Dissertations / Theses on the topic 'Human bioactivity'

The insulin-like growth factors, IGF-I and IGF-II, are ubiquitous polypeptide molecules that have mitogenic and metabolic actions in a wide variety of cell types, and consequently play a major role in mammalian growth and development. Unlike many other peptide hormones, IGF levels and their bioactivity are highly dependent upon the secretion of a family of six specific binding proteins, named IGFBPs. In this thesis, we have developed a normal human fibroblast in vitro cell culture model to investigate both factors that affect IGFBP secretion, the mechanisms behind such regulation, and also the effect of IGFBP modulation upon subsequent IGF-I mitogenic activity. Firstly, we have examined the possible role that the recently discovered IGFBP proteases may have in determining the effect of IGF-I on IGFBP-3 abundance in fibroblast conditioned media. We show that IGF-I increased IGFBP-3 when assessed by ligand blotting, but did not increase immunoreactive IGFBP-3, a discrepancy that could be explained by further data showing an inhibitory effect of IGF-I on the activity of the fibroblast IGFBP-3 protease. Thus, IGF-I protection of IGFBP-3 from enzymatic degradation may help explain the post-transcriptional, non-receptor mediated 'stimulation' of IGFBP-3 by IGF-I in these cells. We also show for the first time the ability of a number of cytokines to regulate IGFBP secretion, perhaps indicating a novel pathway of communication between these immune cell molecules and the IGFs. The inflammatory cytokine interleukin 113(] L-16) inhibited Hs68 fibroblast IGFBP-3 secretion by down-regulating its gene expression, whilst tumour necrosis factor oc (TNF(x) had a similar inhibitory effect on IGFBP-3 and IGFBP-4 but acted via a post-transcriptional mechanism. The exact nature of the TNF(x effect remains to be determined as no evidence was found to suggest TNF(x increased IGFBP protease activity, or that TNF(x removed IGFBPs from the conditioned media by increasing the proportion immobilised on the cell surface. The inhibition of IGFBP secretion by TNF(x was observed to have marked effects upon the mitogenic activity of IGF-I in these cells, with a five-fold increase in sensitivity to the growth factor seen in a novel cytochemical bioassay. 1 Inhibition of fibroblast IGFBP secretion appeared to be restricted to certain cytokines as IL-6 had no effect, whilst high doses of interferon gamma abolished the TNF(X effect. Conversely, transforming growth factor 6 (TGFB) directly stimulated fibroblast IGFBP-3 secretion, via an increase in gene expression, and subsequently resulted in the reduction in the mitogenic activity of IGF-I. These data indicate that a variety of mechanisms can be employed by a number of factors to elicit changes in fibroblast IGFBP secretion, and that these changes may have direct consequencesin determining IGF-I bioactivity. Such is the importance given to the IGFs in maintaining normal somatic growth, changes in IGFBP secretion may contribute to the altered cellular growth and metabolism seen associated with cytokines in conditions as diverse as chronic infection, rheumatoid arthritis, cancer and the wound healing process.

