Dissertations / Theses on the topic 'In vitro gastrointestinal assay'

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2011
Made available in DSpace on 2012-10-25T16:31:35Z (GMT). No. of bitstreams: 1 289039.pdf: 1645045 bytes, checksum: 04fc5f82cc2565abe48b6acb5256d1ff (MD5)
O ensaio PAMPA tem demonstrado sua versatilidade desde 1998, sendo utilizado para avaliar a permeabilidade passiva transcellular de fármacos/compostos, e tem ganhado espaço por ser de baixo custo, muito rápido e por auxiliar, particularmente, na elucidação dos mecanismos de transporte, em conjunto com os ensaios que utilizam células Caco-2. Para padronizar este ensaio no Laboratório de Virologia Aplicada da UFSC, dois modelos: o PAMPA TGI, variante Double-Sink e o PAMPA Pele foram selecionados por mimetizar, respectivamente, a absorção de fármacos/compostos através do trato gastrointestinal e da pele,sendo que estas duas vias foram escolhidas pela fácil adesão do paciente aos tratamentos por via oral e tópica. Inicialmente, para demonstrar a funcionalidade dos modelos em estudo, foram selecionados fármacos de alta e baixa permeabilidade, classificados segundo o Sistema de Classificação Biofarmacêutica. Além dos fármacos utilizados para a padronização, alguns cardenolídeos e o composto galato de pentila foram selecionados para testar os modelos padronizados. Para a quantificação das amostras nos compartimentos aceptores e doadores das placas usadas nos experimentos, foram desenvolvidos e validados métodos analíticos por espectrofotometria no UV, segundo critérios preconizados pela ANVISA e ICH. Os resultados obtidos na validação analítica demonstraram que tais métodos foram suficientemente específicos, lineares, precisos e exatos para quantificar as amostras. A partir dos resultados obtidos nos modelos PAMPA TGI, variante Double-Sink e PAMPA Pele, foram calculados os coeficientes de permeabilidade efetiva (Log Pe) para os fármacos/compostos testados, que permearam através das membranas lipídicas usadas e, desta forma, foi possível correlacioná-los com dados da literatura, quando existentes. O galato de pentila apresentou alta permeabilidade nos dois modelos avaliados; já os cardenolídeos apresentaram baixa permeabilidade nos dois modelos, exceto a digitoxigenina, que permeou através da membrana usada no ensaio PAMPA TGI, variante Double-Sink. A integridade das membranas lipídicas usadas nos dois modelos foi avaliada com corantes marcadores de baixa permeabilidade, Azul de Cresil Brilhante (ACB) e Lucifer Yellow (LY), e foi possível demonstrar a integridade e a uniformidade destas membranas, pela baixa passagem do ACB e pela rejeição do LY.
PAMPA assay has demonstrated its versatility since 1998, and it has been used to assess the passive transcellular permeability of drug/compounds, and has gained importance because of its low cost, fast making profile, and also for its particularly help on the elucidation of transport mechanisms together with the assays that make use of caco-2 cells. In order to standardize this assay in the Laboratorio de Virologia Aplicada UFSC, two models: variant Double-Sink PAMPA-GIT and Skin PAMPA were selected by its mimetizing effect on drugs absorption through the gastrointestinal tract and skin, respectively. These two routes were chosen because of easy patient adherence to topical and oral treatment. Initially, to demonstrate the studying models functionality, drugs with high and low permeability were selected, as classified by the Biopharmaceutics Classification System. In addition to the drugs used for standardization, some cardenolide compounds and pentyl gallate were selected to test the standard models. To quantify the samples in compartment acceptors and donors of the plates used in the experiments were developed and analytical methods were validated by UV spectrophotometry, according to the criteria recommended by ICH and ANVISA. The results obtained in the analytical validation showed that these methods were sufficiently specific, linear, precise and accurate to quantify the samples. From the results obtained in PAMPA GIT model, variant Double-Sink PAMPA and skin, the effective permeability coefficients were calculated (log pe) for the drugs / compounds tested that permeated through the lipid membranes used and thus could correlate them with the literature data. The pentyl gallate showed high permeability in the two models evaluated, the cardenolide already had low permeability in the two models, except the digitoxigenin, that permeated through the membrane used in the PAMPA assay TGI, variant Double-Sink. The integrity of the lipid membranes used in both models was assessed with colored markers of low permeability, brilliant cresyl blue (ACB) and Lucifer Yellow (LY), and it was possible to demonstrate the integrity and uniformity of these membranes, the low pass and the ACB rejection of LY.

Melanoblasts are the embryonic precursors of melanocytes, the pigment producing cells of the skin and hair. Melanoblasts are of key interest to developmental biologists for numerous reasons, including their ability to migrate throughout the body from a single origin in the neural crest (NC). Current methods for the study of the melanocyte lineage are limited by the heavy reliance on animal models. To challenge this, a platform of in vitro tools were designed to replace and complement current studies. A major obstacle is the transition from 2D cultures, which provide only limited behavioural information, to 3D models which are able to recapitulate the environmental conditions. 3D cultures are regularly created using tissue samples and synthetic matrices for attachment, but building a model from cell lines only has not been achieved. A co-culture model using immortalised keratinocyte (COCA) and melanoblast cell lines proved unsuitable for observing developmental processes, due to lack of movement at high cell densities, but may be practical in pigmentation research. Other methods were explored to examine melanoblast behaviour, including the use of cell derived matrices (CDMs) integrated with melanoblast cell lines, and aggregates formed by hanging drop (HD) culture. CDMs were successfully generated from the COCA line, as well as NIH3T3 fibroblasts which has been shown previously. These structures are denuded of cells to leave the deposited extracellular matrix (ECM) components intact, representative of the dermal (fibroblast) and epidermal (keratinocyte) layers of the skin. HDs were prepared from cultured melanoblast cell lines, and form tight aggregates which disseminate when plated, in a manner similar to the dissemination of cells from the NC in explant cultures. The receptor tyrosine kinase KIT and its ligand (KITL), are vital for melanoblast development. Previous study of this signalling complex has often focussed on the haematopoietic lineage and spermatogenesis, where they perform essential roles. KITL is expressed in a membrane localised form found on the surface of keratinocytes thought to promote melanoblast/melanocyte survival, and a soluble isoform found sequestered in the ECM which promotes cell migration. Cell lines expressing fluorescently tagged KIT and KITL were created to visualise their interactions using live-cell confocal imaging. Firstly, cell lines were generated to perform co-culture experiments with KIT and KITL, and we showed that these constructs are able to interact by uptake of KITL into KIT cells. Secondly, tandem fluorescent protein timers of KIT and KITL were generated which were used to observe protein kinetics. We showed that these protein timers can be manipulated using cycloheximide to block protein production, or by increasing ligand availability. These protein timers reveal that soluble KITL (sKITL) has a faster turnover than membrane bound KITL (mKITL), and that in all three proteins, there is distinct change in spatial localisation as the proteins age. Using a novel melanoblast reporter mouse, Pmel-CMN, primary mouse melanoblasts between E12.5 and E14.5 were isolated for RNA sequencing. This time period is the earliest reported for melanoblast isolation for use in gene expression analysis. We show that within this time course, there are significant changes in the RNA expression profiles, including decreasing expression of other NC cell markers, and huge increasing expression of pigmentation genes. To assess the biological relevance of using in vitro assays, cells of the immortalised melanoblast cell line, melb-a, were cultured under different conditions and examined via RNA sequencing. Results reveal differences in several areas between primary cells and those in culture, including loss of melanocyte specificity. The different tools described in this thesis provide a platform on which to study various aspects of cell behaviour, including migration, morphology and cell adhesion at both the individual cell and population levels.

Dietary fats are mainly transported by the intestine in lipoproteins: chylomicrons (CMs) and very low density lipoproteins (VLDLs). Unfortunately, studies of the intestinal absorption of dietary fat have been hampered by the lack of an adequate in vitro model system. As an in vitro model Caco-2 cells are able to secrete lipoproteins. We investigated the possible factors that may affect the secretion of CMs through the ultracentrifugation technique. The dose-dependent effects of oleic acid, mono-olein, egg lecithin, collagen matrix, and the effect of cell differentiation on CM secretion were then tested. We found that oleic acid, lecithin, and cell differentiation are critical for CM secretion by Caco-2 cells. To further confirm that our optimal condition is, in fact, favorable for efficient CM production, we compared it with control groups. We observed that our condition led to more efficient CM secretion as determined by the TGs, ApoB, and TEM analysis.

