La phosphorylation est une modification post-traductionnelle modulant l’activité, la conformation ou la localisation d’une protéine et régulant divers processus. Les kinases et phosphatases sont responsables de la dynamique de phosphorylation et agissent de manière coordonnée. L’activation anormale ou la dérégulation de kinases peuvent conduire au développement de cancers ou de désordres métaboliques. Les récepteurs tyrosine kinase (RTKs) sont souvent impliqués dans des maladies et la compréhension des mécanismes régissant leur régulation permet de déterminer les effets anticipés sur leurs substrats. Dans ce contexte, le but de cette thèse est d’identifier les évènements de phosphorylation intervenant dans la voie de l’insuline chez la drosophile impliquant un RTK : le récepteur de l’insuline (InR). La cascade de phosphorylation déclenchée suite à l’activation du récepteur est conservée chez le mammifère. Afin d’étudier le phosphoprotéome de cellules S2 de drosophile, nous avons utilisé une étape d’enrichissement de phosphopeptides sur dioxyde de titane suivie de leur séparation par chromatographie liquide (LC) et mobilité ionique (FAIMS). Les phosphopeptides sont analysés par spectrométrie de masse en tandem à haute résolution. Nous avons d’abord démontré les bénéfices de l’utilisation du FAIMS comparativement à une étude conventionnelle en rapportant une augmentation de 50 % dans le nombre de phosphopeptides identifiés avec FAIMS. Cette technique permet de séparer des phosphoisomères difficilement distinguables par LC et l’acquisition de spectres MS/MS distincts où la localisation précise du phosphate est déterminée. Nous avons appliqué cette approche pour l’étude des phosphoprotéomes de cellules S2 contrôles ou traitées à l’insuline et avons identifié 32 phosphopeptides (sur 2 660 quantifiés) pour lesquels la phosphorylation est modulée. Étonnamment, 50 % des cibles régulées possèdent un site consensus pour la kinase CK2. Une stratégie d’inhibition par RNAi a été implémentée afin d’investiguer le rôle de CK2 dans la voie de l’insuline. Nous avons identifié 6 phosphoprotéines (CG30085, su(var)205, scny, protein CDV3 homolog, D1 et mu2) positivement régulées suite à l’insuline et négativement modulées après le traitement par RNAi CK2. Par essai kinase in vitro, nous avons identifié 29 cibles directes de CK2 dont 15 corrélaient avec les résultats obtenus par RNAi. Nous avons démontré que la phosphorylation de su(var)205 (S15) était modulée par l’insuline en plus d’être une cible directe de CK2 suite à l’expérience RNAi et à l’essai kinase. L’analyse des données phosphoprotéomiques a mis en évidence des phosphopeptides isomériques dont certains étaient séparables par FAIMS. Nous avons déterminé leur fréquence lors d’études à grande échelle grâce à deux algorithmes. Le script basé sur les différences de temps de rétention entre isomères a identifié 64 phosphoisomères séparés par LC chez la souris et le rat (moins de 1 % des peptides identifiés). Chez la drosophile, 117 ont été répertoriés en combinaison avec une approche ciblée impliquant des listes d’inclusion. Le second algorithme basé sur la présence d’ions caractéristiques suite à la fragmentation de formes qui co-éluent a rapporté 23 paires isomériques. L’importance de pouvoir distinguer des phosphoisomères est capitale dans le but d’associer une fonction biologique à un site de phosphorylation précis qui doit être identifié avec confiance. / Phosphorylation is a reversible post-translational modification that modulates protein activity, and can impart conformational changes and affect translocation of their protein substrates. Kinases and phosphatases are responsible for the dynamic of changes in protein phosphorylation and act in a coordinated manner. Abnormal activation or misregulation of kinase activity can lead to the development of cancers and metabolic disorders. Tyrosine kinase receptor (RTK) associated signaling pathways are often implicated in numerous diseases and the further understanding of mechanisms affecting their regulation is necessary to determine their activity and effects anticipated on their substrates. In this context, the primary objective of this thesis is to study the phosphorylation events arising from the activation of the insulin receptor (InR) following stimulation of drosophila S2 cells with insulin. The phosphorylation cascade triggered after InR activation is conserved in mammals. In order to study the phosphoproteome of drosophila S2 cells, we enriched phosphopeptides on titanium dioxide (TiO2) stationary phase prior to their separation by liquid chromatography (LC) and ion mobility (FAIMS) mass spectrometry (MS). Phosphopeptides were then analysed by tandem MS at high resolution. We first compared the benefits of FAIMS to conventional LC-MS, and observed a 50% increase in the number of identified phosphopeptides when using ion mobility. FAIMS enables the separation of phosphoisomers that are typically unresolved by LC, enabling high confidence assignment of modification sites via distinct MS/MS spectra. This approach was used to profile phosphorylation changes taking place between control and insulin-treated drosophila cells and enabled the identification of 32 phosphopeptides (out of 2 660 quantified) showing differential regulation. Interestingly, 50% of the regulated targets have a CK2 consensus site. These preliminary experiments were followed-up by RNAi mediated inhibition of CK2 and revealed that 6 phosphoproteins (CG30085, su(var)205, scny, protein CDV3 homolog, D1 and mu2) were positively modulated after insulin stimulation and negatively regulated after CK2 RNAi treatment. Using in vitro kinase assay, we identified 29 direct CK2 targets, of which 15 were correlated with results from the CK2 RNAi experiment. We demonstrated specifically that the su(var)205 (S15) is regulated by insulin and is a direct CK2 target based on RNAi and kinase assays. Our phosphoproteomics data also highlighted the presence of isomeric phosphopeptides, several of which could be distinguished using FAIMS. We developed two algorithms to determine the occurrence of phosphoisomers in large scale studies. The first algorithm based on differences in retention times between isomers identified 64 candidates in mouse and rat phosphoproteome datasets corresponding to less than 1% of all identified phosphopeptides. We also identified 117 isomer candidates in drosophila using a targeted LC-MS/MS approach with inclusion lists. The second algorithm is based on the presence of characteristic fragment ions present in MS/MS spectra of co-eluting or partially resolved species and allowed the identification of 23 isomeric pairs. The ability to distinguish phosphoisomers in large-scale phosphoproteome datasets is of significance to correlate phosphorylation events taking place on specific residues with biological activities.