In vitro, la formation de structures de type tubulaire avec des cellules endothéliales de veine ombilicale humaine (HUVEC) a été étudiée en combinant la fonctionnalisation de la chimie des matériaux et le développement de la géométrie tridimensionnelle. Le polycarbonate (PC) a été utilisé comme modèle pour le développement de l'échafaud. Le film de polysaccharide naturel, basé sur un dépôt alternatif couche par couche (LbL) d’acide hyaluronique (HA) et de chitosane (CHI), a d’abord été appliqué sur une surface PC et caractérisé en termes de croissance d’épaisseur microscopie à balayage lascar (CLSM). Cette première fonctionnalisation se traduit par un revêtement complet de la couche PC. Une biofonctionnalisation supplémentaire avec un peptide adhésif (RGD) et deux peptides angiogénétiques (SVV et QK) a été étudiée, immobilisant ces peptides sur le groupe carboxylique de HA précédemment déposé, en utilisant la chimie bien connue du carbodiimide. La version marquée de chaque peptide a été utilisée pour caractériser l’immobilisation et la pénétration des peptides dans les couches de polyélectrolytes, aboutissant à une greffe réussie avec une pénétration complète dans toute l’épaisseur du LbL. Des tests in vitro ont été effectués à l'aide de cellules HUVEC pour évaluer leur efficacité d'adhésion et leur activité métabolique sur la LbL avec et sans immobilisation de peptides, ce qui a permis d'améliorer l'activité préliminaire lorsque des combinaisons de peptides sont utilisées. Enfin, les micro-canaux PC (μCh) ont été développés et caractérisés pour la première fois, et les autres expériences ont été réalisées sur un micromètre de 25 μm de largeur, fonctionnalisé avec une architecture (HA / CHI) 12,5 (PC-LbL) avec des peptides RGD et QK -RGD + QK) ou avec des peptides RGD et SVV (PC-RGD + SVV). Notre première expérience de tubulogénèse a montré de manière surprenante la formation de structures de type tubulaire déjà après 2h d'incubation en utilisant la combinaison double-peptides, mais uniquement avec PC-RGD + QK. Les tubes étaient également présents après 3 et 4 heures de culture. L'expérience de co-culture avec des péricytes humains dérivés du placenta (hPC-PL) montre comment la stabilisation des tubes a été améliorée après 3 et 4 heures également pour l'échantillon de PC-RGD + SVV. Globalement, notre matériel bio-fonctionnel avec les peptides PC-RGD + QK et PC-RGD + SVV permet la formation d'une structure de type tubulaire à la fois dans une expérience de monoculture et de co-culture
In vitro, tubular-like structures formation with human umbilical vein endothelial cells (HUVECs) was investigated by combining material chemistry functionalization and three-dimensional geometry development. Polycarbonate (PC) was used as a template for the development of the scaffold. Natural polysaccharide’s film based on alternate layer-by-layer (LbL) deposition of hyaluronic acid (HA) and chitosan (CHI), was first applied to PC surface and characterized in terms of thickness growth both, in dry conditions using ellipsometry, and confocal lascar scanning microscopy (CLSM). This first functionalization results in a complete coating of the PC layer. Further biofunctionalization with one adhesive peptide (RGD) and two angiogenetic peptides (SVV and QK) was investigated, immobilizing those peptides on the carboxylic group of HA previously deposited, using the well-known carbodiimide chemistry. The labeled version of each peptide was used to characterize the peptides’ immobilization and penetration into the polyelectrolytes layers, resulting in a successful grafting with complete penetration through the entire thickness of the LbL. In vitro tests were performed using HUVECs to assess their adhesion efficiency and their metabolic activity on the LbL with and without peptide immobilization, resulting in a preliminary improved activity when peptide-combinations is used. Finally, PC micro-channels (μCh) were first developed and characterized, and the rest of the experiments were performed on μCh of 25μm width, functionalized with (HA/CHI)12.5 architecture (PC-LbL) with RGD and QK peptides (PC-RGD+QK) or with RGD and SVV peptides (PC-RGD+SVV). Our first tubulogenesis experiment surprisingly showed the formation of tubular-like structures already after 2h of incubation using the double-peptides combination but only using PC-RGD+QK the tubes were present also after 3 and 4 hours of culture. The co-culture experiment with human pericytes derived from placenta (hPC-PL) demonstrates how the stabilization of the tubes was improved after 3 and 4 hours also for the PC-RGD+SVV sample. Globally our bio-functional material with PC-RGD+QK and PC-RGD+SVV peptides allow the formation of tubular-like structure in both mono and co-culture experiment