Medical imaging using Terahertz frequency radiation is in its infancy. Optical adjuncts to enhance the diagnostic precision of white light endoscopy are not new and almost all wavelengths of the electromagnetic spectrum have, at some point, been investigated to this end. The ultimate aim of an endoscopic technique is to identify 100% of mucosal lesions and to classify them with 100% specificity. Methods: This thesis examined the sensitivity and specificity of current technology to accurately identify colonic pathology using a diagnostic precision analysis of published data. The effect of biomaterials such as blood, mucus and faeces on Terahertz radiation was assessed using human tissue samples from health volunteers. The ability to discriminate pathological from normal colonic mucosa was assessed using terahertz radiation to interrogate excised samples of human colon. The physical nature of variation between pathological and normal colonic mucosa was assessed using histological markers in an attempt to identify the cause of terahertz radiation contrast. Results: Current technology has a sensitivity of 95% and a specificity of 78% in a non- inflamed colon. Terahertz pulsed imaging is a sensitive and specific technique for in vitro classification of colonic mucosal pathology. TPI may be a useful adjunct in the presence of inflamed mucosal tissue. Although there were too few data from the present study to quantify any potential benefit. The effects of blood, stool and mucus on TPI are similar but less pronounced than water. Immunohistochemical analysis has demonstrated an association between vascular and lymphatic density with neoplasia. This may be a mechanism for TPI discrimination of colonic pathology. TPI could in the future contribute to a minimally invasive method of in vivo diagnosis of colonic dysplasia or malignancy. Larger scale and in vivo trials of the technology are necessary to further investigate the potential clinical benefit.

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 86-88).
The gastrointestinal system plays a vital role in the functioning of the human body, processing food into useable energy, controlling homeostasis, and serving as the front line of the immune system. The intestines are aided in their many functions by the gut microbiome, a collection of 100 trillion anaerobic bacteria cells that live inside the GI tract. Although they play an essential part in the organ system, they remain little-represented in in vitro gastrointestinal models because of the difficulty of replicating the anaerobic conditions of the intestines. We constructed an in vitro model capable of growing aerobic epithelial intestinal cells along with anaerobic microbes in the same bioreactor. A device called the apical flow module seals a 12-well transwell and provides an inlet and outlet port into the apical chamber. Media is deoxygenated using nitrogen bubbles before it is pumped using a nitrogen-actuated pneumatic pump block. Microbes are injected into the anaerobic fluid through a rubber septum injection port before the fluid flows into the sealed transwell. Effluent is collected in sterile tubes at a controlled height so as to regulate the apical side pressure. Oxygen is provided to the basolateral human epithelial cells through basolateral circulation achieved using a pneumatic circulation plate. Preliminary testing confirms our ability to control the oxygen in all parts of the system and to grow cocultures of human and bacteria cells. Epithelial cells grown in our bioreactor show signs of behaving more similarly to cells in vivo when exposed to the conditions present in our system, providing researchers with an oxygen-controlled gastrointestinal in vitro model.
by Steven John Holcomb.
S.M.

Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

The FRAME KB cytotoxicity assay is an ill vitro test for basal cytotoxicity which measures the sub-lethal inhibition of cell growth by toxic substances. Exponentially growing 3T3-Ll mouse fibroblasts are exposed to a range of concentrations of a test substance for 72 hours, then relative cell number is estimated by the protein/kenacid blue dye-binding method. The assay was evaluated for its ability to predict parameters of in vivo acute lethal potency. In vivo/in vitro comparisons were performed for a set of miscellaneous chemicals and for a set of metal compounds. The degree of correlation was closer for the metal compounds than for the unrelated set, in the in vitro/mouse i.p. LDso comparison. The cytotoxicity assay was more useful than metal "softness" (a physico-chemical parameter) for predicting metal compound toxicity ill vivo. An investigation of the ill vitro toxicities of a group of commercial chemicals and formulations revealed very poor ill vivo/in vitro correlations. Some were toxic to the 3T3-Ll cells, yet of very low toxicity to rats. This was partly due to the poor solubility of some of the substances, which probably caused their virtual non-toxicity to rats by oral dosage. Chemical volatility is another methodological problem for ill vitro assays. A simple modification of the FRAME KB cytotoxicity assay was successfully developed in order to prevent the underestimation of the cytotoxicities of volatile liquids. The assay also demonstrated potential use for providing data for the safety assessment of surfactants and toiletry formulations. It is emphasised that the FRAME KB cytotoxicity assay should never be used in isolation, but as part of a battery of tests chosen for a particular type of toxicity and/or type of chemical or formulation. The F9 embryonal carCInoma cell line was evaluated for its potential usefulness in in vitro toxicity testing. F9 cells were induced to differentiate morphologically and biochemically, and it was found that cells in different stages of differentiation did not respond in the same way to toxic chemicals.

O Glutarldeído é usado como desinfetante de endoscópios, mas por ser irritante, deve ser substituído por outro produto alternativo. A água ácida eletrolisada (AAE) possui efeito bactericida, tecnologia essa desenvolvida no Japão para lavadoras de endoscópios. No nosso estudo, a contaminação endoscópica após o seu uso clínico foi examinada através de cultura para bactérias, micobactérias e fungos, tanto antes quanto após a desinfecção com glutaraldeído por 20 minutos ou água ácida eletrolizada por 7 minutos. As colônias de bactérias foram identificadas e contadas após 48 horas de incubação a 37o C. A contaminação microbiana dos colonoscópios foi detectada após 30 (trinta) procedimentos endoscópicos, contudo o tratamento com a AAE conseguiu erradicar todos os microorganismos. A atividade microbiana da AAE mostrou-se ser similar ao glutaraldeído a 2%. Os resultados indicam que a AAE é um eficiente desinfetante depois da limpeza mecânica dos colonoscópios, podendo ser usado nas unidades de endoscopias como uma alternativa ao glutaraldeído
Efficient disinfection is important given the multiplicity of bacterial exposures to equipment used in endoscopy. Glutaraldehyde is used as a disinfectant for endoscopes, but is an irritant and so should be replaced by an alternative. Electrolized cid water (EAW) has bactericidal effect, and an endoscopic washing device using EAW has been developed in Japan. In our study, endoscopic contamination after clinical use was examinated by culture for bacteria, mycobacteria and fungi before and after exposing to glutaraldehyde (20min) and electrolized acid water (7min). The bacterial colonies were identified and counted after 48 hours of incubation at 37oC. Microbial contamination of colonoscopes was detected after 30 endoscopic procedures, but the treatment of the endoscope with EAW eradicated the microbes. The microbicidal activities of EAW was similar to that of glutaraldehyde. These results indicate that EAW is effective disinfectant after mechanical cleaning of colonoscopes, and can, therefore, be used in the endoscopy unit as an alternative to glutaraldehyde

In vitro cell biology assays are routinely used to study cancer spreading, drug design and tissue repair. However, issues associated with reproducibility are reported in literature. In this thesis we investigate the overlooked source of variability that affects the reproducibility of cell biology assays, using a combined mathematical and experimental approach. By calibrating mathematical models to experimental data, we find that the initial degree of confluence significantly affects cell motility. Following the similar approach, we identify the two-phase growth in scratch assays. We then propose a proliferation mechanism for lattice-based, random walk models, which accounts for biologically more realistic crowding effects. At last, we use a lattice-based, random walk model to mimic the passaging process and find that the passage number could significantly affect the wound closure in scratch assays.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.
Includes bibliographical references.
The lack of correlation between in vitro and in vivo gene delivery experiments presents a significant obstacle in the progress of gene therapy studies by preventing the extrapolation of successful cell culture results into animals. This phenomenon has also been documented in the specific case of liver where standard hepatocyte culture systems fail to reliably predict the in vivo performance of gene delivery vectors. This is possibly a consequence of the loss of differentiated phenotype that these cells undergo when they are dissociated from their in vivo environment and cultured in vitro. This problem underscores the necessity for better in vitro models that can mimic the physiological environment and responses of in vivo liver tissue. This thesis aimed at developing an alternative in vitro gene delivery assay based on the Tissue-Engineered Liver Microreactor, a culture system designed to facilitate the morphogenesis of three-dimensional tissue-like structures from isolated liver cells under continuous perfusion, maintain cell viability and hepatic functionality for long-term culture periods and enable repeated in situ observation with microscopy. We developed experimental assays to non-invasively detect and quantify gene delivery efficiency in the 3D environment of the microreactor culture based on the application of 2-photon microscopy and spectroscopy.
(cont.) These techniques provide a convenient platform for comparative analysis of different vectors. Our main objective was to compare the gene delivery efficiency of an adenoviral vector (Ad5-CMV-EGFP) in the microreactor system and 2D hepatocyte monolayer culture. Quantitative assays were developed based on Real-Time PCR and RT-PCR to measure the levels of Ad vector uptake and transgene expression. The Ad mass transport in both systems was mathematically modeled to estimate the Ad uptake constant as a basis for comparison of delivery efficiency. This parameter was found to be significantly higher in the microreactor system, suggesting a more efficient mechanism of Ad internalization. Moreover, gene expression was measured in terms of transgene mRNA levels; the ratio of gene expression relative to Ad uptake was estimated as the basis for comparison of vector transcription efficiency. No significant difference was found between the 2 systems. These results provide some evidence that a more physiological culture system can yield different information (potentially more relevant to the in vivo situation) compared to standard in vitro culture.
by Artemis Kalezi.
Ph.D.