It is widely known that surfaces play an important role in numerous biological processes and technological applications. Thus, being able to modify surface properties provides an opportunity to control many phenomena occurring at interfaces. One way of controlling surface properties is to adsorb a polymer film onto the surface, for example through layer-by-layer (LbL) deposition of polyelectrolytes. This simple but versatile technique enables various polymers, proteins, colloidal particles etc. to be incorporated into the film, resulting in a multifunctional coating. Due to recent legislations and a consumer demand for more environmentally friendly products, we have chosen to use natural polymers (biopolymers) from renewable resources. The focus of this thesis has been on the adsorption of biopolymers and their layer-by-layer formation at solid-liquid interfaces; these processes have been studied by a wide range of techniques. The main method was the quartz crystal microbalance with dissipation monitoring (QCM-D), which measures the adsorbed mass, including trapped solvent and the viscoelastic properties of an adsorbed film. This technique was often complemented with an optical method, such as ellipsometry or dual polarization interferometry (DPI), which provided information about the “dry” polymer or protein adsorbed mass. From this combination, the solvent content and density of the layers was evaluated. In addition, the surface force apparatus (SFA), X-ray photoelectron spectroscopy (XPS), total internal reflection fluorescence (TIRF), and fluorescence resonance energy transfer (FRET) were utilized, providing further information about the film structure, chemical composition, and polymer inter-layer diffusion. Adsorption studies of the glycoprotein mucin, which has a key role in the mucousal function, showed that despite the net negative charge of mucin, it adsorbed on negatively charged substrates. The adsorbed layer was highly hydrated and the segment density on the substrate was low. We showed the importance of characterizing the mucin used, since differences in purity, such as the presence of albumin, gave rise to different adsorption behaviours in terms of both adsorbed amount and structure. The adsorbed mucin layer was to a large extent desorbed upon exposure to the anionic surfactant sodium dodecyl sulfate (SDS). In order to prevent desorption, we demonstrated that a protective layer of the cationic polysaccharide chitosan could be adsorbed onto the mucin layer and that the mucin-chitosan complexes resisted the desorption normally induced by association with SDS. Moreover, the association between chitosan and SDS was examined at the solid-liquid interface, in the bulk, and at the air-water interface. In all these environments chitosan-SDS complexes were formed and a net charge reversal of the complexes from positive to negative was observed when the concentration of SDS was increased. Furthermore, the LbL deposition method could be used to form a multilayer-like film by alternate adsorption of mucin and chitosan on silica substrates. The LbL technique was also applied to two proteins, lysozyme and β-casein with the aim of building a multilayer film consisting entirely of proteins. These proteins formed complexes at the solid-liquid interface, resulting in a proteinaceous layer, but the build-up was highly irregular with an increase in adsorbed amount per protein deposition cycle that was far less than a monolayer.Continuing with chitosan, known to have antibacterial properties we assembled multilayers with an anti-adhesive biopolymer, heparin, to evaluate the potential of this system as a coating for medical implants. Multilayers were assembled under various solution deposition conditions and the film structure and dynamics were studied in detail. The chitosan-heparin film was highly hydrated, in the range 60-80 wt-% depending on the deposition conditions. The adsorbed amount and thickness of the film increased exponential-like with the number of deposition steps, which was explained by inter-diffusion of chitosan molecules in the film during the build-up. In a novel approach, we used the distant dependent FRET technique to prove the inter-layer diffusion of fluorescent-labelled chitosan molecules within the film. The diffusion coefficient was insignificantly dependent on the deposition pH and ionic strength, and hence on the film structure. With the use of a pH sensitive dye buried under seven chitosan-heparin bilayers, we showed that the