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1
Alias, Nadiawati. "Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection." Thesis, University of St Andrews, 2014. http://hdl.handle.net/10023/4479.
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In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van't Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases.
2
Hopkins, Adam P. "Molecular and biochemical characterisation of SiaP as a sialic acid binding protein component of a TRAP transporter of sialic acid." Thesis, University of York, 2010. http://etheses.whiterose.ac.uk/1030/.
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Sialic acid utilisation plays an important role in the growth and persistence of the obligate human mucosal pathogen Haemophilus influenzae, which causes respiratory tract infections, septicaemia and meningitis. Like many other bacteria, H. influenzae can use host-derived sialic acids as carbon, nitrogen and energy sources, but also as a terminal modification on the LPS to better evade the human immune system. H. influenzae takes up exogenous sialic acid via a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM. This possesses an extracytoplasmic substrate binding protein (SBP), SiaP, which binds the substrate in the periplasm and delivers it to the specific membrane permease, SiaQM. SiaP contains two globular domains, which close around the substrate upon binding. Here, the mechanism of sialic acid binding by SiaP is investigated using site-directed mutagenesis of residues in the ligand binding site and analogues of sialic acid. These, and several mutations on the surface of SiaP, were investigated for their effect on transport by SiaPQM in vitro, using SiaQM reconstituted into proteoliposomes, and in vivo, using expression of siaPQM in an E. coli strain lacking its native sialic acid transporter, NanT. It is demonstrated that stabilisation of the carboxylate group of sialic acid by the totally conserved Arginine-147 is important for high-affinity ligand binding, but is not essential for transport. Mutation of Aparagine-150 to Aspartate abolishes the function of the transporter without affecting ligand binding, suggesting the existence of a critical interaction between the components of the transporter. The catabolism of the sialic acid analogues was also examined in E. coli expressing different sialic acid transporters. This indicates that a wide variety of sialic acid analogues are potential carbon sources in many pathogenic bacteria.
3
Jiménez-Castells, Carmen 1982. "Capture and identification of carbohydrate-binding proteins by SPR and CREDEX-MS." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7237.
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Carbohydrate-binding proteins of non-immunological origin -lectins- have been recognized over the last decades as decisive players in numerous biological processes, ranging from cellcell communication, fertilization, pathogen-cell adhesion to metastasis. Consequently, there is an increasing interest in finding powerful and nanosized tools to screen for these molecules and to study their carbohydrate interactions in detail. Here, two complementary approaches are described to characterize lectin-carbohydrate interactions with high sensitivity, low sample consumption, and without the need for sample labelling: SPR and CREDEX-MS. In SPR, we have developed an approach where the sugar is immobilized onto a sensor surface through a tailor-made peptide module that allows (1) to capture the lectin, (2) to characterize the interaction through kinetic and thermodynamic parameters, and (3) to identify the interacted protein by mass spectrometry. In CREDEX-MS, based on proteolytic excision of proteincarbohydrate complexes and mass spectrometric analysis, the peptides comforming the carbohydrate binding domain are identified.Las lectinas (proteínas de origen no inmune capaces de reconocer azúcares) se han revelado en las últimas décadas como participantes cruciales en multitud de procesos biológicos, tales como la comunicación célula-célula, la fertilización, la adhesión del patógeno a la célula y la metástasis, entre muchos otros. Por lo tanto, existe un gran interés en el desarrollo de técnicas analíticas potentes para el estudio de las interacciones lectina-carbohidrato. En este trabajo, se describen dos aproximaciones complementarias mediante las cuales se pueden caracterizar las interacciones lectinas-azúcar con gran sensibilidad, poca utilización de muestra y sin la necesitad de ningún marcaje. En la técnica basada en resonancia de plasmón superficial (SPR), el azúcar es inmovilizado sobre una superficie a través de un módulo peptídico, lo cual permite (1) capturar la lectina, (2) caracterizar su interacción mediante parámetros cinéticos y termodinámicos y (3) identificar posteriormente la proteína mediante espectrometría de masas. Complementariamente, la técnica CREDEX-MS, basada en la excisión proteolítica del complejo proteína-azúcar y posterior análisis por espectrometría de masas, nos permite identificar los péptidos que forman parte del dominio de unión al azúcar.
4
Zhang, Mai. "Exploring the effect of sialic acid binding properties on Siglec function /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3190162.
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5
Freeman, Sylvie. "Studies on CD33 and sialoadhesin : sialic acid binding receptors of the haemopoietic system." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318544.
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6
Ciccotosto, Silvana. "The preparation and evaluation of N-acetylneuraminic acid derivatives as probes of sialic acid-recognizing proteins." Monash University, Dept. of Medicinal Chemistry, 2004. http://arrow.monash.edu.au/hdl/1959.1/9649.
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Yao, Yujing [Verfasser]. "Influence of sialic acid modification on HIV GP120 binding and Syncytia formation / Yujing Yao." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1176639129/34.
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8
Toro, Imre. "Crystal structures of two nucleic acid-binding proteins." Thesis, Open University, 2000. http://oro.open.ac.uk/58089/.
