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Polimorfismo G22A do gene ADA e abortamento espontâneo recorrente: ausência de associação.Nunes, Daniela Prudente Teixeira 16 December 2010 (has links)
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Previous issue date: 2010-12-16 / Adenosine deaminase (ADA), an enzyme coded by ADA gene (20q13.11) acts in adenosine metabolism and it is involved in the modulation of the immune response. ADA gene G22A polymorphism originates two co-dominants alleles ADA*01 and ADA*02 and influences the level of ADA enzyme in the organism. Apparently it has a fundamental role in gestational maintenance. The ADA*02 allele has been associated as protector effect against recurrent spontaneous abortion (RSA) in European Caucasian women. Aim: To investigate if ADA gene G22A polymorphism is associated with occurrence of RSA in Brazilian women. Methods: After obtaining the written consent 311 women were selected to compose two groups: G1 with previous history of RSA (n=129) and G2 without previous history of RSA (n=182). Genomic DNA was isolated from peripheral blood using commercial kits. The PCR-RFLP method was used to identify ADA gene G22A polymorphism. p>0005 was considered statistically significant. Results: The frequencies of ADA*01;*01, ADA*01;*02 and ADA*02;*02 genotypes were similar in both groups (G1 and G2) with no statistically significance differences observed (p = 0,7170; x2 = 0,6653; GL = 2). ADA*01 and ADA*02 alleles frequencies were 95,6% and 4,4% in G1 group and 94,9% and 5,1% in G2 group, respectively (p = 0,8433; OR = 1,179; CI 95%: 0,5340 2.601). Conclusion: The results suggest that ADA alleles ADA*01 and ADA*02 are not associated with RSA. It xvi is possible that the reduction of ADA levels resulting from the presence of at least one ADA*02 allele do not have a role against abortion in Brazilian women. / Polimorfismo G22A do gene ADA e abortamento espontâneo recorrente: ausência de associação Introdução: A adenosina deaminase (ADA), uma enzima codificada pelo gene ADA (20q13.11), atua no metabolismo da adenosina e modula a resposta imune. O polimorfismo G22A deste gene origina os alelos co-dominantes ADA*01 e ADA*02 e influencia o nível de expressão da enzima ADA, que possui papel fundamental na manutenção da gestação. O alelo ADA*02 tem sido associado a um efeito protetor contra o abortamento espontâneo recorrente (AER) em mulheres caucasianas européias. Objetivo: Investigar se o polimorfismo G22A do gene ADA se associa à ocorrência de AER em brasileiras. Métodos: Após obtenção do Termo de Consentimento Livre e Esclarecido (Parecer CEP FAMERP 308/2008), 311 mulheres foram selecionadas para compor dois grupos: G1 com histórico de AER (N=129) e G2 sem histórico de AER (N=182). O DNA genômico foi extraído a partir de sangue periférico com o uso kit comercial. O polimorfismo G22A do gene ADA foi identificado com o uso do método PCR-RFLP. O valor p>0,005 foi considerado significante. Resultados: As frequências dos genótipos ADA*01;*01, ADA*01;*02 e ADA*02;*02 foram semelhantes entre os grupos e não apresentaram diferenças estatisticamente significantes (p = 0,7170; χ2 = 0,6653; GL = 2). As frequências dos alelos ADA*01 e ADA*02 em G1 foram iguais a 95,6% e 4,4%; em G2, 94,9% e 5,1%, respectivamente (p=0,8433; OR=1,179; IC 95%: 0,5340-2.601). Conclusões: Os resultados sugerem que xiv
os alelos ADA*01 e ADA*02 do gene ADA não estão associados ao AER. É possível que a redução nos níveis da ADA resultantes do alelo ADA*02 não apresente um efeito protetor contra o AER em brasileiras.
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Structural Studies On Enzymes From Salmonella Typhimurium Involved In Propionate Metabolism: Biodegradative Threonine Deaminase, Propionate Kinase And 2-Methylisocitrate LyaseSimanshu, Dhirendra Kumar 09 1900 (has links)
I formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics
Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an
attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems.
By this time (beginning of 2002), I had finished my first semester and a major
part of the course work, so we decided to start a new project focusing on some of the
unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his
Ph. D. from the lab, helped me in literature work and in finding targets for structural
studies. Finally, we decided to target enzymes involved in the propionate etabolism.
The pathways for propionate metabolism in Escherichia coli as well as Salmonella
typhimurium were just established and there were no structural information available for
most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and
succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle.
Most of the enzymes involved in 2-methylcitrate pathway were not characterized
biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize
these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected
a complete diffraction data to a resolution of 2.5 Å which was later on extended to a
resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems.
Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the
plausible rationale for different substrate specificities of these two enzymes. Due to
unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good
diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we
decided to target other enzymes involved in propionate metabolism.
