Studies were carried out on microsomes of the digestive gland of the common mussel, Mytilus edulis L. Cytochrome P-450 specific content, and the specific contents or activities of other mixed-function oxidase (MFO) components, and the oxidative activities benzo[a]pyrene hydroxylase (BPH) and NADPH-independent 7-ethoxycoumarin O-deethylase (ECOD), all varied seasonally. To varying extents, correlations were seen between changes in these parameters, and changes in the mussel reproductive cycle and/or the seasonal variation in water temperature. The existence of P-450 isoenzymes was indicated by asynchrony in the seasonal changes in BPH and NADPH-independent ECOD activities relative to the changes in P-450 specific content, and by seasonal changes in cytochrome P-450 A max and the microsomal protein profile on SDS PAGE. Indications of P-450 isoenzymes were also obtained from purification studies, and from kinetic studies of NADPH-independent ECOD activity (multiphasic kinetics seen with 7-ethoxycoumarin concentration). The purification scheme essentially comprised sodium cholate solubilization, (NH4)2SO4-protein fractionation and affinity and ion-exchange column chromatographic steps. Two cytochrome P-450 peaks were obtained on DEAE-sephacel ion-exchange chromatography (KC1 elution), the overall purification for the major peak being x 20, with a yield of 5%. The final detergent-free P-450 preparation was largely in the low-spin state, and had a monomeric molecular weight of 53.0 Kd. Ligand-binding experiments were performed on partially-purified cytochrome P-450 preparations. Type II spectra were obtained with N-substituted imidazole compounds, metyrapone and pyridine, but with compounds giving type I spectra with mammalian cytochromes P-450 (testosterone, 7-ethoxycoumarin, a-naphthoflavone and SKF 525-A), reverse type I spectra were seen. In vitro MFO metabolic activity toward possible xenobiotic and endogenous substrates, was limited, and surprisingly, largely NADPH-independent. NADPH-independent ECOD activity was susceptable to modulators of mammalian MFO activity, and indicated to be cytochrome P-450-mediated. Mussel microsomal fraction and microsomal-extract were shown to inhibit rat MFO activities and hexobarbital binding to rat cytochrome P-450. These and other results are discussed in terms of a possible "endogenous blocking" of the substrate-binding site of the mussel P-450, and in terms of possible mechanisms of cytochrome P-450 catalytic action in addition to monooxygenation, such as peroxidation. Mussel cytochrome P-450 specific content was relatively unaffected by a variety of mammalian model P-450 inducers, with the exception of a small elevation with exposure to 3-methylcholanthrene (3MC). In contrast, NADPH-cytochrome c (P-450) reductase activity was slightly more responsive. An increase in NADPH-independent ECOD activity with 3MC-exposure was seen at one time of the year, but not at other times.

Field and laboratory studies on the morphology, gut contents, ingestion rates and digestion efficiency of Pollicipes pollicipes were combined to obtain estimates of the likely range and quality of materials required to sustain this species . Orientation of P. pollicipes on the shores of south; west Portugal appeared to be determined by microtopography. Animals generally faced into the wave backwash. Orientation could be temporarily altered by torsion of the peduncle in response to changes in flow direction, permitting more efficient filter feeding. Cirral and mouthpart morphology suggested that P. pollicipes, Capitulum mitella and Lepas anatifera were omnivores. Size-related changes in cirral morphology made small juveniles better equipped than adults to feed on small particles. Cirral activity of P. pollicipes was investigated. Very slow rhythmic cirral extension (or 'beating') was observed in all P. pollicipes, J but only in relatively still water; once flow rates exceeded 14 cm s all barnacles exhibited prolonged cirral extension. The 'beat' rate was temperature dependent in most animals and larger animals exhibited a lower extension frequency than juveniles. It was concluded that 'beating' was primarily respiratory in function and not a feeding mechanism. The gut contents of wild P. pollicipes included animal and algal material but little inorganic matter, -.,. Small organic material predominated in small juveniles while large organic material predominated in adults. The rates of faecal production and growth were much higher in barnacles feeding on zooplankton than on algae and although algal cells were ingested in high numbers, the energy intake was so low that animals barely maintained their body weight. Digestive efficiencies varied with diet but little with barnacle size. A wide range of digestive enzymes were identified in P. pollicipes and L. anatifera suggesting that a variety of foods may be digested. Specific enzyme activity was low, characteristic of more carnivorous animals.

