A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors

Purpose: PI-88 is a mixture of highly sulfated oligosaccharides that inhibits heparanase, an extracellular matrix endoglycosidase, and the binding of angiogenic growth factors to heparan sulfate. This agent showed potent inhibition of placental blood vessel angiogenesis as well as growth inhibition in multiple xenograft models, thus forming the basis for this study. Experimental Design: This study evaluated the toxicity and pharmacokinetics of PI-88 (80-315 mg) when administered s.c. daily for 4 consecutive days bimonthly (part 1) or weekly (part 2). Results: Forty-two patients [median age, 53 years (range, 19-78 years); median performance status, 1] with a range of advanced solid tumors received a total of 232 courses. The maximum tolerated dose was 250 mg/d. Dose-limiting toxicity consisted of thrombocytopenia and pulmonary embolism. Other toxicity was generally mild and included prolongation of the activated partial thromboplastin time and injection site echymosis. The pharmacokinetics were linear with dose. Intrapatient variability was low and interpatient variability was moderate. Both AUC and C-max correlated with the percent increase in activated partial thromboplastin time, showing that this pharmacodynamic end point can be used as a surrogate for drug exposure, No association between PI-88 administration and vascular endothelial growth factor or basic fibroblast growth factor levels was observed. One patient with melanoma had a partial response, which was maintained for >50 months, and 9 patients had stable disease for >= 6 months. Conclusion: The recommended dose of PI-88 administered for 4 consecutive days bimonthly or weekly is 250 mg/d. PI-88 was generally well tolerated. Evidence of efficacy in melanoma supports further evaluation of PI-88 in phase II trials.

The chromatographic separation of carbohydrate mixtures

The separation performance of a semicontinuous counter-current chromatographic refiner (SCCR7), consisting of twelve 5.4 cm id x 75cm long columns packed with calcium charged cross-linked polysytrene resin (KORELA VO7C), was optimised. An industrial barley syrup was used containing 42% fructose, 52% glucose and 6% maltose and oligosaccharides. The effects of temperature, flow rates and concentration on the distribution coefficients were evaluated and quantified by deriving general relationships. The effects of flow rates, feed composition and concentration on the separation performance of the SCCR7 were identified and general relationships between them and the switch time, which was found to be the controlling parameter, were developed. Fructose rich (FRP) and glucose rich (GRP) product purities of 99.9% were obtained at 18.6% w/v feed concentrations. When a 66% w/v feed concentration was used and product splitting technique was employed, the throughput was 32.1 kg sugar solids/m3 resin/hr. The GRP contained less than 4.5% fructose, the FRP was over 95% pure, and the respective concentrations were 22.56 and 11.29% w/v. Over 94% of the glucose and 95.78% of the fructose in the feed were recovered in the GRP and FRP respectively. By recycling the dilute product split fractions, the GRP and FRP concentrations were increased to 25.4 and 12.96% w/v; the FRP was 90.2% pure and the GRP contained 6.69% w/v fructose. A theoretical link between batch and semicontinuous chromatographic equipments has been determined. A computer simulation was developed predicting successfully the purging concentration profiles at `pseudo-equilibrium', and also certain system design parameters. An important further aspect of the work has been to study the behaviour of chromatographic bioreactor-separators. Such batch systems of 5.4cm id and lengths varying between 30 and 230cm, were used to investigate the effect of scaling up on the conversion of sucrose into dextran and fructose in the presence of the dextransucrase enzyme. Conversions of over 80% were achieved at 4 hr sucrose residence times. The crude dextransucrase was purified using centrifugation, ultrafiltration and cross-flow microfiltration techniques. Better enzyme stability was obtained by first separating the non-solid impurities using cross-flow microfiltration, and then removing the cells from the enzyme immediately before use by continuous centrifugation.

