Rozprawy doktorskie: „TTrx”

1

Gregory, Mary Sarah-Jane, i n/a. "Thioredoxin and Oxidative Stress". Griffith University. School of Health Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040301.082639.

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The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.

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2

Gregory, Mary Sarah-Jane. "Thioredoxin and Oxidative Stress". Thesis, Griffith University, 2004. http://hdl.handle.net/10072/367183.

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The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.
Thesis (Masters)
Master of Philosophy (MPhil)
School of Health Sciences
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3

Kerkmann, Heiko. "Differentielle Interaktionen hochpotenter Lokalanästhetika mit TTX-sensitiven und TTX-resistenten Natriumströmen an Spinalganglienzellen der erwachsenen Ratte". Wettenberg : VVB Laufersweiler, 2005. http://deposit.d-nb.de/cgi-bin/dokserv?idn=978034708.

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4

Iakovleva, Irina. "Selection of transthyretin amyloid inhibitors". Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-123939.

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Amyloidosis is a group of clinical disorders caused by the aggregation of specific proteins into abnormal extracellular deposits. Today, 31 different proteins have been linked to amyloid diseases including transthyretin-related amyloidosis (ATTR). ATTR occurs through the aggregation of either wild-type plasma protein transthyretin (TTR) or a mutated form. TTR is a homotetramer that under normal circumstances functions as a carrier of thyroxine and retinol binding protein. The aggregation cascade requires dissociation of the tetramer into monomers, and preventing this dissociation represents a potential mode of intervention. Interestingly, small molecules, referred as kinetic stabilizers, can bind to TTR’s thyroxine-binding site (TBS) and such molecules are currently being used as a therapeutic approach to impair tetramer dissociation. The efficacy of TTR stabilization is directly correlated to the binding affinity of the ligand to TBS. However, the binding of the ligand to TTR in vivo can be affected by other plasma components resulting in poor efficacy. Thus, the selectivity of ligands is an important parameter. We have designed an assay where the ability to stabilize TTR can be directly evaluated in plasma and we have investigated the stabilizing effect of nine potential TTR binders (Paper I). The results, surprisingly, revealed that the binding affinity of molecules has a poor correlation to its selectivity. However, the nature of protein-ligand complex formation can also be described by enthalpic (∆H) and entropic (∆S) energy contributions. ∆H represents the change in chemical bonds and frequently requires a higher order of orientation compared to the ∆S component, which mainly represents the hydrophobic effect via the exclusion of water. We hypothesized that ligands possessing high ΔH in binding to their co-partner would also be more specific in a complex environment such as plasma. By applying a thermodynamic analysis using isothermal titration calorimetry, we found that the selectivity in plasma correlates well with the ∆H contribution and might, therefore, be a better predictor for selectivity. Luteolin was found to be a highly selective stabilizer of TTR and was investigated further (Paper II). The ligand displayed a significant rescuing effect in both cell culture and animal models. However, luteolin undergoes rapid enzymatic degradation in the liver and this impairs its use as a potential therapeutic drug. To attempt to circumvent this issue, we modified the most exposed hydroxyl group thus rendering the molecule inert towards glucuronidation (Paper III). The substitutions resulted in higher stability in the face of hepatic degradation molecules, but they also affected the selectivity in a negative manner. The screening for new TTR stabilizers resulted in the discovery of tetrabromobisphenol A, which displayed a very high selectivity (Paper IV). This study also included a comparison with the drug Vyndaqel™ which currently is in clinically use, and showed how the dosage could be altered to acquire a better level of saturation and possibly also a better clinical effect. Taken together we present new molecules with the ability to stabilize TTR, and these can serve as scaffolds for the design of new drugs. We present a method to measure the efficacy of a TTR-stabilizing drugs in a complex matrix and as well as a way to adjust the dosage of existing drugs. We also show that the selectivity of a drug is affected by the relative proportion of ∆H and ∆S, and this is of interest for drug design in general.

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5

Kerkmann, Heiko [Verfasser]. "Differentielle Interaktionen hochpotenter Lokalanästhetika mit TTX-sensitiven und TTX-resistenten Natriumströmen an Spinalganglienzellen der erwachsenen Ratte / Heiko Kerkmann". Gießen : Universitätsbibliothek, 2015. http://d-nb.info/1069740705/34.

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6

Pullmann, Martin. "Dynamische Blockierung TTX-resistenter Natriumkanäle durch Lokalanästhetika". [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969813872.

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7

Panenic, Robert. "TTX-induced disuse of mammalian skeletal muscle". Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59523.

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Previous reports of the effects of tetrodotoxin (TTX)-induced muscular disuse have demonstrated alterations in muscle force, speed, and fatiguability that might suggest changes in the quality of contractile proteins. These studies were extended to the effects of TTX-induced disuse on the Ca$ sp{2+}$-activation characteristics of myofibrillar ATPase of the rat gastrocnemius. Atrophic responses after TTX treatment were as previously reported with a significant decrease in left gastrocnemius weight (g) compared to the control-pump (C) group (1.25 $ pm$ 0.04 for C vs 0.72 $ pm$ 0.04 for TTX, X $ pm$ SEM, p $ leq$ 0.01). Myofibrillar protein yield (mg$ cdot$g$ sp{-1}$ wet weight) was also depressed (92.8 $ pm$ 4.6 for C vs 70.3 $ pm$ 3.7 for TTX; p $ leq$ 0.01). Maximum ATPase of myofibrils (nmol Pi$ cdot$mg$ sp{-1} cdot$min$ sp{-1}$) was decreased (424 $ pm$ 46 for C vs 199 $ pm$ 27 for TTX, p $ leq$ 0.01). Furthermore, the Hill n which reflects the cooperative aspects of Ca$ sp{2+}$-activation of the myofibrillar ATPase was significantly depressed (1.58 $ pm$ 0.07 for C vs 1.29 $ pm$ 0.09 for TTX; p $ leq$ 0.05) after TTX treatment. The results of the present study suggest that muscle perturbations that result from TTX-induced disuse are at least partially related to changes in the myofibrillar fraction.

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8

Kerkmann, Heiko [Verfasser]. "Differentielle Interaktionen hochpotenter Lokalanästhetika mit TTX-sensitiven und TTX-resistenten Natriumströmen an Spinalganglienzellen der erwachsenen Ratte / vorgelegt von Heiko Kerkmann". Wettenberg : VVB Laufersweiler, 2005. http://d-nb.info/978034708/34.

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9

Azevedo, Ana do Carmo Ramalho Moreira. "Familial amyloid polyneuropathy: TTR sequencing and "in silico" analysis". Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/15608.

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Mestrado em Biomedicina Molecular
Familial amyloid polyneuropathy (FAP) or paramiloidosis is an autosomal dominant neurodegenerative disease with onset on adult age that is characterized by mutated protein deposition in the form of amyloid substance. FAP is due to a point alteration in the transthyretin (TTR) gene and until now more than 100 amyloidogenic mutations have been described in TTR gene. FAP shows a wide variation in age-at-onset (AO) (19-82 years, in Portuguese cases) and the V30M mutation often runs through several generation of asymptomatic carriers, before expressing in a proband, but the protective effect disappear in a single generation, with offspring of late-onset cases having early onset. V30M mutation does not explain alone the symptoms and AO variability of the disease observed in the same family. Our aim in this study was to identify genetic factors associated with AO variability and reduced penetrance which can have important clinical implications. To accomplish this we genotyped 230 individuals, using a directautomated sequencing approach in order to identify possible genetic modifiers within the TTR locus. After genotyping, we assessed a putative association of the SNPs found with AO and an intensive in silico analysis was performed in order to understand a possible regulation of gene expression. Although we did not find any significant association between SNPs and AO, we found very interesting and unreported results in the in silico analysis since we observed some alterations in the mechanism of splicing, transcription factors binding and miRNAs binding. All of these mechanisms when altered can lead to dysregulation of gene expression, which can have an impact in AO and phenotypic variability. These putative mechanisms of regulation of gene expression within the TTR gene could be used in the future as potential therapeutical targets, and could improve genetic counselling and follow-up of mutation carriers.
A Polineuropatia amiloidótica familiar (FAP) ou paramiloidose é uma doença neurodegenerativa autossómica dominante com início na vida adulta sendo caracterizada pela deposição da proteína mutada na forma de substância amilóide. A FAP é devida a uma mutação pontual no gene transtirretina (TTR) e até agora mais de 100 mutações amiloidogénicas foram descritas neste gene. A FAP apresenta uma grande variação na idade de início (AO) (19-82 anos, nos casos portugueses) e a mutação V30M pode segregar através de várias gerações de portadores assintomáticos, antes de se expressar num probando. No entanto, este efeito protetor pode desaparecer numa única geração, com os filhos de casos tardios a apresentarem um início precoce. A mutação V30M não explica por si só os sintomas e a variabilidade da AO observada dentro de uma mesma família. O nosso objetivo neste trabalho foi identificar fatores genéticos associados com a variabilidade da AO e a penetrância reduzida. De modo a cumprir este objetivo genotipámos 230 doentes, por sequenciação automática, para identificar possíveis modificadores genéticos dentro do locus da TTR. Após a genotipagem, investigamos uma possível associação dos SNPs encontrados com a AO e realizamos uma intensiva análise in silico de modo a perceber uma possível regulação da expressão génica. Apesar de não termos encontrado nenhuma associação entre os SNPs e a AO, encontrámos resultados não descritos e muito interessantes na análise in silico dado termos observado algumas alterações a nível do mecanismo de splicing, ligação de fatores de transcrição e ligação de miRNAs. Todos estes mecanismos quando alterados podem levar à desregulação da expressão do gene, o que pode ter um impacto na AO e variabilidade fenotípica. Estes mecanismos hipotéticos da regulação da expressão génica no gene da TTR podem ser úteis para no futuro serem aplicados como potenciais alvos terapêuticos, beneficiando o aconselhamento genético e o follow-up dos portadores da mutação.

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Lobato, Luísa Maria Correia Lopes. "A nefropatia na Polineuropatia Amiloidótica Familiar de Tipo Português (TTR V30M)". Doctoral thesis, Instituto de Ciências Biomédicas Abel Salazar, 2003. http://hdl.handle.net/10216/63699.

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Lobato, Luísa Maria Correia Lopes. "A nefropatia na Polineuropatia Amiloidótica Familiar de Tipo Português (TTR V30M)". Tese, Instituto de Ciências Biomédicas Abel Salazar, 2003. http://hdl.handle.net/10216/63699.

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Häfner, Sibylle. "Strukturelle und physikochemische Determinanten von Substanzen für die Blockade TTX-resistenter Natriumkanäle". [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969261888.