Submitted by JOSÉ RICARDO BARBOZA SILVA null (jose.ricardo_medvet@hotmail.com) on 2018-03-10T14:57:32Z No. of bitstreams: 1 Silva, J.R.B., 2018. Dissertação vs final arquivo.pdf: 2884438 bytes, checksum: 1aeb3fe55c567c7423c443a4d83a7ae2 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-03-12T13:06:22Z (GMT) No. of bitstreams: 1 silva_ jrb_me_botfca.pdf: 2796813 bytes, checksum: 80083164ac1f3e931aed7c335545c884 (MD5) / Made available in DSpace on 2018-03-12T13:06:22Z (GMT). No. of bitstreams: 1 silva_ jrb_me_botfca.pdf: 2796813 bytes, checksum: 80083164ac1f3e931aed7c335545c884 (MD5) Previous issue date: 2018-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Na abordagem dos bovinos com enfermidades digestivas, durante o exame físico, exames complementares são necessários, de forma a retificar a suspeita clínica, contribuir para o estabelecimento do prognóstico e decisão clínica. Tais exames compreendem a avaliação do perfil hematológico, bioquímico, líquido peritoneal, ruminal, exame ultrassonográfico abdominal e laparotomia exploratória. Este último por se tratar de uma abordagem cirúrgica convencional, está associado as complicações e custos inerentes a técnica. Neste cenário a videolaparoscopia se apresenta com uma possibilidade para reduzir as complicações trans e pós-operatórias. O objetivo deste trabalho foi revisar e comparar a utilização da videolaparoscopia, ultrassonografia e demais métodos diagnósticos das enfermidades digestivas dos bovinos. Foram utilizados 7 bovinos mestiços, atendidos na Clínica de Bovinos de Garanhuns, unidade hospitalar da UFRPE. Realizou-se exame físico, ultrassonografia abdominal, videolaparoscopia exploratória, coleta de amostras de sangue, para a realização de hemograma, determinação plasmática da proteína total e fibrinogênio, e do soro foram realizadas análises dos indicadores bioquímicos: Proteína total, albumina, globulinas, relação albumina/globulina, glicose, L-lactato, uréia, creatinina, as atividades enzimáticas da aspartato aminotransferase (AST), gama glutamiltransferase (GGT), creatino quinase (CK) e lactato desidrogenase (LDH). Foi coletado líquido peritoneal e realizada análise físico-química e bioquímica. Diante dos resultados, observamos que a videolaparoscopia e ultrassonografia abdominal auxiliam na determinação do diagnóstico e prognóstico precisos das enfermidades digestivas. / 2016/2006-4