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The Crystal Structure of Sl Nuclease from Aspergillus oryzae S 1 nuclease from Aspergillus oryzae is a glycoprotein of 32 kDa molecular weight. The protein has two enzymatic activities: it is an endo-exonuclease with high specificity for single stranded nucleic acids, and it has an additional 3' -nucleotidase activity. S 1 nuclease is widely used in molecular biology as a single-strand specific nuclease due to its high stability and efficiency. It cleaves single-stranded regions of nucleic acids producing 5' -nucleotides without significant side-reactions. The crystal structure of S 1 nuclease has been determined to 1.7 A resolution by molecular replacement based on the known structure of PI nuclease from Penicillinum citrinum, which has 49 % sequence identity compared to S 1. The overall fold and the active site of S 1 nuclease is basically identical to that of PI nuclease, and also very similar to Phospholipase C from Bacillus cereus and alpha-toxin from Clostridium perfringens. The characteristic feature of this family of enzymes is a trinuclear zinc cluster in their active sites. A BLAST search in the sequence databases revealed several other protein sequences from bacteria, protozoa and plants possessing an approximately 30 % sequence identity compared to S 1 nuclease, but showing an almost complete conservation of structurally and functionally important residues. Soaking and co-crystallisation experiments with substrate analogues have been carried out in order to obtain an enzyme-substrate complex. These efforts have not resulted in the structure determination of any complexes under crystallisation conditions: no binding of substrate has been observed. Nevertheless, an enzyme mechanism has been proposed based on structural data of S 1 nuclease and nucleases with similar active sites. The Crystal Structure of an Sm-Related Protein from Archaeoglobus fulgidus In eukaryotes Sm and Sm-like proteins are the core components of the small nuclear ribonucleoprotein particles (snRNPs), which are involved in a variety of functions including rRNA processing, tRNA maturation and pre-mRNA processing. The Sm proteins are 70 to 120 amino acids long and share a common bi-partite signature sequence. The spliceosome, where the transesterification reaction of splicing occurs, is assembled by several snRNPs named after their constituting snRNA: U1, U2, U4, U5 and U6. An snRNA contains a short single stranded, uridine rich sequence motif, where the Sm proteins bind, but the three-dimensional arrangement of the Sm proteins and the mode of binding is unknown. In humans there are seven different canonical Sm proteins, which according to biochemical and electron microscopic studies seem to form a seven membered ring in vitro. Recently two crystal structures of human Sm protein dimers have been published. Interestingly Sm-related protein sequences have been found in the available genomic database of various Archaebacteria based on sequence homology. In contrast with eukaryotes only one or two Sm-related protein sequences have been identified in one organism. Their function is currently unknown, since analogous pre-mRNA splicing does not occur in Archaebacteria. Two Sm-related proteins of Archaeoglobus fulgidus have been cloned and expressed as fusion proteins. One of them called AF-Sm2 has been o crystallised utilising ammonium sulphate as precipitant and solved to 1.95 A resolution by SIRAS using a single mercury derivative. AF-Sm2 crystallises in a hexagonal space group (P6) and contains one molecule per asymmetric unit. The 77 residue long protein has a very similar fold compared to the solved human Sm protein structures: a short N-terminal a-helix followed by a five stranded, strongly bent, U-shaped ~-sheet resulting in a barrel-like overall fold. Six AF-Sm2 molecules form a ring in the crystal structure mediated by extensive hydrophobic and hydrogen-bonding interactions. Gel filtration experiments have indicated a pH dependence of oligomerisation in accordance with the crystallisation experiences. Currently the target of the Sm-related proteins of Archaeoglobus fulgidus and the stochiometry of oligomerisation in vivo is completely unknown.
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Shum, Bennett Oh Vic St Vincent's Clinical School UNSW. "Regulation of allergic asthma by fatty acid-binding proteins." Awarded by:University of New South Wales. St. Vincent's Clinical School, 2007. http://handle.unsw.edu.au/1959.4/27002.
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Fatty acid-binding proteins are small intracellular proteins with poorly defined functions in intracellular fatty acid transport. The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. Using Affymetrix microarrays, we found that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by airway epithelial cells. aP2 expression was markedly increased following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13, and downregulated by the Th1 cytokine IFN-gamma. Regulation of aP2 mRNA expression by Th2 cytokines was dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2 deficient mice in a model of allergic airway inflammation, and found that infiltration of leukocytes, especially eosinophils, into the airways was highly aP2 dependent. T cell priming and peritoneal allergy was unaffected by aP2 deficiency suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-haematopoietic cells, most likely airway epithelial cells, as the site of aP2 action in allergic airway inflammation. Expression of the pro-inflammatory cytokines MCP-1 and IL-6 was impaired in cytokine activated aP2 deficient airway epithelial cells, while levels of the anti-inflammatory arachidonic acid metabolite 15-HETE was increased, providing a mechanism for the reduced airway inflammation in aP2 deficient mice. In addition to the immune functions of aP2, we found that the related fatty acid-binding protein mal1 was also upregulated by IL-4/IL-13 in airway epithelial cells, and mal1 deficient mice were protected against airway eosinophilia. Significantly, in comparison to single aP2 deficiency, mice with combined aP2-mal1 deficiency had augmented protection against airway inflammation, and bone marrow chimera experiments demonstrated that aP2-mal1 deficiency affected both non-haematopoeitic and haematopoeitic cells. In T cell priming experiments, aP2-mal1 deficiency resulted in defective cytokine profiles in antigen recall responses, suggesting compromised sensitisation to antigen as one mechanism for aP2-mal1 action in airway inflammation. Together, our data therefore demonstrates the crucial roles of fatty acid-binding proteins in airway epithelium, T cell priming and airway inflammation, and provides a new link between fatty acid signalling and allergy.
10
Rowlinson, Marie-Claire. "The fatty acid and retinol binding proteins of nematodes." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289452.
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CRACCO, Laura. "Fatty acid-binding proteins as markers of brain injury." Doctoral thesis, Università degli Studi di Verona, 2009. http://hdl.handle.net/11562/337348.
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Le Fatty Acid-Binding Proteins (FABPS) sono proteine
intracellulari in grado di legare gli acidi grassi a
catena lunga con elevata affinità, regolandone il
trasporto all’interno delle cellule. Nell’uomo sono
state finora identificate 9 diverse isoforme, ciascuna
con un diverso profilo di espressione a livello di
tessuti. La proteina heart-type FABP (H-FABP) da anni è
considerata uno tra i più sensibili ed efficienti
marcatori di danno cerebrale e al miocardio, ma la sua
elevata espressione in differenti tessuti non la rende
un marker specifico di un particolare disordine.