We looked into the literature for the metabolic pathways by which propionate is
synthesized in the Salmonella typhimurium and finally decided to target enzymes present
in the metabolic pathway which converts L-threonine to propionate. Formation of
propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme
towards propionate than acetate.
One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron
density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies
suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments.
By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally
successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme.
The structural studies carried out on these three enzymes have provided structural
as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future.
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Extrato de semente de Syzygium cumini (L.) Skeels reduz o dano renal e hepático provocado pela exposição aguda ao metilmercúrio em ratos neonatos / Syzygium cumini seed extract prevent the renal and liver impairment caused by acute methylmercury treatment in neonatal ratsAbdalla, Faida Husein 25 January 2010 (has links)
Methylmercury (MeHg) is a potent neuro and nephrotoxicant in several animal species including humans, particularly during their development. The purpose of this study was to investigate the effects of aqueous seed extract of Syzygium cumini (L.) Skeels (Scc) on the acute MeHg treatment in neonatal rats. Neonatal rats (P2) received orally a single dose of MeHg (10 mg/kg) and also two doses of Scc. After two days, the effects of this treatment were investigated in the cerebral cortex, hippocampus, kidney, liver and urine samples of rats. We observed that N-Acetyl-β-d-glucosaminidase (NAG) activity in the kidney and urine was higher in MeHg-group when compared with the control group. Similarly, the lipid peroxidation levels were higher in the liver and kidney as well as the Adenosine deaminase (ADA) activity increased in the hippocampus, kidney and liver. These results indicate that increased NAG and ADA activities, as well as thiobarbituric acid reactive species (TBARS) levels may play a critical role in MeHg nephrotoxicity. The most relevant finding in our investigation was that acute MeHg treatment in neonatal rats caused liver and renal impairment and Scc was able to prevent such effects. It appears that mechanisms related to scavenging activity of Scc could be involved with its protection effect.
Key words: Methylmercury; Syzygium cumini; N-Acetyl-β-d-glucosaminidase; rat; adenosine deaminase / O metilmercúrio (MeHg) é um agente tóxico potente tanto para o sistema nervoso central como para o renal. Provoca danos nos seres humanos e em ratos, particularmente durante o estágio de desenvolvimento. Neste estudo, ratos em desenvolvimento (P2) receberam uma dose única de MeHg (10 mg/kg), por via oral e/ou duas doses do extrato aquoso de sementes de Syzygium cumini (L.) Skeels (Scc). Após dois dias (P4), foram investigados os efeitos deste tratamento no córtex cerebral, hipocampo, rim, fígado e urina. Observamos que a atividade da N-acetil-β-d-glicosaminidase (NAG) nos rins e na urina foi maior no grupo que recebeu MeHg, quando comparado com o grupo controle. Da mesma forma, os níveis de peroxidação lipídica foram maiores no fígado e rim e a atividade da adenosina deaminase (ADA) encontrou-se elevada no hipocampo, rins e fígado, nos ratos tratados com MeHg. Estes resultados indicam que o aumento da atividade da NAG e da ADA, bem como os níveis das espécies reativas ao ácido tiobarbitúrico (TBARS), desempenham um papel importante como marcadores de nefrotoxicidade causada pelo MeHg. Assim, o achado mais relevante na nossa investigação foi a de que o tratamento agudo com MeHg em ratos neonatos pode provocar nefrotoxicidade e a administração do Scc pode reverter estes efeitos provavelmente devido as propriedades antioxidantes de Scc.
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Molecular mechanisms controlling immunoglobulin class switch recombination / Mécanismes moléculaires régulant la commutation isotypique des immunoglobulinesSchiavo, Ebe 30 September 2013 (has links)
Lors des réponses immunitaires, le répertoire des lymphocytes B est diversifié par l’hypermutation somatique (HMS) et la commutation isotypique (CI), dépendant d’«activation-induced cytidine deaminase» (AID), qui introduit des lésions dans les gènes Ig. Une déficience d’AID cause un défaut d’HMS et de CI; par contre, une délétion du domaine C-terminal d’AID cause un défaut spécifique de la CI, suggérant que ce domaine interagit avec des facteurs spécifiques de la CI. Pour identifier ces facteurs, nous avons étudié une immunodéficience présentant un défaut de la CI non lié à la carence d’AID ni à un défaut d’HMS. De plus, les cassures de l’ADN ne sont pas détectées au niveau des gènes Ig suggérant qu’AID n’est pas correctement ciblée dans ces loci. Nous avons identifié et analysé des candidats : Spt6, les cohésines et le complexe Smc5/6. Dans les cellules B activées, AID interagit avec Spt6, Spt5, l’ARN polymérase II et le complexe PAF. Par contre, les cohésines pourraient réguler la structure du locus IgH lors de la CI et la voie de réparation des cassures de l’ADN générées pendant la CI. Ces résultats contribuent à une meilleure compréhension des étapes de la CI. / During immune responses, B cell repertoire is diversified through somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR require activation-induced cytidine deaminase (AID), which induces DNA damage. While AID deficiency abrogates SHM and CSR, C-terminal truncations impair CSR without affecting SHM and it has been proposed that AID C-terminal domain associates with CSR-specific factor(s). In order to identify these factors, we studied a human CSR-specific immunodeficiency, characterized by normal SHM and AID expression. B cells from these patients do not display DSBs at switch (S) regions, suggesting that they might lack an AID-binding factor(s) required to target AID to S regions during CSR. Through a multi- approach strategy, we identified and analyzed candidate factors, including Spt6, the cohesin complex and the Smc5/6 complex. We show that, in B cells poised to undergo CSR, AID is in a complex with Spt6, Spt5, the RNA polymerase II and the PAF complex while cohesins might regulate the 3D structure of the IgH locus and the pathway of DSBs repair at the Ig S regions. Our work thus contributes to a better understanding of the CSR reaction.