Hyperspectral (HS) imaging combines spectral measurement of a pixel with 2D imaging technology. It is capable to provide a series of images containing both spectral and spatial information, and has been widely used in medical domain. However, most researches on medical HS imaging are regarding ex-vivo biopsy or skin and oral mucosa. The study on HS imaging regarding in-vivo disease lags far behind. In this thesis, we developed a novel flexible HS endoscope system. It is capable to obtain a series of HS images in vivo in a non-contact way among the wavelength range of 405 – 665 nm. After a lot of time-consuming modifying and debugging work, this new system has high stability and convenience to be applied in clinic now. We evaluated this system in clinic. First, we got ethics approval for clinical trials. Then, we obtained HS images regarding gastrointestinal (GI) diseases inside patients using this system. As far as we know, this type of in-vivo image data has not been reported in previous literatures. Thus using these HS images, we built a database for GI mucosa. Next, we analyzed some typical HS images tentatively. The method of Recursive Divergence is implemented to extract valuable and diagnostic information from these HS images. The results prove the effect and applicability of this new HS endoscope system, which has shown the great potential to be used as a platform and guidance for further medical studies. To further apply the analysis results in clinic, we propose a novel Adaptive Narrow-Band Imaging (ANBI) method based on band selection of HS images of a specific type of disease. It is expected that the new technique has higher accuracy, sensitivity, and specificity compared to conventional Narrow-Band Imaging (NBI) technique. In this thesis, we also discuss the future direction of the system improvement. Especially, to improve light intensity and signal-noise-ratio of HS images in wide-field view, we propose a new imaging method using broad- and overlapped-band filters. Although this method only performs greatly on the foundation of accurate image registration, we hope to apply it in our system in the future.

No description available.

Human populations are at risk of exposure to constitutive androstane receptor (CAR) activators present in a range of substances, including pharmaceuticals, plasticizers and crop protection agents. What exposure to CAR activators means for human health is uncertain. Activation of CAR in rodents is associated with liver hyperplasia, increased proliferation and eventual hepatocarcinoma; however the effect in human hepatic cells is unclear. There are two methods by which a compound can achieve activation of CAR; directly or indirectly via cellular signalling pathways. Phenobarbital is a prototypical activator of CAR and does so in an indirect manner via suppression of epidermal growth factor receptor (EGFR) signalling. Direct activation of CAR in rodents also causes hepatocellular carcinoma but the human outcome is less clear. We have carried out microarray and miRNA analysis of CITCO (a potent and selective hCAR ligand) treated primary human hepatocytes. To mitigate the well documented effect of primary hepatocyte dedifferentiation primary hepatocytes were cultured in dynamic three dimensional culture in vitro. Gene expression changes indicate that direct activation of hCAR causes the promotion of a pro-proliferative and anti-apoptotic phenotype. The miRNA expression profile is crucially different to rodent data that is currently published. Despite the pro-proliferative phenotype shown there is no evidence that primary human hepatocytes proliferate in response to direct activation of CAR by CITCO. This leaves the possibility that a proliferative response may be observed in vivo or that the changes in gene expression are solely a human physiological adaptation to direct hCAR activation by CITCO and no proliferation would occur. The effect on human health and liver toxicity is unclear but this body of work has provided data that may be used to further understand the mechanistic effects of direct hCAR activation in human hepatocytes. A more complete understanding of this will help to inform the toxic potential of direct hCAR activation in vivo.

Cryptosporidiosis is a common infectious diarrhoeal disease of mammalian livestock and humans worldwide. The etiological organisms responsible are intestinal apicomplexans of the genus Cryptosporidium, including C. parvum, that infect intestinal epithelial cells. Immunocompromised or malnourished hosts develop severe life-threatening disease. Immunological elimination of Cryptosporidium requires CD4+ T cells and IFN-γ. Nevertheless, studies have shown innate immune responses have a significant protective role. Importantly, in T cell-deficient mice, IFN-γ is important for control of C. parvum infection. In innate immunity natural killer (NK) cells are major producers of IFN-γ and are activated by cytokines including type I IFNs but the roles of these components in immunity to Cryptosporidium infection have not been investigated. Therefore, the purpose of this project was to study the involvement of type I IFNs and NK cells in immunity to C. parvum employing in vitro and in vivo (murine) infection models. Enterocytes were shown capable of the production of type I IFNs in response to C. parvum infection. These cytokines directly inhibited parasite development in epithelial cells. Also, in neonatal SCID mice the level of infection increased after treatment with anti-type I IFN neutralising serum. A higher level of infection was observed in Rag2-/-γc-/- mice deficient in T, B and NK cells in comparison to Rag2-/- mice with a normal NK cell population and early mortality during chronic infection of adult animals was associated with the absence of NK cells. Using cultures of SCID mouse splenocytes, NK cells were the main source of IFN-γ in response to C. parvum antigen stimulation. However, IFN-γ was also found to have a protective role in Rag2-/-γc-/- mice, implying cells other than lymphocytes produce this cytokine. In conclusion, this is the first study to indicate important protective roles for type I IFNs and NK cells in innate immunity against C. parvum.