Un’altra isoforma espressa nel cervello, chiamata BFABP
(brain-type FABP), potrebbe invece rappresentare
un marcatore specifico di danno cerebrale, essendo la
sua espressione limitata al tessuto nervoso.
Questo lavoro ha avuto come scopo la generazione di
anticorpi in grado di riconoscere la proteina B-FABP in
modo specifico e selettivo.
Il progetto si è articolato in due fasi. Nella prima
parte del lavoro la proteina ricombinante B-FABP è
stata espressa in E. coli e purificata mediante due
successive cromatografie. Essa è stata quindi inoculata
in conigli New Zealand e in topi BalbC, ottenendo dopo
diverso tempo 2 antisieri e 8 diverse cellule ibridoma.
Gli anticorpi presenti nel siero e nel surnatante delle
cellule ibridoma in coltura hanno evidenziato diversi
gradi di affinità nei confronti delle due isoforme, BFABP
e H-FABP; solamente una cellula ibridoma è stata
in grado di produrre un anticorpo specifico e selettivo
per la B-FABP ma purtroppo i successivi passaggi di
subclonaggio della cellula hanno evidenziato una
progressiva diminuzione dell’attività anticorpale, non
permettendo l’utilizzo di questo reagente per
l’individuazione della proteina stessa in campioni
biologici.
Nella seconda parte del lavoro si è quindi cercato di
analizzare e superare le problematiche riscontrate
nella prima fase mediante un nuovo approccio che, con
l’ausilio di software specifici, ha mirato
all’identificazione di due regioni potenzialmente
antigeniche della proteina stessa. Due peptidi
sintetici, di sequenza identica alle regioni
identificate, sono stati inoculati in conigli New
Zealand con l’ottenimento di due anticorpi policlonali,
pAb 2979/2980 e pAb 2981/2982. Questi anticorpi si sono
rivelati specifici e selettivi verso l’isoforma B-FABP
e non hanno evidenziato reazioni di cross-reazione
verso H-FABP.
Si può quindi affermare come l’approccio sperimentale
utilizzato abbia contribuito al raggiungimento
dell’obiettivo, la produzione di un anticorpo specifico
per B-FABP umana. Questo risultato suggerisce possibili
scenari nell’utilizzo futuro degli anticorpi, in
tecniche ELISA e western blot, per valutare se la
proteina B-FABP possa essere considerata un marcatore
ideale, specifico di danno neuronale, in disordini a
diversa eziologia (ischemica, infettiva o
degenerativa).Fatty acid–binding proteins (FABPs) are abundant intracellular proteins that bind long-chain fatty acids with high affinity. 9 different FABPs, with tissuespecific distribution, have been identified so far. The primary role of all the FABP family members is regulation of fatty acid uptake and intracellular transport. Heart-type FABP (H-FABP) is considered one of the most sensible and efficient markers of heart and brain damage, but to date the cross-reactivity limits its specific use. Brain-type FABP (B-FABP) might be considered as a good marker of brain damage, being expressed only in the nervous tissue. The aim of this work was to generate a diagnostic reagent specific for human B-FABP, not cross-reacting with H-FABP. The work has been articulated in two parts: in the first one, recombinant protein B-FABP was expressed in E. coli cells and purified by two subsequent chromatographies. The protein was then utilized as immunogen in New Zealand rabbits and BalbC mice, obtaining two antisera and 8 different hybridoma cells. The antibodies in the rabbit serum and in culture supernatant of hybridoma cells displayed different degrees of affinity for B-FABP and H-FABP; only one hybridoma cell was able to produce antibodies specific and selective for brain-type FABP, but the antibody low-titer level and the activity decrease after subsequent subcloning steps affected its application on B-FABP detection in biological fluids. To overcome the limits encountered in the first part of work, a computer-assisted approach on B-FABP was employed in order to identify the potentially most antigenic regions of the protein. Two synthetic peptides were produced with the selected sequences and inoculated in rabbits. Two polyclonal antibodies were obtained, pAb 2979/2980 and pAb 2981/2982. They showed specific and selective reactivity for human B-FABP, without crossreactions with H-FABP. Taken together, our experimental approach was effective for the generation of specific α-B-FABP antibodies; these results suggest the antibodies obtained could be utilized in ELISA and immunoblot analyses in order to value B-FABP as marker of neurological disorders with ischemic, infective and degenerative etiology.
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Abu-Izneid, Tareq, and n/a. "The Synthesis and Evaluation of Functionalised Carbohydrates as Probes of Tumour Metastasis." Griffith University. Institute for Glycomics, 2005. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20061019.111424.