The field of genetic toxicology has recently undergone reform which has limited or banned the use of animal models within a number of different industries (cosmetics). Consequently, greater emphasis has been placed on developing novel, highly sensitive, in vitro test systems which can generate robust data to aid regulatory hazard and risk assessment. The main aims of this project were i) to develop a highly sensitive and specific, high throughput mammalian in vitro PIG-A gene mutation assay to enable quantitative dose response modelling and further investigate the potential use of in vitro data within human health assessment, ii) Investigate the genotype to phenotype relationship, a potentially delaying step within future OECD guideline drafting for the current in-vivo Pig-a mutation assay and iii) help develop and optimise a preliminary comprehensive human PIG-A bio-monitoring platform. During in-vitro and ex-vivo PIG-A assay development, flow cytometry was the fundamental technique utilised. Multiple additional laser excitation platforms were evaluated for use, including Amnis ImageStream TM and laser scanning confocal. Proteomic as well as genomic techniques were used during the supplementary investigations surrounding assay development, with microbiological groundings throughout. The finalised in-vitro assay protocol was established within human, metabolically active, MCL-5 cells. Using the refined assay design, proof of principle experimentations were able to show the potential for future quantitative work and the general promise with this novel approach. The genotype to phenotype relationship validation is currently still on-going following the preliminary work described herein and recent publications. The ex-vivo human PIG-A assay platforms were shown to require further optimisation in terms of sensitivity, excluding red blood cells, but showed good aptitude for future use. Currently it looks promising that further refinement could lead to a comprehensive high content, high-throughput assay system with the potential to be used within future hazard and risk assessment.

A malária é uma doença parasitária que afecta 214 milhões de pessoas no mundo inteiro. Uma das maiores dificuldades na luta contra este parasita reside no combate aos seus estadios esporogónicos. São necessários novos compostos que bloqueiem a transmissão do parasita entre seres humanos e o mosquito vector da doença. A redução natural decorrente da transição de gametócitos maduros no hospedeiro humano através de fertilização na refeição sanguínea do mosquito até ao oocisto no mosquito representa um potencial alvo de intervenção. Nesta tese, foi desenvolvido e optimizado um ensaio in vitro que permite determinar a actividade de compostos nos diversos estadios de desenvolvimento do ciclo esporogónico de P. berghei. Este processo inclui a formação de oocinetos e oocistos e maturação de oocistos. Um total de dez compostos, atovaquona, azitromicina, ciclohexamida, cloroquina, dihidroartemisinina, lumefantrina, halofantrina, pirimetamina, pironaridina e thiostrepton foram testados in vitro em termos da sua actividade sobre gametócitos, oocinetos e oocistos. A actividade dos compostos e a validade do ensaio no desenvolvimento de oocistos foi confirmada in vivo em mosquitos Anopheles stephensi. Foram identificados a atovaquona, ciclohexamida e o thiostrepton como sendo altamente potentes na redução da transformação de gametócitos para oócinetos e no desenvolvimento de oocistos. No geral, este ensaio inovador é rápido, relativamente fácil e pouco dispendioso, permitindo fazer um rastreio de uma biblioteca abrangente de moléculas para determinar o seu efeito nas fases de desenvolvimento de Plasmodium no mosquito, minimizando a necessidade de um modelo in vivo. É urgente desenvolver e validar ensaios de elevada produtividade permitindo que novas bibliotecas de compostos sejam testadas não só para P. berghei mas também para P. falciparum e P. vivax. Estes ensaios podem identificar compostos para estudos pré-clinicos permitindo encontrar drogas com actividade que bloqueia a transmissão quer em campo quer numa situação clínica. No futuro, novas combinações de fármacos devem ter atividade não só contra a fase sanguínea dos parasitas, mas também destinar-se a bloquear a transmissão.
Malaria is a parasitic disease that affects 214 million people worldwide. One of the greatest struggles of the fight against this parasite resides in its sporogonic stages. Transmission blocking compounds are necessary to prevent transmission of the Plasmodium parasite between humans and mosquitoes. The natural bottleneck created by the transitions from a mature gametocyte in the human host, through fertilization in the mosquito blood meal, to the oocyst in the mosquito, presents a potential target for intervention. In this thesis, we have developed and optimized an in vitro assay for evaluating drug activity in each of the stages of the sporogonic cycle of P. berghei. It includes ookinete and oocyst formation, and oocyst development. A total of ten compounds, atovaquone, azithromycin, chloroquine, cycloheximide, dihydroartemisinin, lumefantrine, halofantrine, pyrimethamine, pyronaridine, and thiostrepton were tested in vitro for their activity on gametocytes and ookinetes and oocysts. The assay was validated in vivo in Anopheles stephensi mosquitoes. We have identified atovaquone, cycloheximide and thiostrepton as three very potent molecules leading to a significant impairment on gametocyte to ookinete conversion and of oocyst development. Overall, this innovative assay is a fast, easy and affordable method for screening large libraries of molecules at a wide range of concentrations for their effect on the development stages of Plasmodium parasites in the mosquito, minimizing the need for an in vivo model. It is urgent to develop and validate high throughput assays allowing for new libraries of compounds to be tested against not only P. berghei, but also P. vivax and also P. falciparum. These assays could be indicative of compounds for preclinical studies to find drugs blocking transmission in the field and in a clinical situation. In the future, new drugs combinations should have not only blood stage activity, but also transmission blocking components.

Collective cell migration is critical for various physiological and pathological processes. In vitro wound healing assay has been widely used to study collective cell migration due to its technical simplicity and ability of revealing the complexity of collective cell migration. This project studies the function and importance of leader cells, the cells pulling cell monolayer migrating into free space, in endothelium and skin epithelial regeneration via plasma lithography enhanced in vitro wound healing assay. Despite leader cells have been identified in in vitro wound healing assays, little is known about their regulation and function on collective cell migration. First, I investigated the role of leader cells in endothelial cell collective migration. I found that the leader cell density is positively related with the cell monolayer migration rates. Second, we used this knowledge to study the effects of arsenic treatment on skin regeneration via in vitro wound healing assay. We found that low concentration of arsenic treatment can accelerate the keratinocyte monolayer migration. We further found that arsenic affected cell migration by modulating leader cell density through Nrf2 signaling pathway. As a conclusion of these studies, we evaluated the function of leader cells in collective cell migration, and elucidated the mechanism of arsenic treatment on skin regeneration.

A biological assay is designed to set up a rapid and robust drug-screening system on a small scale. An assay is considered as a single unit of a platform to screen various compounds for aiding in drug discovery. Each assay is carried out in a 96-well plate, each of whose wells consists of the biological component called the Spheroids. The value of each assay lies in it facilitating for versatile screening applications. The spheroid is considered as a micro-structural product. And the addition of various compounds for testing is performed in each well (consisting of the spheroids). The focus has been to put forth the production principles and validation strategies to run the biological assay and test its efficacy to be used for screening in high volumes. The assay development illustrates processing and validation techniques. The goal is to develop optimized standards to process the assay, addressing various quality control issues, from the raw material to the end-product stage. Such an approach also brings interesting analogies of biological process in a manufacturing scenario. The developed system incorporates a value stream approach, by pulling the product from the customer end. The process involves simply encapsulating HUVECs (Human Umbelical Vein Endothelial cells) from the raw material stage, culturing to form the spheroid and transferring the component to assemblage in a 96-well format undergoing stages of heat treatments. The small scale screening system allows the use of small amounts of drug, which is especially essential for new drug synthesis or in rapid decision making to find out any unknown potent compounds. The design of optimal processes in product development of the spheroid assay is illustrated. Thus in light of the value of this assay, developing the production system has been pivotal so as to produce quality spheroids in the 96-well plate formats. The quantification of the stimulatory and inhibitory effects of the different agents is required to help understand the complex biological behavior involved. The goal is to validate the data using image analysis software. The image analysis helps determine the quantification to be accurate, objective, and consistent. The quality of the product is tested by the reproducibility and robustness of the assay.