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13

Dreimann, Marc. "Therapeutika des neuropathischen Schmerzes blockieren den TTX-resistenten Natriumkanal des peripheren nozizeptiven Systems". [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963032496.

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Donato, Micheline Freire. "Purificação, caracterização bioquímica e eletrofisiológica da toxina Mic6c7NTX da Peçonha da Serpente Micrurus ibiboboca (Merrem, 1820)". Universidade Federal da Paraíba, 2008. http://tede.biblioteca.ufpb.br:8080/handle/tede/6863.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Snake venoms contain a complex arsenal of protein bio-active components, many of these being neurotoxins (NTXs). These snakes have high neurotoxic activity venom, corresponding to the Elapidae family, which includes coral snakes (Micrurus) whose venom contains circa 90-95% of low molecular mass protein components. Among these, several are postsynaptic neurotoxins or α- NTXs (MM = 6-9 kDa). The Micrurus ibiboboca (Merren, 1820) is a snake of the Elapidae family witch is quite common in the Northeast of Brazil. In spite of the great diversity of species of Micrurus, scarce works involving the nervous system with isolated and pure toxins of those serpents has been developed in level biochemical, pharmacological and electrophysiological. The aim of this study was to purify the toxin Mic6c7NTX of the Micrurus ibiboboca venom, characterize to biochemically and electrophysiologically the toxin Mic6c7NTX in the peripheral nervous system (PNS) of rats, evaluating alterations in the record of the Compound Action Potential (CAP) of the isolate nerve and the toxin activity on the voltage-dependent sodium channels (Nav) in the neurons of the dorsal root ganglion (DRG). The venom was extracted from the Micrurus ibiboboca collected in Paraiba State (Brazil). Initially, electrophysiological tests (current clamp method) using the single sucrose gap technique were accomplished with crude venom (100μg/mL). It was observed that in this concentration the crude venom caused reduction in the CAP amplitude (25%). This neurotoxity led into an intriguing question: what components of the venom would promote to reduction in the excitability of the nerve? Based upon this question, I decided to purify the venom throughout the Liquid Chromatography of the High Performance (HPLC) of the Cation Exchange Chromatography (CIEX) and the Reverse Phase Chromatography (RPC). The molecular mass (MM) of the raw toxin was determined by mass-spectrometry (MALDI-QTOF/ MS) and N-terminal sequence by means of Edman s Degradation. The search for similarity with other toxins was accomplished against proteomic data bank. The CIEX profile showed 19 fractions and the highest peak fraction was used for the second dimension. The toxin Mic6c7NTX obtained by RPC showed elution in 26.7%of the acetonitrile (ACN) and MM 7.047.56Da. The obtained partial N-terminal sequence showed 31 aminoacid residues. The search for similarity of structure and function showed great similarity (65%) with other short chain α-NTXs Australian elapids snakes. The electrophysiological studies (single sucrose gap technique) showed that the toxin Mic6c7NTX (1 μM) reduced the excitability of the isolate nerve similarly to the reduction observed in the crude venom about 21%. Other CAP parameters such as despolarization speed (DSCAP), repolarization time (τCAP) and peak of time (PTCAP) did not show alterations. This suggests that the toxin may be affecting the Nav channels. For the confirmation of that hypothesis experiments were accomplished with whole cell patch-clamp technique in DRG neurons. This results showed that the toxin Mic6c7NTX (1 WM) abolished completely the current of Nav channels sensitive the tetrodotoxin (TTX-S). Also the Nav channels TTX resistant (TTX-R) were investigated in the presence of the Mic6c7NTX toxin previously using TTX (100 nM). This results showed that the toxin Mic6c7NTX (100 nM) abolished completely the current of Nav channels TTX-R and IC50 = 30nM. However, reversion of this blocking was not observed. The present study biochemically and electrophysiologically characterized an α-NTX of the Micrurus ibiboboca elapid snake. Furthermore, it showed a potent toxin with affinity Nav channels TTX-S and TTX-R of the PNS. This is the first α-NTX isolated and identified of the venom from the Micrurus ibiboboca (Merrem, 1820) snake.
As serpentes da família Elapidae possuem uma peçonha com alta atividade neurotóxica e capacidade de letalidade. Fazem parte dessa família as serpentes corais americanas (gênero Micrurus) com suas peçonhas contendo cerca de 90-95% de componentes protéicos, sendo na sua maior parte neurotoxinas com baixa massa molecular (6-8 kDa), podendo ser destacadas as neurotoxinas com ação pós-sinápticas ou α-Neurotoxinas (α-NTX). A Micrurus ibiboboca (Merrem, 1820) é uma serpente da família Elapidae, comum na região Nordeste. Apesar da grande diversidade de espécies do gênero Micrurus sp., escassos trabalhos envolvendo atividade de toxinas isoladas e puras destas peçonhas e sistema nervoso têm sido desenvolvidos em nível bioquímico, farmacológico ou eletrofisiológico. O objetivo desse estudo foi purificar a toxina Mic6c7NTX da peçonha de M. ibiboboca, caracterizar bioquímicamente e investigar com ferramentas eletrofisiológicas a ação da toxina no Sistema Nervoso Periférico (SNP) de ratos avaliando alterações no Potencial de Ação Composto (PAC) do nervo isquiático isolado e a atividade da toxina nos canais para sódio dependentes de voltagem (Nav) em neurônios do gânglio da raiz dorsal (DRG). A peçonha da M. ibiboboca foi extraída de serpentes coletadas no Estado da Paraíba (Brasil). Inicialmente, ensaios eletrofisiológicos com o método de current clamp utilizando a técnica de single sucrose gap foram realizados com a peçonha bruta (100 Wg/mL). Os resultados mostraram que a peçonha bruta nessa concentração promoveu redução na amplitude do PAC (25%). Esse efeito da toxina na excitabilidade do nervo levantou o questionamento: Que componentes da peçonha estariam causando essa diminuição da excitabilidade? A peçonha foi purificada por meio de Cromatografia Líquida de Alta Performance (HPLC), de troca catiônica (CIEX) e fase reversa (RPC). Na sequência, os picos da CIEX foram submetidos à RPC e posteriormente analisados por espectrometria de massas (MALDI-TOF/MS) que detectou a massa molecular da toxina Mic6c7NTX de 7.047,56 Da. Em seguida, foi determinado o seu N-terminal por Degradação de Edman que apresentou 31 resíduos de aminoácidos e serviu de estudo para a bioinformática na busca por similaridade em banco de dados proteômicos com outras toxinas protéicas, demonstrando que a toxina Mic6c7NTX apresentou similaridade (65%) com α-NTXs de cadeia curta de serpentes elapídicas australianas. Posteriormente, foi investigado o efeito da toxina isolada no SNP. Os estudos eletrofisiológicos em single sucrose gap demonstraram que a toxina Mic6c7NTX (1 WM) reduziu a excitabilidade do nervo isolado de forma similar à observada pela peçonha bruta. Não foram observadas alterações significantes em outros parâmetros do PAC, como velocidade de despolarização (VDPAC), tempo de repolarização (τPAC) e tempo de pico (PTPAC), sugerindo que a toxina atuasse num sítio de ligação específico dos [Escreva uma citação do documento ou o 11 canais Nav no SNP. Para a confirmação dessa hipótese foram realizados experimentos de voltage clamp com a técnica de whole cell patch-clamp em cultura primária de neurônios DRG da medula espinhal de ratos. Os resultados mostraram que a toxina Mic6c7NTX (1 WM) aboliu completamente as correntes dos canais Nav sensíveis à tetrodotoxina (TTX-S). Também foi investigado o efeito da toxina sobre a população de canais Nav resistentes à TTX (TTX-R), utilizando previamente TTX (100 nM) para bloquear os canais Nav TTX-S. Os registros com a toxina Mic6c7NTX (100 nM) demonstraram um bloqueio total da corrente nos canais Nav TTX-R dos DRGs e uma IC50 da toxina em torno de 30 nM. Também foi observado que essa toxina se liga aos canais Nav de forma lenta e irreversível. O presente estudo caracterizou bioquímica e eletrofisiologicamente uma α-NTX da serpente elapídica Micrurus ibiboboca. Farmacologicamente, trata-se de uma potente toxina com afinidade aos canais Nav TTX-S e TTX-R do SNP. Essa é a primeira α-NTX isolada e caracterizada da peçonha da serpente Micrurus ibiboboca (Merrem, 1820).

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Andersson, Karin, M. Pokrzywa, Ingrid Dacklin i Erik Lundgren. "Inhibition of TTR aggregation-induced cell death : a new role for serum amyloid P component". Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-65622.

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BACKGROUND: Serum amyloid P component (SAP) is a glycoprotein that is universally found associated with different types of amyloid deposits. It has been suggested that it stabilizes amyloid fibrils and therefore protects them from proteolytic degradation. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we show that SAP binds not only to mature amyloid fibrils but also to early aggregates of amyloidogenic mutants of the plasma protein transthyretin (TTR). It does not inhibit fibril formation of TTR mutants, which spontaneously form amyloid in vitro at physiological pH. We found that SAP prevents cell death induced by mutant TTR, while several other molecules that are also known to decorate amyloid fibrils do not have such effect. Using a Drosophila model for TTR-associated amyloidosis, we found a new role for SAP as a protective factor in inhibition of TTR-induced toxicity. Overexpression of mutated TTR leads to a neurological phenotype with changes in wing posture. SAP-transgenic flies were crossed with mutated TTR-expressing flies and the results clearly confirmed a protective effect of SAP on TTR-induced phenotype, with an almost complete reduction in abnormal wing posture. Furthermore, we found in vivo that binding of SAP to mutated TTR counteracts the otherwise detrimental effects of aggregation of amyloidogenic TTR on retinal structure. CONCLUSIONS/SIGNIFICANCE: Together, these two approaches firmly establish the protective effect of SAP on TTR-induced cell death and degenerative phenotypes, and suggest a novel role for SAP through which the toxicity of early amyloidogenic aggregates is attenuated.

Epub 2013 Feb 4.

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Jampala, Raghavendra. "Synthesis of AG10 analogs and optimization of TTR ligands for Half-life enhancement (TLHE) of Peptides". Scholarly Commons, 2017. https://scholarlycommons.pacific.edu/uop_etds/2975.