Epidemiological evidence indicates that increased consumption of whole grain foods is associated with decreased incidence of chronic diseases. The mechanisms underlying these effects are poorly understood but may be due to the antioxidant effects of phenolics or other components. The first postprandial study assessed the effects of consumption of minimally processed wheat bran and aleurone fractions on ferulic acid responses and antioxidant measures in humans. Results showed that both fractions significantly increased urinary total phenolics and plasma and urinary ferulic acid, and that plasma MetSO was significantly reduced after consumption of aleurone. The second postprandial study, which assessed the effects of consumption of aleurone, incorporated into breads, with or without iron addition, showed significant increases in plasma and urinary ferulic acid, and significant decreases in plasma MetSO; the addition of iron did not influence these responses. The third study, which assessed the effects of longer term consumption of aleurone enriched products on antioxidant, inflammatory and other disease risk factors in a parallel, four week intervention study, showed significant decreases in hs CRP and LDL-cholesterol, but other lipids and biomarkers of inflammatory status were unaffected. Furthermore, there were no significant effects on fasting plasma ferulic acid, or antioxidant, anthropometric or blood glucose measures. Overall, the results indicated that wheat bran and aleurone fractions can impact on postprandial ferulic acid levels, and antioxidant measures, and that longer term consumption of wheat aleurone enriched products can ameliorate inflammatory and lipid biomarkers. Future studies should further assess these effects, and extend this work to other components and mechanisms underlying the health benefits of whole grains.

Anthocyanins, the class of flavonoid responsible for giving a red hue to many berries, have been associated with a decreased risk of cardiovascular disease. However, numerous intervention studies feeding anthocyanin-rich foods report limited (<1%) bioavailability of the parent anthocyanins in vivo. Due to the instability of anthocyanins at neutral pH, it is postulated that degradation products and metabolites of anthocyanins may be responsible for the perceived bioactive effects. The aims of the present thesis were: (1) To model and establish analytical methods for the extraction and quantification of putative anthocyanin metabolites in urine, serum and faecal samples. (2) To identify and explore the pharmacokinetics of anthocyanin metabolites via the analysis of urine, serum and faecal samples from two human interventions, feeding either (a) 500 mg of isotopically (13C5) labelled anthocyanin or (b) 500 mg elderberry anthocyanins for 12 wks. (3) To explore the impact of acute (500 mg) versus chronic (500 mg/day for 12 wks) anthocyanin consumption on their metabolism and (4) To investigate the anti-inflammatory activity of six anthocyanin metabolites at physiologically relevant concentrations (0.01 μM to 10 μM) using human umbilical vein endothelial cells (HUVECs). Following the consumption of 500 mg elderberry anthocyanins, 28 anthocyanin metabolites were identified in urine and 21 in plasma, with the phenolic metabolites within plasma identified at 45 fold higher levels than their parent compounds. Similar results were observed within the 13C-labelled anthocyanin intervention, where 17 13C-labelled compounds were identified in serum and 31 in urine. However, chronic consumption of anthocyanins had no impact on the formation of the metabolites. The cardiovascular bioactivity of anthocyanins may be linked to the antiinflammatory activity of their metabolites. IL-6 and VCAM-1 are cytokines and adhesion molecules integral to the initiation and progression of inflammation. In vitro, anthocyanin metabolites reduced CD40L and TNF-α stimulated expression of the inflammatory markers, sVCAM-1 and IL-6, indicating that the anti-inflammatory effects of anthocyanins are likely attributed to their metabolites. In conclusion, the present thesis provides a new understanding into the metabolism and bioactivity of anthocyanins, which should provide an informative insight into how the consumption of higher intakes of anthocyanins may contribute to optimising human health.