We reported the primary structure, tissue distribution, and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. The ovine intestinal oPepT1 cDNA was 2,829 bp long encoding a protein of 707 amino acid residues with an estimated molecular size of 79 kDa, and a pI of 6.57. The cDNA contained a 79-bp 5' untranslated sequence and a 630-bp 3' untranslated sequence. The proposed oPepT1 protein was 77.9, 81.3, and 82.6 percent identical to PepT1 from rabbit, rat, and human, respectively. High stringency northern blot analysis demonstrated that oPepT1 is expressed strongly in the small intestine, at lower levels in the omasum, and at much lower levels in the rumen, but is not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide amino acid nitrogen for the ruminant in a nutritionally significant manner. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. Overall, the in vitro transport characteristics of oPepT1 expressed in oocytes were similar to those of PepT1 from other species. The transport process is electrogenic and pH-dependent, but independent of Na+, Cl-, and Ca2+. It displayed a broad substrate specificity that transported neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (Kt) ranging from 20 mM to .6 mM for dipeptides and .15 to 3.0 mM for tripeptides. No responses were detected from tetrapeptides or free amino acids. Although many of the properties displayed by oPepT1 were similar to those of PepT1 from other species, some differences were noted. First, the isoelectric point of oPepT1 was lower than that of others, but the oPepT1 protein appeared to have the same biological activity as that of others at a physiological pH. Second, more potential phosphorylation sites for protein kinases were present in oPepT1. Third, compared with PepT1 from other species, oPepT1 has more negatively charged amino acids at its C-terminus.
Ph. D.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Genetic toxicity tests used for regulatory screening must be rigorously validated to ensure accuracy, reliability and relevance. Hence, prior to establishment of an internationally-accepted test guideline, a new assay must undergo multi-stage validation. An in vitro transgene mutagenicity assay based on an immortalized cell line derived from MutaMouse lung (i.e., FE1 cells) is currently undergoing formal validation. FE1 cells retain a lacZ transgene in a λgt10 shuttle vector that can be retrieved for scoring of chemically-induced mutations. This work contributes to validation of the in vitro transgene (lacZ) mutagenicity assay in MutaMouse FE1 cells. More specifically, the work includes an intra-laboratory variability study, and a follow-up study to assess the endogenous metabolic capacity of FE1 cells. The former is essential to determine assay reliability, the latter to define the range of chemicals that can be reliably screened without an exogenous metabolic activation mixture (i.e., rat liver S9). The intra-laboratory variability assessment revealed minimal variability; thus, assay reproducibility can be deemed acceptable. Assessment of metabolic capacity involved exposure of FE1 cells to 5 known mutagens, and subsequent assessment of changes in the expression of genes involved in xenobiotic metabolism; induced transgene mutant frequency (±S9) was assessed in parallel. The results revealed that the FE1 cell line is capable of mobilising several Phase I and Phase II gene products known to be involved in the bioactivation of mutagens. Collectively, the results presented support the contention that the FE1 cell mutagenicity assay can be deemed reliable and reproducible. Consequently, the assay is an excellent candidate for continued validation, and eventual establishment of an OECD (Organization for Economic Cooperation and Development) Test Guideline.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Myosin II is the molecular motor responsible for muscle contraction. It transforms the chemical energy in ATP into mechanical work while interacting with actin filaments in so called cross-bridge cycles. Myosin II or its proteolytic fragments e.g., heavy meromyosin (HMM) can be adsorbed to moderately hydrophobic surfaces in vitro, while maintaining their ability to translocate actin filaments. This enables observation of myosin-induced actin filament sliding in a microscope. This “in vitro motility assay” (IVMA) is readily used in fundamental studies of actomyosin, including studies of muscle contraction. The degree of correlation of the myosin II function in the IVMA with its function in muscle depends on how the myosin molecules are arranged on the surface. Therefore a multi-technique approach, including total internal reflection spectroscopy, fluorescence interference contrast microscopy and quartz crystal microbalance with dissipation, was applied to characterize the HMM surface configurations. Several configurations with varying distributions were identified depending on the surface property. The most favorable HMM configurations for actin binding were observed on moderately hydrophobic surfaces.   The effects on actomyosin function of different cargo sizes and amount of cargo loaded on an actin filament were also investigated. No difference in sliding velocities could be observed, independent of cargo size indicating that diffusional processive runs of myosin II along an actin filament are not crucial for actomyosin function in muscle. Furthermore, a tool for accurate velocity measurements appropriate for IVMAs at low [MgATP] was developed by utilizing the actin filament capping protein CapZ. These improvements allowed an investigation of the [MgATP]-velocity relationship to study possible processivity in fast skeletal muscle myosin II.  It is shown that the [MgATP]–velocity relationship is well described by a Michaelis-Menten hyperbola.  In addition, statistical cross-bridge modeling showed that the experimental results are in good agreement with recent findings of actomyosin cross-bridge properties, e.g., non-linear cross-bridge elasticity. However, no effect of inter-head cooperativity could be observed.   In conclusion, the described results have contributed to in-depth understanding of the actomyosin cross-bridge cycle in muscle contraction.

The principal aim of the research described in this thesis was to develop and validate an in vitro micronucleus assay in low passage Chinese hamster Luc2 cells capable of detecting numerical and structural chromosome changes. Chromosome loss was inferred by indirect visualisation of human CREST antikinetochore antibodies bound to centromeres in chemically-induced micronuclei of cytochalasin-B arrested binucleate cells. Ten core chemicals were selcted due to their known or suspected effects on components of the cell division apparatus. These chemicals were colchicine, vinblastine, pyrimethamine, diazepam, chloral hydrate, thiabendazole, hydroquinone, econazole nitrate, thimerosal and cadmium chloride. In addition, 5-Azacytidine was selected to determine if disruption of the centremere would inhibit kinetochore visualisation. Three food additives were also selected for study in the micronucleus kinetochore assay to detect indirect acting mutagens with the use of an external metabolic activation system. Six of the core chemicals induced micronuclei in Chinese hamster Luc2 cells. All six chemicals increased levels of micronuclei which were positive for kinetochore antibody labelling and hence chromosome loss. Similar results were also seen with 5-Azacytidine. The results of these studies show that the cytochalasin-B Mn/k assay is a cost effective, simple and rapid alternative to classical cytogenetic assays for the detection of chemically induced aneuploidy. Future work requires the development of a permanent staining technique to visualise kinetochores and greater standardisation between assay protocols published in the literature. These problems need to be resolved before the assay is accepted by regulatory bodies for routine testing of environmental chemicals.

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2007.
Includes bibliographical references (leaf 20).
Maintaining protein function at the biological-inorganic interface is a critical challenge for bionanotechnology. Specifically, nanoparticle-protein conjugates must be designed to interact with binding partners with biologically-relevant thermodynamics. Towards developing a nanoparticle-tagging system that minimizes interference with normal protein function, here we design and begin development of an assay to assess complex formation between nanoparticle-immobilized proteins and soluble binding partners. Two chaperone proteins, importin-a and importin-3 mediate classical nuclear transport, an essential and highly conserved example of protein complex formation in eukaryotic cells. Together, these two proteins form a chaperone complex that recognizes a nuclear localization signal (NLS), which is a short peptide sequence. Here, we synthesize and purify a fluorescently-labeled importin-a and a positive control for complex formation, which consists of bovine albumin serum (BSA) covalently conjugated to a fluorophore and NLS. Using these two fluorescent molecules, we can perform Forster Resonance Energy Transfer (FRET) experiments to study the kinetics and thermodynamics of these protein interactions. The development of this system will be used in future tests with the NLS-conjugated fluorescent gold nanoparticles.
by Lara Elise Rosenbaum.
S.B.