dye remained highly sensitive to the charge of the outermost layer. From complementary QCM-D data, we suggested that an increase in the energy dissipation does not necessarily indicate that the layer structure becomes less rigid. / Det är välkänt att ytor spelar en viktig roll i många biologiska processer och tekniska tillämpningar. Att kunna modifiera en ytas egenskaper ger därför en möjlighet att kunna kontrollera många fenomen som sker på ytor. Ett sätt att kontrollera ytegenskaperna är genom att adsorbera en polymerfilm på ytan, till exempel genom att växelvis adsorbera olika polyelektrolyter (LbL-teknik). Denna enkla men mångsidiga teknik möjliggör att många olika material kan införlivas i filmen, vilket resulterar i en multifunktionell beläggning. På grund av dagens lagstiftning och konsumenters ökade efterfrågan på miljövänliga material beslutade vi oss för att använda biologiska polymerer (biopolymerer) i detta projekt. Fokus i den här avhandlingen har varit på adsorption av biopolymerer och deras LbL-formation på gränsytan vätska-fast fas, där adsorptionsförloppet och det adsorberade skiktet bestående av biopolymerer studerats med en mängd olika tekniker. Huvudtekniken var kvartskristallmikrovåg med energidissipations-registrering (QCM-D), som mäter massan inklusive inkorporerat vatten, samt de viskoelastiska egenskaperna hos ett adsorberat skikt. Som komplement till denna teknik användes ofta optiska metoder, till exempel ellipsometri och ”dubbel polarisationsinterferometri (DPI)”, två tekniker som endast mäter massan av de adsorberade biopolymererna. Genom denna kombination av metoder kunde massan av inkorporerat vatten i filmen och filmens densitet bestämmas. Dessutom användes ytkraftsapparaten (SFA), röntgenfotoelektronspektrometri (XPS), och fluorescens-spektroskopiteknikerna TIRF och FRET i några undersökningar för att erhålla information om skiktens struktur, kemiska sammansättning och polymerernas diffusion inom skiktet.Adsorptionsstudier av glycoproteinet mucin, som har en central roll i funktionen av slemhinnan, avslöjade att trots att mucinet har en negativ nettoladdning adsorberade det ändå på negativt laddade substrat. Det adsorberade lagret var väldigt hydratiserat och hade en låg andel mucin i direkt kontakt med ytan. Vi påvisade vikten av att noga undersöka mucinet som användes, eftersom olika renhet, till exempel i form av förekomsten av albumin gav upphov till olika adsorptionsbeteende gällande både adsorberad mängd och struktur. En stor andel av det adsorberade mucinlagret desorberade när det exponerades för den anjoniska tensiden natriumdodecylsulfat, SDS. Vi visade att ett skyddande lager av den katjoniska polysackariden chitosan kunde adsorberas på mucinet och att mucin-chitosan-komplexen inte desorberade när SDS tillsattes. Därtill studerades växelverkan mellan chitosan och SDS på gränsytan vätska-fast fas, i bulken och på luft-vattengränsytan. Komplex av chitosan-SDS bildades i samtliga miljöer och en nettoladdningsomsvängning från positiv till negativ observerades när koncentrationen av SDS ökades.Vidare kunde LbL-tekniken nyttjas för att skapa ett multilagerlikt skikt genom att alternerande adsorbera mucin och chitosan på kiseldioxidsubstrat. Denna teknik användes även med två proteiner, lysozym och β-kasein, med målet att skapa ett multilager bestående av endast proteiner. Dessa proteiner bildade komplex på gränsytan vätska-fast fas i form av ett blandat proteinlager, men uppbyggnaden var väldigt oregelbunden med en ökning i adsorberad mängd per proteindeponeringscykel som var avsevärt mindre än ett monolager.Inom området för biomaterial utgör de antibakteriella och antihäftande egenskaperna hos chitosan respektive heparin en lovande blandning för beläggningar av medicinska implantat. Baserat på detta konstruerade vi multilagerfilmer av chitosan och heparin med olika deponeringslösningar och undersökte dynamiken och filmens struktur i detalj. Chitosan-heparin-filmen var starkt hydratiserad, bestående av cirka 60-80 vikt-% vatten beroende på deponeringsbetingelserna. Den adsorberade mängden och tjockleken på filmen ökade nästan exponentiellt med antal deponeringar, vilket förklarades med chitosanets förmåga att diffundera genom filmen under uppbyggnaden. Med ett nytt angreppssätt använde vi FRET för att bevisa diffusionen av fluorescerande färgmärkt chitosan i filmen under uppbyggnaden. Diffusionskoefficienten var i princip oberoende av pH och jonstyrka under deponeringen och följaktligen av filmens struktur. Genom att använda ett pH-känsligt färgämne begravt under sju biskikt av chitosan-heparin visade vi att färgämnet i hög grad påverkades av laddningen på det yttersta lagret. Från QCM-D-data lade vi fram teorin om att en ökning av energidissipationen för ett lager inte nödvändigtvis indikerar att lagrets struktur har blivit mindre styvt. / QC 20100729

