The cholesterol-lowering effect of mixed linkage (1→3) (1→4)-β-D-glucans (β-glucan) from barley has been documented, yet the underlying mechanism responsible for this action and factors influencing it, such as physicochemical properties of β-glucan and genetic background of an individual, remain unclear.As a component of dietary fibre, β-glucan also has the potential to shift the gut microbial community, however, whether alterations in the gut microbiota are associated with the physiological effects of β-glucan have yet to be determined. This study was designed to assess the effects of β-glucan molecular weight (MW) and dose on loweringserum cholesterol levels and to elucidate its mechanism of action in human subjects. Additionally, this study examined gene-diet interactions as well as changes in the gut microbiota profile following consumption of barley foods. In a controlled four phase crossover trial, mildly hypercholesterolemic but otherwise healthy subjects (n =30) were randomly assigned to receive breakfasts containing 3g high MW (HMW), 5g low molecular weight (LMW), 3g LMW barley β-glucan or a control diet with wheat and rice (WR control), each for 5 weeks. The washout period between the phases was 4 weeks. The consumption of 3g/d HMW diet lowered total cholesterol (TC) compared with WR control diet (P =0.0046), but not the LMW diet at either 3g/d or 5g/d. Individuals with the SNP rs3808607-G allele of CYP7A1 had greater TC reduction in response to 3g/d HMW β-glucan diet compared to the individuals carrying homozygous TT alleles (P<0.01). Cholesterol absorption and synthesis were not changed, but bile acid synthesis increased by 3g/d HMW diet compared to the control. Consuming 3g HMW/d β-glucan altered gut microbiota at the phylum and genus levels and the impacted microbial members was correlated with favorable shifts of cardiovascular disease risk factors. In conclusion, physicochemical properties of β-glucan play critical roles in the cholesterol-lowering effect and gut microbiota alteration ability of β-glucan. The results suggest the increasing bile acid synthesis rather than inhibiting cholesterol absorption and synthesis is the mechanism responsible for the cholesterol reducing property of β-glucan.The altered microbiota profile by HMW β-glucan is associated with its physiological effect. / February 2016

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The traditional use of antibiotics in aquaculture as growth promoters has been limited due to the negative effects caused by these drugs. As an alternative to the use of these drugs has been sought manipulation of the microbiota of the gastrointestinal tract of aquatic animals through the use of oligosaccharides and dietary fibers with prebiotic potential. Thus, this study aimed to apply different methodologies to obtain Dietary Fiber Concentrates (DFC) = mucilage (MG); pectin (PN) and β-glucan + manan (βG + M) and evaluate the prebiotic potential of these supplements in the diet of juvenile jundiá (Rhamdia quelen). The determination of the nutritional composition of the ingredients revealed that the predominant component in all DFCs were dietary fiber and insoluble fiber. The DFC that had higher extraction yield was βG + M (19.81 ± 8.54%), followed by pectin (14.54% ± 2.72), and mucilage (7.18 ± 1.54%). The composition of mucilage and pectin had a greater diversity of monosaccharides, since the βG+M consisted primarily of mannose (74.5%) and glucose (24.3%). The supplementation of DFC in jundiás diet was assessed for eight weeks through study of growth, body nutrient deposition, digestive enzymes, biochemical and metabolic parameters, responses to stress and immune and intestinal morphology. The jundiás supplemented with DFCs achieved higher growth than the control group and similar to animals supplemented with 5 g kg-1 commercial prebiotic (CP 5). Most somatic parameters and whole fish proximate composition were influenced by supplementation of DFCs. The supplementation of pectin promoted lower activity of digestive enzymes in relation the control group. The animals supplemented with DFC obtained positive changes in biochemical parameters. Furthermore, jundiás showed no response to application of the stressor, maintaining basal cortisol levels. The fish supplemented with DFCs had higher hepatic glycogen stores in relation the control group. Moreover, supplementation with DFCs increased the height of intestinal villi of jundiá. However, these values were lower for the animals of the group PC 5. For thickness of the epithelium this variable was higher in the control group compared to animals supplemented with β-glucan+Manana. / O uso tradicional de antibióticos na aquicultura como promotores de crescimento tem sido limitado em função dos efeitos negativos promovidos por estes medicamentos. Como alternativa ao uso destas drogas, tem se buscado a manipulação da microbiota do trato gastrointestinal dos animais aquáticos através da utilização de oligossacarídeos e de fibras alimentares com potencial prebiótico. Neste sentido, este estudo teve como objetivo aplicar metodologias para obtenção de diferentes Concentrados de Fibras Alimentares (CFAs) = Mucilagem (MG); Pectina (PN) e β-Glicana+Mananas (βG+M) e avaliar o potencial prebiótico destes suplementos na dieta de juvenis de jundiá (Rhamdia quelen). A determinação da composição nutricional dos ingredientes revelou que os componentes predominantes em todos os CFAs obtidos foram fibra alimentar total e fibra insolúvel. O CFA que apresentou maior rentabilidade de extração foi a βG+M (19,81%±8,54), seguida da Pectina (14,54%±2,72) e Mucilagem (7,18%±1,54). A composição da Mucilagem e Pectina obtiveram maior diversidade de monossacarídeos, já a βG+M consistiu basicamente de manose (74,5%) e glicose (24,3%). A suplementação dos CFAs na dieta de jundiás foi avaliada durante oito semanas, através de estudo de crescimento, deposição corporal de nutrientes, enzimas digestivas, parâmetros bioquímicos e metabólicos, resposta ao estresse e imunológica e morfometria intestinal. Os jundiás suplementados com os CFAs obtiveram crescimento superior em relação ao grupo controle e similar aos animais suplementados com 5 g kg-1 de prebiótico comercial (PC 5). A maioria dos parâmetros somáticos e de composição centesimal de peixe inteiro foram influenciados pela suplementação dos CFAs. A suplementação de Pectina promoveu menor atividade das enzimas digestivas em relação ao grupo controle. Os animais suplementados com os CFAs obtiveram alterações positivas nos parâmetros bioquímicos avaliados. Além disso, os jundiás não mostraram resposta à aplicação do agente estressor, mantendo os níveis de cortisol basal. Os peixes suplementados com os CFAs obtiveram maiores estoques de glicogênio hepático em relação ao grupo controle. Além do mais, a suplementação com os CFAs promoveu aumento na altura de vilos intestinais dos jundiás. Porém, estes valores foram menores em relação aos animais do grupo PC 5. Para espessura do epitélio (EE) esta variável foi maior no grupo Controle comparado aos animais suplementados com β- glicana + Manana.