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Sialyltransferases, CMP-sialic acid synthetases and CMP-sialic acid transport proteins play a crucial role in the construction of cell surface glycoconjugates. These proteins also have a pivotal role to play in a number of diseases, including cancer. The sialyltransferase enzymes are responsible for transfering sialic acids from the donor substrate (CMP-sialic acid) to growing cell surface glycoconjugate chains within the Golgi apparatus. The CMP-sialic acid synthetase enzymes are responsible for the synthesis of the CMP-sialic acid, the donor substrate of the sialyltransferases in the nucleus, while the CMP-sialic acid transport proteins are responsible for transporting CMP-sialic acid from the Cytosol to the Golgi apparatus. When these proteins function in an abnormal way, hypersialylation results, leading to an increased level of sialylation on the cell surface. This increased level of sialylation aids in the detachment of primary tumour cells due to an increase in the level of overall negative charge, causing repulsion between the cancer cells. Therefore, the sialyltransferase enzymes, CMP-sialic acid synthetases and CMP-sialic acid transport proteins are intimately involved in the metastatic cascade associated with cancer. Chapter 1 provides a general introduction of cancer metastasis, discussing the roles of three target proteins (CMP-sialic acid synthetases, CMP-sialic acid transport proteins and sialyltransferases), as well as discussing their substrate specificities, with an emphasis on their involvements in cancer metastasis. The Chapter concludes with an overview of the types of compounds intended to be utilised as probes or inhibitors of these proteins. Chapter 2 describes the general approach towards the synthesis of CMP-Neu5Ac mimetics with a sulfur linkage in the presence of a phosphate group in the general structure 38. The precursor phosphoramidite derivative 45 was prepared and isolated in a good yield using Py.TFA. Unfortunately, the target compound 38 could not be prepared. Chapter 3 describes an alternative strategy wherein S-linked sialylnucleoside mimetics, of the general structure 39, with a sulfur linkage, but no phosphate group, between the sialylmimetic and the ribose moiety in the base is targeted. A series of these S-linked sialylnucleoside mimetics were successfully prepared. Cytidine, uridine, adenosine and 5-fluorouridine nucleosides were used to create a library of different nucleosides and with structural variability also present in the sialylmimetic portion. This small 'library' of 15 compounds was designed to shed light on the interaction of these compounds with the binding sites of the sialyltranferase, CMP-sialic acid synthetase and/or CM-sialic acid transport protein. Approaches towards the synthesis of O-linked sialylnucleoside mimetics of the general structure 40 are described in Chapter 4. Several methodologies are reported, as well as protecting group manipulations, for successful preparation of these sialylnucleoside mimetics. Cytidine and uridine were employed as the nucleosides, thus allowing a direct comparison between the O- and S-linked sialylnucleoside mimetics in biological evaluation. It appears from these synthetic investigations that gaining access into the O-linked series is not as straightforward as for the S-linked series, with alternative protecting group strategies required for the different nucleosides. The biological evaluation of some of the compounds reported in Chapters 3 and 4 is detailed in Chapter 5. The sialylnucleoside mimetics were evaluated, by 1H NMR spectroscopy, for their ability to inhibit CMP-KDN synthetase. In addition, an initial 1H NMR spectroscopic-based assay was investigated for inhibition studies of α(2,6)sialyltranferase in the absence of potential inhibitors. The final chapter (Chapter 6) brings together full experimental details in support of the compounds described in the preceding Chapters.
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Abu-Izneid, Tareq. "The Synthesis and Evaluation of Functionalised Carbohydrates as Probes of Tumour Metastasis." Thesis, Griffith University, 2005. http://hdl.handle.net/10072/367269.
Full textAbstract:
Sialyltransferases, CMP-sialic acid synthetases and CMP-sialic acid transport proteins play a crucial role in the construction of cell surface glycoconjugates. These proteins also have a pivotal role to play in a number of diseases, including cancer. The sialyltransferase enzymes are responsible for transfering sialic acids from the donor substrate (CMP-sialic acid) to growing cell surface glycoconjugate chains within the Golgi apparatus. The CMP-sialic acid synthetase enzymes are responsible for the synthesis of the CMP-sialic acid, the donor substrate of the sialyltransferases in the nucleus, while the CMP-sialic acid transport proteins are responsible for transporting CMP-sialic acid from the Cytosol to the Golgi apparatus. When these proteins function in an abnormal way, hypersialylation results, leading to an increased level of sialylation on the cell surface. This increased level of sialylation aids in the detachment of primary tumour cells due to an increase in the level of overall negative charge, causing repulsion between the cancer cells. Therefore, the sialyltransferase enzymes, CMP-sialic acid synthetases and CMP-sialic acid transport proteins are intimately involved in the metastatic cascade associated with cancer. Chapter 1 provides a general introduction of cancer metastasis, discussing the roles of three target proteins (CMP-sialic acid synthetases, CMP-sialic acid transport proteins and sialyltransferases), as well as discussing their substrate specificities, with an emphasis on their involvements in cancer metastasis. The Chapter concludes with an overview of the types of compounds intended to be utilised as probes or inhibitors of these proteins. Chapter 2 describes the general approach towards the synthesis of CMP-Neu5Ac mimetics with a sulfur linkage in the presence of a phosphate group in the general structure 38. The precursor phosphoramidite derivative 45 was prepared and isolated in a good yield using Py.TFA. Unfortunately, the target compound 38 could not be prepared. Chapter 3 describes an alternative strategy wherein S-linked sialylnucleoside mimetics, of the general structure 39, with a sulfur linkage, but no phosphate group, between the sialylmimetic and the ribose moiety in the base is targeted. A series of these S-linked sialylnucleoside mimetics were successfully prepared. Cytidine, uridine, adenosine and 5-fluorouridine nucleosides were used to create a library of different nucleosides and with structural variability also present in the sialylmimetic portion. This small 'library' of 15 compounds was designed to shed light on the interaction of these compounds with the binding sites of the sialyltranferase, CMP-sialic acid synthetase and/or CM-sialic acid transport protein. Approaches towards the synthesis of O-linked sialylnucleoside mimetics of the general structure 40 are described in Chapter 4. Several methodologies are reported, as well as protecting group manipulations, for successful preparation of these sialylnucleoside mimetics. Cytidine and uridine were employed as the nucleosides, thus allowing a direct comparison between the O- and S-linked sialylnucleoside mimetics in biological evaluation. It appears from these synthetic investigations that gaining access into the O-linked series is not as straightforward as for the S-linked series, with alternative protecting group strategies required for the different nucleosides. The biological evaluation of some of the compounds reported in Chapters 3 and 4 is detailed in Chapter 5. The sialylnucleoside mimetics were evaluated, by 1H NMR spectroscopy, for their ability to inhibit CMP-KDN synthetase. In addition, an initial 1H NMR spectroscopic-based assay was investigated for inhibition studies of ?(2,6)sialyltranferase in the absence of potential inhibitors. The final chapter (Chapter 6) brings together full experimental details in support of the compounds described in the preceding Chapters.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
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Claude, Janine [Verfasser]. "The implication of microglial sialic acid-binding immunoglobulin-like lectin-E (Siglec-E) in neuroinflammation / Janine Claude." Bonn : Universitäts- und Landesbibliothek Bonn, 2014. http://d-nb.info/1049984579/34.