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The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidosis, which is most commonly, caused by aggregation of Immunoglobulin (Ig) light chains or transthyretin (TTR) in the cardiac muscle, represent an important and often underdiagnosed cause of heart failure. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. AG10 is a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of TTR in serum samples obtained from patients with amyloid cardiomyopathy. The oral bioavailability and selectivity of AG10, makes it a very promising candidate to treat TTR amyloid cardiomyopathy. Understanding the reason behind the potency of AG10 would be beneficial for designing stabilizers for other amyloid diseases. This would be possible by designing and synthesizing structural analogues of AG10. Here we report the synthesis, characterization and analysis of AG10 analogs and the comparison of the in vitro activities of the synthesized analogs. The tremendous therapeutic potential of peptides has not been fulfilled and potential peptide therapies that have failed far outnumber the successes so far. A major challenge impeding the more widespread use of peptides as therapeutics is their poor pharmacokinetic profile, due to short In vivo half-life resulting from inactivation by serum proteases and rapid elimination by kidneys. Extending the In vivo half-life of peptides is clearly desirable in order for their therapeutic potential to be realized, without the need for high doses and/or frequent administration. Covalent conjugation of peptides to macromolecules (e.g. polyethylene glycol or serum proteins such albumin) has been the mainstay approach for enhancing the In vivo half-life of peptides. However, the steric hindrance and immunogenicity of these large macromolecules often compromises the In vivo efficacy of the peptides. Recently, our laboratory established the first successful reversible method of extending the half-life of peptides using serum protein TTR. The approach involved the use of a TTR Ligand for Half-life Extension (TLHE-1) which binds to TTR with high specificity and affinity. We have shown that our technology extends the half-life of multiple peptides without seriously affecting their activity. Our main objective here is to modify the structure of TLHE1 using linkers with different length and composition to optimize its affinity and selectivity for TTR in human serum.

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Feldman, Chris R. "Evolutionary Genetics of Tetrodotoxin (TTX) Resistance in Snakes: Tracking a Feeding Adaptation from Populations Through Clades". DigitalCommons@USU, 2008. https://digitalcommons.usu.edu/etd/159.

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Understanding the nature of adaptive evolution has been the recent focus of research detailing the genetic basis of adaptation and theoretical work describing the mechanics of adaptive evolution. Nevertheless, key questions regarding the process of adaptive evolution remain. Ultimately, a detailed description of the ecological context, evolutionary history, and genetic basis of adaptations is required to advance our understanding of adaptive evolution. To address some of the contemporary issues surrounding adaptive evolution, I examine phenotypic and genotypic changes in a snake feeding adaptation. Adaptations can arise through fixation of novel mutations or recruitment of existing variation. Some populations of the garter snakes Thamnophis sirtalis, T. couchii, and T. atratus possess elevated resistance to tetrodotoxin (TTX), the lethal toxin of their newt prey. I show that TTX resistance has evolved independently through amino acid changes at critical sites in a voltage-gated sodium channel protein (Nav1.4) targeted by TTX. Thus, adaptive evolution has occurred multiple times in garter snakes via de novo acquisition of beneficial mutations. Detailing the genetic basis of adaptive variation in natural populations is the first step towards understanding the tempo and mode of adaptive evolution. I evaluate the contribution of Nav1.4 alleles to TTX resistance in two garter snake species from central coastal California. Allelic variation in Nav1.4 explains 29% and 98% of the variation in TTX resistance in T. atratus and T. sirtalis, respectively, demonstrating that Nav1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of trait change and coevolution. Patterns of convergent evolution are cited as some of the most compelling examples of the strength of natural selection in shaping organismal diversity. Yet repeated patterns may tell us as much about the constraints that restrict evolution as about the importance of natural selection. I present data on convergent molecular adaptations in parallel arms races between diverse snakes and amphibians from across the globe. Six snake species that prey on TTX bearing amphibians have independently acquired amino acid changes in Nav1.4. The derived mutations are clustered in two portions of the gene, often involving the same sites and substitutions. While a number of amino acid changes can make Nav1.4 insensitive to TTX, most of these negatively impact or abolish the ion-conducting function of the protein. Thus, intramolecular pleiotropy likely prevents most replacements from becoming fixed and imposes limits on protein evolution.

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Belous, Gregg R. "Novel Machine Learning Techniques for Left Ventricular Analysis in Echocardiography". Thesis, Griffith University, 2020. http://hdl.handle.net/10072/400568.

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Transthoracic (TTX) echocardiography (echo) is vital for the diagnosis and treatment of heart disease. It is also essential for the determination of appropriate therapeutic procedures and monitoring disease progression and response. An important requisite of TTX echo is the quantitative assessment of left ventricular (LV) size and function from its manually traced endocardial border. However, the diagnostic accuracy of this routine task is often adversely affected by artifact and signal dropout, producing substantial observer variability and uncertainty in clinical diagnosis. With the advent of machine learning, computer aided detection (CAD) systems have addressed organ segmentation through robust landmark localization techniques and a reliance on anatomical shape prior models to guide the segmentation process. Shape priors models are most effective when shape variations can be captured by a parametric distribution, and suffcient training data is available. However, in the absence of these conditions, results are invariably much poorer. In addition, the problem of insuffcient training data not only presents challenges to shape prior models, but also classification algorithms, as well. Stress echo (SE) is a widely used functional test for the detection of obstructive coronary artery disease. The interpretive process involves the careful comparison of pre- and post-exercise echo sequences across a number of echocardiographic views. This is a time consuming and highly subjective task. While CAD systems have been shown to be feasible for automated reporting in other areas of medical imaging, they have not been applied to the task of identifying abnormal stress echocardiograms. An automated approach would not only be a useful adjunct to physicians reporting SE, but also aid in physician training of SE reporting. Motivated by these challenges, this thesis presents four novel machine learning techniques for LV analysis in echo. Firstly, two anatomical shape prior models are proposed: online relational manifold learning (ORML) and dual subspace segment projection learning (DSSPL). ORML is formulated to address the challenge of modelling complex shape subspaces. ORML serves to learn a mapping function between a low dimension image manifold and shape manifold. However, different to existing subspace learning approaches, ORML leverages the input image to target more contextually relevant regions between both manifold structures, leading to robust LV shape inference for volume prediction, and the formulation of a shape prior model through a more principled shape selection strategy. ORML demonstrates improved segmentation performance over current benchmark methods, and shows an excellent level of agreement with an expert. DSSPL addresses the challenges of modelling complex shape variations under the scenario of high dimension low sample size (HDLSS) training data. It serves to compose shapes from an ensemble of shape segments where each segment is formed using two subspaces: global shape subspace and segment-specific subspace, each necessary for extracting global shape patterns and local patterns, respectively. This ensures general shape plausibility in regions of signal drop-out or missing boundary information, and also more localized flexibility. The reconstructive properties of DSSPL reduces information loss and leverages the subspaces to provide contiguous shapes without any post-processing. Comprehensive experimental analysis is performed on three databases from different medical imaging systems across X-Ray, MRI, and echo. DSSPL outperforms all compared benchmarks in terms of its shape generalization ability and segmentation performance. The third method proposed is dual subspace discriminative projection learning (DSDPL), which addresses the challenge of image classification, also under the HDLSS training data scenario. Unlike traditional projection learning frameworks that assume discriminative features share a common subspace, DSDPL instead serves to decompose original high dimensional data, via learned projection matrices, into class-shared and class-specific subspaces. The learned projection matrices are jointly constrained with l2;1 sparse norm and LDA terms while the reconstructive properties reduce information loss. Regression-based terms are also included to facilitate a more robust classification approach, using extracted class-specific features for better classification. Results show improved classification accuracy with DSDPL over current benchmark subspace learning methods and deep learning models. The fourth method proposed is deep stage-coupled attentive feature extraction (DSCAFE) for identifying abnormal stress echocardiograms. DSCAFE is a deep neural network model that consists of stage-coupled attentive feature extraction (SCAFE) blocks for extracting the most salient information from connected echo sequences. SCAFE blocks are composed of 3D residual network streams and dual-attention gated mechanisms, which provide more targeted focus across each echo sequence by also taking into account the observed features from the corresponding view at the opposing exercise stage. A recurrent neural network feature aggregation strategy is then employed to model the extracted low dimension spatio-temporal features for more accurate classification. When compared against an expert reviewer, DSCAFE achieved a concordance of 86.5% from a clinical SE dataset. This research draws on machine learning knowledge across a diverse range of domains. While results show definitive improvements over current benchmark methods for LV analysis, the proposed methods are also adaptable to a wide range of computer vision tasks.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Eng & Built Env
Science, Environment, Engineering and Technology
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Kerkmann, Heiko [Verfasser]. "Modell und Berechnung von Konzentrations-Inhibitionskurven hoch- und niedrigpotenter Substanzen am TTX-resistenten Natriumkanal der erwachsenen Ratte / Heiko Kerkmann". Gießen : Universitätsbibliothek, 2013. http://d-nb.info/1065394853/34.

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Grzymala-Lubanski, Bartosz. "Anticoagulation treatment in patients with a mechanical heart valve". Doctoral thesis, Umeå universitet, Institutionen för folkhälsa och klinisk medicin, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-128355.

