No description available.

Thesis (Ph.D.)--Flinders University, Dept. of Chemistry. / Typescript bound. Includes bibliographical references: (leaves 217) Also available online.

Development of the rodent somatosensory cortex is well characterised and involves activity-dependent mechanisms that occur during the first postnatal week. Glutamate is a key neurotransmitter responsible for signalling events that result in formation of cortical barrels - aggregates of cells in the cortex corresponding to whiskers on the face pad. The molecular mechanisms that occur downstream of glutamate signalling are not fully understood and data here contributes to the unveiling of some of these mechanisms. Transgenic mice with deletions of genes that encode members of the post-synaptic complex associated with NMDARs were used to understand the role of individual genes in the formation of barrels. SynGAP, a ras GTPase activating protein (GAP) that negatively regulates the ERK-MAPK pathway downstream of NMDARs is required for the formation of barrels and data here agrees with other findings that the ras GAP NF1 has a similar role. Examination of RICS, a RhoGAP and Dusp6 - a phosphatase that inactivates ERK reveals that neither are necessary for the formation of barrels. This finding adds to previous data postulating that barrels form in an ERK-independent manner (Watson et al., 2006, Barnett et al., 2006). MAGUKs are important scaffolding molecules in the PSD and bind NMDARs to downstream signalling molecules such as SynGAP. Two of these MAGUKs SAP102 and PSD-95 have roles in hippocampal plasticity, and learning and memory and Sap102 mutations result in a form of X-linked human retardation (Tarpey et al., 2004). Deletion of either gene does not cause defects in the development of barrels, perhaps due to compensation mechanisms already described in hippocampus (Vickers et al., 2006 Cuthbert et al., 2007). Double knockout mice die by P3 and analysis of all other mutants revealed a defect in the formation of barrels and segregation of TCAs in Sap102-/y Psd-95+/-. Surprisingly this defect was not seen in Sap102+/- Psd-95-/- mice, agreeing with previous findings that SAP102 is better able to compensate for loss of PSD-95 (is up-regulated) than PSD-95 is for SAP102. An explanation for this effect may lie with the fact that Sap102 is X-linked and therefore females that are heterozygote for Sap102 are mosaic with a population of cells expressing SAP102 and a population not expressing SAP102. Using β-Galactosidase antibody to label one population of cells, female mice that had two populations of cells were examined. In these mice one population of cells were Sap102-Psd-95+/-, and did not previously segregate into normal barrels and the other population were Sap102+Psd-95+/- and should segregate normally. Both populations of cells segregated normally, indicating that the cells expressing SAP102 were rescuing the cells not expressing SAP102 by a cell non-autonomous mechanism. The final part of this thesis focuses on the role of glutamate-dependent signalling pathways in the regulation of CSPGs- key extracellular matrix proteoglycans that regulate the termination of the sensitive period. Analysis of 3 overlapping but distinct subsets of chondroitin-sulphate proteoglycans (CSPGs) reveals that expression of each of the three is different throughout development. After 2-3 weeks perineuronal nets (PNNs) labelled with Cat-315 and Cat-316 are visible and locate to specific regions within the cortical barrel-field. To determine whether the formation of PNNs is regulated by proteins involved in glutamate signalling, expression of the three CSPG subsets was analysed in mice with barrel defects due to mutations of Plcβ1, Mglur5, Syngap and Prkar2b. Interestingly, Prkar2b mutant adults but no other mutants have reduced Cat-315-PNNs, indicating that PKARIIβ regulates pathways that lead to formation of Cat-315-PNNs in adulthood. Cat-315 has previously been found to be regulated in the cortex of visually deprived cats and the cortex of whisker-trimmed mice, indicating that specific subsets of CSPGs are regulated by neuronal activity. Molecular pathways that lead to expression of Cat-315 positive PNNs involve PKARIIβ and the formation of PNNs may be an important step in the plasticity of circuits in barrels. Taken together, these results demonstrate that an important part of molecular signalling downstream of glutamate enabling barrels to form is played by molecules that maintain structure inside the synapse and outside the cell.

