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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Contribution to the study of uremic toxins in the context of chronic kidney disease / Contribution à l'étude des urémique toxines dans le contexte de la maladie rénale chronique

Yi, Dan 28 June 2018 (has links)
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.
22

Protein engineering to explore and improve affinity ligands

Linhult, Martin January 2003 (has links)
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.
23

Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery

Benkestock, Kurt January 2008 (has links)
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
24

Protein engineering to explore and improve affinity ligands

Linhult, Martin January 2003 (has links)
<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>
25

Virus retentive filter paper for processing of plasma-derived proteins

Wu, Lulu January 2020 (has links)
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
26

Production and Evaluation of a Bombesin Analogue Conjugated to the Albumin-Binding Domain and DOTA for Prostate Cancer Radiotherapy / Produktion och utvärdering av en bombesinanalog konjugerad till en albuminbindande domän och DOTA för radioterapi i prostatacancer

Landmark, Fredrika January 2021 (has links)
Prostate cancer is one of the most common types of cancer worldwide and claims hundreds of thousands of lives annually. Currently the most common treatment for prostate cancer is external beam radiotherapy, however, this treatment comes with serious side effects since it lacks selectivity for the cancer cells. Therefore, less harmful treatments are needed and sought for, such as targeted treatments that are intended to only affect cancer cells and thereby reduce the side effects. Targeted treatments require a target that differentiates the cancer cells from healthy cells. A promising target candidate that has gained attention in recent years is gastrin releasing peptide receptor (GRPR), a protein commonly overexpressed in prostate cancer cells. Furthermore, a targeting molecule intended to bind to the target is also required. For this purpose, the bombesin analogue RM26, a high affinity GRPR binder, shows promise. Previous studies have led to the development of RM26-conjugates for the purpose of targeted prostate cancer radiotherapy. In these conjugates RM26 has been linked to a DOTA-chelator for radiolabeling, and an albumin binding domain (ABD) to prolong the conjugate’s half-life in vivo by binding to human serum albumin (HSA). The idea is that the RM26-conjugate will bind to both HSA in the blood and to GRPR on the prostate cancer cells and eliminate the cancer cells with the radiation from the radionuclide attached to the DOTA-chelator. Although these earlier studied conjugates have been very promising some improvements of certain aspects need to be achieved, mainly to improve the biodistribution with retained GRPR binding affinity. Therefor the purpose of this project was to produce three new versions of previous RM26- conjugates and evaluate if they are suitable for further prostate cancer therapy studies. The three RM26-conjugates were developed with primarily recombinant expression in E. coli cells and solid phase peptide synthesis (SPPS). The characterization phase in this project was carried out with mainly five different methods: matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization- mass spectrometry (ESI-MS), circular dichroism (CD), surface plasmon resonance (SPR) and flow cytometry. The results showed that all three new RM26-conjugates were possible to produce and yielded final products corresponding to the expected molecular weights. Furthermore, the results indicate that all three RM26-conjuagtes are stable and maintain their structural properties under in vivo- temperatures and that they have high binding affinity for HSA. Further studies need to be conducted before drawing any certain conclusions regarding GRPR binding affinity. / Prostatacancer är en av de mest vanligt förekommande cancertyperna världen över och skördar hundratusentals liv årligen. I nuläget är extern strålbehandling det vanligaste terapialternativet mot prostatacancer, men denna behandling kommer med allvarliga biverkningar på grund av att den saknar selektivitet för cancerceller. Därför finns ett stort behov av mindre skadliga behandlingsformer, såsom riktade behandlingar som endast är avsedda att påverka cancerceller och därigenom minska biverkningarna. Riktade behandlingar kräver ett mål som skiljer cancercellerna från friska celler. En lovande målkandidat som har uppmärksammats de senaste åren är gastrinfrisättande peptidreceptor (GRPR), ett protein som vanligtvis överuttrycks i prostatacancerceller. I tillägg så krävs också en målsökande molekyl avsedd att binda till målet. För detta ändamål visar bombesinanalogen RM26, en GRPR-bindare med hög affinitet, sig vara lovande. Tidigare studier har utvecklat RM26-konjugat för målinriktad strålbehandling av prostatacancer. Dessa konjugat består av en RM26-peptid bunden till en DOTA-kelator för radioinmärkning och en albuminbindande domän (ABD) för att förlänga konjugatens halveringstid in vivo genom att binda till humant serumalbumin (HSA). Syftet med RM26- konjugaten är att de ska binda till både HSA i blodet och GRPR på prostatacancercellerna, och därmed eliminera cancercellerna med strålning från den radioinmärkta DOTA-kelatorn. Även om de tidigare RM26-konjugaten har varit mycket lovande krävs det att vissa förbättringar av några aspekter uppnås, främst affiniteten för GRPR. Syftet med detta projekt var därför att producera tre nya versioner av tidigare RM26-konjugat och utvärdera ifall de uppvisar tillfredsställande egenskaper. De tre RM26-konjugaten utvecklades primärt rekombinant i E. coli-celler och fastfas- peptidsyntes (SPPS). Karaktäriseringsfasen i detta projekt genomfördes med huvudsakligen fem olika metoder: MALDI-TOF-MS, elektrosprejjonisering-masspektrometri (ESI-MS), cirkulär dikroism (CD), ytplasmonresonans (SPR) och flödescytometri. Resultaten visade att alla tre nya RM26-konjugat var möjliga att producera och gav slutprodukter motsvarande de förväntade molekylvikterna. Vidare indikerar resultaten att alla tre RM26-konjugat är stabila och bibehåller sina strukturella egenskaper under in vivo-temperaturer och att de har hög affinitet för HSA. Ytterligare studier bör utföras innan säkrare slutsatser kan dras angående GRPR-bindningsaffinitet.
27

