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Controlled folding and conformational switching in metal-mediated DNA constructsPereira, Fernanda Antonia 12 August 2016 (has links)
No description available.
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Caracterização estrutural e funcional de duas Nucleosídeo Fosforilases de Schistosoma mansoni / Structural and functional characterization of two Nucleoside Phosphorylase from Schistosoma mansoni.Souza, Juliana Roberta Torini de 18 August 2016 (has links)
As doenças parasitárias são uma das maiores causas de morte em países em desenvolvimento, e recebem pouca ou nenhuma atenção das indústrias farmacêuticas para o desenvolvimento de terapias. Causada pelo parasita Schistosoma mansoni a esquistossomose mansônica afeta aproximadamente 259 milhões de pessoas no mundo sendo aproximadamente 6 milhões somente no Brasil. O S. mansoni não possui a via \"de novo\" para a biossíntese de bases púricas e depende integralmente da via de salvação para o suprimento dessas bases, portanto, essa via é um alvo em potencial. Agentes capazes de bloquear a atividade das enzimas participantes desta via atuam de forma inespecífica e são quase sempre tóxicos ao homem e por isso o estudo minucioso das pequenas diferenças encontradas entre as enzimas do hospedeiro e do parasita são de extrema importância. Uma diferença marcante entre a via de salvação de purinas do parasita e do hospedeiro humano é a presença de atividade para adenosina fosforilase, que no parasita é exercida por duas entidades distintas: pela enzima Metiltioadenosina fosforilase de S. mansoni (SmMTAP) e por uma enzima até então desconhecida. A enzima SmMTAP naturalmente converte 5\'-deoxi-5\'-metiltioadenosina (MTA) em adenina livre, mas ao contrário do que é visto no hospedeiro, no parasita essa enzima atua preferencialmente na conversão de adenosina. Substituições encontradas no sítio ativo dessa enzima, podem explicar tamanha preferência pelo substrato alternativo, revelando mecanismos distintos da enzima humana. A enzima Purina nucleosídeo fosforilase de S. mansoni (SmPNP) converte inosina e guanosina à hipoxantina e guanina, respectivamente, mas não possui atividade catalítica para adenosina. No entanto, no genoma de S. mansoni é descrita uma isoforma para a SmPNP (SmPNP2), cuja atividade catalítica é desconhecida e, portanto, essa enzima pode também atuar na conversão de adenosina juntamente com a SmMTAP. Assim, os objetivos deste trabalho foram realizar estudos bioquímicos da ação da enzima SmMTAP e realizar a caracterização estrutural e funcional da enzima SmPNP2. Para isso, foram realizadas mutações no sítio ativo da SmMTAP (S12T, N87T, Q289L, S12T/N87T e S12T/N87T/Q289L), as mutantes da SmMTAP juntamente com a enzima SmPNP2 foram clonadas, expressas de forma heteróloga e purificadas. Foram realizados ensaios de cristalização e cinéticos por espectrofotometria utilizando um sistema acoplado. A atividade da SmPNP2 foi ainda avaliada por calorimetria e HPLC. Foram determinadas as constantes catalíticas da forma nativa e para os cinco mutantes da SmMTAP para cinco diferentes substratos. Foi determinada atividade catalítica da SmPNP2 por 3 diferentes substratos: adenosina, inosina e citidina, as constantes catalíticas foram determinadas para os três substratos. Foram obtidos cristais para os mutantes da SmMTAP e da SmPNP2, que foram submetidos à difração de raios X nas linhas I04-1 e I02 do laboratório de radiação síncrotron Diamond Light Source (DLS). Foram resolvidas 9 estruturas dos mutantes da SmMTAP e 4 da proteína SmPNP2. Os dados cinéticos, juntamente com os dados estruturais, permitiram compreender mecanismos catalíticos e de interação das proteínas estudadas, complementando o conhecimento do metabolismo do parasita Schistosoma mansoni e revelando alvos em potencial para o desenvolvimento de fármacos específicos. / The parasitic illness are the leading cause of deaths in developing countries, and receives little or no attention from drug companies to develop therapies. The schistosomiasis is caused by Schistosoma mansoni parasite and affects approximately 259 million people worldwide with 6 million only in Brazil. The Schistosoma mansoni