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EFEITO DO EXTRATO DE SYZYGIUM CUMINI, IN VITRO, NA ATIVIDADE DE ENZIMAS QUE DEGRADAM NUCLEOTÍDEOS E NUCLEOSÍDEOS DE ADENINA E ÉSTERES DE COLINA E SOBRE O PERFIL OXIDATIVO EM PACIENTES COM DIABETES MELLITUS TIPO 2 / EFFECT OF SYZYGIUM CUMINI EXTRACT, IN VITRO, ON THE ACTIVITY OF ENZYMES THAT DEGRADE ADENINE NUCLEOTIDES AND NUCLEOSIDES AND ESTERS OF CHOLINE AND ON THE OXIDATIVE PROFILE IN PATIENTS WITH TYPE 2 DIABETES MELLITUSBona, Karine Santos de 01 February 2011 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diabetes mellitus (DM) is a metabolic disorder of multiple etiology characterized by chronic hyperglycemia resulting from deficiency of production and / or insulin action. This state of hyperglycemia may cause a variety of cardiovascular, renal, neurological and eye complications. Adenosine deaminase (ADA), ecto-5 'nucleotidase (5'NT) and Acetylcholinesterase (AChE) are important enzymes responsible for regulating the levels of adenosine (ado) and acetylcholine (ACh) respectively, and changes in their activities have been demonstrated in various diseases, including Diabetes. Syzygium cumini is a plant mostly used for the treatment of DM and presents hypoglycemic, anti-inflammatory, antipyretic and antioxidants properties. The aim of this study was to verify the effect of aqueous leaf extract of Syzygium cumini (ASc) in 100 and 200μg/mL concentrations, in vitro, on enzymes 5'NT in platelets, ADA in erythrocytes and platelets and AChE in erythrocytes, as well as on parameters of oxidative stress in samples of Type 2 diabetic patients. The results showed an increase in the activity of ADA and 5'NT in platelets from diabetic (n=30) compared to the control group (n=17), as well as in the levels of thiobarbituric acid reactive species (TBARS). ASc at concentrations of 100 and 200 μg / mL was able to reverse these effects. Correlations between 5 NT activity and triglycerides levels, as well as between ADA activity and glucose levels were also found in this work. An increase in the activity of enzymes ADA and AChE in erythrocytes of patients with type 2 diabetes (n=30) compared to the control group (n=20), as well as changes in parameters of oxidative stress, such as increased levels of TBARS and decrease in superoxide dismutase (SOD) activity and levels of non-protein sulfhydryl groups (NP-SH) in these cells also were observed. Likewise, ASc reduced the ADA and AChE activities and lipid peroxidation, and reversed the effect of the evaluated oxidative parameters. Still, there were found significant positive correlations between levels of Vitamin C and protein sulfhydryl groups (P-SH), plasma glucose and levels of P-SH and NP-SH, levels of P-SH and ADA activity, besides a negative correlation between TBARS and NP-SH levels. Therefore, it is possible to suggest that the ASc was able to promote a compensatory response in the platelet function and may act in the maintenance of adenosine levels and vasodilatation and thereby, contributes to the maintenance of the vascular integrity which is important in the hyperglycemic state. It is also possible that ASc might modulate the levels of ACh, interfering with oxidative stress and / or inflammatory processes from the diabetic state. So far, these results confirm the already known antioxidants properties of Syzygium cumini, which makes this compound present significant effects on the cellular metabolism, as well as the reduction and prevention of cardiovascular disease risk in diabetics. / O Diabetes mellitus (DM) é uma disfunção metabólica de múltipla etiologia caracterizado por hiperglicemia crônica resultante da deficiência da produção e/ou ação da insulina. Esse estado de hiperglicemia pode provocar uma série de complicações cardiovasculares, renais, neurológicas e oculares. A adenosina deaminase (ADA), ecto-5 nucleotidase (5 NT) e acetilcolinesterase (AChE) são importantes enzimas responsáveis por regular os níveis de adenosina (ado) e acetilcolina (ACh), respectivamente, e alterações nas suas atividades têm sido demonstradas em várias doenças, incluindo o DM. O Syzygium cumini é uma das plantas mais utilizadas no tratamento do DM, apresenta propriedades hipoglicêmicas, antiinflamatórias, antipiréticas e antioxidantes. O objetivo deste estudo foi verificar o efeito do extrato aquoso das folhas de Syzygium cumini (ASc), nas concentrações de 100 e 200 