Background: Colorectal cancer is the result of dysregulation within classic regulatory pathways in epithelial stem-cell(s): the precursors giving rise to all other intestinal lineages. The resulting cancer stem-cell (CSC) generates tumours utilising its innate properties, e.g. self-renewal and lineage plasticity. CSCs appear to persist within a tumour as a distinct subtype responsible for local recurrence/metastasis. Therefore, therapies targeting colorectal CSCs may lead to improved cancer-specific outcome measures. The PROM-1/CD/AC133 cell surface marker has been associated with colorectal CSCs and its expression is reported as an independent negative prognostic marker. Therefore, this thesis sought to investigate the role of PROM-1/CD/AC133 in colorectal cancer. Methods: Tissue-culture, RT-qPCR, IHC, Western blotting, siRNA, PCR-array and FACS analyses were used to quantify and profile mRNA/protein expression patterns. Results: PROM-1/CD/AC133 was widely expressed in patient-matched colorectal tumour, adjacent normal epithelium, vascular invasion, lymph node metastases with significantly decreased expression in liver metastases. Furthermore, PROM- 1/CD/AC133 expression was not found to enrich colorectal cancer cell line populations for additional stem cell phenotypes (expression of ABCB1/ABCG2/BMIJ Murphy 1/CD44/LGR5/MSI-1). The data confirm the presence of alternative splice variants of PROM-1, and show that transcripts specifying PDZ binding predominate in colorectal cancer cell lines. Concomitantly, siPROM-1 was shown to modulate the expression of several key transcripts in colorectal tumourigenesis as well as regulate signal transduction pathways including the central cancer, colorectal cancer, NF-kB and p53 signalling cascades. Conclusions: PROM-1/CD/AC133 does not identify rare colorectal cancer cells responsible for tumourigenesis. However, it is associated with the regulation of signalling networks associated with cell growth, differentiation and apoptosis suggesting a potential role for this marker in colorectal tumourigenesis. The identification of specific target genes and signalling pathways in this thesis provides a springboard for further investigations into the functional role of this marker in colorectal cancer, with the potential for better treatments for this disease.

Objectives: Tolerance is a hyporesponsive state caused by repeated exposure to a stimulus. In the intestine, dysregulation of tolerance to luminal stimuli may lead to chronic and deleterious inflammation, such as characterizes Inflammatory Bowel Disease. The role of T-cells in immune tolerance is well known, but that of the epithelium requires investigation. Epithelial tolerance is gene-specific and differentially regulated, but the role of and involvement of epigenetics in tolerance regulation is unknown. We hypothesized that prior stimulation may cause epithelial cells to become hyporesponsive (tolerized) and that modification of histone methylation may alter the response to pro-inflammatory stimulation. The aim of this work was to examine if known inhibitors of histone methylation modifying enzymes affected the expression of CXCL8 in response to IL-1β. Methods: CXCL8 production of intestinal epithelial cells was measured by ELISA after stimulation with the pro-inflammatory stimuli P3CK and IL-1β and small molecule epigenetic inhibitors. The CXCL8 production of cells stimulated with a pro-inflammatory stimulus was compared to pre-stimulated cells after a second stimulus. CXCL8 production of IL-1β-pre-stimulated cells was also compared to CXCL8 production when these cells were incubated with epigenetic inhibitors. The effects of these inhibitors on histone methylation levels were examined by Western blotting for the global effect and by ChIP-qPCR for specific effects at the CXCL8 locus. Results: Intestinal epithelial cells stimulated with pro-inflammatory stimuli produced a large CXCL8 response. Pre-stimulation significantly decreased CXCL8 production after a second stimulus. The time-course of CXCL8 expression was measured to ensure that CXCL8 expression due to pre-stimulation was over before the second IL-1β-stimulation. In the presence of specific epigenetic inhibitors, pre-stimulation by IL-1β did not reduce CXCL8 production after a second IL-1β- stimulation. The specific effect of these inhibitors on the epigenetic signature at the CXCL8 locus was confirmed by ChIP. Thus, histone methylation modification disrupted tolerization of intestinal epithelial cells to a pro-inflammatory stimulus. Conclusion: The inflammatory response of the intestinal epithelium can be tolerized by prior stimulation with pro-inflammatory cytokines. Tolerization is lost after incubation with inhibitors known to modify histone methylation status, indicating for the first time, the involvement of histone methylation in this phenomenon.