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / O ensaio foi realizado no Setor de Suinocultura do Centro de CiÃncias AgrÃrias do Departamento de Zootecnia da Universidade Federal do CearÃ, com o objetivo de avaliar o desempenho zootÃcnico, os indicadores do metabolismo do nitrogÃnio, o bem-estar animal e o custo de produÃÃo de leitÃes no prÃodo de creche, submetidos a dietas à base de sorgo-soja enriquecidas com caseÃna + lactose isoladas. O experimento teve a duraÃÃo de 42 dias, sendo dividido em duas fases experimentais, a saber: fase 1(21-42 dias) e fase 2 (42 a 63 dias). Foram utilizados 40 animais de linhagem comercial, desmamados com idade aproximada de 21 dias, apresentando um peso mÃdio de 4,76kg. O delineamento experimental utilizado foi em blocos ao acaso, com 4 tratamentos e 5 repetiÃÃes, apresentando 2 animais por repetiÃÃo. Foi realizado um desdobramento fatorial 2X2, sendo 2 cereais energÃticos (milho e sorgo) e 2 suplementos lÃcteos (leite desnatado em pà e caseÃna + lactose). A comparaÃÃo entre as mÃdias foi realizada pelo teste de Duncan a 5%. As dietas experimentais foram isoenergÃticas, isoprotÃicas e isonutrientes para lisina e met+cis. Na fase 1 foi utilizado 21 % de PB e 3350 kcal de EM ⁄ kg. e na fase 2, 18,5 % de PB e 3250kcal de EM ⁄ kg. As dietas experimentais foram em nÃmero de 4 por fase, da seguinte forma: T1 (milho +LDP), T2 (milho+caseÃna +lactose), T3 (sorgo+LDP) e T4 (sorgo+caseÃna+lactose). Foi concluÃdo que a substituiÃÃo do milho pelo sorgo granÃfero e a inclusÃo da lactose + caseÃna à viÃvel quanto ao desempenho zootÃcnico. A utilizaÃÃo do sorgo e da caseÃna+lactose nas dietas para leitÃes desmamados nÃo ocasionou diferenÃas significativas para os indicadores do metabolismo do nitrogÃnio nas duas fases experimentais. Os tratamentos nÃo influenciaram os indicadores do bem-estar animal. NÃo foram registradas interaÃÃes significativas (P>0,05) entre os alimentos energÃticos e os alimentos lÃcteos, nas fases 1 e 2 do perÃodo de creche, para o desempenho zootÃcnico e para os indicadores do bem-estar animal. A melhor resposta econÃmica para a produÃÃo de leitÃes no perÃodo de creche foi obtida com a dieta contendo sorgo + caseÃna +lactose (T4). / The research was developed in the Division of Swine Pro duction, Department of Animal Science, Agricultural Science Center, Federal Un iversity of CearÃ, with the objective of evaluating the animal performance, the occurrence diarrhea, the nitrogen metabolism indicators, animal well-being, and the production cost of piglets submitted to diets containing sorghum-soybean me al and isolated casein+lactose. The assay had the duration of 42 days and it was divided in two phases: 1 (21-42 days of age) and 2 (42-63 days of age ) during the nursery period. The experiment used a total of 40 piglets of a commercial line, weaned at 21 days of age (average weight of 4,76 kg). The exper iment followed a randomized blocks design, with 4 treatments, 5 repetitio ns and 2 animals per repetition, unfolded in a factorial 2 x 2 design, comp osed by two energetic feeds (corn and sorghum) and two milk products (dried skim milk a nd casein + lactose). Mean values were compared through the Duncan's tests 5 %. The diets were isoenergetics, isoproteics and isonutritives for lysine and methionine + cystine. In phase 1, a level of 21% crude protein (CP) and 3350 kcal ME was used and in phase 2, a level of 18,5 % crude protein (CP) and 32 50 kcal ME was used. The experimental diets were in number of 4 for phase: T 1 (corn+ dried skim milk), T 2 (corn + casein + lactose), T 3 (sorghum + dried skim milk) and T 4 (sorghum + casein + lactose). The use of sorghum and casein + lactose in weaned pig diets did not cause significant differences for nitrogen met abolism indicators, in both phases of assay. The treatments did not influence the w ell-being indicators. Related to animal performance and well-being no signi ficant interactions were registered (P>0,05) among the factors, during phases 1 a nd 2 of the nursery xxi period. The best economical answer for production of pi glets in the nursery period was obtained with the diet containing sorghum + casein + lactose (T4).

