies sowel as die totale gemeenskap van bakterieë geproduseer word tydens inkubasie in verskillende media. Vergelyking van die verbindings wat in die interdigitale afskeiding geïdentifiseer is met die metaboliete wat in verskillende media geproduseer is, het ook deel van die studie uitgemaak. Die vlugtige metaboliet ekstrakte van die individuele bakteriese spesies en van die totale gemeenskap is chemies gekarakteriseer deur middel van laeresolusie gaschromatografie-massaspektrometrie. Die volgende groepe verbindings is onder andere in die vlugtige metaboliet ekstrakte geïdentifiseer: • Sure - Alifaties (versadig en onversadig) • Alkohole - Alifaties (versadig en onversadig) • Aldehiede - Alifaties (versadig en onversadig) • Aromatiese verbindings • Ketone - Alifaties (versadig en onversadig) • Pirasiene • Dimetieldisulfied • Skwaleen en cholesterol Verskeie kwalitatiewe verskille is gevind tussen die verbindings wat geïdentifiseer is in die vlugtige metaboliet ekstrakte van die mikroorganismes onderskeidelik in TSB medium en MSM geïnkubeer. Opvallend was byvoorbeeld die voorkoms van pirasiene in gevalle waar mikroorganismes in TSB medium geïnkubeer is, terwyl hierdie groep verbindings afwesig was wanneer MSM gebruik is. Onderlinge kwalitatiewe verskille is ook gevind tussen die verbindings wat geïdentifiseer is in die metaboliet ekstrakte van die individuele bakteriese spesies en die totale gemeenskap van bakterieë, wanneer in TSB medium sowel as in MSM geïnkubeer is. Vergelyking van die verbindings in die interdigitale afskeiding en in die metaboliet ekstrakte van die mikrobiese gemeenskappe, het getoon dat 'n aantal verbindings wat in MSM geproduseer is, ooreenstem met verbindings wat in die interdigitale afskeiding geïdentifiseer is. Daar is gevind dat hierdie verbindings versadigde en onversadigde sure en aldehiede en skwaleen is. Met TSB as medium was daar slegs een ooreenstemmende verbinding.

Orientador: Odair Correa Bueno / Resumo: Atualmente, a tribo Attini compreende 45 gêneros e entre eles estão as formigas-cortadeiras cultivadoras de fungo, pertencentes aos gêneros Atta e Acromyrmex, que causam grandes danos econômicos a agricultura devido ao intenso corte de materiais vegetais frescos para cultivo do seu fungo simbiôntico Leucogaricus gongylophorus. Essas formigas apresentam diferentes mecanismos de defesa para proteger seus ninhos contra organismos competidores. Dentre esses mecanismos está a defesa humoral e celular (inata e adquirida) do sistema imune interno e a defesa imune externa, que inclui qualquer característica atuando no ambiente capaz de melhorar sua proteção contra patógenos, sendo esta caracterizada em formigas pela remoção física de patógenos e pela secreção de compostos antimicrobianos advindos de glândulas exócrinas e simbiontes bacterianos. Poucos estudos buscam integrar os diferentes fatores envolvidos na capacidade defensiva das formigas a fim de compreender estratégias fisiológicas adquiridas para proteger a colônia. Diante disso, a presente pesquisa teve por objetivo investigar a defesa da formiga-cortadeira Atta sexdens contra patógenos. Para tanto, realizamos a revisão sistemática dos mecanismos de defesa das formigas discutidos na literatura, a qual nos direcionou para investigação da defesa química externa dessa espécie de formiga. Os ensaios conduzidos foram de atividade enzimática, análise cromatográfica e testes de inibição de fungos patógenos e antagonistas. Verificam... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently Attini tribe comprises 45 genera and among them are fungus-growing leaf-cutting ants belonging to the genera Atta and Acromyrmex. They cause great economic damage to agriculture due to the intense cutting of fresh plant material for cultivation of its symbiotic fungus Leucogaricus gongylophorus. These ants present different defense mechanisms to protect their nests against competing organisms. Among these mechanisms is the humoral and cellular defense (innate and acquired) of the internal immune system and the external immune defense. This last one includes any hereditary characteristic acting in the environment and being capable of improve its protection against pathogens. It is characterized in ants by the physical removal of pathogens and by secretion of antimicrobial compounds from exocrine glands and bacterial symbionts. Few studies have sought to integrate the different factors involved in ants' defensive capacity in order to understand physiological strategies acquired to protect the colony. In this way, the present research had the goal to investigate the defense of the ant-cutter Atta sexdens against pathogens. For this, we performed the systematic review of ant defense mechanisms discussed in the literature, which directed us to investigate the external chemical defense of this ant species. The tests carried out were of enzymatic activity, chromatographic analysis and tests of inhibition of pathogenic and antagonistic fungi. We have observed that secretion... (Complete abstract click electronic access below) / Mestre