Optimization of immunotherapeutic relevant ABD-derived affinity proteins for prolonged serum half-life

Bergström, Ebba January 2022 (has links)
Marknaden för proteinbaserade läkemedel, de så kallade biologiska läkemedlen, är idag en industri som omsätter miljarder. Ett vanligt sätt att utveckla dessa läkemedel på är med hjälp av monoklonala antikroppar då de kan binda till sitt mål med hög specificitet. Däremot begränsas denna teknik av en lång och dyr produktion som dessutom kräver däggdjursbaserade uttrycksystem. En alternativ teknik till de monoklonala antikropparna är att använda små proteiner som enkelt kan produceras i bakterier till en låg kostnad. Dock begränsas denna metod av de små proteinernas korta cirkuleringstid i blodet. I ett tidigare projekt, har ett litet protein vid namnet ABDderived affinity ProTein (ADAPT) på cirka 7 kDa, utvecklats för att kunna binda till både humant serumalbumin (HSA) för att förlänga cirkulationstiden i blodet och Interleukin 17c (IL17c) som är ett pro-inflammatorisk cytokin. Studien visade dock att ADAPT proteinet inte samtidigt kunde binda till de båda molekylerna tillräckligt effektivt. Syftet med denna uppsats är därför att undersöka om det nämnda proteinet kan optimeras genom så kallad multimering och/eller manipulering av bindningssätet för HSA i syfte att åstadkomma en effektiv och mer långvarig cirkulationstid i blodet samtidigt som det binder sig till sitt mål, IL17c. Tio nya versioner av ADAPT proteinet har utvecklats genom att klona och transformera proteiner till en högt producerande Escherichia coli (E. coli) stam. Proteinerna har sedan producerats och renats fram. Det kunde observeras att proteinerna hade den önskade renheten för att kunna karaktäriseras. Vidare var det möjligt att se att proteinerna hade sin önskade molekylvikt och erhöll sin förväntade struktur som en alfahelix. Proteinernas smältpunkter hade förbättrats eller var liknande jämfört med det ursprungliga proteinet. Dessutom kunde alla proteiner återgå till sin ursprungliga struktur efter upphettning. Utvärderingen av proteinernas bindningskapacitet, med original proteinet som referens, visade på en ökad affinitet till sitt mål, IL17c, för två dimerer och trimeren samt en jämförbar affinitet för två av monomererna med ett manipulerat bindingssäte till HSA. Interaktion till HSA var jämförbar med den ursprungliga ADAPT molekylen för alla nya varianter förutom monomererna med ett manipulerat bindingssäte och dimeren med två manipulerat bindingssäten till HSA. Evaluering av de nya proteinernas kapacitet att binda samtidigt till HSA och IL17c visade att det var gynnsamt med en dimereiserad molekyl då det skapade en distans mellan molekylerna och dess bindningssäten. Vidare kunde det också visas att ordningen som molekylerna interagerade med varandra påverkade proteinernas simultana bindning. / The market for protein-based drugs, or the so-called biopharmaceuticals, is a multibillion-dollar industry today. In the development of protein-based drugs it is common to use monoclonal antibodies (mAbs) due to their ability to bind to its target with high specificity. However, therapeutical development of mAbs is limited by its long and expensive production in mammalian expression system. An alternative to mAbs are the so-called alternative scaffolds which are small proteins that can be produced in bacteria at lower costs. Although a drawback with the latter proteins is their short serum half-life. A small scaffold protein, ABD-Derived Affinity ProTein (ADAPT) of approximate 7 kDa was earlier engineered to obtain bispecific affinity, to Human Serum Albumin (HSA), to extend its half-life, as well as to the pro-inflammatory cytokine, Interleukin 17c (IL17c). Unfortunately, it was shown that the simultaneous binding was not efficient enough for its desired purpose. The aim with this project was therefore to investigate if the previous mentioned binder could be optimized by multimerization and/or manipulation of the HSA binding site for an efficient half-life extension. By generating ten new designs of the ADAPT variants, it was observed that the new variants had stable alpha helical structures and an improved or similar melting temperature as the original variant. The evaluation of the target binding displayed an improved affinity to the target, IL17c, for two of the dimeric versions as well as for the trimer and a comparable affinity for two of the monomers with a manipulated HAS binding site. The interaction to HSA was comparable to the original ADAPT for all binders except from the monomers with impaired HSA binding and the dimer with two impaired HSA binding sites. The evaluation of the simultaneous binding showed that it was favored by dimerization when a distance between the two molecule and their binding surfaces was added. Moreover, it could also be seen that the order of binding events had an impact on the simultaneous binding.
28