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

The effect of gestational and lactational age on the human milk metabolome

Human milk is the ideal nutrition source for healthy infants during the first six months of life and a detailed characterisation of the composition of milk from mothers that deliver prematurely (<37 weeks gestation), and of how human milk changes during lactation, would benefit our understanding of the nutritional requirements of premature infants. Individual milk samples from mothers delivering prematurely and at term were collected. The human milk metabolome, established by (NMR) spectroscopy, was influenced by gestational and lactation age. Metabolite profiling identified that levels of valine, leucine, betaine, and creatinine were increased in colostrum from term mothers compared with mature milk, while those of glutamate, caprylate, and caprate were increased in mature term milk compared with colostrum. Levels of oligosaccharides, citrate, and creatinine were increased in pre-term colostrum, while those of caprylate, caprate, valine, leucine, glutamate, and pantothenate increased with time postpartum. There were differences between pre-term and full-term milk in the levels of carnitine, caprylate, caprate, pantothenate, urea, lactose, oligosaccharides, citrate, phosphocholine, choline, and formate. These findings suggest that the metabolome of pre-term milk changes within 5-7 weeks postpartum to resemble that of term milk, independent of time of gestation at pre-mature delivery.

Identification and characterization of a glycosulfatase-encoding gene cluster in Bifidobacterium breve UCC2003

Bifidobacteria constitute a specific group of commensal bacteria, typically found in the gastrointestinal tract (GIT) of humans and other mammals. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. In the current study, we investigated glycosulfatase activity in a bacterial nursling stool isolate, B. breve UCC2003. Two putative sulfatases were identified on the genome of B. breve UCC2003. The sulfated monosaccharide N-acetylglucosamine-6-sulfate (GlcNAc-6-S) was shown to support growth of B. breve UCC2003, while, N-acetylglucosamine-3-sulfate, N-acetylgalactosamine-3-sulfate and N-acetylgalactosamine-6-sulfate, did not support appreciable growth. Using a combination of transcriptomic and functional genomic approaches, a gene cluster, designated ats2, was shown to be specifically required for GlcNAc-6-S metabolism. Transcription of the ats2 cluster is regulated by a ROK-family transcriptional repressor. This study represents the first description of glycosulfatase activity within the Bifidobacterium genus. Bifidobacteria are saccharolytic organisms naturally found in the digestive tract of mammals and insects. Bifidobacterium breve strains utilize a variety of plant and host-derived carbohydrates which allow them to be present as prominent members of the infant gut microbiota as well as being present in the gastrointestinal tract of adults. In this study, we introduce a previously unexplored area of carbohydrate metabolism in bifidobacteria, namely the metabolism of sulfated carbohydrates. B. breve UCC2003 was shown to metabolize N-acetylglucosamine-6-sulfate (GlcNAc-6-S) through one of two sulfatase-encoding gene clusters identified on its genome. GlcNAc-6-S can be found in terminal or branched positions of mucin oligosaccharides, the glycoprotein component of the mucous layer that covers the digestive tract. The results of this study provide further evidence of this species' ability to utilize mucin-derived sugars, a trait which may provide a competitive advantage in both the infant and adult gut.

Fasciola hepatica surface tegument: glycoproteins at the interface of parasite and host

Fasciola hepatica, commonly known as liver fluke, is a trematode which causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterisation of FhTeg glycosylation using lectin microarrays to characterise carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. While some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components which could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

SACN Carbohydrates and Health Report

Carbohydrates are a major source of energy in the diet. Classified according to their chemistry, carbohydrates can be divided into sugars (monosaccharides and disaccharides), polyols, oligosaccharides (malto-oligosaccharides and non-digestible oligosaccharides) and polysaccharides (starch and non-starch polysaccharides). However, this classification does not allow a simple translation into nutritional effects since each class of carbohydrates has overlapping physiological properties and effects on health. Carbohydrates can also be classified according to their digestion and absorption in the human small intestine. Digestible carbohydrates are absorbed and digested in the small intestine; non-digestible carbohydrates are resistant to hydrolysis in the small intestine and reach the large intestine where they are at least partially fermented by the commensal bacteria present in the colon. There is no universal definition of the term ‘dietary fibre’; broadly speaking, it refers to some or all of the constituents of non-digestible carbohydrates and may also include other quantitatively minor components (e.g.lignin) that are associated with non-digestible carbohydrates in plant cell walls.  