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Zhu, Danni. "Characterization of nicotinic acid adenine dinucleotide phosphate (NAADP) binding proteins." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:cb3309e7-ad14-4c0b-a24c-c42d95b52845.
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Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent intracellular second messenger capable of inducing calcium release from acidic organelles and is responsible for many important physiological activities. It has been recognized that an intermediate protein is required for NAADP to mediate its calcium-mobilizing effect through two-pore channels (TPCs). However, the identity of the intermediate NAADP binding protein remains unknown. This thesis aims to identify the elusive NAADP binding proteins to further elucidate the molecular mechanism of NAADP signalling pathway. The first part of this thesis describes the characterization of NAADP binding protein using a chemistry approach, which includes crosslinking study in mouse embryonic fibroblasts (MEFs) and affinity isolation of binding protein via a NAADP affinity column. Crosslinking introduced significant interference to NAADP binding in MEFs. Radioligand binding assays revealed that NAADP binding in MEFs were predominantly in the membrane fraction and no high affinity binding site was detected. Affinity isolation of binding proteins by NAADP affinity columns was carried out using mouse liver cytosol. However, no high-affinity binding protein was isolated through this approach. The second part of this thesis describes the identification of NAADP binding proteins via a combination of techniques including protein chromatography, photoaffinity labelling, mass spectrometry, virtual screen and label-free ligand binding assays. A sequential chromatography was conceived and optimized, which achieved a more than 2300-fold enrichment of high-affinity binding proteins. Photoaffinity labelling of the enriched fractions by [32P]-5-azido-NAADP revealed a 27 kDa band in SDS-PAGE gel. Subsequent mass spectrometry analysis generated a list of 35 candidates. Virtual screening of candidates by AutoDock Vina and CLC Drug Discovery Workbench predicted Carbonyl Reductase 1 (CBR1), Thiopurine S-Methyltransferase (TPMT) and Cytochrome b5 Reductase 3 (CYB5R3) as promising NAADP binding protein candidates. Further experimental validation by bio-layer interferometry (BLI) and microscale thermophoresis (MST) confirmed that TPMT and CBR1 are NAADP binding proteins. CBR1 binds NAADP and NADP with similar affinities. On the other hand, TPMT showed higher affinity and high selectivity to NAADP as no binding of NADP was observed. The identification of NAADP binding protein suggests new possibility of NAADP functionality and may provide new insights into the mechanism of action of NAADP signalling pathways.
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Mussabekova, Assel. "Evaluating antiviral activity of nucleic acid binding proteins across species." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ006.
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Le projet a permis d’identifier des répertoires de protéines interagissant avec différentes espèces d’acides nucléiques caractéristiques des virus chez cinq espèces animales (Homme, souris, poulet, drosophile, nématode). Ces protéines représentent des candidats pour remplir des fonctions de récepteurs de l’immunité innée ou de molécules antivirales. Certaines d’entre elles ont été conservées au cours de l’évolution, ce qui m’a permis de tester leur fonction dans la drosophile. J’ai réalisé un crible impliquant des infections avec cinq virus différents sur 100 protéines conservées. Ce crible m’a permis d’identifier huit protéines dont l’inhibition impacte la réplication virale. Deux d’entres elles, CG5641 et Zn72D, sont nécessaires pour la réplication des virus de type picornavirus (CrPV). Le candidat le plus intéressant identifié est cependant la protéine Tao, dont l’inhibition entraîne une augmentation de la réplication de virus appartenant à plusieurs familles, chez la drosophile et dans les cellules de mammifèresAntiviral response largely relies on the recognition of viral nucleic acids. The aim of the project was to characterize the range of nucleic acid binding proteins in the context of viral infection in flies. We identified a wide repertoire of proteins, which recognize viral nucleic acids in five species (human, mouse, chicken, fruit fly and roundworm). Among these proteins, there are ones, which are conserved in insects and humans, and therefore their function can be easily studied in the fruit fly model. Afterwards, we have performed a large screen in flies to study more precisely the function of 100 proteins in infection with 5 different viruses. We have found eight promising candidates as a result of this screen. We identified two Drosophila proteins CG5641 and Zn72D, which are also present in humans, as proviral factors. We also identified a protein Tao, which is conserved in humans, and is antiviral against several types of viruses
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Torchia, Enrique C. "The role of intracellular bile acid binding proteins in bile acid transport and cytoprotection." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ60354.pdf.
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Kopatz, Jens Christopher [Verfasser]. "Microglial sialic-acid-binding immunoglobulin-like lectin-H (Siglec-H) and Siglec-11 in neuroinflammation / Jens Christopher Kopatz." Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/1081423676/34.
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Kopatz, Jens [Verfasser]. "Microglial sialic-acid-binding immunoglobulin-like lectin-H (Siglec-H) and Siglec-11 in neuroinflammation / Jens Christopher Kopatz." Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/1081423676/34.
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So, Po-Lin. "The role of cellular retinoic acid binding proteins in zebrafish embryogenesis." Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264167.
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Reinhart, Gregory Allen. "Nutrient regulation and developmental expression of porcine fatty acid binding proteins /." The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487677267728138.
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Aldhamen, Yasser A. "All-trans retinoic acid downregulates CCAAT/enhancer binding proteins in human bronchial epithelial cells." Connect to Online Resource-OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1187302036.
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Thesis (M.S.)--University of Toledo, 2007"In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 37-48, 62-84.
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Nieuwenhoven, Franciscus Arnoldus van. "Heart fatty acid-binding proteins role in cardiac fatty acid uptake and marker for cellular damage /." Maastricht : Maastricht : Rijksuniversiteit Limburg ; University Library, Maastricht University [Host], 1996. http://arno.unimaas.nl/show.cgi?fid=6268.
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Campbell, Fiona M. "Long-chain fatty acid transport by the human placenta : the role of fatty acid-binding proteins." Thesis, University of Aberdeen, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363738.