μg/mL, in vitro, sobre as enzimas 5 NT em plaquetas, ADA em eritrócitos e plaquetas e AChE em eritrócitos, bem como sobre parâmetros de estresse oxidativo em amostras de pacientes diabéticos Tipo 2. Os resultados demonstraram um aumento na atividade das enzimas ADA e 5 NT em plaquetas de diabéticos (n=30) em relação ao grupo controle (n=17), assim como nos níveis de espécies reativas ao ácido tiobarbitúrico (TBARS). ASc, nas concentrações de 100 e 200 μg/mL foi capaz de reverter estes efeitos. Correlações entre a atividade da 5 NT e os níveis de triglicerídeos, bem como entre a atividade da ADA e os níveis de glicose também foram encontradas nesse trabalho. Um aumento na atividade das enzimas ADA e AChE em eritrócitos de pacientes com Diabetes tipo 2 (n=30) em relação ao grupo controle (n=20), além de alterações nos parâmetros de estresse oxidativo, como aumento nos níveis de TBARS e redução na atividade da enzima Superóxido Dismutase (SOD) e nos níveis de grupamentos sulfidrílicos não protéicos (NP-SH) nessas células também foram observados. Igualmente, ASc reduziu a atividade das enzimas ADA e AChE e a lipoperoxidação, e reverteu o efeito dos parâmetros oxidantes avaliados. Ainda foram encontradas correlações positivas significativas entre os níveis de Vitamina C e grupamentos sulfidrílicos protéicos (P-SH), glicose plasmática e níveis de P-SH e NP-SH, níveis de P-SH e atividade da ADA, além de correlação negativa entre os níveis de TBARS e NP-SH. Portanto, é possível sugerir que o ASc foi capaz de promover uma resposta compensatória na função plaquetária, podendo atuar na manutenção dos níveis de adenosina e na vasodilatação e, assim, contribuindo para a manutenção da integridade vascular importante no estado hiperglicêmico, tendo em vista o papel cardioprotetor exercido pela mesma. Também é possível que ASc possa modular os níveis de ACh, interferindo no estresse oxidativo e/ou nos processos inflamatórios provenientes do estado diabético. Ao mesmo tempo esses resultados corroboram com as já conhecidas propriedades antioxidantes de Syzygium cumini, o que faz com que esse composto apresente efeitos significativos no metabolismo celular, bem como na redução e prevenção do risco de doença cardiovascular em diabéticos.
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Species-specific DNA markers for improving the genetic management of tilapiaSyaifudin, Mochamad January 2015 (has links)
The tilapias are a group of African and Middle Eastern cichlid fish that are widely cultured in developed and developing countries. With many different species and sub-species, and extensive use of interspecies hybrids, identification of tilapia species is of importance in aquaculture and in wild populations where introductions occur. This research set out to distinguish between tilapia species and sub-species by retrieving species-specific nuclear DNA markers (SNPs) using two approaches: (i) sequencing of the coding regions of the ADA gene; and (ii) next-generation sequencing, both standard RADseq and double-digest RADseq (ddRADseq). The mitochondrial DNA (mtDNA) marker cytochrome c oxidase subunit I (COI) was used to verify tilapia species status. ADA gene sequence analysis was partially successful, generating SNP markers that distinguished some species pairs. Most species could also be discriminated using the COI sequence. Reference based analysis (RBA: using only markers found in the O. niloticus genome sequence) of standard RADseq data identified 1,613 SNPs in 1,002 shared RAD loci among seven species. De novo based analysis (DBA: based on the entire data set) identified 1,358 SNPs in 825 loci and RBA detected 938 SNPs in 571 shared RAD loci from ddRADseq among 10 species. Phylogenetic trees based on shared SNP markers indicated similar patterns to most prior phylogenies based on other characteristics. The standard RADseq detected 677 species-specific SNP markers from the entire data set (seven species), while the ddRADseq retrieved 38 (among ten species). Furthermore, 37 such SNP markers were identified from ddRADseq data from a subset of four economically important species which are often involved in hybridization in aquaculture, and larger numbers of SNP markers distinguished between species pairs in this group. In summary, these SNPs are a valuable resource in further investigating hybridization and introgression in a range of captive and wild stocks of tilapias.