Pequenos e médios laticínios têm grandes dificuldades no planejamento da produção para maximizar o lucro. Além disso, a forma atual de precificar o leite cru no Brasil desestimula o produtor a melhorar sua qualidade, pois valoriza mais seu volume do que seus componentes sólidos. A composição do leite cru é muito importante para os laticínios, pois ela afeta diretamente o rendimento de produção e a qualidade dos produtos lácteos. Técnicas de otimização, como programação linear (PL), ajudam a resolver problemas relacionados à decisão do mix de produtos, além de fazer análise econômica dos recursos. Em razão disso, foi desenvolvido em PL um modelo para maximização da margem de contribuição total (MCT = receita total das vendas - custos e despesas diretas totais) e precificação do leite cru através da determinação do mix ótimo de produtos lácteos. O modelo foi simulado em cenários diários de alta (Janeiro) e baixa (Julho) disponibilidade de matéria prima comparando as soluções ótimas com resultados reais de um laticínio do sudeste do Brasil. Foram realizadas análises de sensibilidade dos componentes nutritivos de dois tipos de leite cru de diferentes composições (LC1, leite cru proveniente de um fornecedor, e LC50, leite cru proveniente de 50 fornecedores) para determinar seus preços-sombra. Foram observados preços-sombra para o quilograma da caseína em Janeiro e Julho e para o litro do LC50 em Julho. A MCT ótima e os preços calculados de ambos os tipos de leite cru foram maiores em Janeiro devido à maior disponibilidade de matéria prima. Em ambos os cenários, os preços calculados de ambos os tipos de leite cru foram maiores que os praticados pelo laticínio e o lucro antes de juros, impostos, depreciação e amortização (LAJIDA) diário real foi maior que o ótimo. A embaladora foi um recurso limitante atuante na produção do queijo Minas Frescal nos mixes ótimos. A relação entre margem de contribuição unitária (MCU = preço unitário de venda - custo e despesa diretos unitários) e quantidade necessária de matéria prima por unidade de produto e a disponibilidade de recursos são determinantes na definição do mix de produtos lácteos e da MCT do laticínio. A precificação do leite cru pelo modelo proposto remunera o produtor em função da quantidade de seus componentes. O laticínio pode maximizar seu LAJIDA planejando melhor seu mix de produtos com PL e remunerando melhor seus fornecedores em função da qualidade do leite cru. / Small and medium-sized dairies face great difficulties in production planning to maximize profit. In addition, the current way of pricing the raw milk in Brazil discourages producers to improve its quality because its volume values more than its solid components. The raw milk composition is very important for the dairies because it directly affects the yield and quality of dairy products. Optimization techniques, such as linear programming (LP), aid solve problems related to the decision of the product mix and perform economic analyzes of resources. For this reason, a model was developed in LP to maximizing total contribution margin (TCM = total sales revenues - total variable costs and expenses) and pricing the raw milk by determining the optimal mix of dairy products. The model was simulated in two daily scenarios of high (January) and low (July) availability of raw material comparing the optimal solutions with actual results from a dairy plant in Southeastern Brazil. Sensitivity analyzes of the nutritional components of two kinds of raw milk of different compositions (RM1 and RM50) were performed to determine their shadow-prices. Shadow-prices were observed for the kilogram of casein in January and July and for the liter of RM50 in July. The optimal TCM and the calculated prices of both types of raw milk were higher in January due the increased availability of raw materials. In both scenarios, the calculated prices of both types of raw milk were higher than those paid by the dairy plant and the actual daily earnings before interests, taxes, depreciation and amortization (EBITDA) were greater than the optimum ones. The wrapper was an active limiting resource in the production of Frescal Minas cheese in optimal mixes. The relationship between unit contribution margin (UCM = unit sales price - unit variable cost and expense) and required amount of raw material per unit of output and resource availability are crucial in defining the mix of dairy products and the dairy TCM. The proposed raw milk pricing model pays the producer by the quantity of its components. The dairies can maximize their EBITDA planning their product mixes with LP and remunerating their suppliers based on the quality of raw milk.

L'objectif de la thèse a été d'élucider le processus de réhydratation des caséines micellaires et des protéines solubles dans un milieu complexe et opaque : le lait. L'influence de l'état d'hydratation de ces ingrédients laitiers en fonction du temps (5, 120, 180, 240, 300, 480, 900 et 1440 min de réhydratation) sur les propriétés rhéologiques, texturales, physiques ainsi que la microstructure des gels laitiers acides a également été étudiée. Il en résulte que le processus de réhydratation des caséines micellaires diffère de celui des protéines solubles, et est extrêmement long avec trois étapes : une étape de mouillage des particules, suivie d'une étape de gonflement caractérisée par une augmentation de la taille des particules et enfin une étape de dispersion marquée par la diminution de la taille des particules. La réhydratation des protéines solubles est caractérisée par une grande rapidité, avec deux phases : le mouillage et la phase de dispersion (superposée). D'autre part, l'allongement de la durée de réhydratation des caséines micellaires est associé à une augmentation du point de gélification ainsi qu'à une nette amélioration des