Oats is a crop that contains a high amount of fiber, protein and fat, but like all other crops it contains mostly starch. In this study the focus has been oat flours and brans with different b-glucan content. The health benefits of b-glucan, a soluble fiber are well documented and a correlation between intake of b-glucan with high molecular weight and a low glycemic response has been observed. Food with a low glycemic index can lower the risk for diseases like type 2 diabetes, cardiovascular diseases and obesity. Also a connection between intake of b-glucan with high molecular weight and a reduction of LDL-cholesterol has been observed. b-glucans from oat absorb water and build a viscous gel, which make them an interesting component when developing new products, as a fat replacer in for example meat products and pastries. To optimize the use of flours and brans with a modified b-glucan content in new applications, the water absorption was measured with a method called Solvent Retention Capacity and the viscosity with a Rapid Viscosity Analyzer (RVA). The results showed that a higher amount of b-glucan in the flour or bran, a higher water holding capacity (WHC) was observed. The WHC for oat flour with a b-glucan content at 2% was calculated to 73±7%, while the WHC for oat bran with a b-glucan content at 28%, was calculated to a WHC of 880±45%. A comparison of different flours and brans indicates that dietary fiber, where b-glucan have the greatest impact on the WHC. The result from the RVA indicates that a flour with a combination of a high b-glucan content (0.24g) and high starch content (3.72g) leads to a high viscosity 12700 cP, compared to other flours or brans with either a lower b-glucan content (0.12g) or lower starch content (0.12g) gives lower final viscosity, 5390 and 780 cP. The result also indicates that other factors such as a smaller particle size and a higher temperature during the heating step (95°C instead of 64°C) might give a higher viscosity.