Les ressources marines constituent un réservoir considérable de substances actives, en particulier, de peptides bioactifs appelés cryptides. Les cryptides, qui sont initialement dissimulés au cœur des protéines, sont libérés lors de la digestion ou lors de procédés protéolytiques industriels. Ces cryptides pourraient procurer des bienfaits physiologiques ou assurer une protection contre des pathologies telles que celles du syndrome métabolique. Dans ce contexte, nous nous sommes intéressés à l’action de certains cryptides marins sur des cibles impliquées dans l’hypertension, le diabète et l’obésité. Nous avons pu mettre en évidence que certains cryptides pouvaient cibler in vitro plusieurs facteurs de risques associés au développement des anomalies du syndrome métabolique
Marine products represent an important source of active substances, in particular bioactive peptides called cryptides. Cryptides are hidden within the sequence of a parent protein and are released during digestion or industrial proteolytic processes. These cryptides could provide physiological benefit or protection against diseases such as those of metabolic syndrome. In this context, we investigated the action of some marine cryptides on hypertension, diabetes and obesity. We demonstrated that some cryptides can target in vitro several factors associated with the development of metabolic syndrome

碩士
國立臺灣大學
植物學研究所
91
Abstract : Echinacea spp. is widely and heavily used as a medicinal herb or food supplement for stimulating immune function in many American and European countries. Three major species have been studied for candidate bioactivity in pharmacological and immunological effects, e.g., on macrophages or other immune cells. However, little or no evidence has been reported for possible effects of Echinacea on dendritic cells (DC). Dendritic cells are professional antigen-presentation cells (APC) and play an important role in innate as well as adaptive immunities. In this study, we analyzed the immune-associated surface proteins (CD markers) and the differential gene/genomic expression patterns of dendritic cells treated with or without Echinacea extract, using flow cytometry and DNA microarray techniques. Our preliminary results show that Echinacea root extract apparently can stimulate the expression of CD83 in dendritic cells with or without co-treatment with lipopolysaccharide. On the other hand, when treated with Echinacea stem plus leaf extract, CD83 expression apparently get inhibited. Furthermore, by using a home-made, mini-DNA microarray ( containing 225 genes) analysis system, we have been able to detect distinct patterns for the expression of specific immune or other functional activity associated mRNA species. About 10 genes, including those encoding NAFTp, IL-7R, Cybr, Jun, MCP-1, ICAM-1, Erm, Wnt-1, CCR-1 and CCR-9, were up- or down-regulated within 4 to 16 h post treatment. Since this mini-chip genes were selected and organized for immune related or other known functions, bioinformatics analyses are being carried out to evaluate these responsive genes for possible significance in immune or cellular-signaling mechanisms. Previous studies reputed that Echinacea may be associated with anti-inflammatory activity. We show in this study that Echinacea can stimulate or suppress certain specific activities of dendritic cells at the gene and/or protein expression level. In addition, based on these results and leads, bioactivity-guided assays of specific phytocompounds from this medicinal herb can now be systematically tested, by bio-organic partition/fractionation, for identifying phytocompound groups, single compounds or reconstituted formulations from Echinacea plant extracts as candidate therapeutics or health care supplements.

Red raspberries, as an excellent source of dietary antioxidants, were investigated for their effect on oxidative stress in healthy adults. Study 1 measured effects of chronic exposure in a parallel, multi-dose intervention. Subjects consumed one-cup red raspberries (1cR) daily for two-weeks, then were randomized to consume 1cR, 2cR or 4cR for additional two-weeks (n=8, by group). There was a reduction in TBARS, indicating a decrease in lipid peroxidation, after two-weeks of intervention in the 1cR group, but effects were not significant at week 4, or for other treatment groups. Study 2 measured effects of acute exposure using a cross-over design. Subjects (n=8) consumed single treatments of 1cR, 2cR, 4cR, bread and bread plus vitamin C. Post-prandial oxidative stress responses were complex and appeared related to calorie and antioxidant load. Overall there was no clear relationship between red raspberry consumption and protection against oxidative stress.

Recombinant human interferon α-2b (rhIFNα-2b) is a widely used therapeutic protein for the treatment of viral infections such as hepatitis. Being a therapeutic protein it is only active in its native conformation so that it is important to investigate possible pathways of degradation when producing it. In this work rhIFNα-2b was subjected to four different stress conditions and the resulting products characterized with fluorescence spectroscopy, fluorescence anisotropy, circular dichroism, dynamic light scattering and scanning electron microscopy. The results showed that rhIFNα-2b loses its native conformation in all conditions in which it was tested and there was formation of aggregates. It was also made a bioactivity assay where we saw that the protein had biological activity before and after the stress conditions.