Linear growth retardation affects up to 40% of children with Crohn’s disease (CD). It is caused by a combination of under\nutrition, and by a direct effect of cytokines on the axis linking human growth hormone (GH), insulin like growth factor\1 (IGF\1) and the growth plate of growing bones. In particular,the inflammation causes a functional insensitivity to GH, resulting in low circulating IGF\1. Current management is synonymous with the optimal management of childhood CD:to eliminate inflammation,and maintain remission. However, there is currently no consensus as to how to treat growth failure in patients whose inflammation remains intractable to treatment. This thesis examined the hypothesis that successful treatment of inflammation would improve linear growth in children with CD. We performed a series of retrospective studies examining a cohort of children aged ≤17 years with CD treated at Bart’s and The London Children Hospital. We determined the height standard deviation scores (SDS) of patients at diagnosis and investigated growth outcomes following treatments used to induce and maintain remission. These were:exclusive enteral nutrition (EEN), thiopurines and infliximab. Finally, as a potential new therapy, we considered the use of exogenously administered recombinant human IGF\1 (rhIGF\1) to restore plasma IGF\1 levels. We performed an open\labelled pharmacokinetic (PK) study of rhIGF\1 in 8 children with CD and growth failure. 9% of our patients had height SDS of \2 SDS at diagnosis; a four\fold increase compared to the normal age matched population. In addition, symptom duration negatively correlated with height SDS at diagnosis (r=\0.06; p=0.02). From 89 5 patients receiving EEN for primary induction of remission, 62.9% (56/89) of patients achieved complete remission. Over the course of 5 years, responders to EEN grew significantly better than non\responders (change in height SDS +0.18 [0.12] vs to \ 0.37 [0.13] respectively; p=0.005). This occurred despite no differences in duration of remission or treatment escalation between groups. 51% (26/51) patients treated with thiopurines were in remission at 12 months. These patients had improved growth (median change height SDS [IQR] 0.08 [\0.06 – 0.19] vs \0.24 [\0.61 \ \0.07] in non\responders; p=0.001). 39.2% (20/51) of these patients started anti\TNF therapy. However, over 12 months, we found that this conferred no improvement in growth (median [IQR] at 0 months\0.38 [\1.73 to \0.10] as compared to \0.61 [\ 1.72 to \0.09] at 12 months; p=0.43), irrespective of response to treatment. Subcutaneous treatment with 120 μg/kg twice daily rhIGF\1 restored plasma levels of IGF\1 in our patients, albeit it in some to high levels (median [range] concentration +2.09 [\1.27 to +5.21]). We were able to develop a PK mathematical model from these results to determine a more appropriate dosage. We found that in addition to patient’s age and weight, PCDAI also needs to be taken into consideration when determining dose. In summary, growth retardation is very common finding in paediatric CD. Response to EEN and thiopurines seems to result in beneficial effects upon growth. However, a number of children continued to have poor growth despite treatment. Our results show it is possible to increase circulating IGF\1 concentrations with exogenous injections, and develop a mathematical model to devise a dose to use in further trials.