No description available.

No / Rapid exposure of cardiac muscle to high concentrations of caffeine releases Ca 2+ from the sarcoplasmic reticulum (SR). This Ca 2+ is then extruded from the cell by the Na +/Ca 2+ exchanger. Measurement of the current carried by the exchanger ( I Na/Ca) can therefore be used to estimate of the Ca 2+ content of the SR. Previous studies have shown that caffeine, however, can also inhibit K + currents. We therefore investigated whether the inhibitory effects of caffeine on these currents could contaminate measurements of I Na/Ca. Caffeine caused partial inhibition of the inward rectifier K + current ( I K1): the outward current at ¿40 mV was 1.15±0.24 pA/pF in control and decreased to 0.34±0.15 pA/pF in the presence of 10 mmol/l caffeine ( P<0.05, n=15). This was similar to the effect of caffeine on the holding current observed at ¿40 mV in the absence of K + channel block and could therefore account for the contaminating effects of caffeine observed during measurements of I Na/Ca. Moreover, caffeine also partially inhibited the transient outward ( I to) and the delayed rectifier ( I K) K + currents.

O objetivo desse estudo foi avaliar o pH, a liberação de íons cálcio, a atividade antimicrobiana in vitro de cimento de aluminato de cálcio (EndoBinder) com diferentes radiopacificadores (óxido de bismuto, óxido de zinco e óxido de zircônia) e a biocompatibilidade do cimento acrescido de óxido de bismuto como agente radiopacificador, em comparação ao mineral trióxido agregado (MTA). Para a avaliação do pH e a liberação de íons cálcio, 25 amostras (n=5) de 2,0 x 10 mm foram obtidas de EndoBinder Puro (EBP), EndoBinder com Óxido de Bismuto (EBOB), EndoBinder com Óxido de Zinco (EBOZn), EndoBinder com Óxido de Zircônia (EBOZr) e Mineral Trióxido Agregado (MTA), que foram imersas em 10 ml água destilada e deionizada (pH=6,9). Após 2, 4, 12, 24, 48 horas, 7, 14 e 28 dias, foi medido o pH e a quantidade de íons cálcio da água onde as amostras foram imersas. Para a determinação in vitro da atividade antimicrobiana dos cimentos (EBP, EBOB, EBOZN, EBOZr e MTA), foram obtidas 15 amostras de cada material (n=3) de 5,0 x 5,0 mm colocadas em contato com 5 tipos de microrganismos: Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 10231) e a Candida glabrata (ATCC 2001) em placas de petri com Agar (Muller Hinton e Sabouraud Dextrose) deixadas em temperatura ambiente por 2 horas para a pré-difusão e depois incubadas a 37°C por 24 horas. Utilizou-se uma régua milimetrada com precisão de 0,5 mm para realizar as medidas dos halos de inibição em torno dos corpos de prova. A biocompatibilidade dos materiais foi avaliada a partir da implantação de tubos de polietileno em tecido subcutâneo de 15 ratos machos, sendo 1 implante escapular e 1 pélvico, num total de 2 tubos por animal (n=5). Como controle, foi utilizada a lateral do tubo de polietileno. Após 7, 21 e 42 dias os animais foram sacrificados e os tecidos processados para a obtenção dos cortes histológicos e posterior análise histopatológica. Os resultados do ensaio de pH, liberação de cálcio e de atividade microbiológica foram submetidos à análise estatística (2-way ANOVA, Bonferroni, p<0,05). Os resultados de biocompatibilidade foram classificados quanto à severidade da reação inflamatória e quantificados em relação aos eventos presentes nas áreas pré-determinadas. Ao final de 28 dias não houve diferença estatística (p>0,05) entre nenhum grupo, tanto para o pH quanto para a liberação de íons cálcio. MTA, EBP e EBOB apresentaram atividade antimicrobiana quando em contato com todos os microrganismos, e EBOZr e EBOZn apenas quando em contato com Staphylococcus aureus e Candida glabrata. A reação inflamatória observada com os implantes subcultâneos diminuiu no decorrer do período de análise, porém ao final de 42 dias MTA ainda apresentou uma discreta reação inflamatória, situação não encontrada com o EB. Desta forma, concluiu-se que EB apresentou propriedades químicas e biológicas semelhantes ao MTA, além de apresentar menor reação tecidual, sendo biocompatível quando testado em tecido subcutâneo de ratos. / The purpose of this study was to evaluate the pH, calcium ion release, in vitro antimicrobial activity of a calcium aluminate cement (EndoBinder) with different radiopacifiers (bismuth oxide, zinc oxide, or zirconium oxide) and the biocompatibility of calcium aluminate cement with bismuth oxide radiopacifier in comparison with the mineral trioxide aggregate (MTA). For the evaluation of pH and calcium ion release 25 sample (n=5) measuring 2,0 x 10 mm were obtained from EndoBinder Pure (EBP), EndoBinder with bismuth oxide (EBOB), EndoBinder with zinc oxide (EBOZn), EndoBinder with zirconium oxide (EBOZr) and mineral trioxide aggregate (MTA) wich were immersed in a flask containing 10 ml of deionized and distilled water (pH=6,9). After 2, 4, 12, 24, 48 hours and 7, 14, 28 days, meassurements of the pH and calcium ion release from the water where the samples were immersed were assessed. To determine the in vitro antimicrobial activity of the cements (EBP, EBOB, EBOZN, EBOZr and MTA) 15 samples of each material (n=3) measuring 5,0 x 5,0 mm were placed in contact with five types of microorganisms: Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 10231) e a Candida glabrata (ATCC 2001) .The MullerHinton agar (MH) diffusion method was employed and the plates were kept at room temperature for 2 hours for pre diffusion and then incubated at 37 C° for 24 hours. Zones of inhibition around the specimens were measured using a 0.5mm precision millimeter ruler. The biocompatibility of the test materials was evaluated from the implantation of 2 polyethylene tubes in the subcutaneous tissue of 15 male rats (n=5), 1 being scapular (EBOB) and 1 pelvic (MTA). The external tube wall was considered as the control group. After 7, 21 and 42 days animals were sacrificed, obtaining 5 tissue samples per group at each time interval of analysis then histological analysis was performed. The test results for pH, calcium ion release and in vitro antimicrobial activity were submitted to statistical analysis (two-way ANOVA, Bonferroni, p <0.05). The results of biocompatibility were classified according to severity of inflammatory reaction and quantified in relation to present events in pre-determined areas. After 28 days there was no statistical difference (p> 0.05) between any group for both the pH and calcium ions release. MTA, EBP and EBOB showed antimicrobial activity against all microorganisms and EBOZr and EBOZn only against Staphylococcus aureus and Candida glabrata. The inflammatory reaction observed with subcutaneous implants decreased during the period of analysis however, after 42 days MTA still had a discrete inflammatory reaction, this situation was not found with EBOB. Thus, it was concluded that EB has been shown to have similar biological and chemical properties compared with MTA, it also showed less tissue reaction and biocompatibility when tested in rat subcutaneous tissue.