parasite does not possess the \"de novo\" pathway for purine bases biosynthesis and depends entirely on salvage pathway for its purine requirement, therefore this pathway is a potential target. Compounds able to block the enzymes from this pathway, are not specific and are often toxic to humans, thus the thorough study about the particularity found between enzymes from host and parasite are extremely important. A notable difference between human and parasite metabolism, is the activity existence to Adenosine phosphorylase that in parasite is carried out by two distinct entities: by Methylthioadenosine phosphorylase (SmMTAP) and by a hitherto unknown enzyme. The SmMTAP enzyme, naturally converts 5\'-deoxy-5\'-methylthioadenosine (MTA) to free adenine and in opposition to host, in the parasite this enzyme acts manly in adenosine conversion. Substitutions found in the active site from SmMTAP, can explain the huge preference by alternative substrate and to expose a distinct mechanisms from human enzyme. The Purine nucleoside Phosphorylase from S. mansoni (SmPNP) converts inosine and guanosine to hypoxanthine and guanine, respectively, but it not possess catalytic activity to adenosine conversion. However in the S. mansoni genome there is a isoform to SmPNP, whose activity is unknown, thus is possible that SmPNP2 enzyme also can to convert adenosine. This study aimed to perform biochemical studies to investigate the SmMTAP enzyme action and perform the structural and functional characterization of SmPNP2. For this propose was made site-directed mutagenesis (S12T, N87T, Q289L, S12T/N87T e S12T/N87T/Q289L). The SmMTAP mutants and SmPNP2 enzyme were cloned, expressed by heterolog process and purified. Were perform kinetic assays by spectrophotometric method in a coupled system. The SmPNP2 activity was also available by calorimetry and HPLC methods. Were determined the catalytic constants to wild and mutants SmMTAP to five different substrate. Was determinated to SmPNP2 catalictical activity and kinetics parameters to three substrate: adenosine, inosine e cytidine. Were obtained crystals from SmMTAP mutants and SmPNP2 enzyme, those crystals were submitted to X-rays diffractions in the I04-1 and I02 beamlines from Diamond Light Source (DLS). Nine structures were obtained from SmMTAP mutants and four from SmPNP2 enzyme. The kinetics and structural data allowed understanding the catalytic and interaction mechanisms about the protein studied, supplementing the knowledge around Schistosoma mansoni metabolism and reporting potential targets for the specific drugs development.
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DISCOVERY OF NOVEL MURAYMYCIN ANTIBIOTICS AND INSIGHT INTO THE BIOSYNTHETIC PATHWAYCui, Zheng 01 January 2018 (has links)
New antibiotics with novel targets or mechanisms of action are needed to counter the steady emergence of bacterial pathogens that are resistant to antibiotics used in the clinic. MraY, a promising novel target for antibiotic development, initiates the lipid cycle for the biosynthesis of peptidoglycan cell wall, which is essential for the survival of most, if-not-all, bacteria. MraY is an enzyme that catalyzes the transfer and attachment of phospho-MurNAc-pentapeptide to a lipid carrier, undecaprenylphosphate. Muraymycins are recently discovered lipopeptidyl nucleoside antibiotics that exhibit remarkable antibiotic activity against Gram-positive as well as Gram-negative bacteria by inhibiting MraY. We conducted a thorough examination of the metabolic profile of Streptomyces sp. strain NRRL 30473, a known producer of muraymycins. Eight muraymycins were isolated and characterized by a suite of spectroscopic methods, including three new members of muraymycin family named B8, B9 and C5. Muraymycins B8 and B9, which differ from other muraymycins by having an elongated fatty acid side chain, showed potent antibacterial activity against Escherichia coli ∆tolC mutant and pM IC50 against Staphylococcus aureus MraY. Muraymycin C5, which is characterized by an N-acetyl modification of the disaccharide’s primary amine, greatly reduced its antibacterial activity, which possibly indicates this modification is used for self-resistance.