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The placenta is thought to play a vital role in the transfer of essential fatty (EFA) and their long-chain polyunsaturated derivatives (LCPUFA) from mother to the fetus. There is a preferential accumulation of these fatty acids from maternal to fetal tissues. However, little was known about the manner in which these nutrients preferentially traversed the placenta. This study investigated part of this placental transport mechanism. The results from these investigations demonstrated that the preferential transport of LCPUFA to the fetal circulation may at least be partially mediated by a preferential uptake system in the placenta involving a 40 kDa, placental membrane fatty acid binding protein (p-FABPpm). This protein was found exclusively in the maternal facing microvillous membranes. It was characterised as different from previously identified ubiquitous FABPpm by virtue of having a different pl value, different amino acid composition, no aspartate aminotransferase activity and a higher binding affinity for LCPUFA over non-essential fatty acids. The human choriocarcinoma cell line (BeWo) expressed a protein immunoreactive to anti-p-FABPpm anti-serum. This anti-serum inhibited the binding of LCPUFA to placental membranes and the uptake of LCPUFA by BeWo cells, to a greater degree than it inhibited the uptake and binding of non essential fatty acids. In addition to p-FABPpm the existence of multiple types of both cytosolic (L-FABP and H-FABP) and membrane (FAT and FATP) fatty acid-binding proteins was demonstrated in placental cells. These proteins could play important roles in both the uptake of fatty acids by the placenta and in controlling the fate of fatty acids inside placental cells.
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Robertson, Timothy Allen. "Development and validation of statistical potential functions for the prediction of protein/nucleic-acid interactions from structure /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9268.
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Li, Huiying. "Adipocyte fatty acid-binding protein : a link between inflammation and vascular dysfunction /." Click to view the E-thesis via HKUTO, 2010. http://sunzi.lib.hku.hk/hkuto/record/B44248714.
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Li, Huiying, and 李慧颖. "Adipocyte fatty acid-binding protein: a link between inflammation and vascular dysfunction." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44248714.
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Erol, Erdal. "Heart- and liver-type fatty acid binding proteins in lipid and glucose metabolism." Diss., Texas A&M University, 2004. http://hdl.handle.net/1969.1/1148.
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Heart-type Fatty Acid-Binding Protein (H-FABP) is required for high rates of skeletal muscle long chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild type and H-FABP null mice were fed a standard chow or high fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% (P<0.01) at 0.02 mU/ml of insulin, but did not cause a significant effect at 2 mU/ml insulin; skeletal muscle triglyceride and long chain acyl-CoA (LCACoA) levels remained normal. With the fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% (P<0.01) at 2 mU/ml insulin, thus partially preventing insulin resistance, and completely prevented the threefold (P<0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCACoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulinindependent) soleus muscle deoxyglucose uptake by 28% (P<0.05). These results establish a close relationship of FABP-dependent lipid pools with insulin sensitivity, and indicate the existence of a non-acute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake.
Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of chain LCFA for oxidation and for peroxisome proliferator-activated receptor (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced β-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism.
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Krisinger, Michael J. "Membrane binding properties of prothrombin and other gamma-carboxyglutamic acid-containing coagulation proteins." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31087.
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Haemostasis is a highly regulated, fundamental, physiological process featuring numerous peripheral membrane proteins. Of these, the membrane and calcium binding properties of the vitamin K-dependent proteins are dependent on a common N-terminal γ-carboxyglutamic acid (Gla)-containing domain. Previous work on Gla proteins has provided a wealth of affinity and kinetic membrane binding information. These studies have employed a number of biophysical techniques using artificial phosphatidylserine-containing model membranes. However, many aspects of the membrane binding interaction, in terms of mechanism and modulation by protein cofactor remain obscure. This thesis examines two methods for studying the membrane binding properties of human plasma derived Gla proteins with emphasis on prothrombin.
In Chapter 3 differential centrifugation combined with immunoaffinity detection was used to quantify the effect the cofactor Factor Va had on the membrane binding affinity of prothrombin for membrane. Factor Va bound to anionic phospholipid membrane undoubtedly enhanced the membrane binding affinity of prothrombin relative to prothrombin binding in the absence of the cofactor. Thus, these results indicate that Factor Va can recruit prothrombin or prethrombin 1, a Gla-domain less fragment of prothrombin, to the membrane surface, plausibly contributing to its cofactor function.
In Chapters 4 and 5, surface plasmon resonance (SPR) was used to evaluate the Ca²⁺-specific binding properties of a number of Gla proteins to immobilized membranes. Membrane affinity, molar binding preference and kinetics controlling complex formation and complex breakdown
varied widely between Gla proteins. The comparative results obtained by SPR indicate that the majority of homologous Gla proteins bind membranes with a complex mechanism which may involve membrane induced protein dimers. Unlike prothrombin, the binding profiles for fragment 1 and fragment 1.2 fitted closely to a one-site binding model. Apparent biphasic association and biphasic dissociation phases were observed for prothrombin and commonly amongst the other Gla proteins at a wide range of protein concentrations including physiological concentrations. For prothrombin, dimerization appears to be specific to the protease domain as neither fragment 1 nor fragment 1.2 displays such binding complexities. It is possible that dimerization increases the half-life of membrane-bound Gla proteins thereby promoting their participation in complex assembly and function.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Kokorelis, Steve H. "Biochemical Analysis of Putative Single-Stranded Nucleic Acid Binding Proteins in Porphyromonas gingivalis." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4833.