Résumé : Chez lorge, la dégradation des polysaccarides des parois cellulaires et de lamidon est de première importance dans le contexte de sa valorisation brassicole. La présente recherche doctorale a été entreprise dans le but didentifier des marqueurs de sélection utilisables lors de lamélioration génétique de lorge de printemps pour ses qualités brassicoles. Des gènes candidats ont dabord été choisis sur base de leur expression dans les tissus de la graine en germination. Leur position cartographique a été déterminée et située par rapport à des marqueurs QTL connus de la qualité brassicole. Exploitant des variétés cultivées de qualités contrastées, une recherche de polymorphismes nucléotidiques au niveau de ces gènes a été réalisée. Tous les gènes savèrent polymorphes, certaines des variations de séquence conduisant à des modifications de la structure primaire de la protéine encodée. Plusieurs caractères technologiques ont été retenus et définis chez les variétés étudiées, en exploitant les données de la littérature : la teneur en extrait, la viscosité, la teneur en β-glucanes du mout, latténuation finale apparente et le pouvoir diastasique. Plusieurs caractères biochimiques ont été étudiés chez ces mêmes variétés et ont ciblé quatre activités enzymatiques impliquées dans la dégradation des polysaccharides pariétaux et de réserve. Le pouvoir discriminant et la simplicité du test dactivité amylase, ainsi que sa relation avec des critères technologiques importants, en font un test prometteur en vue de faciliter la sélection variétale. La relation entre les sites polymorphes de lADN et les caractères technologiques et biochimiques identifient une soixantaine de sites dont les polymorphismes semblent associés à au moins lun des caractères. Sur tous les sites polymorphes, treize sites sont choisis pour leur potentiel en tant que marqueurs de sélection. Ils distinguent en effet un allèle favorable et un allèle défavorable pour plus dun critère brassicole. Ces treize sites sont localisés sur des gènes de (1-3,1-4)-β-glucanase, un gène de (1-3)-β-glucanase, un gène de (1-4)-β-xylan endohydrolase et un gène dα-amylase. La mise en évidence de ces allèles peut être réalisée par des tests PCR simples (« allèles spécifiques ») et relativement peu coûteux, dont six ont été mis au point au terme de la recherche./ Summary: In barley, the degradation of cell wall polysaccharides and starch is of utmost importance for its malting quality. The aim of this thesis is the identification of selection markers useful in the breeding of spring Barley cultivars for improved malting quality. Candidate genes were first chosen based on their expression profile in tissues of germinating seeds. Their mapping positions were determined and compared with known QTLs for malting quality. Varieties with contrasted malting qualities were searched for DNA polymorphisms for each of these genes. All genes proved to be polymorphic, some of the sequence variations leading to changes in the primary structure of the encoded protein. Technological traits were chosen and used to characterize the varieties based on literature data: extract yield, viscosity, β-glucan content of the wort, final apparent attenuation and diastatic power. Biochemical traits were also investigated on the same varieties and focused on four enzyme activities implicated in cell wall polysaccharides and starch degradation. The Discriminant power and ease of the amylase test, as well as its relation with technological traits, make it a promising selection test in breeding programs. The relationship between the DNA polymorphisms and biochemical and technological traits reveals around sixty polymorphisms displaying apparent relationships with at least one trait. Thirteen out of them were chosen for their potential as selection markers. They are located on (1-3,1-4)-β-glucanase genes, on one (1-3)-β-glucanase gene, on one (1-4)-β-xylan endohydrolase gene and on one α-amylase gene. Detection of these alleles can be achieved by simple and inexpensive PCR tests ( allele specific), and six assay protocols have been set up at the completion of this research.

L’immunothérapie anti-cancéreuse vise à éliminer les cellules tumorales en stimulant les propres cellules du système immunitaire du patient. Dans ce projet, nous avons développé une nouvelle stratégie d’immunothérapie visant à cibler directement des immunostimulants a la surface des cellules tumorales par ligation bio-orthogonale. Nous utilisons, pour marquer spécifiquement les cellules tumorales, leur métabolisme particulier et très actif qui permet d’intégrer à leur surface, dans leurs glycanes, des azido sucres capables de se lier en grand nombre avec divers immunostimulants portant des groupements réactifs adéquats (= glyco-ingénierie métabolique). Pour la ligation aux glycanes, nous employons la chimie bio-orthogonale qui est basée sur l’utilisation de 2 groupements mutuellement réactifs, tous 2 absents des milieux biologiques et qui peuvent se coupler rapidement très sélectivement et donc pratiquement sans réactions secondaires dans des conditions douces compatibles avec une application in vivo. Notre choix d’immunostimulants s’est porte sur les oligonucléotides de type CpG (puissants immunostimulants) et sur les β-glucanes qui, en combinaison avec des anticorps thérapeutiques, stimulent la phagocytose et n’entrainent pas une secretion importante de cytokines. Ainsi, après avoir determiné les meilleures conditions d’incorporation des azido sucres et mis au point le couplage des immunostimulants à différents groupements bioorthogonaux, nous sommes parvenus à montrer in vitro la fixation des immunostimulants à la surface de différentes lignées tumorales. Des tests immunologiques in vitro et une étude in vivo ont ensuite permis de valider l’effet des immunostimulants fixés à la surface des cellules tumorales. Nous avons ainsi observe sur une série de souris, un ralentissement de développement tumoral en présence d’un puissant immunostimulant fixé sur les cellules tumorales. / Cancer immunotherapy uses the patient's own immune system to fight cancer. In this research project, we propose a new strategy for immunotherapy: binding immunostimulants in situ to the tumor cell surface using bio-orthogonal chemistry. For that purpose we use the particular and active metabolism of tumor cells to introduce by metabolic glycoengineering into their cell surface glycans, azido sugars capable of binding many different immunostimulants carrying adequate reactive groups. The biorthogonal chemistry allowing this specific ligation is based on the use of two mutually reactive groups both naturally absent from biological systems and which can be coupled selectively and very quickly in conditions totally compatible with living organisms. Our choice of immunostimulants consists, on one hand, of CpG oligonucleotides (powerful general immunostimulants) and on the other hand of β-glucans (phagocytosis stimulants used in combination with therapeutic antibodies without causing strong cytokine secretion). We determined the best conditions for the introduction of azido sugars into cell glycans of different tumor models and tried different biorthogonal groups and reaction conditions to obtain the best immunostimulant coupling to the surface of various tumor cell lines. Then, we performed in vitro immunological tests and in vivo studies in mice in order to validate the effect of the association between immunostimulants and tumor cells on the immune response against tumors. Thereby, we observed on a group of mice, reduced tumor growth when the strong immunostimulant CpG was fixed onto tumor cell surface.