propriétés physiques, texturales et rhéologiques des gels : augmentation de leur fermeté et de leur force, diminution de la synérèse et de la formation de grumeaux. La durée de réhydratation des protéines solubles n'a pas d'influence sur ces paramètres. En revanche, leur dénaturation (par chauffage à sec) est associée à une dégradation des propriétés texturales des gels acides. Finalement, il s'avère que les gels acides formulés à partir des protéines solubles sont de meilleure qualité texturale (à l'exception de la formation de grumeaux) que ceux préparés à partir des caséines micellaires / The main objectives of this work were to elucidate the rehydration mechanism of the two major milk proteins (micellar casein and whey protein) into a complex and opaque medium such as milk and to assess the influence of hydration state (defined as a function of rehydration length after 5,120,180,240,300, 480, 900 and 1440 minutes of rehydration) on the rheological, textural, physical properties and microstructure of the obtained acid milk gels. Whereas, micellar casein presented a long rehydration process into milk characterized by three stages: a wetting, swelling and dispersion phase, whey protein displayed a quick rehydration process characterized by an overlapping of wetting and dispersion phase. Furthermore, an extended rehydration time of micellar casein powder into the milk base was associated with a postponed onset of gelation and enhanced physical, textural as well as rheological properties of the obtained acid milk gels characterized by increases in gel firmness, strength, and decreases in syneresis susceptibility and grains formation. In contrast, acid milk gels prepared with whey protein powder exhibited comparable overall textural properties regardless the different rehydration times. Nevertheless, denaturation of whey protein powder (by dry heating) was associated with a deterioration of the textural properties of the acid milk gels. Finally, acid gels prepared with whey proteins displayed better overall textural quality than those prepared with micellar casein (except for grains formation)

The diploma thesis is dealing with the determination of caseins in lyophilised samples of milk using the method of capillary electrophoresis (CE). This method was optimised and verified by Ivana Micikova in her diploma thesis in 2009. The theoretical part of the diploma thesis gives information about the chemical compound of cow's milk. Caseins and their usage in the food and non-food industries are described in a separate unit. Impact of both the nutritional and non-nutritional factors on the composition of the milk is discussed further. The description and the principles of the CE and a list of the methods of CE used in the last several years are stated at the end of this part. The experimental part is focused on the preparation of the solutions, process of casein isolation and its analysis. It summarises the setting of the experiment, sampling and sample processing and the composition of the feeding mixtures. The diploma thesis has arisen on the basis of the cooperation of the Institute of Food Science and Biotechnology, the Faculty of Chemistry, Brno University of Technology and the Research Institute for Cattle Breeding, Ltd., Department of Animal Nutrition Physiology in Pohorelice.

La localisation de l'ARNm est un mécanisme post-transcriptionnel régulant l'expression des gènes qui donne un contrôle précis sur la production spatiale et temporelle des protéines. Des milliers de transcrits dans un large éventail d'organismes ou de types cellulaires se sont avérés localisés dans un compartiment sous-cellulaire spécifique. La levure bourgeonnante Saccharomyces cerevisiae est l'un des organismes modèle les plus étudiés pour comprendre le processus de localisation de l'ARNm. Plus de trente ARNm sont activement transportés et localisés à l'extrémité du bourgeon de la levure bourgeonnante. Dans cet organisme, la localisation des transcrits à l'extrémité du bourgeon, tels que l'ARNm ASH1, dépend de la protéine de liaison à l'ARN She2, qui interagit directement avec les éléments de localisation dans ces ARNm durant leur transcription. She2 est une protéine liant l’ARN non-canonique, qui s’assemble en tétramère pour pouvoir lier l’ARN. Lorsque le complexe ARNm-She2 est exporté vers le cytoplasme, celui-ci interagit avec la protéine She3 et la myosine Myo4, qui transportent le complexe vers le bourgeon. Une fois qu'un ARNm est correctement localisé, sa traduction est activée pour permettre la synthèse locale de sa protéine. Les mécanismes régulant la localisation des ARNm sont encore très peu connus. Cependant, plusieurs évidences suggèrent que la machinerie de localisation peut être régulée par des modifications post-traductionnelles. Dans notre étude, en utilisant une colonne de purification de phosphoprotéines, nous avons constaté que She2 est une phosphoprotéine. Nous avons utilisé une approche de phosphoprotéomique pour identifier les résidus phosphorylés dans She2 in vivo. Nous avons identifié plusieurs nouveaux phosphosites qui affectent la capacité de She2 à favoriser l'accumulation asymétrique de la protéine Ash1. Fait intéressant, plusieurs phosphosites sont présents aux interfaces de dimérisation et de tétramérisation de She2. En nous concentrant sur la position T109, nous montrons qu'un mutant phosphomimétique T109D inhibe l'interaction She2-She2 et diminue l'interaction de She2 avec ses cofacteurs Srp1, She3 et l’ARNm ASH1. Fait intéressant, la mutation T109D réduit considérablement l'expression de She2 et perturbe la localisation de l'ARNm ASH1. Nos résultats montrent que le contrôle de l'oligomérisation de She2 par phosphorylation représente un mécanisme qui régule la localisation de l'ARNm dans la levure bourgeonnante. Dans le but d’identifier la ou les