Dentre os microrganismos que a compõem a microbiota intestinal, sobressai Escherichia coli, que possui como principal nicho ecológico o intestino grosso de humanos. Sua importância destaca-se em fazer parte dos microrganismos comensais pioneiros na colonização da mucosa intestinal e também o seu papel patogênico causando doenças intra e extra intestinais. O objetivo deste estudo foi avaliar a resposta imune contra antígenos totais de Escherichia coli. Os resultados desse estudo podem servir como subsídio para o entendimento da resposta imune sistêmica a um microrganismo presente na mucosa. Os antígenos totais de E. coli foram obtidos após lise com solução de guanidina a 8M. Realizou-se a diálise e a dosagem de proteínas. Foi realizado a caracterização eletroforética em gel de poliacrilamida e perfil antigênico por Western Blotting. Avaliou-se a presença de anticorpos IgG total e IgA sérica específicos em 30 soros de humanos e também a resposta de células mononucleares de sangue periférico humano (PBMC) através da metabolização de MTT. Os resultados obtidos demonstraram que a suspenção de antígenos obtida era composta por várias proteínas e o teste de Western Blotting revelou que estas foram reconhecidas por anticorpos presentes nos soros de humanos. Foi possível detectar a presença de anticorpos IgG total e IgA sérica contra os antígenos de E. coli por ELISA. No ensaio de viabilidade e proliferação celular pelo MTT, observou-se que houve proliferação celular em diferentes concentrações do antígeno e a viabilidade não foi inferior a 70%. Os resultados sugerem que os antígenos oriundos de E. coli podem induzir respostas imunes locais e sistêmicas.
Among the microorganisms that make it up the intestinal microbiota, stands out Escherichia coli, which has as main ecological niche, the large human intestine. Its importance stands out in being part of the pioneers commensal microorganisms on the colonization of the intestinal mucosa also his pathogenic role causing extra intra and intestinal diseases. The objective of this study was to evaluate the immune response against total antigens of Escherichia coli. The results of this study could aid to understanding systemic immune response to a commensal microorganism that lives in the mucosa. Total E. coli antigens were obtained after lysis with 8M guanidine solution. After dialysis, protein assay was carried out. It was performed electrophoretic characterization of the antigens using polyacrylamide gel electrophoresis and the antigenic profile by Western blotting. We evaluated the presence of total IgG and IgA specific antibodies in 30 human sera. It also assessed the human response of peripheral blood mononuclear cell (PBMC) by MTT metabolization. The results obtained demonstrated that the antigens were composed of various proteins and Western Blotting showed that antigen proteins were recognized by antibodies present in human serum. It was possible to detect the presence of total IgG and IgA antibodies against E. coli antigens by ELISA. In viability assay evaluated by the MTT metabolization by PBMC, it found that cell proliferation occurred at different antigen concentrations, and viability was not less than 70 %. The results suggest that the antigens from E. coli can induce local and systemic responses.
Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