Thesis (M.A.)--East Carolina University, 2009. / Presented to the faculty of the Department of Anthropology. Advisor: Charles R. Ewen. Title from PDF t.p. (viewed May 25, 2010). Includes bibliographical references.

In order to process information, neurons must connect together to form a neuronal circuit. The formation of neuronal circuits is dependent on synaptic activity through glutamate receptors and downstream molecules within the post-synaptic density (PSD). The pathways downstream of glutamate receptors play an important role in maintaining appropriate synapses and forming neural circuits; mutations in genes that encode PSD proteins disrupt these pathways and are associated with many forms of intellectual disability in humans (Grant 2012). The development of neuronal circuits relies on two key developmental events; neurons must first send out axons locally and to disparate brain regions and then, neurons must form connections with the dendrites of target neurons. The rodent trigeminal system is a neuronal circuit that processes somatosensory information from whiskers on the facepad via nuclei in the brainstem to the thalamus and ultimately the cerebral cortex. Brain regions that comprise the trigeminal system are organised in a manner that topographically recapitulates the whisker pattern; each whisker on the rodent facepad corresponds to a physiological and anatomical unit in the primary somatosensensory cortex (S1) called a barrel. This topographical organisation creates a pattern consisting of thalamocortical axons (TCA) clustered into distinct whisker-related bundles and layer IV spiny stellate cells which segregate around the outside of TCA bundles. Three different anatomical patterns can be identified within the mouse S1 by labelling the cell soma, axons or the extracellular matrix. This strict organisation makes the rodent S1 an excellent model for discerning the proteins involved in neural circuit formation, and by screening genetic mutants for S1 patterning defects the molecular pathway involved in setting up neuronal circuits can be elucidated. Furthermore an understanding of these pathways may provide insight into how neuronal networks are disrupted in human intellectual disability. In the first data chapter, the expression profile of three neurofilament subunits were characterised in order to develop a method of identifying anatomical defects in barrel morphology. The precise organisation of the rodent S1 can also be used as a method to identify the cellular localisation of neurofilament subunits ex vivo. Neurofilaments are polymers formed from three subunits identified by their relative molecular weight. By using the unique patterning of S1, each neurofilament subunit shows a unique spatialtemporal expression pattern in the somatosensory cortex. Two neurofilaments subunits; the medium and the heavy neurofilament subunits can be used to identify TCA which can be used as an indicator of anatomical defects in barrel patterning. In chapter 4 neurofilament labelling was used in conjunction with other histological techniques to investigate S1 organisation in mice lacking synapse associated protein 102 (SAP102). SAP102 is a PSD scaffolding molecule that binds to both NMDA receptor subunits and SynGAP, a synaptic GTPase activating protein, furthermore it is associated with X-linked mental retardation in humans (Tarpey et al., 2004; Zanni et al., 2010). Mutant mice lacking functional NMDA receptors or PSD proteins such as SynGAP show defects in S1 pattern formation (Barnett et al., 2006; Iwasato et al., 2000; Wijetunge, Till, Gillingwater, Ingham, & Kind, 2008). SAP102 null mutants (SAP102-/y) have defects in