In addition to the discovery of new muraymycins, eleven enzymes from the biosynthetic pathway were functionally assigned and characterized in vitro. Six enzymes involved in the biosynthesis of amino ribofuranosylated uronic acid moiety of muraymycin were characterized: Mur16, a non-heme, Fe(II)-dependent α-ketoglutarate: UMP dioxygenase; Mur17, an L-threonine: uridine-5′-aldehyde transaldolase; Mur20, an L-methionine:1-aminotransferase; Mur26, a low specificity pyrimidine nucleoside phosphorylase; Mur18, a primary amine-requiring nucleotidylyltransferase; Mur19, a 5-amino-5-deoxyribosyltransferase. A one-pot enzyme reaction was utilized to produce this disaccharide moiety and its 2′′-deoxy analogue. Two muraymycin-modifying enzymes that confer self-resistance were functionally assigned and characterized: Mur28, a TmrB-like ATP-dependent muraymycin phosphotransferase, and Mur29, a muraymycin nucleotidyltransferase. Notably, Mur28 preferentially phosphorylates the intermediate, aminoribofuranosylated uronic acid, in the muraymycin biosynthetic pathway to produce a cryptic phosphorylated-dissacharide intermediate. Mur23 and Mur24 were assigned as two enzymes that modify the cryptic phosphorylated intermediate by attachment of an aminopropyl group. Mur24 catalyzes the incorporation of butyric acid into the phosphorylated-disaccharide. Following the incorporation, Mur23 catalyzes a PLP-dependent decarboxylation. Finally, Mur15, which belongs to the cupin family, is functionally assigned as a non-heme, Fe(II)-dependent α-ketoglutarate dioxygenase that catalyzes the β-hydroxylation of a leucine moiety in muraymycin D1 to form muraymycin C1. Mur15 can also hydroxylate the γ-position of leucine moiety to muraymycins with fatty acid chain in β-position.
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Drug transporters in the nasal epithelia and their contribution in drug deliveryAl-Ghabeish, Manar I. 01 December 2014 (has links)
The nasal route has primarily been used to deliver drugs for the treatment of local diseases such as nasal infections, nasal congestion and allergies. The nasal route can also be used as a non-invasive alternative route to deliver drugs systemically when a rapid onset of action and/or avoidance of hepatic metabolism are desired. Moreover, there is a growing interest in the use of this route for direct transport of drugs from the nose to the brain. Most of the drugs that have been studied for nasal delivery are either small molecules which are lipophilic enough to passively diffuse through the nasal epithelia or macromolecules where bioavailabilities less than 1% are clinically effective and acceptable. This study focused on identifying carrier proteins or transporters in the nasal mucosa that could improve the absorption of specific drug substrates across the nasal respiratory and olfactory epithelia.
The presence of drug transporters in the nasal mucosa of humans and commonly used animal models were investigated. DNA microarray results for nasal samples from humans and two commonly used models, mice and rats, were obtained from GenBank and were analyzed in collaboration with the University of Iowa Center for Bioinformatics and Computational Biology. While cow tissues are frequently used in in-vitro nasal permeability analyses, there is limited information available in GenBank for this species. Both DNA microarray analysis and RT-PCR were performed on bovine nasal explants to determine transporter expression. Good agreement between the microarray and RT-PCR results was observed.
While human and three animal species commonly used as models in nasal drug delivery research (mouse, rat, and cow) show similar patterns of expression for several transporters, interspecies differences in the level of expression were observed. Therefore, the expression level of transporters remains a factor to consider when translating results obtained using animal models to humans.