GRAÇA, José Ronaldo Vasconcelos da. O citrato de sildenafil (Viagra) inibe a motilidade gastrintestinal em ratos acordados e anestesiados e a contratilidade in vitro de tiras isoladas de duodeno de ratos ex vivo. 2005. 99 f. Tese (Doutorado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2005.
Submitted by denise santos (denise.santos@ufc.br) on 2012-11-01T15:41:59Z No. of bitstreams: 1 2005_tese_jrvgraça.pdf: 465862 bytes, checksum: e5978dafa3436ee7141781083ca8f08c (MD5)
Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2012-11-06T13:54:08Z (GMT) No. of bitstreams: 1 2005_tese_jrvgraça.pdf: 465862 bytes, checksum: e5978dafa3436ee7141781083ca8f08c (MD5)
Made available in DSpace on 2012-11-06T13:54:08Z (GMT). No. of bitstreams: 1 2005_tese_jrvgraça.pdf: 465862 bytes, checksum: e5978dafa3436ee7141781083ca8f08c (MD5) Previous issue date: 2005
We evaluated the effect of sildenafil citrate (Viagra®) a vasodilator largely used for the treatment of male erectile dysfunction, on the gastrointestinal motility in rats. Experiments were performed on 175 male, Wistar rats, weighing 200-350g. Four groups of study were done: the sildenafil effects on the: i) Gastric emptying (GE) and gastrointestinal (GI) transit and ii) Intestinal transit (IT) of liquid in awake rats; iii) Gastric compliance in anesthetized rats and iv) Contractility of rat duodenal isolated strips. i) In 64 rats fasted for 24h with previous vascular access (right jugular vein and left carotid artery), we studied the effect of an i.v. injection (0.2mL) of sildenafil (4mg/Kg) or vehicle (0.01N HCl) on GE and GI transit of a liquid meal, as well as on arterial pressure (AP) in a separated group of rats. Animals were gavage-fed with 1.5mL of a test meal (0.5mg/mL of phenol red in 5% glucose). After 10, 20 or 30min, animals were sacrificed and submitted to a laparotomy to obstruct the pylorus, cardia and terminal ileus. The gut was removed and then divided into: stomach and consecutive three small intestine segments (40% proximal; 30% medial and 30% terminal). After processing these segments, the dye retention was determined at 560nm. The percentage of dye retention in each segment permitted to evaluate GE and GI transit. Arterial pressure was continuously monitored by a digital acquisition system during 20min before and 30min after sildenafil injection. We observed a significant increase of gastric retention in sildenafil treated rats at 10, 20, or 30min after the test meal (44,2±2,0 vs 53,2±2,1; 25,4±1,3 vs 37,3±1,6; 20,9±2,5 vs 32,5±2,9%, respectively), as well as a significant GI transit delay. Despite of sildenafil inducing hypotension, AP returned to basal levels 10min afterwards. Acid gastic secretion blocking pre-treatment with omeprazol did not modify the sildenafil effect on gastric retention, GI transit or AP. ii) In another group we evaluated the sildenafil (4mg/Kg) or diluente (0.01N HCl, 0.2mL) effects on the IT in awake rats, fasted for 24h. Animals were studied 3d after the insertion of a silastic cannula (0.6cm ID) into the duodenal bulb. We evaluated the progression of a radioactive liquid test meal fed (10MBq of 99mTc – 1mL of saline 0,9%) administered through the inserted cannula into the small intestine. After 20, 30 or 40min, animals were sacrificed by anesthetic overdose. After laparotomy, we removed and divided the gut in: stomach, five congruent and consecutive segments of the small intestine and the large intestine. Radioactivity counting was obtained in a gamma-chamber collimator. Sildenafil promoted an IT delay (p<0.05), indicated by shifting the center of mass to the proximal portions of the TGI (2.8±0.2 vs 3.3±0.1; 3.0±0.2 vs 3.7±0.1 and 3.4±0.1 vs 4.2±0.2) in relation to control group. iii) Gastric compliance study was performed on 39 anesthetized rats after 24h of fasting. Gastric volume (GV) variations were measured by plethysmography while AP was continuously monitored. We have also observed that GV increased (p<0.05) after sildenafil treatment (3mg/Kg - e.v) (3.08±0.18; 3.10±0.17 and 3.09±0.17mL vs 2.91±0.19mL) at 10, 20 and 30min after drug administation, respectively. Basal AP (105.8±2.28mmHg) dropped by the sildenafil injection (59.8±3.2; 64.8±3.7 and 59.3±4.6mmHg-p<0.05) while vehicule (0.01N HCl) did not change either GV or AP. After splanchnotomy or pre-treatments (e.v.) with methylene blue (3mg/Kg-guanilate cyclase blocker), L-NNA (3mg/Kg - NO synthase blocker) or propranolol (2mg/Kg - ß-blocker) prevented GV increase due to sildenafil; while post-treatment with sodium nitroprusside (1mg/Kg - NO donor) raised it. iv) The in vitro contractility studies were performed on isolated duodenal strips obtained from rats (n=28) killed by cervical dislocation. Duodenal strips were suspended longitudinally in a glass chamber (10mL), filled with Tyrode solution (37oC and pH 7.4). After 1h of stabilization under 1g of initial tension, the spontaneous or induced contractility were continuously recorded by a digital acquisition system. Increasing and cummulative doses of sildenafil (0.1 to 300µmol/L) relaxed (9.6µmol/L of EC50) the duodenal strips. This effect was more intense than those displayed by zaprinast or papaverine (PDEs blockers) (91.6 and 78.5µmol/L of EC50, in this order). Sildenafil showed significant antispasmodic and myorelaxant effects on the duodenal contractions induced by acetylcholine or carbamylcholine (IC50 26.7 and 16.2µmol/L, respectively). Pre-treatment with methylene blue, ODQ (guanilato cyclase blocker) or L-NAME (NO synthase blocker) also prevented these sildenafil effects, but D-NAME (an inactive substrate for NO synthase) did not. Myorelaxant sildenafil effect was reverted by L-arginine (substrate for NO synthase) and contrarily it was largely increased by sodium nitroprusside. Forskolin adenylate cyclase activation pre-treatment also increased the myorelaxant effect of sildenafil. In summary, we have observed that sildenafil slowed down the gastrointestinal motility, delaying GE, GI and intestinal transits of a liquid meal in awake rats; Gastric compliance was also increased in anesthetized rats treated with sildenafil. Sildenafil also exhibited both antispasmodic and myorelaxant effects on isolated strips of duodenum of ex vivo rats. Besides central or peripheral sympathetic nervous system activation, sildenafil possibly acts at the gastrointestinal myocite level by activating the NO/GMPc system.
Estudamos o efeito do citrato de sildenafil (Viagra®), vasodilatador largamente utilizado na terapêutica da disfunção erétil, sobre o comportamento motor do trato gastrintestinal (TGI) de ratos Wistar. Para tanto, utilizamos 175 animais machos, pesando entre 200 a 350g, distribuídos nos quatro seguintes grupos de estudo: efeitos do citrato de sildenafil sobre o i) esvaziamento gástrico (EG) e os trânsitos gastrintestinal (GI) e ii) intestinal de líquido em ratos acordados; iii) a complacência gástrica de ratos anestesiados e iv) a contratilidade de tiras isoladas do duodeno de ratos ex vivo. i) Avaliamos, em 64 ratos acordados sob jejum e livre acesso à água por 24h, o efeito da injeção (0,2mL; e.v.) de sildenafil (4mg/Kg) ou veículo (HCl 0,01N) sobre o EG e o trânsito GI de líquido, bem como sobre a pressão arterial (PA). Mediante gavagem, 1,5mL da refeição-teste (vermelho de fenol - 0,5mg/mL em glicose a 5%) foi injetada no estômago. Depois de 10, 20 ou 30min, sacrificamos os animais e, após laparotomia, obstruímos o piloro, o cárdia e o íleo terminal. Removemos e dividimos o TGI em: estômago e segmentos consecutivos do intestino delgado (40% iniciais; 30% mediais e 30% terminais). Após o processamento destas porções viscerais, determinamos as absorbâncias das amostras a 560nm. A retenção fracional de vermelho fenol em cada segmento permitiu o cálculo do EG e trânsito GI. Em um grupo separado de animais, a PA foi monitorada continuamente por meio de um sistema digital de aquisição de dados durante 20min antes e 30min após o tratamento com sildenafil ou diluente. Comparado ao grupo controle, houve aumento significativo da retenção gástrica (44,2±2,0 vs 53,2±2,1; 25,4±1,3 vs 37,3±1,6; 20,9±2,5 vs 32,5±2,9%) nos animais tratados com sildenafil e sacrificados aos 10, 20, ou 30min, respectivamente, bem como retarde significativo no trânsito GI. Embora o sildenafil tenha provocado hipotensão, a PA retoma níveis basais logo após 10min. O pré-tratamento com omeprazol (bloqueador da secreção ácida estomacal) não modificou o efeito do sildenafil sobre os valores de retenção gástrica e intestinal nem nos níveis de PA. ii) Noutros animais (n=44), sob jejum de 24h e dotados previamente (3d) de uma cânula crônica no bulbo duodenal, estudamos o efeito do sildenafil sobre a progressão ao longo do intestino delgado de uma refeição teste (10MBq de Tecnécio ligado a fitato e diluído em 1mL de salina 0,9%). Decorridos 20, 30 ou 40min da injeção (0,2mL e.v.) de sildenafil (4mg/Kg) ou diluente (HCL 0,01N), sacrificamos os animais e, após laparotomia e remoção do TGI, dividimo-o em: estômago, cinco segmentos congruentes e consecutivos de intestino delgado e o intestino grosso. A contagem da radiatividade foi determinada num colimador de gama-câmara. O sildenafil promoveu retarde (p<0,05) do TI, indicado pelos retardes dos centros geométricos da refeição de 2,8± 0,2 vs 3,3± 0,1; 3,0± 0,2 vs 3,7± 0,1 e 3,4± 0,1 vs 4,2± 0,2 em relação ao grupo controle, aos 20, 30 ou 40min. iii) Os estudos de complacência gástrica foram conduzidos em 39 ratos anestesiados, sob jejum de 24h. As variações do volume gástrico (VG), foram medidas por pletismografia, enquanto a PA foi monitorada continuamente por um sistema digital de aquisição de dados. Em relação aos valores basais (2,91±0,19mL) o sildenafil (3mg/Kg – e.v.) aumentou (p<0,05) o VG após 10, 20 e 30min (3,08±0,18; 3,10±0,17 e 3,09±0,17mL). A PA basal (105,8±2,28mmHg) caiu significativamente com o sildenafil (59,8±3,2; 64,8±3,7 e 59,3±4,6mmHg) enquanto o diluente (HCl 0,01N) não modificou seja o VG ou a PA. O pré-tratamento mediante esplancnotomia ou injeção e.v. com azul de metileno (3mg/Kg-bloqueador da guanilato ciclase), L-NNA (3mg/Kg-bloqueador da NO sintetase) ou propranolol (2mg/Kg-ß-bloqueador) preveniram o aumento do VG pelo sildenafil; já o pós-tratamento com nitroprussiato de sódio (1mg/Kg - e.v.) o ampliou significativamente. iv) Avaliamos ainda o efeito do sildenafil sobre a contratilidade de tiras isoladas do duodeno de ratos ex vivo (n=28), sacrificados por deslocamento cervical. Tiras dissecadas do duodeno foram suspensas longitudinalmente em cuba de vidro (10mL), plena de solução de Tyrode (37oC e pH 7,4), e submetidas a uma tensão inicial de 1g. Após 1h de estabilização, a contratilidade espontânea ou induzida das tiras foi registrada continuamente por um sistema digital de aquisição de dados. O sildenafil em doses crescentes e cumulativas (0,1 a 300µmol/L) relaxou (EC50 de 9,6µmol/L) o duodeno, mais até que o zaprinaste ou a papaverina (bloqueadores de FDEs) (EC50 91,6 e 78,5µmol/L, nesta ordem). Observamos ademais que o sildenafil inibiu as contrações induzidas por acetilcolina ou carbacol (IC50 26,7 e 16,2µmol/L, respectivamente). Já o pré-tratamento com azul de metileno, ODQ (bloqueador da guanilato ciclase) ou L-NAME (bloquedor da NO sintetase), mas não o D-NAME (isômero inativo da NO sintetase) preveniram o efeito do sildenafil. O efeito mio-relaxante do sildenafil foi ampliado pela L-arginina (substrato do NO sintetase) ou nitroprussiato de sódio (doador de NO). O pré-tratamento com forskolina (estimulador da adenilato ciclase) também aumentou o efeito mio-relaxante do sildenafil. Em resumo, observamos que o sildenafil diminui a motilidade gastrintestinal, retardando o EG, os trânsitos GI e intestinal de líquido em ratos acordados; aumenta a complacência gástrica em ratos anestesiados além de apresentar efeitos antiespasmódico e mio-relaxante sobre tiras isoladas de duodeno de ratos ex vivo; por estimulação do sistema nervoso simpático e tendo como provável mecanismo de ação ao nível do miócito gastrintestinal a via do NO/GMP cíclico.