synaptic plasticity and are slow to learn on behavioural tasks (Cuthbert et al., 2007), however it is unclear how the loss of SAP102 may disrupt neural networks. SAP102-/y were found to have a reduction in brain mass compared to wild-type littermates, but cortical thickness and patterning of S1 is unchanged. SAP102-/y have fewer TCA reaching the cortex compared to littermates; furthermore SAP102-/y mutants have layer specific defects in the density of dendritic spines. These data suggest that in the absence of SAP102 connectivity in the S1 is altered by layer specific changes in synapses number and fewer axons innervating cortical layer IV. In the final experimental chapter (chapter 5) the combined role of SAP102 and Postsynaptic density protein 95 (PSD95) in S1 patterning was investigated. SAP102 and PSD95 are the main members of the membrane-associated guanylate kinase (MAGUK) family expressed during early cortical development. These proteins share a similar protein structure, perform similar functions at the synapse (Elias & Nicoll, 2007) and have been shown to compensate for each other in vitro (Elias, Elias, Apostolides, Kriegstein, & Nicoll, 2008). Genetic mutants lacking both SAP102 and PSD95 are not viable and do not survive beyond birth (Cuthbert et al., 2007; Petrie, 2008). Therefore in order to investigate the combined role of these proteins a novel approach was developed that utilises X-inactivation to produce mosaic animals containing cells that lack both proteins. The distribution of cells containing the same X-chromosome was investigated and found to be evenly distributed throughout the cortex, demonstrating that this method could be used to investigate allosomal genes. In mosaic animals where approximately half the cells only lack PSD95 and the remaining cells lack both SAP102 and PSD95, double knockout cells are viable and are equally represented in S1. These double knockout cells contribute to normally barrel formation which suggests that SAP102 and PSD95 are not required for barrel formation.

A number of studies have shown that sensory deprivation is associated with selective decreases in GABA, GAD, and GABA receptors, in deprived areas of visual and somatosensory cortex. Those studies focused on layer 4, a recipient of direct thalamocortical sensory input. However, supragranular layers 2/3 have been recently identified as a major locus of functional plasticity in sensory deprivation and long-term potentiation. To examine whether GABAA receptors in layers 2/3 are affected by sensory deprivation, rats had mystacial vibrissae in middle row C or rows ABDE trimmed for 6 weeks beginning in early adulthood. Layers 2/3 above the deprived and adjacent whisker barrels were located in tangential sections, using patterns of radial blood vessels as fiducial marks. In deprived whisker barrel columns, [3H]muscimol binding to GABAA receptors decreased by 12.8% ± 1.2 (P &lt; 0.001) in layers 2/3 and 11.4% ± 1.2 (P<0.001) in layer 4. Altered levels of GABAA α1 subunit (Fritschy et al., 1994) were indicated by reduced optical density of immunostaining, both in deprived layers 2/3 (6.4% ± 0.7; P&lt; 0.001) and in layer 4 (3.4% ± 1.0; P &lt; 0.005). Interestingly, Nissl staining density also decreased in deprived layers 2/3 (12.7% ± 1.8 P &lt; 0.001) and in 4 (6.0 ± 0.7 (P &lt; 0.001). The percent decreases were greater in layers 2/3 than in 4 for both GABAA α1 (P &lt; 0.05) and Nissl substance (P &lt; 0.005). The present results suggest that down-regulation in GABAA receptors may underlie the physiological signs of disinhibition observed in neurons of layer 2/3 and 4 in deprived whisker barrel columns.

No description available.