kinases impliquées dans la phosphorylation de She2, nous avons recherché des motifs de reconnaissance de kinases connues parmi les phosphosites que nous avons identifiés. Nous avons trouvé que les résidus T109, S217 et S224 font partie de sites putatifs de la Caséine kinase II (CKII), suggérant que ces positions seraient susceptibles d'être phosphorylés par cette kinase. Un essai de phosphorylation in vitro a révélé que She2 est phosphorylée par CKII au niveau des résidus S217 et S224, mais pas au résidu T109. Nous avons montré que la phosphorylation de la forme monomérique de She2 par CKII in vitro est augmentée par rapport à la forme sauvage tétramérique. De plus, nous avons observé que le domaine C-terminal de She2, qui contient sa séquence de localisation nucléaire (NLS) est phosphorylé par CKII. Cependant, le rôle de la phosphorylation dans le NLS de She2 demeure inconnu. Dans l'ensemble, nos résultats montrent que les modifications post-traductionnelles sur She2 régulent la localisation de l'ARNm chez la levure. Cette étude permettra d'élucider les mécanismes de contrôle de la localisation de l'ARNm chez la levure et comment des modifications post-traductionnelles sur She2 régulent ce processus. / mRNA localization is a post-transcriptional mechanism regulating gene expression that gives precise control over the spatial and temporal production of proteins. Thousands of transcripts in a wide array of organisms or cell types were shown to localize to specific subcellular compartments. The budding yeast Saccharomyces cerevisiae serves as one of the best model organisms to study the mechanisms of mRNA localization. Over thirty transcripts are actively transported and localized at the bud tip of the budding yeast. In this organism, localization of transcripts to the bud tip, such as the ASH1 mRNA, depends on the RNA-binding protein She2, which is responsible for recognizing localization elements in these mRNAs during transcription. She2 is a non-canonical RNA-binding protein which assembles as a tetramer in order to bind RNA. When the mRNA-She2 complex is exported to the cytoplasm, the protein She3 and myosin Myo4 join the complex to transport it to the bud. Once an mRNA is properly localized, its translation is generally activated to allow the local synthesis of its protein. The mechanisms regulating the localization of mRNAs are still poorly known. Still, several pieces of evidence suggest that post-translational modifications may regulate the localization machinery. Using a phosphoprotein purification column, we found that She2 is a phosphoprotein. We used a phosphoproteomic analysis to identify the phosphorylated residues in She2 in vivo. We identified several new phosphosites that impact the capacity of She2 to promote the asymmetric accumulation of Ash1. Interestingly, several of these phosphosites are present at the dimerization and tetramerization interfaces of She2. Focusing on T109, we show that a phosphomimetic mutant T109D inhibits She2-She2 interaction and decreases the interaction of She2 with its co-interactors Srp1, She3 and ASH1 mRNA. Interestingly, the T109D mutation significantly reduces the expression of She2 and disrupts ASH1 mRNA localization. Altogether, our results show that the control of She2 oligomerization by phosphorylation represents a mechanism that regulates mRNA localization in budding yeast. In order to identify which kinase(s) are involved in She2 phosphorylation, we searched for known kinases recognition motifs among the identified phosphosites. We found that T109, S217 and S224 are putative Casein kinase II (CKII) sites, suggesting that this kinase may phosphorylate these residues. Indeed, an in vitro phosphorylation assay revealed that She2 is phosphorylated by CKII at S217 and S224 but not at T109. We found that the phosphorylation of a monomeric She2 mutant by CKII in vitro is increased compared to the wild-type tetrameric protein. Furthermore, we found that the C-terminal domain of She2, which contains its nuclear localization signal (NLS), is phosphorylated by CKII. However, the biological function of the phosphorylation in the NLS is still unknown. Altogether, our results show that post-translational modifications in She2 regulate mRNA localization in yeast. This study will help elucidate the mechanisms that control mRNA localization in yeast and how post-translational modifications in She2 regulate this process.

To investigate the influence of the internal micellar structure on the course of enzymatic cross-linking especially in the initial phase of the reaction, casein micelles isolated from raw milk via ultracentrifugation were incubated with microbial transglutaminase (mTG) in comparison with non-micellar sodium caseinate. Reactive lysine and glutamine residues were identified using a label-free approach, based on the identification of isopeptides within tryptic hydrolysates by targeted HRMS as well as manual monitoring of fragmentation spectra. Identified reactive sites were furthermore weighted by tracking the formation of isopeptides over an incubation time of 15, 30, 45 and 60 min, respectively. Fifteen isopeptides formed in the early stage of mTG cross-linking of caseins were identified and further specified concerning the position of lysine and glutamine residues involved in the reaction. The results revealed lysine K176 and glutamine Q175 of β-casein as the most reactive residues, which might be located in a highly flexible region of the molecule based on different possible reaction partners identified in this study. Except for the isopeptide αₛ₁ K34–αₛ₂ Q101 in sodium caseinate (SC), all reactive sites were detected in micellar and in non-micellar casein, indicating that the initial phase of enzymatic cross-linking is not affected by micellar aggregation of caseins.

No description available.