Circa l’1% della popolazione mondiale è colpita da celiachia. I celiaci sono costretti a seguire una dieta priva di glutine e molto spesso quest’ultima risulta essere sbilanciata e/o carente in molti nutrienti. Recentemente, l’uso di matrici alternative al frumento, come pseudocereali, legumi e cultivar di riso pigmentate sta riscuotendo grande interesse a causa del loro elevato quantitativo di composti bioattivi (polifenoli). Quindi, considerando l’importanza attuale dei polifenoli sia nella formulazione tecnologica che nella promozione di aspetti salutistici degli alimenti senza glutine, lo scopo di questa tesi è stata basata su: 1) profilazione dei polifenoli in matrici prive di glutine (farine non di frumento, legumi, pseudocereali e frutta secca) e loro proprietà antiossidanti in vitro; 2) valutazione dell’impatto di trattamenti termici e di fermentazioni microbiche sul profilo fenolico di queste matrici prive di glutine; 3) valutazione del ruolo dei polifenoli come inibitori degli enzimi amilolitici; e 4) valutazione del destino dei polifenoli caratterizzanti alimenti senza glutine durante processi in vitro simulanti digestione gastrointestinale e fermentazione fecale. I polifenoli sono stati analizzati sfruttando tecniche di metabolomica mirata/non-mirata.
Around 1% of world population is affected by coeliac disease. Coeliac people are constrained to follow a strict gluten free (GF) diet and very often this latter is unbalanced and lacks in many nutrients. In the last years, the exploitation of alternative crops or underutilized species, such as pseudocereals, legumes and pigmented cereal cultivars, is gaining interest because of their amount and profile of bioactive compounds, such as polyphenols. Therefore, considering the actual importance of polyphenols for both the formulation of GF foods and their health-promoting properties, the current PhD thesis was based on: 1) the profiling of polyphenols in GF raw materials (such as non-wheat flours, legumes, pseudocereals and nuts) and their in vitro antioxidant activities; 2) the evaluation of the impact of different heat treatments and microbial fermentations on the phenolic profile of GF raw materials; 3) the investigation of polyphenols in GF foods as inhibitors of digestive enzymes; and 4) the assessment of the fate of polyphenols characterizing GF foods during simulated in vitro gastrointestinal and fermentation processes. Polyphenols were analysed by means of targeted/untargeted metabolomics-based approaches (i.e., high resolution chromatography and mass spectrometry platforms).

Increased and combined knowledge of food processing, molecular biology, health and nutrition has triggered production of many different types of functional foods and pharmaceutics recently. The efficacy and safety of such products are being assessed prior to marketing by in vivo and/or in vitro studies. Traditional in vivo studies require excessive time, cost and labour, as well as ethical approvals with subject to humans or animals in some instances. Therefore excessive number of runs may be avoided if reliable in vitro system is available. During the course of this study, an in vitro physicochemical upper gastrointestinal tract system (IPUGS), the first of its kind in literature, has been developed to simulate the relevant conditions of the gastrointestinal tract (GIT) as closely as possible to the human physiology with multi-disciplinary approach, combining biology, physiology, gastroenterology, process technology, chemical engineering and automation. The IPUGS is aimed at having a high predictive capability towards the real digestion processes occurring in the human upper GIT which allows for examining of the bioavailability of nutrients and drugs, drug-nutrient interactions, viability of probiotics and case studies of gastrointestinal disorders. Digestion of rice and baby foods have been studied with the IPUGS by UV-spectrophotometer, HPLC, light microscope and pH meter under the conditions of normal state and common gastric disorders, such as gastroparesis, dumping syndrome, Zollinger-Ellison syndrome and hypochlorhydria. By comparing the data from many physiological and clinical sources in the literature, it would seem that the IPUGS was able to generate more reliable data compared to the existing in vitro digestion (mechanical) models in the literature. In future, computer-controlled and computer-recorded data by possibly designing a new software or equations would be desirable to implicate a better understanding of the digestive processes.

Abstract Tumor associated macrophages (TAMs) are linked to the invasion of oral tongue squamous cell carcinoma (OTSCC). We modified THP-1 leukemia cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type macrophages in order to examine their interactions with OTSCC-cells (HSC-3) by using different kinds of in vitro migration and invasion models. We observed that interaction of TAM-resembling M2-type macrophages with HSC-3 cells induced invasion and migration, whereas the influence of M1 macrophages reduced them. Patient response to chemoradiotherapy is highly reliant on the characteristics such as the aggressiveness and stage of the cancer. Therefore, new methods for treatment testing are needed in order to design personalized therapies. We tested the applicability and consistency of human TME mimicking tissue methods for analyzing the effects of chemoradiation using commercial OTSCC cell lines. Based on our trials, both our human uterine leiomyoma tissue -based matrix models provide viable platforms for future in vitro chemoradiotherapy testing. Conventionally pro-tumorigenic activities of matrix metalloproteinase (MMP)9 have been linked with oral squamous cell carcinoma, but recently its tumor-suppressor role has also been revealed. Our study provides strong evidence that MMP9 also has an anti-invasive effect in OTSCC and is a potential mediator of the protective effects of arresten in tongue cancer cells
Tiivistelmä Makrofageilla on yhteys kielen levyepiteelikarsinooman invaasioon eli syöpäkasvaimen tunkeutumiseen ympäröivään kudokseen. Tutkimuksessamme muokkasimme ihmisen THP-1 leukemiasoluja kemiallisesti tulehdusreittejä aktivoiviksi M1-makrofageiksi, kasvaimeen liittyvien makrofagien kaltaisiksi M2-makrofageiksi sekä imidatsokinoliini-käsitellyiksi R848-makrofageiksi. Tarkoituksenamme oli tutkia makrofagien ja kielisyöpäsolujen vuorovaikutuksia erilaisilla in vitro migraatio- ja invaasiomalleilla. Anti-inflammatoristen, syövän etenemistä edesauttavien TAM-makrofagien kaltaisiksi erilaistetut M2-tyypin makrofagit lisäsivät HSC-3 kielikarsinoomasolujen invaasiota ja migraatiota, kun taas M1-tyypin makrofagien vaikutus oli päinvastainen. Potilaan vaste kemosädehoitoon riippuu syöpäkasvaimen ominaisuuksista, kuten syöpäsolujen aggressiivisuudesta ja syövän levinneisyysasteesta. Tämän vuoksi on tarve uusille menetelmille, joiden avulla voidaan ottaa huomioon potilaan sekä syöpätyypin yksilölliset ominaisuudet hoitoa suunniteltaessa. Testasimme syöpäkasvaimen mikroympäristöä mallintavien, ihmiskudokseen perustuvien menetelmien käyttökelpoisuutta ja luotettavuutta kemosädehoidon vaikutusten arvioimiseen. Testiemme perusteella myoomakudokseen pohjautuvat menetelmät voivat auttaa kemosädehoidon vaikutusten testauksessa. Matriksin metalloproteinaasi (MMP) 9:n on pitkään uskottu olevan yksinomaan syövän etenemistä edesauttava molekyyli. Viimeaikaisissa tutkimuksissa on myös havaittu, että MMP9:llä voi olla syövältä suojaavia vaikutuksia. Tutkimme MMP9:n vaikutusta kielisyöpäsoluihin ja havaitsimme, että MMP9:llä on myös invaasiota hillitseviä vaikutuksia. Lisäksi MMP9 saattaa toimia verisuonten muodostumista estävän arresten-molekyylin syövältä suojaavien mekanismien välittäjänä