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Proteins that bind to both DNA and RNA embody the ability to perform multiple functions by a single gene product. These nucleic acid binding proteins in prokaryotes can play a vital role in many cellular processes, including replication, transcription, gene expression, recombination, and repair, to name a few. Nucleic acid binding proteins have unique functional characteristics that stem from their structural attributes that have evolved in a widely-conserved manner. In Escherichia coli (E. coli), the highly-conserved histone-like protein, HU, which predominates as a heterodimer of HUα and HUβ, has been found to bind to both dsDNA and ssDNA. Likewise, RNA-binding proteins contain various structural motifs, many of which are also conserved amongst many bacterial species like the RNA recognition motif. However, in Porphyromonas gingivalis, a periodontal pathogen, the histone-like, HU proteins and the RNA-binding protein (RBP) are not well characterized compared to their respective structures in E. coli. In our study, we sought to characterize and compare the HU proteins and RBP in order to gain a better understanding of their structure and function in the cell. Our data showed the HU proteins predominate as homo-tetramers and RBP as a monomer. We demonstrated single-stranded DNA binding with all three proteins. We found both P. gingivalis HU subunits bind non-specifically to ssDNA but show preferential binding to poly(dG) content, while binding to poly(dA) the weakest. These results show that HUα, HUβ and RBP are novel ssDNA binding proteins in P. gingivalis, indicating an expanded role and function within the cell.
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Seyfried, Nicholas T. "The structure and function of hyaluronan-binding proteins in extracellular matrix assembly." Thesis, University of Oxford, 2004. http://ora.ox.ac.uk/objects/uuid:e1a2cf8f-7ac7-4c5a-bd3f-53d7653e8888.
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The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.
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Smith, Nicholas James. "The clinical utility of FABP in acute coronary syndromes." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391403.
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Chan, Cangel Pui Yee. "A superior early myocardial infarction marker : human heart-type fatty acid-binding protein /." View Abstract or Full-Text, 2002. http://library.ust.hk/cgi/db/thesis.pl?CHEM%202002%20CHAN.
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Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002.Includes bibliographical references (leaves 139-166). Also available in electronic version. Access restricted to campus users.
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Sauer, Anne-Kathrin [Verfasser]. "Analysis of the sialic acid binding activity of the hemagglutinins of influenza viruses and its role in host tropism / Anne-Kathrin Sauer." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2013. http://d-nb.info/1037878744/34.
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Nadassy, Katalin. "Molecular recognition by proteins : structural features of zinc and protein-nucleic acid binding sites." Thesis, University of Stirling, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341227.
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Brailsford, Louis Alex. "Investigating the possible role of fatty acid binding proteins (FABPs) in nociceptive pain processing." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/44886/.
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The transient receptor potential vanilloid 1 (TRPV1) channel protein is activated by lipid metabolites synthesized in the cytosol of nociceptors in response to noxious stimulation. Lipid species include the endocannabinoid anandamide (AEA) and the linoleic acid metabolite 13 (S) hydroxyoctadecadienoic acid (HODE) which act as endogenous TRPV1 ligands (endovanilloids) by evoking TRPV1 mediated Ca2+ entry. Members of the fatty acid binding protein (FABP) family have been widely reported to act as intracellular lipid binding proteins for hydrophobic lipid species in aqueous cytosolic environments. The aim of this thesis was to identify which FABP isotypes could solubilize then shuttle AEA and 13(S)HODE to TRPV1 during nociception. Inhibiting FABP mediated transportation of endovanilloids could represent an alternative approach to analgesia by indirectly antagonizing TRPV1 activity during nociception while avoiding the widely reported negative side effects of direct antagonism. For the first time, it was found that FABP isoforms 5, 7 and 8 were expressed in rat dorsal root ganglia cell preparations. Furthermore, subsequent cell free competitive binding assays confirmed that FABP5, 7 and 8 could all physically bind to AEA and 13(S)HODE albeit with variable affinities. The ability of FABP 5, 7 and 8 to physically associate with TRPV1 and therefore deliver AEA was then assessed in live mammalian cell lines transfected with plasmid DNA constructs expressing recombinant TRPV1 and FABPs. Physical interactions between TRPV1 and FABP5, 7 and 8 were observed in COS-7 cells examined by fluorescence microscopy. However, when increases in intracellular Ca2+ levels were measured in COS-7 cells co-expressing TRPV1 and FABP, in response to treatment with 1µM AEA, the magnitude of AEA evoked Ca2+ influxes were not significantly different to those observed in COS-7 cells not co-expressing the FABPs. This suggested that the FABPs did not functionally associate with TRPV1 and did not deliver AEA to TRPV1 receptors. In conclusion, data in thesis showed that the FABP isoforms expressed in DRG cell preparations could physically associate with lipid species reported to activate TRPV1 during nociception.
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Aldhamen, Yasser. "All-trans retinoic acid downregulates CCAAT/enhancer binding proteins in human bronchial epithelial cells." University of Toledo Health Science Campus / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=mco1187302036.
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Ruberte, Esther. "Retinoic acid receptors and cellular retinoid binding proteins : their transcript distribution during mouse embryogenesis." Université Louis Pasteur (Strasbourg) (1971-2008), 1992. http://www.theses.fr/1992STR13009.
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Nous avons etudie la distribution des arn messagers codant pour les recepteurs de l'acide retinoique et pour les proteines cytoplasmiques lors de l'embryogenese de la souris en utilisant la technique d'hybridation in situ. Nos resultats montrent que les recepteurs nucleaires et les proteines cytoplasmiques ont des distributions specifiques dans les tissus et les organes embryonnaires a des moments ou leur developpement est altere par des taux anormaux de retinoides. La correlation de ces domaines d'expression avec les effets morphogenetiques de la vitamine a suggerent que la cellular retinol binding protein est impliquee dans le proces de transformation du retinol en acide retinoique et que les cellular retinoic acid binding proteins sont responsables du controle des taux d'acide retinoique libre dans les cellules. Le recepteur gamma pourrait jouer un role specifique lors de la chondrogenese et de la formation des epitheliums keratinises tandis que le recepteur beta aurait des fonctions particulieres au cours du developpement du systeme nerveux et des epitheliums des muqueuses respiratoires et digestives
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Petrescu, Anca Daniela. "Ligand binding proteins: roles in ligand transfer and activation of nuclear receptors." Diss., Texas A&M University, 2004. http://hdl.handle.net/1969.1/290.