The nucleoside transporter family was selected for further evaluation of the potential to improve the nasal absorption of substrates. Nucleoside transporters are integral proteins responsible for mediating and facilitating the flux of nucleosides across cellular membranes; they are also known to be responsible for the uptake of nucleoside analog drugs such as anti-cancer and anti-viral agents. RT-PCR and Western blotting were used to verify the presence of two transporter subtypes, ENT1 and CNT3, in the bovine nasal respiratory and olfactory mucosa. The expression level of both transporters in the respiratory mucosa was comparable to that in the olfactory mucosa. Using immunohistochemistry, ENT1 and CNT3 were found to be localized primarily at the apical surface of the nasal epithelial cells. This indicates that the nasal epithelium likely absorbs exogenous nucleosides for intracellular uses such as nucleic acid synthesis and regulating other cellular activities.
The contribution of the nucleoside transporters to the permeation of a nucleoside analogue drug, alovudine, across the nasal epithelia was also studied. The transport of alovudine showed a non-linear increase with increasing donor concentration over the range of 50 to 3000 µM which suggests that nucleoside transporters play a role in its uptake. Polarized transport was not observed suggesting that the facilitative nature of ENT1 plays a major role in alovudine transport. S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT1 inhibitor, incompletely decreased alovudine permeability across the nasal mucosa. This demonstrates that at least one transporter, ENT, plays a significant role in the uptake of this nucleoside drug across the nasal mucosa.
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pH changes localized to the surface of membrane transport proteinsJohnson, Danielle Elaine 06 1900 (has links)
Intracellular pH was monitored at the cytosolic surface of plasma membrane solute transporters (Na+/H+/nucleoside co-transporters, or Cl-/HCO3- exchangers), using pH-sensitive fluorescent proteins (FPs), dual emission green FP (deGFP4) and a monomeric red FP Nectarine (mNect), whose development and characterization are also reported here.
Human concentrative nucleoside transporter, hCNT3, mediates Na+/H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in HEK293 cells. pH changes at the intracellular surface of hCNT3 were monitored by fusing mNect to the cytoplasmic N-terminus of hCNT3 (mNect.hCNT3) or an inactive hCNT3 mutant (mNect.hCNT3-F563C). Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mM uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+. This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported/uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule and intestine, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3.
Microdomains, discrete regions of altered cytosolic solute concentration, are enhanced by rapid solute transport and slow diffusion rates. pH-regulatory membrane transporters, like the Cl-/HCO3- exchanger AE1, could nucleate H+ microdomains, since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. As AE1 drives Cl-/HCO3- exchange, differences in pH, near and remote from AE1, were monitored simultaneously by deGFP4 fused to AE1 (deGFP4.AE1) and mNect.hCNT3-F563C. deGFP4.AE1-mNect.hCNT3-F563C distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the deGFP4.AE1-mNect.hCNT3-F563C distance increased, mNect.hCNT3-F563C detected the cytosolic pH change with a time delay and reduced rate of pH change, compared to deGFP4.AE1. Carbonic anhydrase activity was essential for H+ microdomain formation. H+ diffusion along the plasma membrane was 60-fold slower than to the cytosolic ER-surface. During physiological HCO3- transport, a H+ microdomain 0.3 µm in diameter develops around AE1, which will affect nearby pH-sensitive processes.