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An Examination of Cytosine Deaminase plus 5-Fluorocytosine Suicide Gene Therapy In Combination With Cisplatin Chemotherapy For the Treatment Of Cancer / Suicide Gene Therapy of CancerNethercot, Victoria 08 1900 (has links)
Cancer is a disease characterized by complexity and unpredictability. Consequently, its treatment is difficult and all too often unsuccessful. Almost all cancers are treated with some combination of the traditional anti-cancer armamentarium: surgery, chemotherapy, and radiotherapy. Recently, however, gene therapy has emerged as a promising addition to this existing repertoire. Its application as a single agent, or in combination with other anti-cancer treatments is proving successful in both pre-clinical and clinical settings. In this work I have investigated the combination of a conventional chemotherapy drug, cisplatin, with a type of cancer gene therapy known as cytosine deaminase + 5-fluorocytosine suicide gene therapy. Suicide gene therapy is the intracellular conversion of non-toxic prodrug to its active form by a metabolic enzyme of non-mammalian origin. There are many established enzyme/prodrug combinations, but here the bacterial enzyme cytosine dearninase (CDA) was used to convert inert 5-fluorocytosine (5FC) to highly toxic 5-fluorouracil (5FU). Of the various vector systems for therapeutic gene delivery, adenoviral (Ad) vectors have proven particularly suitable for application to cancer. This work used a first generation adenovirus type 5 vector expressing the enzyme cytosine deaminase (AdCDA) cloned from E. coli. The combination of AdCDA/5FC with cisplatin was chosen because the combination of 5FU and cisplatin, both of which are used extensively in cancer treatment, has proven effective clinically and demonstrates synergy in vitro. This combination was evaluated in murine mammary carcinoma MTIA2 cells, human colorectal carcinoma HT29 cells, HT29pl4 cells, the photofrin resistant sub-line of HT29 cells, and murine melanoma Bl6/FIO cells. The classical clonogenic assay was used to evaluate this combination treatment since it provides an accurate indication of the effectiveness a cancer treatment will have in vivo. AdCDA infected MTIA2, HT29, and HT29pl4 cell lines exhibited a dose response to increasing concentrations of SFC that was significantly different from control vector infected cells. Similarly, uninfected cells demonstrated a dose response to increasing concentrations of cisplatin. The effect of the combination on clonogenic survival, administered in the sequence of a 48 h exposure to SFC followed by 1 h exposure to cisplatin, was greater than additive compared to the effect of the two treatments alone.
F10 cells exhibited a dose response to increasing concentrations of cisplatin. However, it could not be shown reproducibly that AdCDA infected FlO cells exhibited a dose response to SFC that differed significantly from control vector infected cells. Work with the FlO cells was inconclusive regarding the combination treatment, but it rendered information regarding the sensitivity of these cells to what is hypothesized to be an unidentified component present in some preparations of 5FC.
Evaluation of this treatment in vivo, using both murine and human tumor cell lines, will further define the potential of AdCDA/5FC + cisplatin as a clinically relevant cancer treatment. / Thesis / Master of Science (MSc)
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A comparison of different analytes in distinguishing transudate and exudate of pleural effusion, and the use of adenosine deaminase activity in the differentiation of tuberculous and non-tuberculous pleural effusion.January 1998 (has links)