Orientador: Cristiane Yume Koga Ito / Coorientadora: Marcia Hiromi Tanaka / Banca: Luana Marotta Reis de Vasconcellos / Banca: Bruno Mello de Matos / Banca: Soraya Carvalho da Costa / Banca: Daniel Freitas Alves Pereira / Resumo: A Fibrose Cística (FC) é uma doença genética de elevada prevalência global e que causa função anormal das glândulas exócrinas. As alterações nas funções das glândulas salivares podem impactar a saúde bucal que por sua vez podem influenciar a saúde geral. A boca pode representar um reservatório microbiano de potenciais patógenos e colonizadores das vias aéreas, causando infecções crônicas pulmonares. O objetivo deste estudo foi avaliar os impactos da FC na cavidade bucal, saliva e microbioma bucal. Foram incluídos no estudo 50 pacientes com diagnóstico de FC com idades de 3 a 20 anos, divididos em 2 grupos de acordo com o grau de severidade da doença determinado pelo escore de Shwachman-Kulczycki: G1 (baixa severidade) e G2 (alta severidade). Foi também incluído grupo controle pareado ao grupo de estudo quanto ao gênero e idade (G3, n=50). A presença de lesões de cárie foi avaliada. O impacto da FC sobre a saúde bucal foi avaliado por questionário preenchido pelos pais ou responsáveis. Amostra de saliva estimulada foi coletada de todos os pacientes. O microbioma bucal foi avaliado por Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) e metodologias de cultivo, para análise da microbiota potencialmente oportunista e cariogênica. Realizou-se ainda a análise proteômica da saliva e quantificação de imunoglobulinas salivares. Os resultados foram analisados e, de acordo com a distribuição dos dados e avaliação desejada, foram aplicados os testes estatístic... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract : Cystic Fibrosis (CF) is a genetic disease with high global prevalence that causes abnormal function of the exocrine glands. The functional alterations of salivary glands and saliva can impact the oral health and influence general health. Oral cavity may represent a microbial reservoir of potential pathogens that can colonize the airways and cause chronic pulmonary infections. The aim of this study is to evaluate the impact of cystic fibrosis on the oral cavity, saliva and oral microbiome. Fifty CF patients aged from 3 to 20 years were divided into two groups according to the disease severity determined by the Shwachman-Kulczycki score: G1 (low severity) and G2 (high severity). Also, age and gender paired control group was included in the study (G3, n = 50). The occurrence of caries was evaluated. The impact of CF on oral health was evaluated by a questionnaire filled by parents or responsible person. Stimulated whole saliva (WS) samples were collected from all patients. The oral microbiome was analyzed by Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) and by microbiological culture methodologies to evaluate the potential opportunistic and cariogenic microbiota. The proteomic analysis of saliva and quantification of salivary immunoglobulins were carried out. Statistical analysis was performed according to the normality of the data at a significance level of 5%. The applied questionnaire pointed out that oral health did not impact systemic health negatively, according to the parents in all groups. The groups of patients with CF had lower rates of dmft, DMFT, salivary flow rate and initial pH in comparison to the control group. The counts of staphylococcal and yeast from CF groups were significant higher than the controls. All fungal isolates were susceptible to the antifungal agents. Higher incidence of bacterial resistance was ... (Complete abstract click electronic access below) / Doutor

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Au cours de ce travail, nous nous sommes attachés à étudier certaines réponses cellulaires secondaires à l’activation des récepteurs purinergiques P2X dans deux modèles différents, les macrophages péritonéaux et les cellules des glandes sous-maxillaires. Ces cellules contribuent à notre immunité innée, soit tournée vers l’intérieur (macrophages), soit vers l’extérieur (glandes sous-maxillaires).<p><p>Nous avons dans un premier temps confirmé par Western blot et par des dosages de la concentration intracellulaire de calcium ([Ca2+]i) que les deux types de cellules étudiés expriment des récepteurs P2X4 et P2X7 fonctionnels.<p><p>Nous nous sommes alors concentrés sur deux réponses impliquées dans la protection de l’hôte contre les agressions et l’élimination de pathogènes :la production d’espèces réactives de l’oxygène (ROS) ainsi que la sécrétion de la cytokine pro-inflammatoire interleukine-1beta (IL-1beta). Nos résultats montrent que la production de ROS en réponse à l’ATP extracellulaire est secondaire à l’activation d’une NADPH oxydase dans les deux types de cellules. Cette réponse est médiée par les récepteurs P2X7 ainsi que, dans les macrophages, par d’autres récepteurs purinergiques comme par exemple les récepteurs P2X4 et des récepteurs P2Y. Dans les glandes exocrines, contrairement aux macrophages, la protéine kinase C ainsi que ERK1/2 interviennent dans l’activation de