1 Abstract This doctoral thesis is focused on the synthesis and characterization of novel hybrid polysaccharide-based polymers applicable for biomedicine, specifically for a conceptually new bimodal cancer treatment - immunoradiotherapy. For this purpose, polysaccharides β-glucan from Auricularia auricula-judae and κ-carrageenan from Kappaphycus alvarezii, exhibiting immunostimulatory and anticancer activities, were chosen to be grafted with thermoresponsive poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline)s (POXs) (with different graft lengths and grafting densities) that induced a lower critical solution temperature of the final polymers. The thermoresponsive behavior of resulting polymers was studied with temperature-dependent light scattering methods, fluorescence measurements and also nuclear magnetic spectroscopy to select a polymer material with the most suitable properties for the intended application, aiming at a polymer depot formation after the injection of a polymer solution into the body. The chosen polymer, β-glucan-graft-POX with graft length of 2500 Da, was then modified to bear 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid and a fluorescent dye Dyomics-615 at the graft ends and tested first in vitro to investigate its immunostimulatory properties and also the cellular uptake....

Zymosan-induced peritonitis is a model commonly used to study systemic inflammatory response syndrome and multiple organ dysfunction syndrome. However, effects of zymosan on cardiac function have not been reported. We evaluated cardiac responses to zymosan in mice and the role of β-Glucan and dectin-1 in mediating these responses. Temperature and cardiac function were evaluated before and after intraperitoneal (i.p.) injection of zymosan (100 or 500 mg/kg) or saline. Chronotropic and dromotropic functions were measured using electrocardiograms (ECGs) collected from conscious mice. Cardiac inotropic function was determined by echocardiography. High-dose zymosan caused a rapid and maintained hypothermia along with visual signs of illness. Baseline heart rate (HR) was unaffected but HR variability (HRV) increased, and there was a modest slowing of ventricular conduction. High-dose zymosan also caused prominent decreases in cardiac contractility at 4 and 24 h. Because zymosan is known to cause gastrointestinal tract pathology, peritoneal wash and blood samples were evaluated for bacteria at 24 h after zymosan or saline injection. Translocation of bacterial occurred in all zymosan-treated mice (n=3), and two had bacteremia. Purified β-Glucan (50 and 125 mg/kg, i.p.) had no effect on temperature or ECG parameters. However, deletion of dectin-1 modified the ECG responses to high-dose zymosan; slowing of ventricular conduction and the increase in HRV were eliminated but a marked bradycardia appeared at 24 h after zymosan treatment. Zymosan-treated dectin-1 knockout mice also showed hypothermia and visual signs of illness. Fecal samples from dectin-1 knockout mice contained more bacteria than wild types, but zymosan caused less translocation of bacteria. Collectively, these findings demonstrate that zymosan-induced systemic inflammation causes cardiac dysfunction in mice. The data suggest that dectin-1-dependent and -independent mechanisms are involved. Although zymosan treatment causes translocation of bacteria, this effect does not have a major role in the overall systemic response to zymosan.