Le lait écrémé est utilisé depuis plus d’un demi-siècle comme diluant protecteur des spermatozoïdes de mammifères. Depuis quelques années, il existe une demande grandissante pour des diluants exempts de produits d’origine animale. Toutefois, le mécanisme par lequel le lait protège les spermatozoïdes n’est pas connu, ce qui rend difficile de lui trouver un substitut. Les protéines majeures du plasma séminal de taureau, les protéines « Binder of SPerm » (BSP), sont néfastes lors de la conservation de la semence. Les spermatozoïdes sont en contact avec une grande concentration de protéines BSP qui stimulent une extraction continuelle de cholestérol/phospholipides de leur membrane plasmique. Les lipoprotéines de faible densité (LDL) du jaune d’oeuf, un autre composé utilisé dans les diluants, empêcheraient les protéines BSP de se lier à la membrane des spermatozoïdes de taureaux et de stimuler un efflux des lipides membranaires, ce qui les protégerait durant la conservation. Notre hypothèse était que les protéines du lait protègent les spermatozoïdes durant la conservation en séquestrant les protéines BSP. Premièrement, nous avons démontré par filtration sur gel qu’il y a une interaction entre les protéines BSP bovines et les protéines du lait. Le lait écrémé a été fractionné en trois fractions : F1 (alpha-lactalbumine, bêta-lactoglobuline et caséine kappa), F2 (toutes les protéines du lait) et F3 (sels, sucres et petits peptides). Les protéines BSP1 et BSP5 ont une affinité plus grande pour F1 que BSP3, tandis que toutes les protéines BSP ont une affinité pour F2. Le titrage calorimétrique isotherme a permis de confirmer l’interaction entre les protéines BSP et les protéines du lait. L’association entre la protéine BSP1 bovine et les micelles de caséines est caractérisée par une constante d’affinité (Ka) de 3.5 × 10^5 M-1 et un paramètre stoichiométrique (n) de 4,5 BSP1 pour une caséine. L’association entre la protéine BSP1 bovine et l’alpha-lactalbumine (une protéine du sérum principale), est caractérisée par un Ka de 2.4 × 10^5 M-1 et une valeur “n” de 0,8. Ces résultats indiquent que le lait protège les spermatozoïdes bovins en séquestrant les protéines BSP grâce à une interaction protéine : protéine, tandis que le jaune d’oeuf les protège grâce à une interaction protéine : lipoprotéine. Deuxièmement, nous avons démontré par filtration sur gel que les protéines homologues aux BSP bovines retrouvées dans le plasma séminal de porc, d’étalon et de bélier ont une affinité avec les protéines du lait, ce qui suggère que le mécanisme de protection des spermatozoïdes par le lait pourrait être le même chez ces espèces. Troisièmement, nous avons caractérisé l’interaction entre BSP1 bovine et les LDL du jaune d’oeuf qui a un Ka de 3.4 ± 0.4 × 10^6 M-1 et une valeur de « n » de 104 BSP1 pour une particule de LDL, indiquant qu’il existe des différences entre le mécanisme de protection des spermatozoïdes par le lait et le jaune d’oeuf. Nous croyons que les résultats présentés dans cette thèse aideront à créer de nouveaux diluants ne contenant pas de produits d’origine animale afin de cryoconserver les spermatozoïdes des mammifères. / Skim milk is being used as a protective agent for mammalian semen conservation over half a century. Recently, there has been increased interest in developing extenders free of animal products. However, it is difficult to find suitable component in order to replace milk as an extender, because the mechanisms by which milk protect sperm against cooling and freezing damages during the storage is unknown. The Binder of SPerm (BSP) proteins are the major proteins of bull seminal plasma and they are harmful during sperm storage. In fact, sperm would be in contact with a large quantity of BSP proteins that induce a continuous cholesterol and phospholipids efflux from the sperm membrane during storage. When bull sperm is diluted with an extender containing egg yolk, another compound frequently used in extender, the low-density lipoproteins (LDL) present in the egg yolk prevent the binding of the BSP proteins to the sperm membrane, thus, preventing the lipid efflux from the sperm membrane induced by the BSP proteins. Our hypothesis was that milk proteins would protect sperm during storage by binding BSP proteins. First, we demonstrated by gel filtration that bovine BSP proteins could bind the milk proteins. Skim milk was fractionated into three fractions: F1 (alpha-lactalbumin and beta- lactoglobulin, the major whey proteins and kappa-casein), F2 (mainly caseins and all other milk proteins in small amounts) and F3 (salts, sugars and small peptides). Bovine BSP1 and BSP5 have more affinity for F1 as compared to BSP3 and all the BSP proteins have affinity for F2. We confirmed the interaction between bovine BSP proteins and milk proteins by isothermal titration calorimetry. The binding of BSP1 to casein micelles is characterized by an affinity constant (Ka) of 3.5 × 10^5 M-1 and of a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin (one of the major whey proteins) is characterized by a Ka of 2.4 × 10^5 M-1 and a “n” value of 0.8. These results support our contention that milk can protect sperm by preventing the BSP proteins’ binding to the sperm membrane attributable to a protein : protein interaction, while egg yolk sperm protection is attributable to a protein : lipoprotein interaction. Second, our studies showed that the homologous BSP proteins found in the boar, stallion and ram seminal plasma can bind the milk proteins. These results indicate that the mechanism of sperm protection by milk in these species should be similar to the one in bovine species. Third, we characterized the interaction between bovine BSP1 protein and LDL from hen’s egg yolk. The binding was characterized by a Ka of 3.4 ± 0.4 × 10^6 M-1 and a « n » value of 104 BSP1 per LDL particle. Our results indicated that there is difference between the mechanism of sperm protection by milk and egg yolk. We believe that the results presented in this thesis may help to create new extenders free of animal product for mammal sperm preservation in liquid or frozen state.