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Background Every year about 2,500 patients in Sweden undergo surgery for heart valve disease, primarily in the aortic valve. In contrast to the mitral valve, which can be repaired in 70% of the cases, the aortic valve is normally replaced by a mechanical or biological prosthesis. A mechanical heart valve (MHV) necessitates lifelong anticoagulation treatment with a vitamin K antagonist, most commonly warfarin, due to the high thrombogenicity of the prosthesis. The quality of the warfarin treatment is crucial in these patients. Compared to other countries, treatment quality in Sweden is very high; nonetheless, there is always room for improvement. One of the ways to achieve this improvement is to implement computerized dosing assistance. Treatment recommendations for anticoagulation intensity are based on few and old studies, making these recommendations uncertain. There is therefore a need for studies designed to establish the appropriate level of anticoagulation therapy. Aim The aim of these studies was to investigate the efficacy and safety of anticoagulation treatment among patients with mechanical heart valve prostheses in Sweden; to assess whether computerized dosing can increase the treatment quality; to investigate the influence of the treatment quality, measured by Time in Therapeutic Range (TTR) and INR variability, on the risk of complications and, finally, to establish the optimal intensity of anticoagulation treatment in this group of patients. Methods Data were obtained from AuriculA – a national quality registry established in 2006, which currently includes approximately 50% of all patients treated with oral anticoagulation in Sweden. Study II used only data from AuriculA. 769,933 warfarin-dosing suggestions proposed by the dosing algorithm in AuriculA were analysed. Accepted dose suggestions (590,939) were compared with 178,994 manually-changed doses in regard to the resultant INR value, measured as mean error (deviation from target INR) and hit rate (number of INR samples within the target range 2-3). In study III, AuriculA was used to identify patients in Sundsvall and Malmö in the period 2008 – 2011 who were receiving warfarin for a mechanical heart valve prosthesis, as well as to retrieve their INR data. Data on background characteristics and bleedings or thromboembolic complications were manually retrieved from medical records by two investigators. A total of 534 patients with mechanical heart valve prostheses were divided into quartiles based on TTR and were compared regarding the risk of complications. For Studies I and IV, data from AuriculA were merged with the Swedish National Patient Register, SWEDEHEART/ Heart surgery, and the Swedish Cause of Death Register, comprising in total 77,423 patients on warfarin with 217,804 treatment years. Every treatment period registered in AuriculA was given an individual identification number. During the study period a patient could have any number of treatment periods. The number of complications in total and in different patient groups within the study population was investigated. Complications were defined by ICD-10 codes. Major bleeding was defined as an event necessitating hospital treatment and given a discharge diagnosis with one of the ICD-10 codes reflecting bleeding, as listed in the Appendix. Bleeding events were divided into intracranial, gastrointestinal and other bleedings. Thromboembolic complications consist of venous events (deep vein thrombosis, pulmonary embolism, venous stroke) or arterial events (stroke, TIA, acute myocardial infarction, peripheral arterial embolism). Data were analysed using both simple, descriptive statistical methods and various tests such as Mann-Whitney (or two sample Wilcoxon), T-test, Chi 2 test, ANOVA, multivariate analysis with logistic regression and survival analysis with Cox Regression with proportional hazard assumption. Results Treatment quality Mean TTR among all patients in Study I was 76.5% whereas patients with mechanical heart valve prostheses had a TTR of 74.5%. The annual incidence of major bleeding or thromboembolic events among all patients was 2.24% and 2.65%, respectively. The incidence of intracranial bleeding was 0.37% per year in the general population and 0.51% among patients with mechanical heart valve prostheses, who also had a higher bleeding rate in total (3.37% per year). Both the mean and median errors were smaller (0.44 vs. 0.48 and 0.3 vs. 0.4, respectively) and the hit rate was higher (0.72 vs. 0.67) when the dose suggested by the algorithm was accepted, compared to when it was manually changed. TTR In Study III there was no significant difference in the risk of thromboembolism regardless of TTR level. Risk of bleeding in quartiles I and II was more than two times higher than in the quartile with TTR >82.9. In Study IV, lower TTR (≤70%) was associated with a significantly higher rate of complications when compared with TTR >70%. Bleeding risk was higher in the group with lower TTR (HR=2.43, CI 2.02-2.89, p<0.001). After dividing patients into TTR quartiles, the rate of complications in total was significantly higher in quartiles I to III compared with quartile IV, which had the highest TTR. Risk of thromboembolism, major bleeding and death was higher in the first and second quartile compared to the quartile with the highest TTR. INR variability Higher INR variability above mean (≥0.40) was related to a higher rate of complications compared with lower INR variability (<0.40) as shown in Study IV. Bleeding risk was higher in the group with INR variability ≥0.40 (HR = 2.15, CI 1.75-2.61, p<0.001). Comparison of quartile IV, which had the lowest INR variability, with the other three revealed that quartiles I and II, which had the highest INR variability, had significantly worse outcomes for all complications except for thromboembolic events, plus also death in quartile II. TTR and INR variability combined High variability and low TTR combined was associated with a higher risk of bleedings (HR 2.50, CI 1.99-3.15), death (3.34, CI 2.62-4-27) and thrombosis (1.55, CI 1.21-1.99) compared to the best group. Level of anticoagulation Higher warfarin treatment intensity (mean INR 2.8-3.2 vs. 2.2-2.7) was associated with a higher rate of bleedings (HR 1.29, CI 1.06-1.58), death (1.73, CI 1.38-2.16) and complications in total (1.24, CI 1.06-1.41) after adjustment for MHV position, age and comorbidity. Conclusion Warfarin treatment quality is crucial for patients with mechanical heart valve prostheses. Computerized dosing assistance could help maintain high warfarin treatment quality. Well-managed treatment with TTR ≥70% and INR variability below mean <0.40 is associated with a lower risk of serious complications compared with a lower TTR and higher INR variability. No benefit of higher warfarin treatment intensity was found for any valve type or position.

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Lobato, Maria Inês Rodrigues. "Estudo de marcadores moleculares relacionados a cadeia dos retinóides (TTR e RXR[beta] e suas relações com endofenótipos clínicos e dismórficos em esquizofrenia)". reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2004. http://hdl.handle.net/10183/5554.

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Duong, Sun. "Evaluation of novel fluorescent probes for in vivo Transthyretin amyloid using fibrils generated in vitro under varying conditions". Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-154611.

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Transthyretin (TTR) amyloidosis is a disease that appears in three variants. One variant affects the elderly population with heart failure, the other two variants are hereditary and caused by an amino acid substitution in the gene, resulting in polyneuropathy and/or heart issues depending on the amino acid substitution. However, in all three variants, other organs may also be affected with amyloid deposition in the disease course. Amyloid fibrils of TTR (ATTR) contains a mixture of full-length protein and fragments (50-127). Luminescent conjugated oligothiophenes (LCO’s) are novel amyloid binding probes used to stain amyloid fibrils and these amyloid probes have the feature of characterizing the amyloid structure in terms of fluorescence spectra. Apart from LCO’s, a few other amyloid binding probes are used to stain recombinant amyloid transthyretin and native transthyretin for binding studies. The majority of generated TTR aggregates in vitro did not have the characteristic fluorescence spectra when bound to LCO’s and was observed as a clumped gel-like aggregate. The generation of recombinant TTR fibrils in vitro using the mutant TTR-T49M to obtain an aggregation prone fragment (50-127) after being treated with cyanogen bromide had a low yield of in vivo amyloid-like fibrils, but with characteristic LCO spectra. Carpal tunnel ATTR often precedes ATTR deposition in heart tissue. Amyloid transthyretin in carpal tunnel tissues was stained with LCO’s and used as a reference in the comparison against the in vitro generated recombinant amyloid transthyretin fibrils. This project also includes quantification of amyloid transthyretin in a few selected parts of the carpal tunnel tissue using ImageJ. In the long run this method could help in diagnosing TTR amyloidosis.

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Costa, Luiz Augusto de Oliveira. "Estudo de viabilidade técnica, econômica e ambiental preliminar da aplicação da tecnologia de Tratamento Térmico de Resíduos e Materiais Multifásicos-TTRM a resíduos de terra diatomácea de usina genérica de produção de biodiesel". Universidade do Estado do Rio de Janeiro, 2014. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=8130.

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O cenário mundial de matriz energética apresenta dados crescentes de contribuição das energias renováveis. No Brasil, o governo tem realizado esforços para aumento da parcela de combustíveis renováveis, e com isso isso também para o aumento da produção de biodiesel. O principal processo de fabricação de biodiesel com seu polimento em via seca gera quantidades significativas de resíduos, dentre eles, o resíduo de terra diatomácea, com potenciais características de inflamabilidade. Em contrapartida existe a Política Nacional de Resíduos Sólidos com objetivos de não geração, redução, reutilização, reciclagem e redução de periculosidade dos resíduos, e envio para aterro somente de resíduos sem qualquer possibilidade viável de tratamento. O presente trabalho objetiva realizar um estudo de viabilidade técnica, econômica e ambiental preliminar da aplicação da tecnologia de Tratamento Térmico de Resíduos e Materiais Multifásicos - TTRM a resíduo de terra diatomácea - RTD de usina genérica de biodiesel. Foram utilizados como base para o estudo: os resultados dos testes da PETROBRAS e da ALBRECHT realizados em escala de bancada de laboratório e em planta piloto que simularam a aplicação do TTRM ao RTD; premissas técnicas; premissas operacionais; e dados econômicos de referência. Foram estabelecidos cenários específicos para o estudo da aplicabilidade e realizada análise de sensibilidade para os principais fatores da composição dos custos. Observou-se para este estudo preliminar que: na dimensão técnica o TTRM demonstrou ser aplicável; na dimensão econômica, os indicadores são positivos em sua totalidade no cenário esperado, mesmo após análise de sensibilidade com variações de 25% dos principais parâmetros de entrada do estudo de viabilidade; na dimensão ambiental o TTRM demonstrou ser uma alternativa que incorpora os conceitos para uma gestão alinhada com a Política Nacional de Resíduos Sólidos, seja na redução da periculosidade do resíduo, na potencial minimização da geração dos resíduos ou no reuso e reaproveitamento resíduos.
Renewable energy has played an ever increasing role in the global energy matrix. In Brazil, the public administration has shown efforts to increase the share of renewable fuels and the biodiesel production. The Brazilian National Waste Policy has among its goals non-generation, minimization, reuse, recycling and hazard reduction of waste, considering the alternative of landfill disposal only where waste treatment is not viable. However, the final filtering process in biodiesel production can generate a great amount of waste, including diatomaceous earth waste with significant inflammable characteristic. This work conducts a feasibility study of Multiphase Waste and Materials Thermal Treatment technology applied to diatomaceous earth waste generated from a generic biodiesel production plant. The present study has been based on: the results of diatomaceous earth waste thermal treatment tests performed by PETROBRAS and ALBRECHT both on laboratory and pilot scales; Technical and operational assumptions; and economic reference data. Specific scenarios were established to study the applicability and sensitivity analysis where performed for key factors of costs composition. The study concluded that thermal treatment is technically applicable to diatomaceous earth waste. In the economic dimension all indicators were be positive even where variations of 25% were introduced into the main input parameters. In the environmental dimension the Multiphase Waste Thermal Treatment of diatomaceous earth waste proved to be aligned with the Brazilian National Waste Policy, with regard to hazard reduction and minimization of waste generation as well as improving the potential for waste reuse.

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Björnfot, Therese, i Pia Kujala. "Engelsk språkinlärning och läromedel : En kvalitativ läromedelsanalys av Happy – Textbook year 3". Thesis, Mälardalens högskola, Akademin för utbildning, kultur och kommunikation, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-37448.

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Syftet med föreliggande studie är att med utgångspunkt i relevant forskning analysera om läromedel, som används i engelskundervisningen i årskurserna F-3, med tillhörande sångtexter, rim och ramsor stöder elevers språkinlärning i engelska. Metoder som använts i studien var dels en delstudie om vilket läromedel i engelskundervisningen kommunala skolor i Sverige använder i årskurserna F-3. Därutöver gjordes en bild- och textanalys av materialets innehåll samt jämfördes materialets verb med Corpus of Contemporary American English (COCA) (Davies, 2008-) och metoden type/token-ratio (TTR) för att kunna jämföra den inbördes variationen av verb och substantiv i materialet. Resultatet visar att läromedlet stöder språkinlärningen hos elever i de yngre åldrarna. Majoriteten av verben i materialet fanns med bland de 300 mest frekventa verben i COCA (Davies, 2008-) och skillnaden mellan de undersökta ordklasserna var inte lika stora som befarat. Materialet är i linje med och stöds av kursplanen för engelskämnet i Lgr 11 (Skolverket, 2011b), dock med få undantag. Språket i materialet är mestadels skrivet i presens. Slutsatsen som kan dras är att läromedel med tillhörande sånger, rim och ramsor kan stödja elevers språkinlärning i engelska.