Cerveja é uma bebida alcoólica mundialmente popular e a mais consumida no Brasil. Existem diversos estilos de cerveja no mundo, os quais são produzidos por modificações no processo de produção, no uso de diferentes ingredientes, na maturação utilizando barris de madeira e/ou adição de fragmentos de madeira, entre outros. A maturação em madeira pode proporcionar complexidade aromática às bebidas, sendo a madeira de carvalho amplamente utilizada para a maturação de bebidas alcoólicas. O uso dessa madeira na maturação da cerveja é o foco desse trabalho, que maturou cervejas a 0°C, durante três meses, em garrafas de vidro de 600 mL, barris de carvalho e recipientes plásticos com cubos de carvalho, na dose de 3g/L, provenientes de três níveis diferentes de tosta (leve, média, e alta). Das cervejas oriundas dos diferentes tratamentos, foram analisadas graduação alcoólica, pH, acidez total, turbidez, fenólicos totais, cor e amargor; os compostos voláteis (aldeídos, ésteres e álcoois superiores) foram analisados por Cromatografia gasosa (FID) e os compostos fenólicos de baixo peso molecular (ácido gálico, 5-hidroximetil-furfural, furfural, ácido vanílico, ácido siríngico, vanilina, siringaldeído, coniferaldeído e sinapaldeído) por Cromatografia líquida de alta eficiência (HPLC). As cervejas também foram analisadas sensorialmente mediante teste de preferência. A análise dos resultados mostrou que não houve alterações na qualidade da cerveja que pudessem ser atribuídas ao armazenamento com madeira. Os compostos voláteis tiveram pequenas alterações, por outro lado, os compostos fenólicos de baixo peso molecular foram os que apresentaram maiores incrementos no período de três meses de maturação. Não houve diferença na aceitação sensorial entre as cervejas maturadas com cubos de madeira, barril e em garrafas de vidro. Futuros estudos são necessários para que seja possível obter um produto de qualidade que possa satisfazer o consumidor e seja acessível à indústria. / Beer is a very popular alcoholic beverage in the world and the most widely consumed in Brazil. There are many styles of beer in the world that can be produced by changes in the production process, use of various ingredients, maturation using wood barrels and / or addition of wood fragments, and others. Wood maturation can provide aromatic complexity to alcoholic beverages, and the oak wood is widely used. The use of oak in the maturation of beer is the focus of this work. The beers matured at 0 °C for three months in glass bottles of 600 mL, oak barrels and plastic containers with oak cubes at a dose of 3g/L, with three different levels of toasting (light, medium, and high). Beers resulting from the different treatments were analyzed physico-chemically (alcohol content, pH, total acidity, turbidity, total phenolics, color and bitterness), the volatile compounds (aldehydes, esters and higher alcohols) by gas chromatography (FID), the low molecular weight phenolic compounds (gallic acid, 5-hydroxymethylfurfural, furfural, vanillic acid, syringic acid, vanillin, syringaldehyde, coniferaldehyde and sinapaldehyde) by High Performance Liquid Chromatography (HPLC), and sensory. The analysis shows that there were no qualities changes in beer that could be attributed to the storage in contact with oak wood. The volatile compounds had minor changes; the low molecular weight phenolic compounds were those with the greatest increases within three months of maturation. There was no difference in sensory acceptance between beers matured in oak barrel, oak cubes and glass bottles. This work suggests that wood influences sensory beer, but more studies are needed to be able to get a quality product that can satisfy the consumer and is accessible to the industry.