Effects of early nutrition on the wnt and bone morphogenic protein pathways in the developing intestine

Infant formula is consumed by the majority of infants in the United States for at least part of the first year of life. Infant formula lacks many of the bioactive compounds that are naturally occurring in breast milk. Because of this, there has been an increased interest by the companies that manufacture infant formula to include additives that would potentially allow formula to more closely mimic breast milk activity. One such ingredient currently being added to infant formula is prebiotics. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth of specific healthful bacteria in the colon. It is speculated that prebiotics replicate the activity of breast milk oligosaccharides, which through the production of butyrate by intestinal microbiota, may interact with the Wnt/BMP pathways. The Wnt/BMP pathways regulate intestinal stem cells, which determine the growth, development and maintenance of the intestine. Therefore, the objective of this study was to explore the effects that the addition of prebiotics to formula have on the regulation of the Wnt/BMP pathways when fed to neonatal piglets, a model commonly used in the study of infant nutrition. Piglets (n=5) were randomized into sow-reared (SR), fed control formula (F), or fed formula with added prebiotics (F+P). Fructooligosaccharides (FOS) (2 g/L) and polydextrose (PDX) (2 g/L) were chosen as the prebiotics for this study, because this combination had been less studied than other combinations. Ileum and ascending colon were collected at 7 and 14 days-of-age. Dry matter content, pH, and short chain fatty acid (SCFA) content was measured. The mRNA expression of β-catenin, sFRP3, sFRP4, frizzled 6, DKK1 (Wnt pathway), gremlin (BMP pathway), TNF-a, HNF-4α and osteopontin (OPN) was measured by RT-qPCR. Piglets fed the F+P diet had greater acetate concentration and lower pH in the ileum at day 14 and in the colon at day 7 and day 14 than F piglets. Butyrate concentrations were highest in SR with F+P not differing from F in ileum at day 14 and colon at day 7 and day 14. Effects of age were seen in all genes, with the exception of OPN, sFRP-3 and sFRP-4. On day 7, no effect of diet was observed in the ileum, however, mRNA expression of DKK1 and frizzled 6 were greater in F+P than SR (p≤0.05). On day 14, gremlin expression was lower and OPN was greater in the ileum of SR piglets compared to F and F+P. Also on day 14, HNF-4α mRNA expression was greater in both ileum and colon of F+P piglets and sFRP3 mRNA expression was greater in the colon than F or SR . In summary, differences were observed between gene expression of F+P and SR piglet intestines, but the supplementation of 2 g/L scFOS and 2 g/L PDX to formula did not shift expression of genes in the Wnt/BMP pathways to be more similar to SR than F. As the Wnt/BMP pathway is known to exist in a gradient along the crypt-villus axis, with Wnt expression dominating in the crypt region and BMP expression dominating in the villi, it was possible that pooling whole tissue reduced our ability to detect treatment effects that would be concentrated in either region. A method was therefore developed to remove intestinal epithelial cells along the villus-to-crypt axis. Twenty-five-day-old F and SR piglets were euthanized and ileal tissue was collected and placed in a dissociation buffer in a shaking water bath. Exfoliated cells were removed at increasing time points from 5 to 100 minutes in order to remove cells along the villus-to-crypt axis. After the final incubation, remaining mucosal tissue was removed using a sterile glass microscope slide and pooled with the final exfoliated cell isolation. After each cell collection, a section of tissue was fixed in formalin for histomorphological examination. Expression of genes in the Wnt/BMP pathways, along with crypt marker genes (CDK5 and v-myb), were measured in both whole ileal tissue, pooled epithelial cells, and separate epithelial cell isolations from the same piglet. The expression of β-catenin, HNF-4α, TNF-α, TGF-β and the crypt marker v-myb matched the expected villus-to-crypt pattern in cells collected after 10 (incubation 1), 30 (incubation 2) and 60 (incubation 3) minutes. However, expression of expression in cells collected after 100 minutes (incubation 4) was variable, which may be due to the fact that crypt cells were not efficiently removed and the presence of unwanted non-epithelial tissue. Gremlin, OPN, DKK1, sFRP3 and sFRP4 expression was not statistically different along the villus-to-crypt axis. Frizzled 6 and CDK5 did not express as we had predicted, with expression highest towards the villi. In summary, the epithelial cell collection method used was not entirely successful. While much of the gene data suggests that cells were removed along the villus-to-crypt axis through the first three incubations, the last incubation, which involved scraping the tissue, removed non-epithelial components of the mucosa, while leaving the crypts intact. In conclusion, the addition of 2 g/L PDX and 2 g/L scFOS did not cause gene expression of the Wnt/BMP pathways to mirror either F or SR expression. New isolation methods to extract cells along the crypt-villus axis should be considered, including the use of a laser capture microdissection. While this combination of prebiotics did not yield the intended effects, future research should be done on other combinations, such as the inclusion of galactooligosaccharides (GOS), which is commonly added to food products including infant formula.