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Branco, Bárbara da Costa. "Metodologia para deteção e diferenciação entre a estirpe vacinal e estirpes selvagens de Salmonella spp. por RT-PCR". Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/21528.

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Mestrado em Microbiologia
A realização deste trabalho teve como principais objetivos o acompanhamento da rotina laboratorial do Instituto Nacional de Investigação Agrária e Veterinária (INIAV, I.P.) e o desenvolvimento e implementação de nova uma metodologia por PCR em tempo real (RT-PCR) que permita a confirmação de Salmonella após execução da norma ISO 6579:1-2017, e a distinção entre a estirpe vacinal e as estirpes selvagens de Salmonella presentes em amostras provenientes de produção primária. A rotina laboratorial consistia a análise de amostras alimentares, ambientais e de produção primária, inseridas em diferentes atividades (e.g. Plano de Inspeção de Géneros Alimentícios, Plano Nacional de Controlo de Salmonella, Postos de Inspeção Fronteriços e Ensaios Interlaboratoriais). Todas as amostras foram analisadas tendo em conta os parâmetros microbiológicos estabelecidos para a pesquisa de Campylobacter spp., pesquisa de enterotoxinas estafilocóccicas, pesquisa de Escherichia coli STEC/VTEC, pesquisa e contagem de Listeria monocytogenes, contagem de Enterobacteriaceae e de microrganismos a 30°C e pesquisa de Salmonella spp. A vacinação contra Salmonella é uma medida importante no controlo de bandos de frangos, perus, galinhas poedeiras e reprodutoras. Por isso, é essencial detetar no menor tempo possível a presença desta bactéria e consequentemente o seu serotipo, permitindo saber qual a estirpe de Salmonella que está a infetar os bandos. Então, procedeu-se ao desenvolvimento de uma nova metodologia relativamente rápida (i.e., após duas horas), qualitativa (i.e., presença/ausência) para deteção e diferenciação de estirpes de Salmonella através de uma técnica por RT-PCR baseada na utilização de três sondas do tipo TaqMan, contendo uma delas bases de LNA, tornando-a mais específica para deteção do serotipo vacinal. As amostras alimentares são testadas diretamente da purificação de uma colónia em NA do meio XLD que irá para serotipificação. Caso haja fluorescência no canal HEX, significa que foi detetada a sequência do gene ttr, utilizado para testar a presença de Salmonella. No caso de amostras de produção primária, são retiradas cerca de 10 colónias do XLD para NA (i.e., se através do aspeto macroscópico se suspeitar que existem diferentes serotipos) e da placa de XLD que é analisada em Lisboa, para serotipificação, e é retirada uma colónia (já purificada) de modo a confirmar os resultados obtidos, na medida em que os testes de confirmação serológicos e biológicos são realizados a partir desta mesma colónia isolada. Aqui, espera-se que haja fluorescência no canal Cy5 (nhaA-V) que deteta o gene nhaA no caso de a estirpe ser vacinal. Se a estirpe for selvagem, não haverá curva de amplificação na corrida de RT-PCR visto existir uma sequência para estirpes selvagens que compete com os locais de ligação da sonda nhaA-V. Desta forma, e associando ao seguimento das primeiras fases da norma ISO 6579 é possível garantir o isolamento de Salmonella e a eliminação dos falsos positivos. A existência de um controlo interno de amplificação (IAC) que deteta a sequência do plasmídeo pUC19, através do canal ROX, fornece uma ferramenta importante para a indicação de falsos negativos, que possam ser causados por inibição da reação de PCR, provocada pelas diferentes matrizes, ou até por algum erro no funcionamento do termociclador. Assim, foi possível concluir que a nova metodologia permite detetar Salmonella spp., após isolamento em meio XLD através da ISO 6579, e diferenciar a estirpe vacinal das selvagens, uma vez que o aspeto macroscópico obtido apresenta diferenças (i.e., aspeto côncavo no caso da estirpe vacinal e aspeto convexo no caso de estirpes selvagens). Foi ainda possível verificar que o caldo MKTTn favorecia o crescimento da estirpe vacinal e permitia com maior facilidade a sua deteção.
This project’s main goals were to follow the Instituto Nacional de Investigação Agrária e Veterinária (National Institute for Agrarian and Veterinary Research - INIAV, I.P.) laboratory daily routine and the development and implementation of a new methodology in real-time PCR (RT-PCR) that allowed the confirmation of Salmonella after the execution of normative ISO 6579:1-2017, and the distinction between vaccine and wildtype strains of Salmonella present in primary production’s samples. Laboratory daily routine consisted in the analysis of food, environmental and primary production samples, inserted in different activities (e.g., Food Inspection Plan, Airport and Seaport frontier analytical Support, Salmonella National Surveillance Plan and samples of interlaboratory assays. Every sample was analyzed taking into account the microbiological parameters established for the detection of Campylobacter spp., detection of staphylococcal enterotoxins, detection of Escherichia.coli STEC/VTEC, detection and enumeration of Listeria monocytogenes, enumeration of Enterobacteriaceae and plate count technique of microorganisms at 30°C and detection of Salmonella spp.. The vaccination in poultry is an important measure in control of Salmonella in the broilers, turkeys, laying and breeding hens. That is why it is essential to detect as fast as possible the presence of this bacteria and consequently its serotype, allowing us to know which Salmonella’s strain is infecting the flocks. Then, we proceeded to the development of a relatively fast (i.e., after two hours of the PCR run) and qualitative (i.e., presence/absence) new methodology for the Salmonella strains detection and differentiation through RT-PCR technique based on the utilization of three TaqMan probes, one of them containing LNA bases, making it more specific for vaccine serotype detection. The food samples are directly tested from the purification of a colony in NA plate of the XLD medium which will go to the serotyping. In case of a fluorescence detection in the HEX channel, a sequence of the ttr gene was detected, which is used to detect the presence of Salmonella spp.. In case of primary production samples, 10 colonies from XLD medium are inoculated into NA plate (i.e., if there is a suspicion of the existence of different serotypes through the macroscopic appearance) and from the XLD plates which go to Lisbon, for serotyping a colony is purified so it can confirm the obtained results, in so far as the serological and biological confirmation tests are performed from this isolated colony. Here is expected to exist fluorescence in channel Cy5 (nhaA-V) which detects the nhaA gene in case of the vaccine serotype. There will be no amplification curve in the RT-PCR assay in case of wildtype strain since there is a sequence for wildtype strains that compete with specific probes of vaccine strain for similar binding sites during RT-PCR. Therefore, and associating to the follow-up of the ISO 6579 first stages it is possible to assure the Salmonella isolation and the detection of false positive results. The existence of an internal amplification control (IAC) that detects the pUC19 plasmid’s sequence, through the ROX channel, provides an important tool for the indication of false-negative results, that may be caused by the PCR reaction inhibition, caused by the different matrices, or even by an error in the thermocycle operation. Therefore, it was possible to conclude that the new methodology allows to detect Salmonella spp., after isolation in the XLD medium, following ISO 6579, and differentiate the vaccinal strains from the wildtype once since the obtained macroscopic appearance reveals differences, i.e., concave appearance in the vaccinal strain’s case and convex appearance in the wildtype strains. It was also possible to see that the MKTTn broth was the medium which favoured the vaccine strain growth and allowed a easier detection.

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Froder, Hans. "Desenvolvimento de métodos para a quantificação direta de Salmonella sp. por PCR-tempo real e por transcriptase reversa-PCR-tempo real". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/9/9131/tde-25012011-175156/.

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Para obter resultados rápidos e confiáveis que permitam o monitoramento da segurança microbiológica de alimentos, seja pela indústria ou pelos órgãos de fiscalização, diversos métodos alternativos têm sido desenvolvidos para a detecção e quantificação de Salmonella. Os propósitos do estudo foram avaliar a viabilidade de emprego do QIAamp® DNA Stool Mini Kit para extração e purificação de DNA de Salmonella; validar ensaios baseados em PCR-tempo real (PCR-RT) para quantificar o DNA de Salmonella empregando ttr ou tuf e desenvolver um ensaio para quantificar Salmonella baseado na transcriptase reversa-PCR-tempo real (RT-PCR-RT). Para avaliação do QIAamp® DNA Stool Mini Kit empregaram-se fezes coletadas diretamente do reto de animais infectados ou não, sendo estas últimas artificialmente contaminadas e submetidas à extração segundo protocolo do fabricante. As amostras de DNA isoladas foram quantificadas empregando um ensaio Salmonella-específico PCR-RT utilizando como alvo o lócus ttr. O mesmo ensaio foi utilizado para células de Salmonella provenientes de meio de cultura. O ensaio PCR-RT baseado no alvo tuf foi validado empregando-se primeiramente cepas de diferentes sorotipos de Salmonella e de outras Enterobacteriaceae. A seguir sua eficiência foi avaliada para alimentos-modelo (ave e suíno) artificialmente contaminadas com elevada (≈ 6 log UFC/mL) e baixa (≈ 2 log UFC/mL) população de Salmonella Typhimurium DT 104. A validação do método quantitativo de Salmonella por RT-PCR-RT foi realizada primeiramente com células em meio de cultura e posteriormente nos mesmos alimentos-modelo utilizados para PCR-RT. Em ambos os métodos, alíquotas dos alimentos-modelo foram mantidas a 20 ºC e a 8 ºC, sendo examinadas em diferentes tempos pós-inoculação. Como controle empregou-se a enumeração de microrganismos mesófilos totais e de Salmonella por técnicas convencionais. A taxa de recuperação de Salmonella em fezes suínas artificialmente inoculadas, após tratamento com QIAamp® DNA Stool Kit, variou entre 25% a 50%, dependendo da quantidade inicial de células. Empregando o DNA extraído e submetendo-o à PCR-RT para o ttr obteve-se limite de detecção de 2,8 log UFC eq/g de fezes; método que foi menos sensível que o convencional. A quantificação de Salmonella por PCR-RT empregando tuf apresentou limite de detecção menor que 1 log UFC eq. Os resultados obtidos com este método, empregando-se células em meio de cultura ou alimentos-modelo, foram, de maneira geral, ligeiramente inferiores aos do método convencional. A eficiência de amplificação para PCR-RT e tuf foi de 94%. O método RT-PCR-RT apresentou limite de detecção semelhante ao obtido com o ttr (2 log UFC eq) e sua eficiência de amplificação foi de 100%. Observou-se que tuf é expresso na fase logarítmica de multiplicação bacteriana, o que o torna um bom indicador da viabilidade de Salmonella.
In order to get fast and trustworthy results that allow monitoring the microbiological food safety either by industries or governmental agencies, diverse alternative methods have been developed for Salmonella detection and quantification. The purposes of this study were to evaluate the viability of the use of QIAamp® DNA Stool Mini Kit for Salmonella DNA extraction and purification; to validate assays based on real time-PCR (PCR-RT) to quantify Salmonella DNA by using ttr or tuf, and to develop an assay to quantify Salmonella based on reverse transcriptase- PCR-real time (RT-PCR-RT). For QIAamp® DNA Stool Mini Kit evaluation feces taken directly from the rectum of infected or health animals were used, with the former being artificially contaminated. Samples were submitted to DNA extraction, according to manufacturers protocol. The isolated DNA were quantified using a Salmonella-specific PCR-RT targeting the ttr locus. The same assay was used for Salmonella cells originated from culture medium. The PCR-RT assay with tuf as target was first validated employing different Salmonella serovars and other Enterobacteriaceae strains. After, its efficiency was evaluated on food-models (chicken and swine) spiked with high (≈ 6 log CFU/mL) and low (≈ 2 log CFU/mL) Salmonella Typhimurium DT 104 populations. The validation of the quantitative RT-PCR-RT method was first conducted with cells grown in culture medium, and then in the same food-model used for PCR-RT. For both methods aliquots of foodmodels were maintained at 20 ºC and 8 ºC being evaluated at different incubation times. Enumeration of total mesophilic microorganisms and Salmonella based on conventional methods were used as controls. The DNA recovery rate in swine feces artificially inoculated, after QIAamp® DNA Stool Mini Kit treatment, was between 25% to 50% depending the initial amount of cells. Using the extracted DNA and submitting it to PCR-RT for ttr a detection level of 2,8 CFU eq/g of feces was obtained. This method showed lower sensitivity than the conventional. Salmonella quantification by PCR-RT employing tuf showed a detection level lower than 1 log CFU eq. The results obtained with this method and cells suspended in culture medium or in food-model systems were, in general slightly lower that those obtained with the conventional method. The efficiency of amplification for PCR-RT tuf was 94%. Detection limit of RT-PCR-RT was similar to that of ttr (2 log CFU eq) and efficiency of amplification was 100%. tuf was expressed in logarithmic phase of bacteria growth curve showing that it is a good viability indicator for Salmonella.