by Mo-Lung Chen. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 70-75). / Abstract also in Chinese. / ABBREVIATIONS --- p.iv / LIST OF TABLES --- p.v / LIST OF FIGURES --- p.vii / ACKNOWLEDGEMENT --- p.ix / ABSTRACT --- p.xi / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- BACKGROUND --- p.4 / Chapter 2.1 --- Production of pleural fluid --- p.4 / Chapter 2.2 --- Pathophysiology of pleural effusion --- p.5 / Chapter 2.3 --- Separating exudate from transudate --- p.8 / Chapter 2.4 --- Receiver operating characteristic curve --- p.9 / Chapter CHAPTER 3. --- ADENOSINE DEAMINASE --- p.12 / Chapter 3.1 --- Background --- p.12 / Chapter 3.2 --- Differentiation of tuberculous and non-tuberculous pleural effusion --- p.12 / Chapter CHAPTER 4. --- MATERIALS AND METHODS --- p.17 / Chapter 4.1 --- Patients --- p.17 / Chapter 4.2 --- Collection and handling of specimens --- p.17 / Chapter 4.3 --- Diagnostic criteria --- p.18 / Chapter 4.4 --- Methods --- p.19 / Chapter 4.4.1 --- Routine chemistries --- p.19 / Chapter 4.4.2 --- Protein zone electrophoresis --- p.19 / Chapter 4.4.3 --- Adenosine deaminase --- p.19 / Chapter 4.4.3.1 --- Instrumentation --- p.22 / Chapter 4.4.3.2 --- Optimization of reaction time --- p.24 / Chapter 4.4.4 --- Analytical performance --- p.24 / Chapter 4.4.4.1 --- Imprecision --- p.24 / Chapter 4.4.4.2 --- Recovery --- p.26 / Chapter 4.4.4.3 --- Lowest detection limit --- p.26 / Chapter 4.4.4.4 --- Linearity --- p.26 / Chapter 4.4.4.5 --- Interference by ammonia --- p.26 / Chapter 4.4.4.6 --- Interference by turbidity --- p.28 / Chapter 4.4.4.7 --- Interference by haemoglobin --- p.28 / Chapter 4.4.4.8 --- Interference by bilirubin --- p.29 / Chapter 4.4.4.9 --- Storage stability of ADA at -80°C --- p.29 / Chapter 4.4.5 --- Statistical analysis --- p.30 / Chapter CHAPTER 5. --- RESULTS OF OPTIMIZATION AND EVALUATION EXPERIMENTS --- p.31 / Chapter 5.1 --- Optimization of reaction time --- p.31 / Chapter 5.2 --- Analytical performance --- p.31 / Chapter 5.2.1 --- Imprecision --- p.31 / Chapter 5.2.1.1 --- Within-run --- p.31 / Chapter 5.2.1.2 --- Between-run --- p.31 / Chapter 5.2.2 --- Recovery --- p.31 / Chapter 5.2.3 --- Lowest detection limit --- p.34 / Chapter 5.2.4 --- Linearity --- p.34 / Chapter 5.2.5 --- Interference by / Chapter 5.2.5.1 --- ammonia --- p.34 / Chapter 5.2.5.2 --- turbidity --- p.34 / Chapter 5.2.5.3 --- haemoglobin --- p.37 / Chapter 5.2.5.4 --- bilirubin --- p.37 / Chapter 5.2.6 --- Storage stability of ADA at -80°C --- p.37 / Chapter CHAPTER 6. --- TRANSUDATIVE AND EXUDATIVE PLEURAL EFFUSION --- p.39 / Chapter 6.1 --- Results of routine chemistries --- p.39 / Chapter 6.2 --- Decision thresholds by ROC curve --- p.39 / Chapter 6.3 --- Discussion --- p.39 / Chapter 6.4 --- Results of protein zone electrophoresis --- p.49 / Chapter 6.5 --- Discussion --- p.51 / Chapter 6.6 --- Comparison of protein zone electrophoresis and Light's criteria --- p.55 / Chapter 6.7 --- Discussion --- p.55 / Chapter CHAPTER 7. --- TUBERCULOUS AND NON-TUBERCULOUS EXUDATIVE PLEURAL EFFUSION --- p.59 / Chapter 7.1 --- Results of adenosine deaminase assay --- p.59 / Chapter 7.2 --- Combinations of analysis --- p.59 / Chapter 7.3 --- Decision thresholds by ROC curve --- p.64 / Chapter 7.4 --- Discussion --- p.64 / Chapter CHAPTER8. --- GENERAL DISCUSSION --- p.69 / REFERENCES --- p.70
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Estudo associativo entre a esquizofrenia e o polimorfismo G22A no gene da adenosina deaminase (ADA)Dutra, Gustavo Pimentel 09 June 2008 (has links)
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Previous issue date: 2008-06-09 / O sistema purin?rgico, especialmente a adenosina, pode desempenhar um papel na patofisiologia da esquizofrenia. A ativa??o dos receptores de adenosina A1 inibe a libera??o de v?rios neurotransmissores como o glutamato, a dopamina, a serotonina e a acetilcolina, e diminui a atividade neuronal pela hiperpolariza??o p?s-sin?ptica. A adenosina (ADA) participa no metabolismo da adenosina convertendo-a em inosina. O polimorfismo funcional mais freq?ente da ADA (22 G - (seta)A) (ADA1 *2) exibe 20-30% menos atividade enzim?tica em indiv?duos com o gen?tipo G/A do que em indiv?duos com o gen?tipo G/G. Esse polimorfismo foi avaliado em 152 pacientes esquizofr?nicos e 111 controles saud?veis. N?s observamos uma diminui??o significativa na freq??ncia do alelo de baixa atividade ADA1 *2 em pacientes esquizofr?nicos (7 4,6%) em rela??o aos controles (13 17%, p= 0,032, OR= 2,6). Esses resultados sugerem que o alelo ADA1 *2 associado ? baixa atividade da ADA, e conseq?entemente a altos n?veis de adenosina, ? menos freq?ente entre os pacientes esquizofr?nicos.