Activation of the immune system during photodynamic therapy (PDT) and improvement of the effector functions of mAbs - these are the ways to use and enhance the potential of the immune system to fight cancer. Tumor cells lack β-glucan as a surface compo¬nent and can‘t trigger complement receptor 3-dependent cellular cytoto¬xicity and initiate tumor-killing activity during PDT. So, it gave rise to the hypothesis that β-glucan in combination with PDT will produce more effective killing of by iC3b fragment opsonized tumor cells. The human Fc portion is essential for the recruiting of human effector immune cells to produce antitumor effect. Therefore, connection of Fv portion of murine anti-CD7 antibody with Fc portion of human IgG1 can be helpful for such protein to obtain ideal feathers. However, each modification of the monoclonal antibody can cause the lost or decrease in the rate of protein expression and antigen-binding properties. Monoclonal antibody products are unique in their molecules. Because of post-translational modifications that often occur during the fermentation process, the final product is heterogeneous. Therefore, careful characterization of monoclonal antibodies is required in order to assess their identity, purity, potency and safety. Response of Lewis lung carcinoma tumor to PDT modulated by β-glucan was assessed in mice and functional characteristics of novel purified chimeric anti-CD7 antibody was tested in this study. / Imuninės sistemos aktyvinimas taikant fotosensibilizacinę terapiją (FNT) ir monokloninių antikūnų efektorinių funkcijų gerinimas yra vienas iš būdų, kaip naudoti ir sustiprinti organizmo imuninės sistemos veiksnių potencialą kovai su naviku. Navikinės ląstelės membranos paviršiuje nėra β-gliukano, todėl FNT metu negali būti aktyvinamas nuo komplemento receptoriaus 3 priklausomas ląstelės citotoksiškumo mechanizmas bei sukeliama navikinės ląstelės žūtis. Todėl galima tikėtis, kad β-gliukanas ir FNT, taikomi kartu, gali aktyvinti iC3b frag¬mento opsonizuotų navikinių ląstelių žūtį. Norint aktyvinti žmogaus imunines ląsteles priešnavikiniam poveikiui sukelti, pelės monokloniniam antikūnui būtina žmogaus imunoglobulino Fc dalis. Todėl pelės antikūno prieš CD7 vienos grandinės Fv fragmento sujungimas su žmogaus imunoglobulino G1 Fc dalimi baltymui gali suteikti idealias savybes. Tačiau, bet kokia monokloninio antikūno modifikacija gali keisti jo savybes ir, išskiriant baltymą, dėl daugiapakopio proceso ji gali keistis. Todėl, norint įvertinti chimerinio CD7-Fc antikūno identiškumą, grynumą, veiksmingumą bei saugumą, būtina atlikti tyrimus, kuriais būtų galima nustatyti, ar antikūnas išlaiko specifiškumą antigenui, kaip aktyviai jungiasi prie jo, ar aktyvina imuninius mechanizmus. Šio tyrimo metu buvo vertintas pelėms įskiepytos Luiso plaučių karcinomos atsakas į FNT moduliaciją β-gliukanu ir išgrynintas chimerinis antikūnas prieš CD7 bei ištirtos jo funkcinės savybės in vitro.

The ability of the soluble fibre (1->3)(1->4)-β-D-glucan to attenuate postprandial glycemic responses depends on its viscosity which, in turn, depends on molecular weight (MW) and dose. However, the effect of altering viscosity by changing solution volume is unknown. Furthermore, β-glucan solutions may form hard gels when left to age, but the effect of these gels on glycemic responses is unknown. Therefore, the effects of varying the MW and volume of β-glucan solutions and hard gels, on glycemic responses were determined. The results showed that glycemic responses were reduced by increasing viscosity by increasing MW but not by reducing solution volume. Although β-glucan gels reduced the rate of glucose diffusion in vitro, they had no effect on glycemic responses in vivo. Thus, changing solution viscosity through changes in volume does not alter the effect of β-glucan on glycemic response, and β-glucan gels are ineffective at attenuating in vivo glycemic responses.

The ability of the soluble fibre (1->3)(1->4)-β-D-glucan to attenuate postprandial glycemic responses depends on its viscosity which, in turn, depends on molecular weight (MW) and dose. However, the effect of altering viscosity by changing solution volume is unknown. Furthermore, β-glucan solutions may form hard gels when left to age, but the effect of these gels on glycemic responses is unknown. Therefore, the effects of varying the MW and volume of β-glucan solutions and hard gels, on glycemic responses were determined. The results showed that glycemic responses were reduced by increasing viscosity by increasing MW but not by reducing solution volume. Although β-glucan gels reduced the rate of glucose diffusion in vitro, they had no effect on glycemic responses in vivo. Thus, changing solution viscosity through changes in volume does not alter the effect of β-glucan on glycemic response, and β-glucan gels are ineffective at attenuating in vivo glycemic responses.