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Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiaeHuang, Bo January 2007 (has links)
Transfer RNAs (tRNAs) act as adaptor molecules in decoding messenger RNA into protein. Frequently found in tRNAs are different modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U). Although modified nucleosides are present at many positions in tRNAs, two positions in the anticodon region, position 34 (wobble position) and position 37, show the largest variety of modified nucleosides. In Saccharomyces cerevisiae, the xm5U type of modified uridines found at position 34 are 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2´-O-methyluridine, (ncm5Um), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U). Based on the complex structure of these nucleosides, it is likely that their formation requires several synthesis steps. The Elongator complex consisting of proteins Elp1p - Elp6p, and the proteins Kti11p - Kti14p, Sit4p, Sap185p, and Sap190p were shown to be involved in 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side-chain synthesis at position 34 in eleven tRNA species. The proteins Urm1p, Uba4p, Ncs2p, Ncs6p, and Yor251cp were also identified to be required for the 2-thio (s2) group formation of the modified nucleoside mcm5s2U at wobble position. Modified nucleosides in the anticodon region of tRNA influence the efficiency and fidelity of translation. The identification of mutants lacking ncm5-, mcm5-, or s2-group at the wobble position allowed the investigation of the in vivo role of these nucleosides in the tRNA decoding process. It was revealed that the presence of ncm5-, mcm5- or s2-group promotes reading of G-ending codons. The concurrent presence of the mcm5- and the s2-groups in the wobble nucleoside mcm5s2U improves reading of A- and G-ending codons, whereas absence of both groups is lethal to the yeast cell. The Elongator complex was previously proposed to regulate polarized exocytosis and to participate in elongation of RNA polymerase II transcription. The pleiotropic phenotypes observed in Elongator mutants were therefore suggested to be caused by defects in exocytosis and transcription of many genes. Here it is shown that elevated levels of hypomodified tRNALys [mcm5s2UUU] and tRNAGln[mcm5s2UUG] can efficiently suppress these pleiotropic phenotypes, suggesting that the defects in transcription and exocytosis are indirectly caused by inefficient translation of mRNAs encoding proteins important in these processes.
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Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar TyphimuriumChen, Peng January 2004 (has links)
Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described. A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C. cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent. During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.
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Investigation of the Role of Groove Hydration and Charged Nucleosides in DNA Charge TransferOnyemauwa, Frank Okezie 11 August 2006 (has links)
Structural analyses of DNA oligonucleotides indicate the presence of bound water molecules in the major and minor grooves of DNA. These water molecules participate in DNA charge transfer by their reaction with guanosine radical cation to form 7,8-dihydro-8-oxo-guanine (8-oxoG), which when treated with a base leads to DNA strand cleavage. We probed the reaction of guanosine radical cation with water with series of alkyl substituted cytidines and thymidines by incorporating the modified nucleosides into anthraquinone linked DNA duplexes and irradiating them with UV light at 350 nm. The incorporation of these hydrophobic substituents disrupt the DNA spine of hydration, and we have observed that these modifications in the major and minor groove do not effect the trapping or long distance hopping of radical cations in DNA.
The second part of the work reported herein examines the role of charged nucleosides in long range charge transfer in duplex DNA. DNA methylation is a naturally occurring process mediated by enzymes responsible for such functions in biological systems. Hypermethylation of DNA can also occur as a result of environmental alkylating agents leading to mutation of the affected cells. Methylation of the ring nitrogen of a purine base can introduce a positive charge in the ring resulting in the cleavage of the glycosidic bond of the nucleoside.
To understand the role of a charged nucleoside on charge transfer in DNA, we designed and synthesized cationic nucleoside mimics, which were incorporated into anthraquinone-linked DNA strands and irradiated at 350 nm. The presence of the cationic bases on the duplexes inhibits the migrating hole from hopping along the DNA strand, and induces a prominent local structural distortion of the DNA as a result of the charged nucleobase.