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Avalia??o dos efeitos de f?rmacos benzodiazep?nicos sobre o catabolismo de nucleot?deos, nucleos?deos e acetilcolina em enc?falo de zebrafish adulto : (Danio rerio)Altenhofen, Stefani 26 December 2012 (has links)
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Previous issue date: 2012-12-26 / Benzodiazepines, such as diazepam and midazolam, are a widely used class of drugs for anxiety treatment, with anxiolytic, hypnotic, and anticonvulsant properties. The use of zebrafish (Danio rerio) as a model for evaluating pharmacological mechanisms has gained importance due to their rapid development and high sensitivity to drugs. Studies have shown that behavioral parameters were altered in zebrafish after benzodiazepine treatment. Many neurotransmitter systems have been identified in this species, including purinergic and cholinergic system. Purinergic system is characterized by the action of ATP and adenosine on purinoreceptor P2 and P1, respectively. The levels of these molecules are regulated by ectonucleotidases, especially nucleoside triphosphate diphosphohydrolase (NTPDases) and ecto-5'-nucleotidase, which constitute the extracellular cascade for ATP hydrolysis to adenosine. Adenosine can be subsequently deaminated to inosine by action of adenosine deaminase (ADA). ATP is coreleased with other neurotransmitters, including acetylcholine, and has been demonstrated that adenosine can control the release of acetylcholine. Cholinergic system is characterized by the action of acetylcholine (ACh) on muscarinic and nicotinic receptors. The level of this molecule is regulated by acetylcholinesterase (AChE), which catalyzes degradation of ACh into choline and acetate. Since there are few reports relating these enzyme activities and the action mechanism of benzodiazepines, the aim of this study was evaluated the in vitro and ex vivo effects of classical benzodiazepines, such as diazepam and midazolam, on NTPDase, ecto-5'nucleotidase, ADA, and AChE activities in zebrafish brain and gene expression pattern in treatments that induced changes in enzyme activity in the ex vivo experiments. In order to elucidate whether diazepam or midazolam has direct effects on these enzymes, we performed in vitro experiments. Diazepam, at 500 μM, promoted a decrease on ATP hydrolysis (66%), whereas this drug, at 10-500 μM, reduced ADP hydrolysis (40-54%, respectively). Midazolam also decreased ATP (16-71% for 10-500 μM, respectively) and ADP hydrolysis (48-73% for 250-500 μM, respectively), and ecto-ADA activity (26-27.5% for 10-500 μM, respectively). Diazepam and midazolam did not induce significant changes on ecto-5?-nucleotidase activity at the concentrations tested. Concerning to AChE activity, 500 μM diazepam promoted a decrease on ACh hydrolysis (19%), whereas midazolam, at 50-500 μM, reduced AChE activity (18-79%, respectively). For ex vivo experiments, diazepam or midazolam exposures did not alter NTPDase activities in zebrafish brain membranes. AMP hydrolysis was decreased in animals treated with of 0.5 and 1mg/L midazolam (31.5% and 36.1%, respectively) when compared to the control group. However, diazepam was unable to alter ecto-5 -nucleotidase. Both drugs significantly decreased the ecto-ADA activity, whereas diazepam and midazolam reduced the adenosine hydrolysis at a concentration of 1.25 mg/L (30.85%) and 1 mg/L (32.8%), respectively. Diazepam did not alter cytosolic-ADA activity; however, the exposure to 0.1 mg/L midazolam induced a significant increase in cytosolic-ADA (39.9%) when compared with the control group. The gene expression pattern demonstrated that the CD73 transcript levels were increased (41.7%) after treatment with 0.5 mg/L midazolam. Moreover, the changes caused by diazepam and midazolam in the ADA activity are not related to the transcriptional control. Concerning the cholinerg signaling, diazepam decreased ACh hydrolysis at 1.25 mg/L (30.7%) when compared to the control group. Similarly, the exposure to 0.5 mg/L midazolam also changed the enzymatic activity of AChE promoting an increase in the ACh hydrolysis (36.7%). It is possible to suggest that these drugs can induce a direct effect on the enzyme activities, since we observed a decreased on nucleotide and nucleoside hydrolysis after in vitro exposure. In addition, the alteration on AMP hydrolysis, ADA and AChE activities suggest a modulation of extracellular adenosine and ACh levels induced by benzodiazepine