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Scafà, Martina. "Un esempio di Economia Circolare: il riciclo e il riuso dei dispositivi Tessili per Sala Operatoria". Master's thesis, Alma Mater Studiorum - Università di Bologna, 2016.

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Negli ultimi decenni l’attenzione alle tematiche della sostenibilità ambientale e dell’inquinamento globale da parte dell’opinione pubblica e delle imprese è in costante aumento. Una tra le principali fonti di inquinamento è costituita dai rifiuti, e di conseguenza, la loro gestione e il loro smaltimento sono diventate una priorità, non solo per le istituzioni, ma anche per le imprese e per i cittadini. Inoltre la crescita della domanda delle risorse presenti in natura è in costante aumento e l’approvvigionamento di esse si è rivelato essere invece soggetto a significativi limiti. In questo attuale contesto quindi, il modello economico lineare fino ad oggi utilizzato, “take-make-dispose”, non risulta più idoneo. Si è sviluppato così il concetto di “Economia Circolare”. L’obiettivo di questa economia è quello di eliminare il concetto di scarto, il rifiuto deve essere considerato una vera e propria risorsa. In questo sistema tutte le attività, a partire dall’ estrazione fino ad arrivare alla produzione, sono organizzate in modo che i rifiuti di uno diventino risorse per un altro. L’obiettivo della tesi è quello di applicare il concetto di Economia Circolare al mercato dei dispositivi tessili per sala operatoria (DTSO), utilizzando la valutazione del ciclo di vita di un prodotto (analisi LCA) in modo da individuare le fasi più critiche e poter operare dei miglioramenti. Dopo essere stati sottoposti a 70 cicli di lavaggio e sterilizzazione i dispositivi TTR (Tessuti Tecnici Riutilizzabili) non sono più idonei ad essere utilizzati in ambito ospedaliero e vengono smaltiti in discarica , così come vengono smaltiti altri miliardi di rifiuti ogni giorno.Questi tessuti, seppur non conformi agli standard qualitativi richiesti dalle norme ospedaliere, possono essere riciclati o riutilizzati in prodotti di altro tipo. L’obiettivo di questo lavoro è quello di reimmettere questi dispositivi in un ciclo di vita di un nuovo prodotto, in ottica appunto di economia circolare.

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Blasi, Pérez Daniel. "Drug Discovery Targeted to Transthyretin Related Amyloidosis". Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/108283.

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Several drug discovery approaches has been performed to find new compounds able to interact with high affinity with the hormone binding site of the homotetrameric protein transthyretin (TTR), and stabilize this tetramer, becoming drug candidates to treat several rare amyloid diseases associated with TTR. With this aim, several computational workflows and chemico-biological databases have been developed, and in collaboration with two experimental research laboratories of our TTR Consortium (one contributing with the chemical synthesis or acquisition of the designed compounds, and the other contributing with the biological activity assay results for the synthesized or acquired compounds). The specific objectives of this thesis are: a) The generation of a chemico-biological database containing the historical and newly generated results of the TTR Consortium, containing the chemical structures and biological activities of the TTR ligands. b) Explore the possibility of using repurposing techniques applied to the discovery of new TTR inhibitors among the existing drugs, with particular focus on anti-inflammatory drugs, which are known to be good TTR ligands. c) Design of new flavonoid compounds as TTR ligands by means of structure-based drug design. d) Incorporate the Ligand Efficiency Indices analysis (both retrospective and prospective) as a new tool for designing new compounds with increased efficiency as TTR ligands. e) The computational development of a combined predictive/experimental workflow for the analysis of the metabolic stability of TTR ligands, as a tool for improving the prioritized compounds in our in-house database to obtain new compounds with better metabolic and pharmacokinetic properties. Among this thesis those workflows have been developed in order to obtain possible new amyloidogenic inhibitors: a) A computational workflow to obtain TTR ligand fingerprints has been developed, and the application of this workflow to the repurposing of marketed antiinflammatory drugs has delivered 3 compounds as new TTR stabilizers. b) A computational workflow to obtain a TTR-protein structure based pharmacophore has been developed, and the application of this workflow to a database of flavonoid compounds has delivered one compound as a new TTR stabilizer.

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29

Boldbaatar, Batbold. "The role of non-coding genetic variants on transthyretin gene transcription in transthyretin amyloidosis". Thesis, 2021. https://hdl.handle.net/2144/42221.

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The transthyretin-associated amyloidoses are a group of protein-folding disorders caused by deposition of the liver-secreted plasma protein transthyretin (TTR) in various tissues of the body. The sporadic form of the disease is caused by deposition of wild-type TTR whereas the inherited form is caused by deposition of mutated TTR; there are over 100 known amyloidogenic mutations of the TTR gene. The transcriptional regulation of the mouse transthyretin gene has been well studied. Organ-specific modulation of TTR mRNA is achieved by coordinated binding of hepatocyte-specific and ubiquitously expressed transcription factors to regulatory regions in the proximal promoter and upstream enhancer region. The hypothesis of this dissertation is that non-coding genetic sequence variations in the promoter of the transthyretin gene situated upstream of the regulatory regions, alter its transcriptional regulation, contributing to the onset and expression of transthyretin-associated amyloidosis. Previously, we identified a significant association of a non-coding polymorphism of the TTR promoter, rs3764479, with age of onset and survival in patients with ATTRwt amyloid disease. In this dissertation, electrophoretic mobility shift assays (EMSA) were used to investigate transcription factor binding of HepG2 nuclear proteins to short DNA probes with and without rs3764479. These mobility shift studies revealed that HepG2 nuclear extract proteins showed higher affinity to the wild-type TTR sequence than to one containing the rs3764479 SNP. Competition EMSAs suggested SNP-related changes in the binding of transcription factors hepatocyte nuclear factor-1 (HNF1) and hepatocyte nuclear factor-3b may alter TTR gene transcription. To investigate transthyretin gene regulation in V122I ATTR amyloid, the most prevalent TTR gene variant in the United States, the proximal promoter region from patients with V122I ATTR amyloidosis was sequenced and analyzed. In total, 8 SNPs were identified; one (rs955705399) was significantly associated with disease between the two V122I genotype-positive cohorts studied with and without cardiomyopathy. It is postulated that the presence of SNPs could influence gene expression and ultimately disease pathogenesis. In summary, these studies suggest that presence of disease-associated non-coding genetic variations modify transthyretin gene expression by disrupting transcription factor binding and may, in part, explain the clinical heterogeneity seen in patients with transthyretin-associated amyloidoses.

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Macedo, Nídia Sampaio. "TTR-induced cytoskeleton remodelling: a double-edged sword". Master's thesis, 2018. https://hdl.handle.net/10216/117140.

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Macedo, Nídia Sampaio. "TTR-induced cytoskeleton remodelling: a double-edged sword". Dissertação, 2018. https://repositorio-aberto.up.pt/handle/10216/117140.

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Martins, César Fernando de Bessa. "Estudo da expressão da transtirretina (TTR) no cérebro de ratinho". Master's thesis, 2008. http://hdl.handle.net/10400.6/2781.

