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Un groupe d'études épidémiologiques fournit une bonne preuve de la relation inverse associé à la consommation de fruits et légumes et les maladies chroniques important comme maladies cardiovasculaires et certains types de cancers. Après les longues années d'études sur phytomacronutrients, le rôle de phytomicronutrients tels que les polyphénols est désormais très étudiée et appréciée dans le contrôle de ces maladies dégénératives. La présente étude combine les études d'extraction, de synthèse et d'analyse sur les principaux polyphénols des fruits d'agrumes, FLAVANONES. Connaissance de nutritionnels et de santé a augmenté la production d'agrumes en provenance des dernières décennies. Ces productions plus générer des bye-produits. Pour leur utilisation alternative à des antioxydants extraits riches, l'extraction assistée par ultrasons (UAE) des polyphénols en particulier flavanones de l'orange (Citrus sinensis L.) par son peau en utilisant l'éthanol comme solvant de qualité alimentaire a été prouvé son efficacité en comparaison avec la méthode conventionnelle . Un plan composite central (CCD) a révélé que l'approche des conditions optimisées pour UAE ont une température de 40 ° C, une puissance de 150W sonication et un 4:1 (v / v) d'éthanol: ratio de l'eau. En outre, l'activité antioxydante déterminée par les tests DPPH et ORAC a confirmé la pertinence des UAE pour la préparation d'extraits de plantes riches en antioxydants.Les glucuronides de flavanone sont les principaux métabolites phénoliques détectés dans le plasma humain après la consommation d'agrumes. Jusqu'à maintenant, toutes les études sur les cellules liées au cancer ou les maladies cardiovasculaires ont été réalisées soit sur les aglycones ou sur leurs glycosides. Par conséquent, il ya grand besoin de glucuronides flavanone pure pour démontrer le potentiel réel de flavanones dans la prévention de ces maladies. Dans ce travail, glucuronides de naringénine (4'- et 7-O-β-D-glucuronides) et de hespérétine (3'- et 7-O-β-D-glucuronides), les aglycones flavanone majeur dans le pamplemousse et d'orange, respectivement, ont été synthétisés chimiquement par une protection et la déprotection sélective des groupements d'acide glucuronique et de flavanone. La caractérisation structurale complète de composés purifiés a été réalisée par résonance magnétique nucléaire et spectrométrie de masse.L'affinité des quatre glucuronides pour l'albumine sérum d’humaine (HSA) a été testée par leur capacité à éteindre la fluorescence intrinsèque de HSA (Trp, seul résidu de sous-domaine IIA). Leurs constantes de fixation (K) ont été estimées de l'ordre de 30 à 60 × 103 M-1 et comparées à celles de l'aglycones (70 à 90 × 103 M-1). Les enquêtes de la liaison compétitive ou non compétitive de la glucuronides dans la présence de sondes fluorescentes (sarcosine dansyl) nous a permis d'obtenir un aperçu dans les sites de liaison. L'étude a également été étendue aux chalcones hespérétine et naringénine (synthétisés en utilisant des conditions alcalines optimisée), qui sont les précurseurs de biosynthèse des flavanones / A bunch of epidemiological studies provides good evidence on the inverse relationship associated with the consumption of fruits and vegetables and the chronic diseases importantly cardiovascular diseases and some types of cancers. After the long years of study on phytomacronutrients, the role of phytomicronutrients such as polyphenols is now highly studied and appreciated in the control of such degenerative diseases. The present study combines the extraction, synthetic and analytical studies on the major polyphenols of citrus fruits, FLAVANONES.Awareness of nutritional and health facts has increased the production of citrus fruits from last few decades. These higher productions generate higher by-products. For their alternative utilisation to have antioxidants rich extracts, the ultrasound-assisted extraction (UAE) of polyphenols especially flavanones from orange (Citrus sinensis L.) peel by using ethanol as afood grade solvent has been proved its efficiency when compared with the conventional method. A central composite design (CCD) approach revealed that the optimized conditions for UAE were a temperature of 40°C, a sonication power of 150W and a 4:1 (v/v) ethanol:water ratio. Furthermore, the antioxidant activity determined by the DPPH and ORAC tests confirmed the suitability of UAE for the preparation of antioxidant-rich plant extracts. Flavanone glucuronides are the major phenolic metabolites detected in human plasma after consumption of citrus fruits. Up to now all cell studies related to cancer or cardiovascular diseases were conducted either on the aglycones or on their glycosides. Hence, there is great need of pure flavanone glucuronides to demonstrate the real potential of flavanones in the prevention of these diseases. In this work, glucuronides of naringenin (4′- and 7-O-β-D-glucuronides) and hesperetin (3′- and 7-O-β-D-glucuronides), the major flavanone aglycones in grapefruit and orange respectively, have been chemically synthesized by selective protection and deprotection of flavanone and glucuronic acid moieties. The complete structural characterisation of purified compounds were realised by nuclear magnetic resonance and mass spectrometry.The affinity of the four glucuronides for human serum albumin (HSA) was tested via their ability to quench the intrinsic fluorescence of HSA (single Trp residue in sub-domain IIA). Their binding constants (K) were estimated in the range of 30 – 60 × 103 M-1 and compared with those of the aglycones (70 – 90 × 103 M-1). Investigations of competitive or noncompetitive binding of the glucuronides in the presence of fluorescent probes (dansyl sarcosine) allowed us to get some insight in the binding sites. The study was also extended to the hesperetin and naringenin chalcones (synthesised using optimized alkaline conditions), which are the biosynthetic precursors of flavanones