碩士
台南應用科技大學
生活應用科學研究所
100
(I) Colorectal cancer is the leading cause of cancer mortality, and metastasis is responsible for approximately 40% of death in colon cancer patients. Matrix metalloproteinases (MMPs) are key enzymes in the degradation of extracellular matrix, and MMP-2 and -9 are critical for cell migration leading to invasion and metastasis of cancer. The inhibition of MMP-2 and -9 is therefore considered that might depress the occurrence of tumor invasion and metastasis. Rosmarinic acid (RA) is a bioactive polyphenol that widely presented in Rosmarinus officinalis L. However, the literature regarding the effect of RA on invasion of cancer cells is still limited. In this study, we investigated the anti-invasion activity of RA against human colorectal adenocarcinoma Colo205 and HT-29 cells. The cells were treated with 0, 10, 50, 100, or 200 M of RA at 37C for 24 h, and the MMP-2, -9, and uPA activities as well as cell-matrix adhesion, motility, and invasion activities were determined. The MMP-2, -9, TIMP-1 and -2 mRNA levels were assayed by RT-PCR. The molecular signaling was determined by Weastern blot. The results showed that the MMP-2 and uPA activities of Colo205 and HT-29 cells were significantly inhibited by RA at a concentration of > 50 μM. The migration and invasion activities of Colo205 and HT-29 cells were also suppressed after treating with RA at a concentration higher than 50 M. The mRNA level of MMP-2 in HT-29 and Colo205 were significantly inhibited by RA at a concentration of > 50 M. The mRNA levels of TIMP-1 and -2 in HT-29 and TIMP-1 in Colo205 were significantly increased by RA. The signaling of p-ERK and p-p38 in Colo205 and HT-29 were signigicantly inhibited by RA. Furthermore, the activations of NF-κB and AP-1 in colorectal cancer cells were also suppressed by RA. Our results suggest that RA might be a bioactive with potential anti-invasive activity against colorectal cancer cells by inhibiting uPA and MMP-2 activity and reducing motility capabilities through inhibiting the activations of MAPKs signaling pathways and NF-κB and AP-1 transcription factors. (II) Tuberculosis (TB) is a contagious disease which causes a serious public health risk. WHO estimates that there were approximately 9,400,000 new TB cases globally in 2008, and South-East Asia Region accounted for 34% of incident cases. Multidrug-resistant TB (MDR-TB) is a particularly dangerous form of TB, which is responsible for the majority of failures in TB therapy. Mycobacterium tuberculosis is the major pathogeny of TB, and uridine diphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is the major enzyme that involved to the synthesis of cell wall. The inhibition of UGPase might depress the proliferation of the bacilli. Rosmarinic acid (RA), a polyphenol which is widely presented in natural plants of Lamiaceae and Boraginaceae family, has been reported that possesses several biological activities such as anti-virus, anti-inflammation, and anti-bacteria. The aim of this study was to investigate the inhibitory effects of RA on the viability of multidrug-resistant M. tuberculosis, and their impact on UGPase was also evaluated. Because the biological activity of a bioactive sometimes having an association with their antioxidant power, we therefore first determined the antioxidant activity of RA by trolox equivalent antioxidant capacity (TEAC) method. The viability of the bacilli was measured by counting the colony growing on 7H11 agar plates, and the expression of UGPase was performed by RT-PCR. The results showed RA having a strong antioxidant activity, and the viability of M. tuberculosis was reduced by treating with RA at a concentration of > 250 g/ml for 24 h. Furthermore, the expression of UGPase was also decreased by treating with 250 g/ml RA for 24 h. Based on the data mentioned above, it is suggested that RA can be used to inhibit the proliferation of multidrug-resistant M. tuberculosis, and the anti-proliferous activity of RA might be through inhibiting the expression of UGPase.