Effets dynamiques et conformationnels sur le rôle de transport des albumines sériques / Dynamics and conformational effects on the transport role of serum albumins

Paris, Guillaume 05 June 2014 (has links)
L’albumine sérique humaine (HSA) est une protéine connue pour ses propriétés de transport exceptionnelles et son contenu élevé en ponts disulfure. L’étude de sa dynamique conformationnelle représente un défi important dans la compréhension de ses fonctions physiologiques. Le but de notre travail a été d’étudier cette dynamique conformationnelle et de comprendre le rôle des ponts disulfure dans le maintien de la structure native de la protéine. Notre analyse est basée sur des simulations de dynamique moléculaire couplées à des analyses par composantes principales. Outre la validation de la méthode de simulation les résultats fournissent de nouveaux éclairages sur les principaux effets de la réduction des ponts disulfure dans les albumines sériques. Les processus de dépliement/repliement protéique ont été détaillés. La prédiction de la structure réduite d’équilibre a également fait l’objet d’une attention particulière. Une étude détaillée de la dynamique conformationnelle globale de la protéine ainsi que celle des deux sites principaux de complexation a été effectuée. D’éventuels effets allostériques entre ces deux sites ont été recherchés. Les résultats théoriques obtenus ont été discutés avec les données expérimentales disponibles / Human serum albumin (HSA) is a protein known for its exceptional transport properties and its high content of disulfide bridges. The study of the conformational dynamics represents a major challenge in the comprehension of its physiological functions. The aim of our work was to study the conformational dynamics and to understand the roleof disulfide bonds in the stability of the native protein structure. Our analysis is based on simulations of molecular dynamics coupled with principal component analysis. Beyond the validation of the simulation method, the results provide new insights on the main effects of the disulfide bonds reduction in serum albumins. Protein unfolding/refolding processes were detailed. A special attention is paid to the prediction of the reduced structure at the equilibrium. A detailed study of the global protein conformational dynamics as well as the two main binding sites were performed. Possible allosteric effects between these two sites were researched. The theoretical results have been discussed with the available experimental data
29

Bioanalytical Applications of Intramolecular H-Complexes of Near Infrared Bis(Heptamethine Cyanine) Dyes

Kim, Junseok 15 July 2008 (has links)
This dissertation describes the advantages and feasibility of newly synthesized near-infrared (NIR) bis-heptamethine cyanine (BHmC) dyes for non-covalent labeling schemes. The NIR BHmCs were synthesized for biomolecule assay. The advantages of NIR BHmCs for biomolecule labeling and the instrumental advantages of the near-infrared region are also demonstrated. Chapter 1 introduces the theory and applications of dye chemistry. For bioanalysis, this chapter presents covalent and non-covalent labeling. The covalent labeling depends on the functionality of amino acids and the non-covalent labeling relies on the binding site of a protein. Due to the complicated binding process in non-covalent labeling, this chapter also discusses the binding equilibria in spectroscopic and chromatographic analyses. Chapter 2 and 3 evaluate the novel BHmCs for non-covalent labeling with human serum albumin (HSA) and report the influence of micro-environment on BHmCs. The interesting character of BHmCs in aqueous solutions is that the dyes exhibit non- or low-fluorescence compared to their monomer counterpart, RK780. It is due to their H-type closed clam-shell form in the solutions. The addition of HSA or organic solvents opens up the clam-shell form and enhances fluorescence. The binding equilibria are also examed. Chapter 4 provides a brief introduction that summaries the use of capillary electrophoresis (CE), and offers a detailed instrumentation that discusses the importance and advantage of a detector in NIR region for CE separation. Chapter 5 focuses on the use of NIR cyanine dyes with capillary electrcophoresis with near-infrared laser induce fluorescence (CE-NIR-LIF) detection. The NIR dyes with different functional groups show that RK780 is a suitable NIR dye for HSA labeling. The use of BHmCs with CE-NIR-LIF reduces signal noises that are commonly caused by the interaction between NIR cyanine dyes and negatively charged capillary wall. In addition, bovine carbonic anhydrase II (BCA II) is applied to study the influence of hydrophobicity on non-covalent labeling. Finally, chapter 6 presents the conformational dependency of BHmCs on the mobility in capillary and evaluates the further possibility of BHmCs for small molecule detection. Acridine orange (AO) is used as a sample and it breaks up the aggregate and enhances fluorescence. The inserted AO into BHmC changes the mobility in capillary, owing to the conformational changes by AO.
30