Dependendo das etapas de produção, a aguardente de cana de açúcar, pode ser contaminada por Hidrocarbonetos Policíclicos Aromáticos (HPAs) que apresentam propriedades carcinogênicas e/ ou mutagênicas. Neste trabalho se determinou os teores de HPAs em aguardentes acondicionadas em tonéis de carvalho sem tostar e tostados por 1, 2 e 3 minutos. Os dados obtidos em função do tempo de envelhecimento e do tempo de tosta do tonel foram correlacionados. Após períodos variados, as amostras foram coletadas e submetidas a processos de extração em cartuchos de SPE (C18) e analisadas por Cromatografia Líquida de Alta Eficiência (HPLC) com detector de fluorescência. Dos 16 HPAs monitorados pela Agencia de Proteção Americana (USEPA) foram analisados: Naftaleno, Acenafteno, Fluoreno, Fenantreno, Antraceno, Fluoranteno, Pireno, Benzo(a)Antraceno, Criseno, Benzo(b)fluoranteno, Benzo(k)fluoranteno, Benzo(a)pireno, Dibenzo(a,h)antraceno, Benzo(g,h,i)perileno e Indeno(1,2,3 c,d)pireno. Os HPAs nas aguardentes armazenadas, apresentaram um perfil comum, atingindo teores máximos entre o 4° e o 12° dia de armazenamento, começando a diminuir até atingir a valores em alguns casos próximos ao do Branco. Realizaram-se dois experimentos de armazenamento, o 1º experimento ao longo de 553 dias e o 2º experimento por um período de 57 dias. As amostras armazenadas no 2º experimento (tonéis re-utilizados) apresentaram teores inferiores de HPAs em relação ao 1º experimento. A soma dos teores médios máximos dos 15 HPAs, nas amostras de aguardente armazenada no 1º e 2º experimento respectivamente foram: 1 min. de tosta (27,76 -14,45 &micro;/L ), sem tosta (20,34 -9,02 &micro;g/L), 2 min. de tosta (15,45 -10,38 &micro;g/L) e 3 min. de tosta (14,25 -12,89 &micro;g/L). Os valores do Branco para o 1º e 2 º experimento foram: 10,88 &micro;g/L e 6,89 &micro;g/L, respectivamente. Com respeito ao Benzo(a)pireno (BaP), os teores médios máximos alcançados em aguardentes envelhecidas em tonéis tostados por 1, 2 e 3 minutos foram respectivamente: 0,0094 &micro;g/L, 1,401 &micro;g/L, 0,119 &micro;g/L e 0,051 &micro;g/L, atingindo teores maiores que o teor do Branco do BaP (0,014&micro;g/L). / During the different steps of production, the sugarcane spirits can be contaminated with Polycyclic Aromatic Hydrocarbons (PAHs) which have carcinogenic and/or mutagenic properties. The scope this work is to determine the levels of PAHs in aged sugarcane spirits in oak barrels with and without toasting for later correlation of results depending on the aging time and the time of the burning barrel. The samples were stored in barrels without and with toasting for 1, 2 and 3 minutes. After varying periods, samples were collected and subjected to extraction processes using SPE (C18) cartridges and analyzed by using High Efficiency Liquid Chromatography (HPLC) coupled with a fluorescence detector. We analyzed the following PAHs: naphthalene, acenaphthene, fluorene phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene and indeno(1,2,3-c,d)pyrene. PAHs in aged spirits had a common profile, reaching maximum levels between the 4th and 12th day of aging, starting to decrease the values, in some cases, lower than blank. There were two storage experiments, experiments 1 through 553 days and the 2nd experiment for a period of 57 days. Samples stored in the 2nd experiment (re-used barrels) had lower levels of PAHs compared to the 1st experiment. The sum of the maximum average levels of 15 PAHs in the samples spirit stored in the 1st and 2nd respectively experiment were: 1 min. of toast (27.76 -14.45 &micro;/L) without toast (20.34 -9.02 &micro;g/L), 2 min. toasting (15.45 -10.38 &micro;g/L), and 3 min. toasting (14.25 -12.89 &micro;g/L). The blank values for the 1st and 2nd experiment were: 10.88 &micro;g/L and 6.89 &micro;g/L, respectively. With respect to Benzo(a)pyrene (BaP), the maximum average levels achieved in spirit aged in barrels toasted by 1, 2 and 3 minutes were respectively: 0.0094 &micro;g/L, 1.401 &micro;g/L, 0.119 &micro;g/L and 0.051 &micro;g/L, reaching higher levels than the blank content of BaP (0,014 &micro;g/L)