exposure. / F?rmacos benzodiazep?nicos, como diazepam e midazolam, s?o muito usados na pr?tica cl?nica para o tratamento da ansiedade, possuindo propriedades ansiol?ticas, hipn?ticas e anticonvulsivantes. O uso do zebrafish (Danio rerio) como modelo para avaliar mecanismos farmacol?gicos tem ganhado grande import?ncia devido ao r?pido desenvolvimento e alta sensibilidade a drogas que essa esp?cie possui. Estudos t?m demonstrado que par?metros comportamentais mostraram-se alterados em zebrafish ap?s tratamento com benzodiazep?nicos. Muitos sistemas de neurotransmiss?o foram identificados nessa esp?cie, incluindo os sistemas purin?rgico e colin?rgico. O sistema purin?rgico ? caracterizado pela a??o do ATP e adenosina (ADO) nos purinoreceptores P2 e P1, respectivamente. Os n?veis dessas mol?culas s?o regulados pela a??o das ectonucleotidases, especialmente as nucleos?deo trifosfato difosfoidrolases (NTPDases) e a ecto-5 -nucleotidase, que catalisam a hidr?lise do ATP a adenosina. A adenosina pode ser desaminada a inosina pela a??o da adenosina desaminase (ADA). O ATP ? coliberado com outros neurotransmissores, entre eles a acetilcolina, e tem sido demonstrado que a adenosina pode controlar a libera??o de acetilcolina. O sistema colin?rgico ? caracterizado pela a??o da acetilcolina (ACh) nos receptores muscar?nicos e nicot?nicos. O n?vel dessa mol?cula ? regulado pela acetilcolinesterase (AChE), que catalisa a degrada??o da ACh em colina e acetato. Uma vez que existem poucos relatos relacionando esses sistemas enzim?ticos e a a??o de f?rmacos benzodiazep?nicos, o objetivo deste estudo foi avaliar o efeito in vitro e ex vivo do tratamento com f?rmacos benzodiazep?nicos, tais como diazepam e midazolam, sobre a atividade das NTPDases, ecto-5'-nucleotidase, ADA and AChE no enc?falo de zebrafish e o padr?o de express?o g?nica nos tratamentos que induziram altera??es na atividade enzim?tica nos experimentos ex vivo. A fim de elucidar se o diazepam e o midazolam t?m efeitos diretos nessas enzimas, experimentos in vitro foram realizados. Na concentra??o de 500 μM, o diazepam diminuiu a hidr?lise de ATP (66%) e, nas concentra??es de 10-500 μM, este f?rmaco reduziu a hidr?lise de ADP (40-54%, respectivamente). O midazolam tamb?m diminuiu a hidr?lise do ATP (16-71% para 10-500 μM, respectivamente), ADP (48-73% para 250-500 μM, respectivamente) e a atividade da ecto-ADA (26-27,5% para 10-500 μM, respectivamente). Diazepam e midazolam n?o induziram altera??es significativas sobre a atividade da ecto-5?-nucleotidase nas concentra??es testadas. Com rela??o ? atividade da AChE, o diazepam, 500 μM, promoveu uma diminui??o na hidr?lise de ACh (19%) e o midazolam, nas concentra??es de 50-500 μM, reduziu a atividade da AChE (18-79%, respectivamente). Nos experimentos ex vivo, as exposi??es ao diazepam e midazolam n?o alteraram a atividade enzim?tica das NTPDases em membranas cerebrais de zebrafish. A hidr?lise do AMP diminuiu em animais tratados com 0.5 mg/L e 1 mg/L de midazolam (31.5% e 36.1%, respectivamente) quando comparados com o grupo controle. Entretanto, o diazepam foi incapaz de alterar a atividade da ecto-5 -nucleotidase. Ambos os f?rmacos diminu?ram significativamente a atividade da ecto-ADA, sendo que o diazepam e o midazolam reduziram a hidr?lise da adenosina na concentra??o de 1.25 mg/L (30.85%) e 1 mg/L (32.8%), respectivamente. O diazepam n?o alterou a atividade da ADA citos?lica, no entanto a exposi??o a 0.1 mg/L de midazolam induziu um significativo aumento na atividade dessa enzima (39.9%) quando comparado ao grupo controle. O padr?o de express?o g?nica demonstrou que os n?veis de transcritos do CD73 apresentaram-se reduzidos (41,7%) ap?s o tratamento com 0.5 mg/L de midazolam. Com rela??o a sinaliza??o colin?rgica, diazepam diminuiu a hidr?lise da ACh na concentra??o de 1.25 mg/L (30.7%) quando comparado ao grupo controle. Similarmente, a exposi??o ? concentra??o de 0.5 mg/L de midazolam tamb?m alterou a atividade enzim?tica da AChE, promovendo um aumento na hidr?lise da ACh (36.7%). ? poss?vel sugerir que essas drogas podem induzir um efeito direto na atividade enzim?tica, uma vez que foi observada uma diminui??o na hidr?lise de nucleot?deos e nucleos?deos ap?s a exposi??o in vitro. Al?m disso, as altera??es na hidr?lise do AMP e atividade da ADA e da AChE sugerem uma modula??o dos n?veis extracelulares de adenosina e acetilcolina induzidos pela exposi??o aos f?rmacos benzodiazep?nicos.
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