Acute phase proteins and biomarkers for health in chickens

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.

Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm, and testa

Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos.

Optimização das condições de cultura da Saccharomyces Cerevisiae e de outros fungos produtores de oligossacáridos

Este estudo envolve o controlo e a optimização das condições de culturas dos microrganismos: Saccharomyces cerevisiae CCMI 396, S. cerevisiae v. lab., Aspergillus oryzae CCMI 125, Aspergillus japonicus CCMI 443, Fusarium oxysporum CCMI 866, Aspergillus niger CCMI 296 com vista à produção de oligossacáridos. Determinaram-se os parâmetros característicos das culturas de duas diferentes estirpes de Saccharomyces com diferentes fontes de carbono e em diferentes condições ambientais. O perfil de crescimento da S. cerevisiae CCMI 396 foi semelhante nos diferentes meios de cultura estudados, sendo a velocidade específica de crescimento mais elevada no meio com glucose a pH 5 e a 30°C (0,36h-1). A S. cerevisiae v. lab. Teve velocidade específica de crescimento idêntica nas mesmas condições da outra estirpe, no entanto, o perfil de crescimento foi diferente nos outros meios de cultura. Estudou-se o efeito da adição de sumo de laranja ou de tomate ao meio de cultura com sacarose e avaliou-se a evolução glucídica no meio de cultura durante o ensaio por HPLC com detector RI. Determinou-se a frutosiltransferase no sobrenadante e na fracção intracelular e determinou-se a evolução dos oligossacáridos. Numa segunda parte deste trabalho efectuaram-se culturas dos quatro fungos filamentosos com vista a avaliar a capacidade de produção, nomeadamente, de fruto­oligassacáridos. Os resultados mostraram que a espécie Aspergillus japonicus CCMI 443 originou, nas mesmas condições de cultura, valores superiores, sendo a percentagem de produção FOStotais/GluCtotais de 61% para as enzimas intracelulares e 40% para as enzimas no sobrenadante. ABSTRACT; This study involves control and optimization of the cultures of microorganisms: Saccharomyces cerevisiae CCMI 396, S. cerevisiae v. lab., Aspergillus oryzae CCMI 125, Aspergillus japonicus CCMI 443, Fusarium oxysporum CCMI 866, Aspergillus níger CCMI 296 for oligosaccharides production. Were determined the parameters characteristic of the cultures of two different strains of Saccharomyces with different sources of carbon and in different environmental conditions. The growth profile of S. cerevisiae CCMI 396 was similar in different cultures media, but the highest specific growth was obtained in a medium with glucose, pH 5, at 30°C (0.36h-1). S. cerevisiae v. lab. had similar growth profile in a medium with glucose but with others culture media was different. We studied the effect of adding orange juice or tomato to the culture medium with sucrose and evaluated the evolution glucidic in the culture medium during the test by HPLC with RI detector. Fructosyltransferase was determined in the extracellular and the intracellular fractions and determined the evolution of oligosaccharides. ln the second part of this work were carried out cultures of four filamentous fungi in order to assess production capacity, in particular, fructoligosaccharides. The results showed that the specie Aspergillus japonicus CCMI 443 originated in the same culture conditions, higher values and the percentage of production FOStotal/Guctotal of 61% for intracellular enzymes and 40% for extracellular enzymes.