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A transtirretina (TTR) é uma proteína tetramérica, que está presente no plasma e líquido cefalorraquidiano (LCR) de vertebrados. As suas principais funções biológicas conhecidas englobam o transporte da tiroxina (T4) e do retinol (vitamina A) através da formação de um complexo com a proteína de ligação ao retinol (RBP). Adicionalmente, tem sido demonstrado que a TTR é capaz de ligar e sequestrar o péptido beta amilóide (A-Beta), enfraquecendo a sua deposição nos tecidos nervosos, uma característica chave da doença de Alzheimer (AD). Assim, tem sido sugerido um papel protector desta proteína contra o desenvolvimento da AD, suportado por estudos que indicam uma correlação negativa entre os níveis da TTR no LCR e o grau de severidade desta doença neurodegenerativa. O fígado e os plexos coroideus do cérebro estão bem documentados como os principais locais de síntese da TTR, mas recentemente têm surgido evidências que apontam para a expressão desta proteína noutras regiões do cérebro, suscitando alguma controvérsia. Numa tentativa de contribuir para a resolução desta controvérsia foram realizadas experiências de hibridação in situ (ISH) e imunohistoquímica (IHC) num cérebro de ratinho wild-type previamente seriado, de forma a caracterizar a distribuição celular da TTR neste órgão. Os resultados obtidos indicam que para além de uma intensa marcação nas células epiteliais do PC, como esperado, o mRNA da TTR está igualmente presente em regiões específicas do parênquima cerebral, como o cerebelo e o bolbo raquidiano, com possíveis implicações nas propriedades desta proteína no cérebro.
Transthyretin (TTR) is a tetrameric protein which exists in the serum and cerebrospinal fluid (CSF) of vertebrates. It’s main biological known functions comprise the transport of tyroxine (T4) and retinol (vitamin A) through the formation of a complex with the retinol binding protein (RBP). Additionally, it has been shown that TTR is capable to bind and sequester beta amyloid peptide (A-Beta), weakening it’s deposition in nervous tissues, a key feature of Alzheimer’s disease (AD). Consequently, it has been suggested a protective role for this protein against the development of AD, supported by reports that indicate a negative correlation between the levels of TTR in the CSF and the severity degree of this neurodegenerative disease. The liver and the brain choroid plexus (CP) are well documented as the major sites of TTR synthesis, however recently, evidence has emerged that aim to an expression of this protein in others regions of the brain, prompting some controversy. In an attempt to contribute to the resolution of this controversy, in situ hybridization (ISH) and imunohistochemical (IHC) investigations were performed in wild-type mice brain, previously serialized, in order to characterize the cellular distribution of TTR in this organ. The acquired data indicates that besides an intense staining in the PC epithelial cells, as expected, the mRNA of TTR is also present in specific regions of the cerebral parenchyma, such as the cerebellum and rachidian bulb, with possible implications on the properties of this protein in the brain.

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33

Pullmann, Martin [Verfasser]. "Dynamische Blockierung TTX-resistenter Natriumkanäle durch Lokalanästhetika / vorgelegt von Martin Pullmann". 2003. http://d-nb.info/969813872/34.

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Silva, Joana Patrícia Abreu. "Modulators of phenotypic variability in Familial Amyloid Polyneuropathy (TTR-FAP Val30Met)". Master's thesis, 2017. https://repositorio-aberto.up.pt/handle/10216/109157.

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Silva, Joana Patrícia Abreu. "Modulators of phenotypic variability in Familial Amyloid Polyneuropathy (TTR-FAP Val30Met)". Dissertação, 2017. https://repositorio-aberto.up.pt/handle/10216/109157.

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Pen-wen-sen i 沈姵妏. "To examine the conjugal functions of two ttrA homologs in SCP1 and S. coelicolor chromosome". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/p394u7.

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碩士
國立臺北科技大學
生物科技研究所
97
Streptomyces has linear chromosomes and linear plasmids, and terminal proteins are covalently linked at 5’ ends of these DNA. Part of Streptomyces linear plasmids own conjugal ability, they can transfer themselves and chromosomes into recipients. One of related gene of conjugal transfer of Streptomyces linear replicons is ttrA, and previously study showed that ttrA of S. lividans chromosome and SLP2 linear plasmid worked in cis for DNA transfer during conjugation. ttrA homologs are commonly located in the end of linear replicons of Streptomyces, but the linear plasmid SCP1 carries two ttrA homologous, with lower identity, located on the central region. This study was analyzed the function of ttrA homologs of Streptomyces in conjugation by gene replacement. The ttrA of S. coelicolor chromosome showed the different effects on two strains, M145 without plasmids and 3456 with integrated SCP1. M145-

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Chang, Hsiang-Yu, i 張翔喻. "Studies on quantitative gene probe development for pufferfish and genotoxicity of TTX". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/17638139549831038336.

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碩士
亞洲大學
保健營養生技學系碩士班
99
The majority 30 species of Taiwan pufferfish species are born with tetrodotoxin (TTX) and non-economic value due to small quantity of production. Due to fraud or incorrect manufacturing processes, different proportions of unexpected or pufferfish muscle may be incorporated. The volatility of these factors may lead to counterfeit and concerned issue for public food safety. To solve these problems, a rapid qualitative and quantitative method which can accurately and quantitatively authenticate the source fish species under different of production processing should be established as soon as possible. The first study was focused on the gene map analysis of cytochrome b (cyt b) and cytochrome c oxidase I (COI) for Lagocephalus genus and Takifugu genus. The results show the strong genus-specificity and sensitivity on cyt b gene of L. genus and COI gene of T. genus. The further designed genus specific primers and genus Taqman probes are LF-cytb74163 / LR-cytb86890 and T. genus: TF-COI31332 / TR-COI41233, L- probe cytb and T- probe COI of L. genus and T. genus respectively. The amplified length of fragments was 150 base pairs for L. genus and 121 base pairs for T. genus. Specificity and sensitivity of specific primers and genus Taqman probes were also tested. These results demonstrated the primers and Taqman probes were able to discriminate efficiently of L. and T. genus individually and did not have reaction with other species. The detection limit of DNA concentration is 10-2 ng/μL of L. genus and T. genus. Furthermore, apply the above method to identify the commercially products is successfully identify the dry-dressed fish fillets collected before which were all made from pufferfish. The recent collection of these two years from Taiwan’s northern, central, southern and eastern the samples were all not made from pufferfish. It was shown the primers and Taqman probes were able to identificate the processed products and coupled with Real-time PCR is an accurate qualitative analysis technique. Once the TTX was ingested into the body, the toxicity mechanism is blocking the sodium channels entry of sodium ion on neuronal membranes. Suppose low dose-TTX has been ingested that forms a number of different mononucleotide and dinucleotide adducts in DNA, TTX-derived DNA adducts may cause liver cell toxicity. This study actually provided a very important reference on TTX toxicogenomics analysis. In our study, the in vitro test of TTX interacted with a single nucleotide dGMP via the HPLC analysis was done. It was shown that TTX did not have reaction with dGMP. Our data can be used as the basic information of TTX toxicogenomics. To summarize our study actually provided a very important reference on quantitative analysis of sensitive and reliable analyzing method for food safety and especially for adulterated pufferfish products. In addition, the TTX of genotoxicity also provide the basis toxicogenomics information for future related research purposes, and this is also the first study. Our study actually proved groundbreaking and practicability on science research.

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Magalhães, Marisa Monteiro. "Estudo da estabilidade da TTR após o transplante hepático em doentes PAF". Master's thesis, 2016. http://hdl.handle.net/10451/25517.

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Tese de mestrado, Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2016
A Polineuropatia Amiloidótica Familiar (PAF) é uma doença autossómica dominante progressiva caraterizada pela deposição de fibras amilóides formadas pela proteína Transtirretina (TTR). É uma patologia severa e que constitui um paradigma, uma vez que existem observações fenotípicas que sugerem um modelo de patogénese mais complexo do que a simples presença de mutações na cadeia polipeptídica da TTR. De modo a tentar compreender de que forma outros fatores não genéticos podem alterar a progressão desta doença, o presente trabalho apostou na análise de vários estados de progressão da PAF, para o qual se utilizou amostras de plasma de indivíduos portadores heterozigóticos da mutação V30M sintomáticos e indivíduos que sofreram um transplante de fígado cadáver. Foram avaliados os interactuantes da TTR e os níveis de glicação. O trabalho desenvolvido mostrou inequivocamente que o tempo de meia vida da TTR ronda os 3 dias, sendo que ao fim do sexto dia a variante mutada já foi totalmente substituída pela variante WT em indivíduos transplantados com fígado cadáver. Já os interactuantes da TTR identificados neste trabalho em pacientes PAF não transplantados foram a albumina, o retinol-binding protein, urocortino-3 e IgG1, sendo que a albumina e o retinol-binding protein já tinham sido descritos na literatura. Contudo, nos pacientes transplantados e monitorizados estavam apenas presentes a albumina, a IgG1 e o urocortino-3. A execução deste trabalho permite reforçar o envolvimento de fatores não genéticos no modelo de patogénese da PAF, nomeadamente um modelo multifatorial.
Familial Amyloid Polyneuropathy (FAP) is a progressive autosomal dominant disease characterized by the deposition of amyloid fibrils formed by the protein transthyretin (TTR). It is a severe disease and it is a paradigm, since there are phenotypic observations which suggest a more complex model of pathogenesis, with more than the presence of mutations in the polypeptide chain of TTR. In order to try to understand how other non-genetic factors can alter the progression of this disease, this study bet on the analysis of various progression states of PAF, for which was used plasma samples from heterozygous carries of V30M mutation with symptoms and individuals who have undergone a liver transplant. We evaluated the moleculs that interact with TTR and glycation levels. The work clearly showed that the half-life of TTR was around 3 days, and at the end of the sixth day the mutated variant has been completely replaced by the WT variant. The interacting molecules of TTR identified in this study in non-transplanted PAF patients were albumin, retinol-binding protein, urocortino-3 and IgG1, wherein the albumin and retinol-binding protein were already described in the literature. However, in transplanted patients were present only albumin, IgG1 and urocortino-3. The execution of this work reinforces the involvement of non-genetic factor in the pathogenesis modelo f FAP.

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Santos, Luís Miguel Cardoso dos. "Search for early TTR-related biomarkers in a transgenic AD mouse model". Master's thesis, 2013. https://repositorio-aberto.up.pt/handle/10216/71161.

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Abdullahi, Hassan. "Studies of an unusual transthyretin protein (TTR GLU51_SER52DUP) associated with familial amyloidosis". Thesis, 2017. https://hdl.handle.net/2144/23758.

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Transthyretin-related amyloidosis (ATTR) is a disease involving the formation of a misfolded transthyretin (TTR) protein and resulting insoluble aggregates that deposit in extracellular regions of various tissues and organs. There are hereditary forms of the disease, referred to as ATTRm, and more than 100 TTR amyloid-forming mutants have been reported. The major goal of this work was to analyze the biochemical and biophysical properties of a unique and recently identified TTR mutant protein, TTR Glu51_Ser52dup, found in a patient with ATTRm. Unlike other single nucleotide replacements that have been described as amyloidogenic, the gene abnormality in the present case is the first identification of a TTR duplication mutation. The patient with TTR Glu51_Ser52dup exhibited an extremely aggressive form of ATTRm; clinical symptoms included peripheral neuropathy at baseline evaluation and rapid disease progression to early death from pneumonia and congestive heart failure. We hypothesized that the TTR Glu51_Ser52dup variant would be less stable than the wild-type protein and similar in stability to another highly amyloidogenic mutant, TTR L55P; moreover, the highly unstable nature of this TTR variant would provide a basis for understanding the extremely aggressive clinical phenotype observed in this case. Using Escherichia coli (E. coli) as an expression system and an appropriately modified expression vector, we produced histidine-tagged recombinant human TTR Glu51_Ser52dup protein in high yield and purified to homogeneity. Structural and stability studies were performed by circular dichroism (CD) spectroscopy and SDS-PAGE analysis. We demonstrated that TTR Glu51_Ser52dup was less stable than the wild-type or L55P proteins when measured under different types of denaturing conditions, including thermal and chemical stress. The presence of diflunisal, a drug that stabilizes tetrameric TTR and is currently approved for treatment of ATTRm, was also investigated; our results indicated that diflunisal stabilized the TTR Glu51_Ser52dup protein. Collectively, the data obtained from these studies suggest that Glu51_Ser52dup is one of the least stable and most amyloidogenic TTR variant described to date. Future investigations are necessary to determine which specific structural elements of the protein destabilize the TTR tetramer, and precisely characterize the binding of small molecules, including diflunisal, to the protein.