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Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.
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Yeung, Chun-yu. "Adipocyte- and epidermal-fatty acid-binding proteins in relation to obesity and its medical complications." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B44204565.
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Wong, Tak-sui, and 黃德緒. "Adipocyte fatty acid binding protein acts as a suppressor of autophagy contributing to foam cell formation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/206502.
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Background and objectives:
Growing bodies of evidence demonstrate that adipocyte fatty acid binding protein (A-FABP) mediates the pathogenesis of atherosclerosis through its direct impacts on macrophages. Loss-of-function study was conducted by utilizing peritoneal macrophages derived from A-FABP knockout (KO) mice, to investigate the role of A-FABP in autophagy and macrophage foam cell formation.
Key findings:
1. No morphological changes between the peritoneal macrophages derived from A-FABP knockout (KO) or their wild-type (WT) littermates.
2. Foam cell formation was successfully induced by the treatment of acetylated low-density lipoproteins (LDL) in peritoneal macrophages derived from A-FABP WT and KO mice.
3. LDL treatment induces autophagy in peritoneal macrophages from both A-FABP WT and KO mice.
4. The extent of LDL-induced autophagy is reduced in peritoneal macrophages of WT mice and is accompanied by increased lipid droplet accumulation when compared with A-FABP KO mice.
Conclusions:
A-FABP is a suppressor of autophagy and contributes to the attenuation of cholesterol efflux, subsequently resulting in enhancement of lipid droplets accumulation in peritoneal macrophages. A-FABP mediates the formation of macrophage foam cell via the suppression of autophagy. The results suggest that A-FABP is a potential therapeutic target to suspend the progression of atherosclerosis and remit the atherosclerotic lesion.published_or_final_version
Medicine
Master
Master of Medical Sciences
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Ploskon-Arthur, Eliza. "Binding of intermediates by type 1 and type 11 fatty acid synthase acyl carrier proteins." Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521059.
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Yeung, Chun-yu, and 楊振宇. "Adipocyte- and epidermal-fatty acid-binding proteins in relation to obesity and its medical complications." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B44204565.
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Roundell, Jennifer. "Cellular retinoic acid binding protein (CRABP) mRNA expression in splotch mutant mouse embryos." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27397.
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The splotch (sp) mutation has been identified as a mutation in the paired box gene, Pax-3. Heterozygous mice carrying this mutation are phenotypically normal, with the exception of a white spot on their bellies. Homozygous embryos do not live to birth, and suffer from a wide range of developmental defects, all of which result from delayed neural tube closure, or inadequate neural crest cell migration. Most notably, homozygotes have an increased rate of spina bifida with or without exencephaly. Retinoic acid (RA), which has been shown to be very important in the development of a number of systems, was shown to cause a selective mortality of the homozygous splotch embryos when administered maternally at day 9 p.c. (Moase and Trasler, 1987). Since cellular retinoic acid binding protein (CRABP) is localized to the tissues which are affected by both the splotch gene, and retinoic acid teratogenesis, its expression patterns in the developing splotch embryo were examined. It was found that the distribution of CRABP mRNA is normal, but its expression levels are excessive in splotch homozygous day 9 mouse embryos.
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Garza, John Anthony. "Structural and ligand-binding properties of a dual substrate specific enzymes from schizosaccharomyces pombe a dissertation /." San Antonio : UTHSC, 2009. http://learningobjects.library.uthscsa.edu/cdm4/item_viewer.php?CISOROOT=/theses&CISOPTR=45&CISOBOX=1&REC=17.
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Peet, Daniel J. "Protein-bound fatty acids in mammalian hair fibres /." Connect to thesis, 1994. http://eprints.unimelb.edu.au/archive/00000641.
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Wong, Yue-ling, and 黃愉鈴. "The role of adipocyte fatty acid binding protein in the pathogenesis of non-alcoholic fatty liver disease." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45164873.
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Powell, Catherine. "The Roles of Metabolic Proteins, Fatty Acid Binding Protein 5 and S100A7, in Breast Cancer and Obesity." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1426179329.
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Kemp, Hilary A. "A complex of six FAR proteins required for pheromone arrest and mating /." view abstract or download file of text, 2003. http://wwwlib.umi.com/cr/uoregon/fullcit?p3113011.
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Thesis (Ph. D.)--University of Oregon, 2003.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 94-104). Also available for download via the World Wide Web; free to University of Oregon users.
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Pedersen, Sindre Andre. "Metal binding proteins and antifreeze proteins in the beetle Tenebrio molitor : a study on possible competition for the semi-essential amino acid cysteine." Doctoral thesis, Norwegian University of Science and Technology, Department of Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-1504.
Full textAbstract:
In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are involved in these defence systems (Korsloot et al. 2004). Since an organism’s resources may be considered to be limited, the ability to restore homeostasis depends on the severity of the different forms of stress it experiences. It has been proposed that pollutants present in the environment may alter the ability to respond to climatic stressors like e.g. low temperature, desiccation (Holmstrup 2002).
This work deals with the possible consequences of combined stress from metal exposure and low temperature in cold hardy insects. Many of these insects produce so called antifreeze proteins that protect them from lethal freezing. Metallothioneins are metal binding proteins that are considered to be important in detoxification when animals are exposed to metals. Metallothioneins and most forms of antifreeze proteins from insects are known to contain unusually high amounts cysteine. Cysteine is considered to be semi-essential, since it must be derived from the essential amino acid methionine (Choen 2004). Induction of one of these two types of proteins may potentially deplete the cysteine pool and thus reduce the capacity to produce the other type. Alternatively, the animals might have evolved other structures to avoid a potential competition for cysteine. The purpose of the present work was to explore these possible scenarios.
Paper I and II reprinted with kind permission of Elsevier, Sciencedirect.com