碩士
台南應用科技大學
生活應用科學研究所
100
(I) Colorectal cancer is the leading cause of cancer mortality, and metastasis is responsible for approximately 40% of death in colon cancer patients. Matrix metalloproteinases (MMPs) are key enzymes in the degradation of extracellular matrix, and MMP-2 and -9 are critical for cell migration leading to invasion and metastasis of cancer. The inhibition of MMP-2 and -9 is therefore considered that might depress the occurrence of tumor invasion and metastasis. Rosmarinic acid (RA) is a bioactive polyphenol that widely presented in Rosmarinus officinalis L. However, the literature regarding the effect of RA on invasion of cancer cells is still limited. In this study, we investigated the anti-invasion activity of RA against human colorectal adenocarcinoma Colo205 and HT-29 cells. The cells were treated with 0, 10, 50, 100, or 200 M of RA at 37C for 24 h, and the MMP-2, -9, and uPA activities as well as cell-matrix adhesion, motility, and invasion activities were determined. The MMP-2, -9, TIMP-1 and -2 mRNA levels were assayed by RT-PCR. The molecular signaling was determined by Weastern blot. The results showed that the MMP-2 and uPA activities of Colo205 and HT-29 cells were significantly inhibited by RA at a concentration of > 50 μM. The migration and invasion activities of Colo205 and HT-29 cells were also suppressed after treating with RA at a concentration higher than 50 M. The mRNA level of MMP-2 in HT-29 and Colo205 were significantly inhibited by RA at a concentration of > 50 M. The mRNA levels of TIMP-1 and -2 in HT-29 and TIMP-1 in Colo205 were significantly increased by RA. The signaling of p-ERK and p-p38 in Colo205 and HT-29 were signigicantly inhibited by RA. Furthermore, the activations of NF-κB and AP-1 in colorectal cancer cells were also suppressed by RA. Our results suggest that RA might be a bioactive with potential anti-invasive activity against colorectal cancer cells by inhibiting uPA and MMP-2 activity and reducing motility capabilities through inhibiting the activations of MAPKs signaling pathways and NF-κB and AP-1 transcription factors. (II) Tuberculosis (TB) is a contagious disease which causes a serious public health risk. WHO estimates that there were approximately 9,400,000 new TB cases globally in 2008, and South-East Asia Region accounted for 34% of incident cases. Multidrug-resistant TB (MDR-TB) is a particularly dangerous form of TB, which is responsible for the majority of failures in TB therapy. Mycobacterium tuberculosis is the major pathogeny of TB, and uridine diphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is the major enzyme that involved to the synthesis of cell wall. The inhibition of UGPase might depress the proliferation of the bacilli. Rosmarinic acid (RA), a polyphenol which is widely presented in natural plants of Lamiaceae and Boraginaceae family, has been reported that possesses several biological activities such as anti-virus, anti-inflammation, and anti-bacteria. The aim of this study was to investigate the inhibitory effects of RA on the viability of multidrug-resistant M. tuberculosis, and their impact on UGPase was also evaluated. Because the biological activity of a bioactive sometimes having an association with their antioxidant power, we therefore first determined the antioxidant activity of RA by trolox equivalent antioxidant capacity (TEAC) method. The viability of the bacilli was measured by counting the colony growing on 7H11 agar plates, and the expression of UGPase was performed by RT-PCR. The results showed RA having a strong antioxidant activity, and the viability of M. tuberculosis was reduced by treating with RA at a concentration of > 250 g/ml for 24 h. Furthermore, the expression of UGPase was also decreased by treating with 250 g/ml RA for 24 h. Based on the data mentioned above, it is suggested that RA can be used to inhibit the proliferation of multidrug-resistant M. tuberculosis, and the anti-proliferous activity of RA might be through inhibiting the expression of UGPase.