Mestrado em Biotecnologia Industrial e Ambiental
O corpo humano abriga uma comunidade microbiana da qual, parte vive no aparelho digestivo. O cólon é a região com a comunidade bacteriana mais densa, denominada microbiota intestinal. O seu desenvolvimento em bebés pode ser influenciado por um número de fatores, tal como o ambiente intrauterino, tipo de parto e/ou o modo de alimentação. No que diz respeito ao modo de alimentação, o leite materno tem um papel importante na colonização intestinal de bebés através do fornecimento de uma variedade de oligossacáridos. Para bebés que não possam ser amamentados, uma formulação infantil é necessária como substituta e, portanto, deve satisfazer as necessidades nutritivas dos recém-nascidos. A manipulação da microbiota intestinal, recorrendo à adição de probióticos e/ou prebióticos à dieta, tem-se tornado uma prática recorrente. Assim, este estudo teve como objetivo testar um novo oligossacárido do leite humano (NMO), via experiências gastrointestinais in vitro, a fim de proporcionar a melhor formulação possível para substituição do leite humano. Inicialmente, uma experiência de pré-triagem foi realizada para obter informações sobre as potenciais diferenças inter-individuais entre bebés, em resposta à administração de NMO. No global, 7 dos 10 dadores responderam intensamente ao tratamento com NMO, o que resultou numa acidificação do meio. Um efeito bifidogénico foi também observado, com a degradação de NMO ocorrendo através de diferentes cenários de resposta. A escolha do dador 10 foi fundamentada tendo em conta a sua elevada taxa de fermentação e consequente produção de acetato, mas principalmente devido a um intenso efeito bifidogénico, mais especificamente, a um estímulo característico da B. longum subsp. infantis, após administração de NMO. De seguida, um baby M-SHIME® com 5 unidades foi realizado utilizando amostras fecais de um único doador (bebé 10) como inóculo, tornando esta segunda parte do projeto ainda exploratória. Diferentes doses de um "golden standard" (GS) e NMO foram testados. O consumo base-ácido, as concentrações de ácidos gordos de cadeia curta (AGCCs), lactato e amónio e a composição da microbiota foram analisados. Durante o período de tratamento, 3.2 g/L de GS, NMO, ou combinações destes, foram adicionados aos reatores, resultando no aumento dos níveis de consumo de base-ácido e de AGCCs relacionados com a saúde. Um pico no lactato foi observado para as misturas e diminuições dos níveis de marcadores proteolíticos foram também observados. No que diz respeito a mudanças na composição de Bifidobacterium, GS provocou um estímulo de B. longum, enquanto NMO aumentou a abundância de B. longum subsp. infantis, com um efeito dose-resposta claro em ambas as situações. No decorrer do tempo, a administração NMO causou também um aumento dos níveis de Enterobacteriaceae com relação dose-resposta. Das enterobactérias podem também fazer parte alguns agentes patogénicos e, sendo assim, a dosagem de NMO seria recomendada. A dose ótima pode, por conseguinte, ser a dose para a qual existe ainda uma forte estimulação de B. longum subsp. infantis e AGCCs relacionados com a saúde, embora não resultando numa grande expansão de enterobactérias, como, por exemplo, 75% / 25% ou 50% / 50% (GS / NMO).
The human body harbours a microbial community, part of which lives in the gastrointestinal tract. The colon is the region with the densest bacterial community, called gut microbiota. Its development in infants may be influenced by a number of factors, like intra-uterine environment, delivery mode and/or the feeding mode. Regarding the feeding mode, human breast milk plays an important role in early gut colonization of infants. It does so by providing a variety of human milk oligosaccharides. For infants who cannot be breastfed, infant formula is required as a substitute and so, must satisfy the nutritional requirements of infants. Since modulation of gut microbiota resorting to the addition of probiotics and/or prebiotics to the diet is increasingly becoming a recurrent practice, in order to provide infants that do not receive breast-feeding with the best possible alternative formula feeding, this study aimed to test a new human milk oligosaccharide (NMO) via gastrointestinal in vitro experiments. Firstly, a pre-screening experiment was performed to gain information on the potential inter-individual differences among babies in response to NMO administration. Overall, 7 out of the 10 donors responded strongly to the NMO treatment resulting in an acidification of the medium. A bifidogenic effect was also noted, with NMO degradation being found to occur via several different response scenarios. The choice of donor 10 was substantiated based on the strong overall fermentation and corresponding acetate production, but mainly due to a strong bifidogenic effect and thus, most interestingly, a specific stimulation of B. longum subsp. infantis upon NMO administration. Afterwards, a 5 units’ baby M-SHIME® experiment was performed using faecal sample from a single donor (donor 10) as the inoculum, making this second part of the research still exploratory. Different doses of a “golden standard” (GS) and NMO were tested. Base-acid consumption, short chain fatty acids (SCFAs), lactate, ammonium concentrations and microbiota composition were analysed. For the treatment period, 3.2 g/L of GS, NMO or combinations of thereof were supplemented to the vessels resulting in increased base-acid consumption and health-related SCFAs levels. A peak in lactate was observed for the mixtures and minor decreases were also observed on proteolytic markers. With respect to changes in Bifidobacterium composition, it followed that GS stimulated B. longum, while NMO increased the abundance of B. longum subsp. infantis with a clear dose-response effect in both situations. Over time, NMO administration caused an increase of Enterobacteria levels in a dose-related way. Given the fact that Enterobacteria also contain opportunistic pathogens, dosing would be recommended. The optimal NMO dose might therefore be the dose at which there is still a strong stimulation of B. longum ssp. infantis, and health-related SCFAs, although not resulting in a major expansion of Enterobacteria, like for example 75% / 25% or 50% / 50% (GS / NMO).

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciências dos Alimentos, Florianópolis, 2017.
Made available in DSpace on 2017-08-01T04:12:52Z (GMT). No. of bitstreams: 1 348396.pdf: 1675720 bytes, checksum: 0e2ecf374c9b627190e55517e767a98a (MD5) Previous issue date: 2017
Stevia rebaudiana (Bert.) é uma planta que vem atraindo o interesse da indústria e da comunidade científica nos últimos anos devido aos efeitos do extrato de estévia quanto a sua atividade antioxidante, anti-hiperglicêmica, antimicrobiana e adoçante. Dessa forma, a adição de extrato de S. rebaudiana (Bert.) com o intuito de enriquecer produtos como o iogurte torna-se interessante. Os principais objetivos deste trabalho foram avaliar o comportamento do iogurte incorporado de extrato liofilizado de estévia (FSE) quanto aos aspectos físico-químicos, microbiológicos e ao teor de compostos fenólicos totais (CFT) e atividade antioxidante nos dias 1, 15 e 30 de armazenamento a 4 ± 2 ° C, bem como avaliar a atividade antioxidante através dos métodos FRAP e ABTS e a bioacessibilidade dos compostos fenólicos durante a simulação gastrointestinal in vitro após 30 dias de armazenamento. Foram avaliados iogurtes enriquecidos com 0,25 % e 0,5 % (m/m) de extrato de estévia e um iogurte controle (sem adição de extrato). Tanto a adição quanto a quantidade de FSE adicionado no iogurte contribuíram para o aumento da atividade antioxidante, do teor de CFT, dos parâmetros de cor (a* e b*) e do teor de sólidos totais dos iogurtes. Por outro lado, a acidez, o pH, a sinerese e as contagens de S. thermophilus e L. bulgaricus do produto final não foram significativamente afetadas (p > 0,05). A amostra enriquecida com 0,5 % de FSE apresentou os maiores valores para CFT, ABTS e FRAP. Ao longo do tempo de armazenamento, o teor de de sólidos totais, a sinerese, a cor e o teor de CFT das amostras adicionadas de FSE permaneceram estáveis, enquanto que a atividade antioxidante e o pH diminuíram significativamente. Após a simulação da digestão in vitro, a bioacessibilidade dos compostos fenólicos e a atividade antioxidante dos iogurtes enriquecidos aumentou cerca de 2,5 e 7,5 vezes, respectivamente, em relação às frações não digeridas. Assim, comprova-se que o enriquecimento do iogurte com extrato de estévia aumenta as propriedades antioxidantes e o teor de compostos fenólicos do iogurte, podendo ser uma opção promissora para o desenvolvimento de alimentos funcionais.

Abstract : Stevia rebaudiana (Bert.) is a plant that has been attracting the interest of industry and the scientific community in recent years due to the effects of the stevia extract on its antioxidant , antihyperglycemic, antihypertensive, antimicrobial activity and sweetener. In this way, it became interesting to add extracts such as S. rebaudiana (Bert.) in order to enrich products such as yogurt. The main objectives of this work were to evaluate the behavior of the incorporated yogurt of freeze-dried stevia extract (FSE) regarding the physicochemical, microbiological and total phenolic compounds (CFT) and antioxidant activity on days 1,15 and 30 of storage at 4 ± 2 °C, in addition to evaluating the antioxidant activity through the FRAP and ABTS methods and the bioaccessibility of the phenolics during in vitro gastrointestinal simulation at the end of the shelf life (day 30). The yogurts enriched with 0,25 and 0,5 % (w / w) of stevia extract and the control sample (without extract) were evaluated. The incorporation of FSE and the percentage of addition contributed to increase antioxidant activity, CFT, color parameters (increased values for a* and b* and decreased value for L *) and total solids; while the acidity, pH, syneresis and counts of S. thermophilus and L. bulgaricus of the final product were not significantly affected (p> 0,05). The sample enriched with 0.5 % FSE presented the highest values for CFT, ABTS and FRAP. Throughout the storage time, the parameters of total solids, syneresis, color and CFT of the samples added of FSE remained stable, whereas the antioxidant activity and pH decreased significantly. After in vitro digestion simulation, the bioaccessibility of the CFT and the antioxidant activity of the enriched yogurts increased by 2,5 and 7,5 times, respectively, in relation to the undigested fractions. Thus, it is proven that the enrichment of yogurt with stevia extract actually increases the antioxidant properties and the amount of phenolic compounds of the yogurt, being to be a promising option for the development of functional foods.