la NADPH oxydase. <p><p>Par la suite nous avons comparé la régulation de l’expression et de la sécrétion d’IL-1beta par les macrophages et les glandes sous-maxillaires. Nous avons observé que l’IL-1beta est présente dans la salive collectée chez des souris injectées par de la pilocarpine. Des analyses par ELISA, RT-PCR et Western blot montrent que la cytokine est exprimée de manière constitutive par les cellules acineuses et ductales des glandes sous-maxillaires, à un niveau plus élevé que dans les macrophages. Contrairement aux cellules phagocytaires, l’expression de la cytokine dans les cellules des glandes salivaires n’est pas augmentée suite à la stimulation par des lipopolysaccharides. De même, dans ces cellules l’ATP n’a pas provoqué la sécrétion d’IL-1beta malgré l’efflux de K+ secondaire à l’activation des récepteurs P2X7. <p><p>Dans une dernière série d’expériences nous avons évalué les effets du peptide antimicrobien CRAMP sur les macrophages murins. Le CRAMP a inhibé toutes les réponses secondaires à l’activation des récepteurs P2X7 (ouverture du canal cationique, formation de pore, production de ROS, libération d’IL-1beta, d’acide oléique et de lactate déshydrogénase). L’inhibition par le CRAMP de l’augmentation de la [Ca2+]i en réponse à l’ATP n’était pas médiée par les récepteurs aux peptides formylés car les agonistes de ces récepteurs n’ont pas bloqué cette augmentation. Le CRAMP n’a pas eu d’effet sur l’augmentation de la [Ca2+]i secondaire à l’activation des récepteurs P2X4 par une combinaison d’ATP et d’ivermectine.<p><p>Nos expériences ont révélé que les récepteurs P2X7 sont couplés à diverses voies de signalisation dans les macrophages et dans les glandes exocrines. Les voies activées diffèrent en fonction du type de cellules. Nous avons également conclu que les peptides antimicrobiens de la famille de cathélicidines ne sont pas des agonistes universels des récepteurs P2X7.<p><p><p><p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Type I diabetes is an autoimmune disorder characterized by the loss of insulin producing islet cell mass. While daily insulin injection provides an easy means of glycemic control, it does not prevent long-term complications associated with diabetes. Islet transplantation has been suggested as a permanent cure for type 1 diabetes. However, the recurrence of host immunity and shortage of donor islets hinder the prevalence of islet transplantation. Biomaterial strategies provide an alternative route to solving the problems associated with host immune response and shortage of donor islets. One highly recognized platform for achieving these goals are hydrogels, which are hydrophilic crosslinked polymers with tissue-like elasticity and high permeability. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, including encapsulation of pancreatic islets and serving as a material platform for pseudo-islet differentiation. PEG hydrogels formed by mild and rapid thiol-ene photo-click reactions are particularly useful for studying cell behaviors in three-dimension (3D). Thiol-ene PEG-based hydrogels can be rendered biodegradable if appropriate macromer and cross-linker chemistry is employed. However, the influence of hydrogel matrix properties on the survival, growth, and morphogenesis of cells in 3D has not been fully evaluated. This thesis aims at using norbornene-functionalized PEG macromers to prepare thiol-ene hydrogels with various stiffness and degradability, from which to study the influence of hydrogel properties on pancreatic cell fate processes in 3D. Toward establishing an adaptable hydrogel platform for pancreatic tissue engineering, this thesis systematically studies the influence of hydrogel properties on encapsulated endocrine cells (e.g., MIN6 beta-cells) and exocrine cells (PANC-1 cells), as well as human mesenchymal stem cells (hMSC). It was found that thiol-ene photo-click hydrogels provide a cytocompatible environment for 3D culture of these cells. However, cell viability was negatively affected in hydrogels with higher cross-linking density. In contrast to a monolayer when cultured on a 2D surface, cells with epithelial characteristic formed clusters and cells with mesenchymal features retained single cell morphology in 3D. Although cells survived in all hydrogel formulations studied, the degree of proliferation, and the size and morphology of cell clusters formed in 3D were significantly influenced by hydrogel matrix compositions. For example: encapsulating cells in hydrogels formed by hydrolytically degradable macromer positively influenced cell survival indicated by increased proliferation. In addition, when cells were encapsulated in thiol-ene gels lacking cell-adhesive motifs, hydrolytic gel degradation promoted their survival and proliferation. Further, adjusting peptide crosslinker type and immobilized ECM-mimetic bioactive cues provide control over cell fate by determining whether observed cellular morphogenesis is cell-mediated or matrix-controlled. These fundamental studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for pancreatic tissue engineering