Estudo de interação dos flavonóides Isovitexina e 2-Fenilcromona com a Albumina do Soro Humano: abordagem experimental e computacional / Interaction study of flavonoids Isovitexin and 2-Phenylcromone with Human Serum Albumin: experimental and computational approach

Caruso, Ícaro Putinhon [UNESP] 26 August 2016 (has links)
Submitted by ÍCARO PUTINHON CARUSO null (ykrocaruso@hotmail.com) on 2016-09-19T16:20:38Z No. of bitstreams: 1 tese_doutorado_icaro_vf.pdf: 17027642 bytes, checksum: 71d2f869670d044aace5f2ab6326b657 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-09-22T14:26:36Z (GMT) No. of bitstreams: 1 caruso_ip_dr_sjrp.pdf: 17027642 bytes, checksum: 71d2f869670d044aace5f2ab6326b657 (MD5) / Made available in DSpace on 2016-09-22T14:26:36Z (GMT). No. of bitstreams: 1 caruso_ip_dr_sjrp.pdf: 17027642 bytes, checksum: 71d2f869670d044aace5f2ab6326b657 (MD5) Previous issue date: 2016-08-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os flavonóides fazem parte de uma ampla classe de compostos polifenólicos os quais ocorrem naturalmente nas plantas e podem ser encontrados nas sementes, caules, folhas, flores e/ou frutos. Estudos recentes indicam que esses compostos polifenólicos podem apresentar uma variedade significativa de atividades biológicas benéficas para a saúde humana, como por exemplo: antioxidante, anti-inflamatória, antibacteriana, antiviral e anticancerígena. A Albumina do Soro Humano (HSA) é a principal proteína extracelular presente no plasma sanguíneo. A função central dessa proteína é transportar e distribuir ligantes endógenos e exógenos para diferentes alvos moleculares no corpo humano. Por tais aspectos, torna-se importante o desenvolvimento de estudos que caracterizam a interação dos flavonóides com a proteína transportadora HSA. Este trabalho investiga a interação dos flavonóides Isovitexina (ISO) e 2-Fenilcromona (2PHE) com a HSA, utilizando técnicas experimentais de espectroscopia de fluorescência, absorbância UV-Vis, dicroísmo circular (CD) e infravermelho com transformada de Fourier (FT-IR); juntamente com ferramentas computacionais de cálculo {\it{ab initio}}, dinâmica molecular e modelagem molecular. A integração dessas abordagens experimentais e computacionais possibilita caracterizar a formação dos complexos HSA-flavonóides, determinando aspectos físico-químicos como: constantes de afinidade, parâmetros termodinâmicos, número de sítios de ligação, perfil de cooperatividade e resíduos de aminoácidos responsáveis pelas interações proteína-flavonóides (hidrofóbicas e eletrostáticas). / Flavonoids belong to a large class of polyphenolic compounds which occur naturally in plants and can be found seeds, stems, leaves, flowers and/or fruits. Recent studies indicate that these polyphenolic compounds can present a significant variety of beneficial biological activities on human health, such as: antioxidant, anti-inflammatory, antibacterial, antiviral, and anticancer. Human Serum Ambumin (HSA) is the main extracellular protein presents in blood plasma. The core function of this protein is to carry and distribute endogenous and exogenous ligands to different molecular targets in the human body. For these aspects, it is important to develop studies that characterize the interaction of the flavonoids with the carrier protein HSA. This work investigates the interaction of the flavonoids Isovitexin (ISO) and 2-Phenylchromone (2PHE) with the HSA, using experimental techniques of fluorescence, UV-Vis absorbance, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy; along with computational tools of ab initio calculation, molecular dynamics, and molecular modeling. The integration of these experimental and computational approaches allows to characterize the formation of the HSA-flavonoids complexes, determining physicochemical aspects, sucha as: affinity constants, thermodynamic parameters, number of binding sites, cooperativity profile and aminoacid residues responsable for the protein-flavonoids interactions (hydrophobic and electrostatic).

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