L'insuffisance rénale chronique (IRC) est une affection caractérisée par une perte progressive de la fonction rénale. L’IRC est associée à l'accumulation de diverses toxines urémiques. Les toxines urémiques ou solutés de rétention de l'urémie sont des composés qui s'accumulent chez les patients atteints d'IRC en raison d'un défaut de clairance rénale et qui exercent des effets biologiques délétères. Les hémodialyses éliminent mal les toxines urémiques liées aux protéines (PBUT), en raison de leur liaison aux protéines plasmatiques, en particulier la sérumalbumine humaine. En conséquence, les toxines urémiques liées aux protéines s'accumulent chez les patients atteints d'IRC et leur concentration ne peut que difficilement être diminuée chez les patients atteints d'insuffisance rénale terminale (IRT). Mes travaux sont principalement centrés sur les toxines urémiques, en particulier les toxines urémiques liées aux protéines, comme l’indoxyl-sulfate (IS), l'acide phénylacétique (PAA) et le p-crésyl-glucuronide (p-CG); et la zinc-alpha2-glycoprotéine (ZAG) qui est une « middle molécule ». Nous avons étudié le rôle de l'IS dans le développement de la résistance à l'insuline et d'autres troubles métaboliques associés à l'IRC, ainsi que ses effets sur l'inflammation et le stress oxydant. Nous avons exploré les propriétés de liaison du PAA et du p-CG à la sérumalbumine, qui est la plus abondante protéine dans le plasma humain. Enfin, nous avons essayé de développer une nouvelle stratégie d'élimination des PBUT, à l’aide de déplaceurs/compétiteurs chimique. / Chronic kidney disease (CKD) is a condition characterized by progressive loss of kidney function. CKD is associated with the accumulation of various uremic toxins. Uremic toxins or uremic retention solutes are compounds that accumulate in patients with CKD due to impaired renal clearance and exert deleterious biological effects. Protein-bound uremic toxins (PBUT) is poorly removed by hemodialysis because of its binding to plasma proteins, particularly human serum albumin. As a result, protein-bound uremic toxins accumulate in patients with CKD and their concentration can hardly be reduced in patients with end-stage renal disease (ESRD). My work focuses mainly on uremic toxins, particularly protein-bound uremic toxins such as indoxyl-sulfate (IS), phenylacetic acid (PAA) and p-cresyl-glucuronide (p-CG); and zinc-alpha2-glycoprotein (ZAG) which is a "middle molecule". We investigated the role of IS in the development of insulin resistance and other metabolic disorders associated with CKD, as well as its effects on inflammation and oxidative stress. We have investigated the binding properties of PAA and p-CG to serum albumin, which is the most abundant protein in human plasma. Finally, we tried to develop a new strategy to eliminate PBUTs, using chemical displacers / competitors.

<p>This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium <i>Finegoldia magna</i>. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule.</p><p>Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.</p>

In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering. Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface ofStreptococcus. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix. Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue. The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region. In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein. <b>Keywords:</b>binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.

This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule. Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.

Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process. The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available. In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed. The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. / QC 20100705

<p>In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering.</p><p>Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface of<i>Streptococcus</i>. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix.</p><p>Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue.</p><p>The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region.</p><p>In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein.</p><p><b>Keywords:</b>binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.</p>

A new Laser Induced Fluorescence Capillary Zone Electrophoresis (LIF-CZE) bioassay to detect and study the catalytic activity of the sulfur assimilating enzyme commonly found in E. coli species; alkanesulfonate monooxygenase (EC 1.14.14.5) is described for the first time. This technique enables the possibility for direct injection onto a capillary for detection without the need for pre-concentration of sample and with minimal sample preparative steps prior to analysis. In this bioassay, a group of Fischer based cyanine dyes and two Oxazine (Nile red) derivatives were designed for further optimization as key Vis-NIR fluorescent substrate. In developing this technique, the test dyes were first assessed for their photophysical properties, based on four criteria; (1) photostable (2) solvatochromism (3) binding affinity towards both the monooxygenase active site and serum albumin and (4) chemical stability in strong electric field strength. Applying key dye characterization procedures including; molar absorptivity determination, quantum yield determination, photostability, solvatochromism and protein interaction studies it was determined that the Fischer indolium cyanine dyes were most suitable for the method development. The data revealed that under the test conditions, reduced flavin, the oxidative monooxygenase catalytically specifically converts the alkylsulfonate substituted cyanine dyes to the corresponding aldehyde. This new bioassay has proven to be quick, portable, sensitive, reliable and the exhibit the possibility of ‘on-the-spot’ detection; advantages not readily realized with other commonly applied techniques such as PCR, SPR, ELISA and GC used to study bacterial sulfur assimilation processes. In addition, recent literature results proposed by other research groups developing similar techniques showed strong reliance on GC analyses. Those assays involve the use of low molecular weight straight chain non-emissive alkanesulfonate substrates. Once enzyme catalysis occurs the aldehyde is formed becomes rather volatile and requires complex and tedious headspace sampling for GC analyses. This feature limits the in vitro applicability and eliminated the possibility in vivo development. Our goal is to further develop, optimize and present this CZE based bioassay as a suitable alternative to the current trends in the field while creating a more robust and sensitive in vitro monooxygenase detection method with the possibilities of in vivo application.

The studies in the present thesis explored the feasibility of using nanocellulose-based filters in virus removal filtration of plasma-derived proteins.   In Paper I, two-step nanofiltration of commercially available human serum albumin (HSA) product, which was diluted to 10 g L-1 by phosphate buffer saline (PBS) and adjusted pH to 7.4, was performed to remove soluble protein aggregates and reduce filter fouling. The two-step filtration of HSA employed nanocellulose-based filters of varying thickness, i.e. 11 μm and 22 μm filters.  The removal of HSA aggregates during filtration through 11 μm pre-filters dramatically improves the flow properties of the 22 μm filter, enabling high protein throughput and high virus clearance. A distribution of pore sizes between 50 nm and 80 nm, which is present in the 11 μm filter and is absent in the 22 μm filter, plays a crucial part in removing the HSA aggregates. With respect to virus filtration, 1 bar constant trans-membrane pressure filtration shows poor removal ability of ΦX174 bacteriophage (28 nm), i.e., log10 reduction value (LRV) ≤ 3.75, while that at 3 bar and 5 bar achieves LRV[MOU1] [LW2]  &gt; 5 model virus clearance and overall rapid filtration. Removal of protein aggregates during bioprocessing of HSA products is key to improving the filtration flux, which makes it possible to apply virus removal filtration for HSA to ensure its virus safety.   In Paper II, nanofiltration of human plasma-derived intravenous immuno-globulin (IVIG) intermediate (11.26 g L-1, pH 4.9) was carried out to demonstrate high product recovery and high model virus clearance. Virus removal filtration of industrial-grade human IVIG was achieved using 33μm filters at both low (60 Lm-2) and high (288 Lm-2) volumetric load. No changes in IVIG structure were detected and high product recovery was recorded. High virus clearance (LRV ≥ 5-6) was achieved for the small-size model viruses (ΦX174 and MS2 bacteriophages) during the load volume of 60 Lm-2. Side-by-side comparisons with commercial virus removal filters suggest that the nanocellulose-based filter paper presents great potential for industrial bioprocessing of plasma-derived IVIG.   In Paper III, process analytical technology (PAT) approach was employed to identify the critical filter parameters, e.g. thickness, basis weight, pore size, and flux, affecting model virus removal efficiency using filters produced by different hot presses.  The quality parameters were analyzed with ANOVA and Shewhart charts. Compared with other studied parameters, the hydraulic flux appears as the most relevant final product quality attribute of the nanocellulose-based filter paper to reflect the virus removal efficiency. In particular, a 15% higher flux may be associated with a 0.5-1.0 log10 reduced virus clearance (p=0.007). The results are highlight the importance of continued systematic studies in quality assurance using statistical process control tools  [MOU1]Define LRV  [LW2]Defined in the line above