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Stereoselective Synthesis Of Optically Active Cyclitol Precursors Via Chemoenzymatic Method And Synthesis Of A Nucleoside PrecursorOguzkaya, Funda 01 June 2006 (has links) (PDF)
ABSTRACT
STEREOSELECTIVE SYNTHESIS OF OPTICALLY ACTIVE CYCLITOL
PRECURSORS VIA CHEMOENZYMATIC METHOD
AND
SYNTHESIS OF A NUCLEOSIDE PRECURSOR
Oguzkaya, Funda
M.S., Department of Chemistry
Supervisor: Prof. Dr. Cihangir Tanyeli
June 2006, 99 pages
& / #945 / ' / -acetoxylation of & / #945 / ,ß / -unsaturated cyclic ketones was adjusted via Mn(OAc)3 in regioselective manner. Then, PLE hydrolysis was carried out so as to afford enantiomerically enriched & / #945 / ' / -acetoxylated and & / #945 / ' / -hydroxylated cyclic compounds. From our knowledge about the literature and previous works dealing with & / #945 / ' / -hydroxylated products which are easily racemized, protection was directly adjusted via acetylation so as to prevent this possibility. Resulting enantiomerically enriched products were subjected to Upjohn Dihydroxylation to obtain cyclitol precursors and following Luche Reduction of ketone was adjusted so as to obtain corresponding cyclitols.
In addition with such synthetic design, firstly dimethyl cyclopent-3-ene-1,1-dicarboxylate was obtained so as to reach in former manner 3-cyclopentene-1,1-dicarboxylic acid, and in latter manner cyclopent-3-enecarboxylic acid. Resulting compound was converted to 6-iodo-2-oxa-bicyclo[2.2.1]heptan-3-one and followingly to the target nucleoside precursor which is 2-oxa-bicyclo[2.2.1]hept-5-en-3-one.
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The role of Nm23-H1 in uveal melanoma /Bakalian, Silvin. January 2008 (has links)
Uveal Melanoma (UM) is the most common malignant intra-ocular tumor in adults. Despite the high accuracy of clinical diagnosis and advances in local treatment, more than 50% of UM patients develop metastasis within ten years of initial diagnosis. NM23 is a human metastasis suppressor gene. Reduced Nm23-H1 expression is correlated with high metastatic potential in a variety of different cancers including melanoma. C-Met is a receptor tyrosine kinase (RTK) that has been known to stimulate the invasive growth and increase the metastatic potential of cancer cells. Expression of c-Met is correlated with high mortality rate in UM patients. Treatment with CQX-2 inhibitors showed promise as an adjuvant therapy in adenocarcinoma of the colon. A previous report from our laboratory showed that topical treatment with Nepafenac (a CQX-2 inhibitor) delayed the progression of the primary tumor and the formation of metastasis in the experimental rabbit model of UM. / The purpose of this thesis is to investigate the expression levels ofNm23-H1 in UM cell lines with different metastatic potentials, in paraffin embedded tissues from primary tumors of UM patients, and in an experimental rabbit model. In addition, the aim of this thesis is to determine whether treating human uveal melanoma cell lines with Nepafenac would increase the expression levels of Nm23-H1 and decrease the expression levels of c-Met in vitro (UM cell lines) and in vivo (experimental rabbit model). / To achieve our goal, we used several types of assays in our UM cell lines and paraffin embedded tissues from patient samples and experimental rabbit model, including quantitative immunostaining, quantitative Real-time PCR, and small interference RNA (siRNA). / The Real-time PCR results of five human uveal melanoma cell lines showed that expression of Nm23-HI is higher in cell lines with low metastatic potential compared to those with high metastatic potential. The invasive ability of the uveal melanoma cell lines increased after silencing Nm23-H1 expression with siRNA. The increased immunostaining intensity of Nm23-H1 in patient samples is associated with better survival rate. Moreover, treatment with Nepafenac resulted in increase of Nm23-H1 levels and decrease of c-Met levels in both the UM cell lines and the experimental rabbit model. / In conclusion, Nm23-H1 is a potent prognostic marker to predict the survival rate of UM patients and it has the potential to identify high-risk patients. To the best of our knowledge, this is the first study to show that treatment with COX-2 inhibitor causes an upregulation of Nm23-H1 and downregulation of c-Met in UM. Therefore, treatment with COX-2 inhibitors may be a useful strategy as an adjuvant therapy for UM patients.
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