Produção de enzimas quitosanolíticas utilizando Paenibacillus ehimensis e Paenibacillus chitinolyticus para obtenção de quitoologossacarídeos

The acquisition of oligosaccharides from chitosan has been the subject of several studies in the pharmaceutical, biochemical, food and medical due to functional properties of these compounds. This study aimed to boost its production of chitooligosaccharides (COS) through the optimization of production and characterization of chitosanolytic enzymes secreted by microorganisms Paenibacillus chitinolyticus and Paenibacillus ehimensis, and evaluating the antioxidant potential of the products obtained. In the process of optimizing the production of chitosanase were employed strategies Fractional Factorial Experimental Design and Central Composite Rotatable Design. The results identified the chitosan, peptone and yeast extract as the components that influenced the production of chitosanase by these microorganisms. With the optimization of the culture media was possible to obtain an increase of approximately 8.1 times (from 0.043 to 0.35 U.mL U.mL-1) and 7.6 times (from 0.08 U.mL-1 to 0.61 U.mL-1) in the enzymatic activity of chitosanase produced by P. chitinolyticus and P. ehimensis respectively. Enzyme complexes showed high stability in temperature ranges between 30º and 55º C and pH between 5.0 and 9.0. Has seen the share of organic solvents, divalent ions and other chemical agents on the activity of these enzymes, demonstrating high stability of these crude complexes and dependence of Mn2+. The COS generated showed the ability of DPPH radical scavenging activity, reaching a maximum rate of scavenging of 61% and 39% when they were produced with enzymes of P. ehimensis and P. chitinolyticus respectively. The use of these enzymes in raw form might facilitate its use for industrial applications

Non-use of Formal Health Services in Malawi: Perceptions from Non-users

Background The study upon which this paper is based was undertaken to understand users’ and non-users’ perceptions concerning facilitators and barriers to equitable and universal access to health care in resource-poor countries such as Malawi. In this study, non-users of health services were defined as people who were not in need of health services or those who had stopped using them due to significant barriers. Methods A total of 80 interviews with non-users of health services were conducted in Rumphi, Ntchisi, Phalombe and Blantyre Districts of Malawi. Interviews focused on why informants were not using formal health services at the time of data collection. In order to identify non-users, snowballing was used health surveillance assistants, village headmen and community members also helped. One focus group discussion was also conducted with non-users of health services who were members of the Zion Church. Results Informants described themselves as non-users of health services due to several reasons: cost of health services; long distances to health facilities; poor attitude of health workers; belief in the effectiveness of traditional medicines; old age and their failure to walk. Others were non-users due to their disability; hence they could not walk over long distances or could not communicate effectively with health providers. Some of these non-users were complete non-users, namely members of the Zion Church and those who believed in traditional medicine, and they stated that nothing could be done to transform them into users of health services. Other non-users stated that they could become users if their challenges were addressed e.g. for those who were non-users of health services due to poor attitudes of health workers, they stated that if these health workers were transferred they would be able to access health services. Conclusions Public health education targeting both health workers and non-users, ensuring a functional outreach program and addressing other health system challenges such as shortage of drugs and human resources would assist in transforming non-users into users of health services.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.