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Santos, Luís Miguel Cardoso dos. "Search for early TTR-related biomarkers in a transgenic AD mouse model". Dissertação, 2013. https://repositorio-aberto.up.pt/handle/10216/71161.

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He, Chih-Wen, i 何志文. "To examine the conjugal functions of ttrA homologs in S. coelicolor chromosome and linear plasmid SCP1". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/j8pm69.

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碩士
國立臺北科技大學
生物科技研究所
99
Streptomyces processes linear chromosomes and linear plasmids, and most of the linear plasmids can be transferred by conjugation. The mechanism of conjugal transfer for E. coli circular plasmids is not applied to the conjugal system of Streptomyces. Previous research in Streptomyces conjugation had shown that the terminally located ttrA genes on the S. lividans chromosome and SLP2 were involved in conjugation probably acting in cis. Homologs of ttrA exist in the terminal regions of most Streptomyces chromosomes and some linear plasmids. However two ttrA homologs are located in the central region on the linear plasmid SCP1. This study was to understand whether ttrA gene on the S. coelicolor chromosomes and SCP1 were also involved in conjugal transfer. The ttrA gene (SCO0002) of S. coelicolor 3456 (containing integrated SCP1) was deleted and the mutant was mated with S. coelicolor M130 and S. lividans TK54. In mating with M130, the recombinant frequency was decreased by 60 to 176 folds. In mating with S. lividans TK54, the recombinant frequency was decreased by about 1,000 folds. The results indicated that ttrA on the S. coelicolor chromosome was more conjugatant influenced in inter-specifical transfer than intra-specifical transfer. In addition, two the ttrA homologs on SCP1 (SCP1.136 and SCP1.216Ac) were knockout individually. When these mutants were mated with S. coelicolor or S. lividans, the recombinant frequencies were decreased by about 10 folds. Double knockout mutant was also created, and the mutant was mated with S. coelicolor M145, the recombinant frequency was deceased by about 800 times. These results showed that the both ttrA homologs on SCP1 were involved in conjugal transfer.

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Gaiteiro, Cristiana Milhazes. "Role of TTR in A β peptide brain efflux - Impact in Alzheimer's disease". Master's thesis, 2014. https://repositorio-aberto.up.pt/handle/10216/80517.

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Gaiteiro, Cristiana Milhazes. "Role of TTR in A β peptide brain efflux - Impact in Alzheimer's disease". Dissertação, 2014. https://repositorio-aberto.up.pt/handle/10216/80517.

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Gilberto, Samuel Filipe da Graça. "Efeito do fibrinogénio na formação de fibras de TTR em doentes com amiloidose familiar". Master's thesis, 2011. http://hdl.handle.net/10451/8778.

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Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
A Polineuropatia Amiloidótica Familiar (PAF) é uma doença neurodegenerativa hereditária caracterizada pela deposição de transtirretina (TTR) no sistema nervoso periférico. Como consequência, verifica-se perda de sensibilidade nos membros dos pacientes, seguida de atrofia muscular, fraqueza e morte 10 a 20 anos após o início dos sintomas. O desenvolvimento desta doença requer a presença de TTR mutada, conhecendo-se mais de 80 mutações que levam à formação de fibras amilóides. Como mecanismo para a formação destes agregados, estudos in vitro apontam para uma destabilização dos tetrâmeros que contêm variantes da TTR, resultando na formação de monómeros amiloidogénicos. No entanto, existem diversas observações que sugerem um mecanismo mais complexo in vivo, envolvendo factores não genéticos. Por exemplo, indivíduos que possuem a mesma mutação no gene da TTR, incluindo gémeos monozigóticos, não mostram o início da sintomatologia com a mesma idade. Existem ainda diferenças geográficas no que toca à idade de desenvolvimento dos sintomas e na própria morfologia dos agregados. Ao contrário do expectável, indivíduos homozigóticos para genes de TTR mutada não apresentam uma forma mais agressiva da doença. Com um papel central nestas diferenças fenotípicas poderão constar mecanismos de defesa, como é o caso do conjunto de proteínas denominadas chaperones moleculares. Seguindo esta hipótese, observou-se recentemente uma interacção da TTR com o chaperone extracelular fibrinogénio. Curiosamente, o fibrinogénio encontra-se selectivamente modificado em pacientes PAF. Esta modificação pós-traducional, denominada glicação, foi descrita como estando envolvida noutras doenças de natureza semelhante à PAF, como é o caso da doença de Alzheimer ou de Parkinson. Neste trabalho, investigou-se o efeito do fibrinogénio e da glicação na amiloidogénese da TTR. Verificou-se que o fibrinogénio tem a capacidade de prevenir a sua agregação, atingindo-se a actividade máxima numa proporção molar fibrinogénio:TTR de 1:20, sugerindo uma relevância desta interacção in vivo, já que neste caso esta proporção é aproximadamente 1:2. Adicionalmente, observou-se que a glicação do fibrinogénio leva a uma abolição da referida função. Paralelamente, foi realizado um mapeamento dos locais modificados do fibrinogénio com vista a estabelecer a correlação entre os locais modificados e a perda de função deste. Desta forma, foi estabelecido um método optimizado para realizar este mapeamento por espectrometria de massa, tendo-se identificado um total de 60 locais modificados nas três cadeias do fibrinogénio, α, β e γ, com uma cobertura de sequência média de 82%.
Familial Amyloidotic Polyneuropathy (FAP) is an hereditary neurodegenerative disease characterized by the deposition of transthyretin (TTR) in the peripheral nervous system. As a consequence, sensibility loss in the patients’ arms and legs takes place, followed by muscle atrophy and death after 10 to 20 years of the disease onset. The development of this disease requires the presence of a mutated variant of TTR, being described more than 80 mutations that result in amyloid fiber formation. As a mechanism for TTR aggregation, in vitro studies suggest that in these patients TTR tetramer (its native form) is destabilized, resulting in the formation of amyloidogenic monomers. However, several observations suggest a more complex mechanism in vivo, with the implication of non-genetic factors. For instance, patients that share the same mutation in the TTR gene, including monozygous twins, do not show the same disease onset age. There are also geographical differences when it comes to the aggregates’ morphology and also the disease onset age. Opposite from the expected, homozygous individuals to mutated TTR genes do not show a more aggressive form of the disease. With a central role in these phenotypic differences may be defense mechanisms, such as molecular chaperones. Following this hypothesis, recently an interaction between TTR and the extracellular chaperone fibrinogen was described. Surprisingly, fibrinogen is selectively modified in FAP patients. This post-translational modification, termed glycation, was previously described as being involved in other conformational diseases, such as Alzheimer’s and Parkinson’s diseases. In this work, the effect of fibrinogen and glycation in TTR amyloidogenesis was studied by monitoring the aggregation process using spectroscopic methods. It was observed that fibrinogen has the ability to prevent its aggregation, achieving its maximum activity in a molar ratio of 1:20 (fibrinogen:TTR), suggesting a relevance of this interaction in vivo, as in this case the ratio fibrinogen:TTR is approximately 1:2. Moreover, it was observed that fibrinogen glycation abolishes its chaperone function. Additionally, the glycation sites in fibrinogen were mapped, in order to correlate those sites with the loss of fibrinogen’s chaperone function. Therefore, here is also established an optimized method to perform such mapping using mass spectrometry, having been identified a total of 60 modified sites in the three fibrinogen chains, α, β e γ, with an average sequence coverage of 82%.

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Silva, Marina Isabel Oliveira da. "Dissecting the role of cytoskeleton remodelling in a Drosophila model of TTR-induced neurodegeneration". Master's thesis, 2015. https://repositorio-aberto.up.pt/handle/10216/82245.

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Ferreira, Miguel Fernando Alves. "Phenotypic variability in familial amyloid polyneuropathy: TTR modifiers in Caenorhabditis elegans and human models". Doctoral thesis, 2019. https://hdl.handle.net/10216/121022.

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Ferreira, Miguel Fernando Alves. "Phenotypic variability in familial amyloid polyneuropathy: TTR modifiers in Caenorhabditis elegans and human models". Tese, 2019. https://hdl.handle.net/10216/121022.

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Silva, Marina Isabel Oliveira da. "Dissecting the role of cytoskeleton remodelling in a Drosophila model of TTR-induced neurodegeneration". Dissertação, 2015. https://repositorio-aberto.up.pt/handle/10216/82245.

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Yu, Chen Wan, i 陳婉瑜. "Blockade of the TTX-resistant Na+ channel by multivalent cations in dorsal root ganglion neurons". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/66738233537446350819.

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碩士
國立臺灣大學
生理學研究所
90
TTX（tetrodotoxin）is a common animal toxin to block the Na+ channel. According to the sensitivity to the TTX, Na+ channel can be divided into two groups, the TTX-sensitive Na+ channels and the TTX-resistant Na+ channels. TTX-resistant Na+ channels have been found in peripheral nervous systems, and may play an important in the transmission of slow pain. The TTX-resistant Na+ channels have lower affinity to TTX, much slower activation and inactivation rate, and more sensitive to inorganic Ca2+ channel blockers（Cd2+、Co2+、Mn2+、Zn2+、Ni2+、La3+） than TTX-sensitive ones. However, the mechanism underlying the block of the TTX-resistant Na+ channels by the inorganic cations remains unknown. We found that the blocking effect of these cations on the TTX-resistant Na+ channels in dorsal root ganglion neurons are both concentration dependent and flow dependent. The blocking effect is much more manifest in the presence of inward Na+ current than outward Na+ current, with the blocking potency La3+> Zn2+> Ni2+> Co2+>Mn2+. On the other hand, the voltage dependence is Mn2+>Co2+>Ni2+ >Zn2+>La3+. These findings suggest that these cations and Na+ ion can bind to a single-file multi-ion region in TTX-resistant Na+ channels, and these region may contain ion binding sites consisting of carboxyl groups, sulfhydryl groups, and/or oxygen-containing groups.

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