Dependendo das etapas de produção, a aguardente de cana de açúcar, pode ser contaminada por Hidrocarbonetos Policíclicos Aromáticos (HPAs) que apresentam propriedades carcinogênicas e/ ou mutagênicas. Neste trabalho se determinou os teores de HPAs em aguardentes acondicionadas em tonéis de carvalho sem tostar e tostados por 1, 2 e 3 minutos. Os dados obtidos em função do tempo de envelhecimento e do tempo de tosta do tonel foram correlacionados. Após períodos variados, as amostras foram coletadas e submetidas a processos de extração em cartuchos de SPE (C18) e analisadas por Cromatografia Líquida de Alta Eficiência (HPLC) com detector de fluorescência. Dos 16 HPAs monitorados pela Agencia de Proteção Americana (USEPA) foram analisados: Naftaleno, Acenafteno, Fluoreno, Fenantreno, Antraceno, Fluoranteno, Pireno, Benzo(a)Antraceno, Criseno, Benzo(b)fluoranteno, Benzo(k)fluoranteno, Benzo(a)pireno, Dibenzo(a,h)antraceno, Benzo(g,h,i)perileno e Indeno(1,2,3 c,d)pireno. Os HPAs nas aguardentes armazenadas, apresentaram um perfil comum, atingindo teores máximos entre o 4° e o 12° dia de armazenamento, começando a diminuir até atingir a valores em alguns casos próximos ao do Branco. Realizaram-se dois experimentos de armazenamento, o 1º experimento ao longo de 553 dias e o 2º experimento por um período de 57 dias. As amostras armazenadas no 2º experimento (tonéis re-utilizados) apresentaram teores inferiores de HPAs em relação ao 1º experimento. A soma dos teores médios máximos dos 15 HPAs, nas amostras de aguardente armazenada no 1º e 2º experimento respectivamente foram: 1 min. de tosta (27,76 -14,45 &micro;/L ), sem tosta (20,34 -9,02 &micro;g/L), 2 min. de tosta (15,45 -10,38 &micro;g/L) e 3 min. de tosta (14,25 -12,89 &micro;g/L). Os valores do Branco para o 1º e 2 º experimento foram: 10,88 &micro;g/L e 6,89 &micro;g/L, respectivamente. Com respeito ao Benzo(a)pireno (BaP), os teores médios máximos alcançados em aguardentes envelhecidas em tonéis tostados por 1, 2 e 3 minutos foram respectivamente: 0,0094 &micro;g/L, 1,401 &micro;g/L, 0,119 &micro;g/L e 0,051 &micro;g/L, atingindo teores maiores que o teor do Branco do BaP (0,014&micro;g/L). / During the different steps of production, the sugarcane spirits can be contaminated with Polycyclic Aromatic Hydrocarbons (PAHs) which have carcinogenic and/or mutagenic properties. The scope this work is to determine the levels of PAHs in aged sugarcane spirits in oak barrels with and without toasting for later correlation of results depending on the aging time and the time of the burning barrel. The samples were stored in barrels without and with toasting for 1, 2 and 3 minutes. After varying periods, samples were collected and subjected to extraction processes using SPE (C18) cartridges and analyzed by using High Efficiency Liquid Chromatography (HPLC) coupled with a fluorescence detector. We analyzed the following PAHs: naphthalene, acenaphthene, fluorene phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene and indeno(1,2,3-c,d)pyrene. PAHs in aged spirits had a common profile, reaching maximum levels between the 4th and 12th day of aging, starting to decrease the values, in some cases, lower than blank. There were two storage experiments, experiments 1 through 553 days and the 2nd experiment for a period of 57 days. Samples stored in the 2nd experiment (re-used barrels) had lower levels of PAHs compared to the 1st experiment. The sum of the maximum average levels of 15 PAHs in the samples spirit stored in the 1st and 2nd respectively experiment were: 1 min. of toast (27.76 -14.45 &micro;/L) without toast (20.34 -9.02 &micro;g/L), 2 min. toasting (15.45 -10.38 &micro;g/L), and 3 min. toasting (14.25 -12.89 &micro;g/L). The blank values for the 1st and 2nd experiment were: 10.88 &micro;g/L and 6.89 &micro;g/L, respectively. With respect to Benzo(a)pyrene (BaP), the maximum average levels achieved in spirit aged in barrels toasted by 1, 2 and 3 minutes were respectively: 0.0094 &micro;g/L, 1.401 &micro;g/L, 0.119 &micro;g/L and 0.051 &micro;g/L, reaching higher levels than the blank content of BaP (0,014 &micro;g/L)