O objetivo desse estudo foi avaliar o pH, a liberação de íons cálcio, a atividade antimicrobiana in vitro de cimento de aluminato de cálcio (EndoBinder) com diferentes radiopacificadores (óxido de bismuto, óxido de zinco e óxido de zircônia) e a biocompatibilidade do cimento acrescido de óxido de bismuto como agente radiopacificador, em comparação ao mineral trióxido agregado (MTA). Para a avaliação do pH e a liberação de íons cálcio, 25 amostras (n=5) de 2,0 x 10 mm foram obtidas de EndoBinder Puro (EBP), EndoBinder com Óxido de Bismuto (EBOB), EndoBinder com Óxido de Zinco (EBOZn), EndoBinder com Óxido de Zircônia (EBOZr) e Mineral Trióxido Agregado (MTA), que foram imersas em 10 ml água destilada e deionizada (pH=6,9). Após 2, 4, 12, 24, 48 horas, 7, 14 e 28 dias, foi medido o pH e a quantidade de íons cálcio da água onde as amostras foram imersas. Para a determinação in vitro da atividade antimicrobiana dos cimentos (EBP, EBOB, EBOZN, EBOZr e MTA), foram obtidas 15 amostras de cada material (n=3) de 5,0 x 5,0 mm colocadas em contato com 5 tipos de microrganismos: Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 10231) e a Candida glabrata (ATCC 2001) em placas de petri com Agar (Muller Hinton e Sabouraud Dextrose) deixadas em temperatura ambiente por 2 horas para a pré-difusão e depois incubadas a 37°C por 24 horas. Utilizou-se uma régua milimetrada com precisão de 0,5 mm para realizar as medidas dos halos de inibição em torno dos corpos de prova. A biocompatibilidade dos materiais foi avaliada a partir da implantação de tubos de polietileno em tecido subcutâneo de 15 ratos machos, sendo 1 implante escapular e 1 pélvico, num total de 2 tubos por animal (n=5). Como controle, foi utilizada a lateral do tubo de polietileno. Após 7, 21 e 42 dias os animais foram sacrificados e os tecidos processados para a obtenção dos cortes histológicos e posterior análise histopatológica. Os resultados do ensaio de pH, liberação de cálcio e de atividade microbiológica foram submetidos à análise estatística (2-way ANOVA, Bonferroni, p<0,05). Os resultados de biocompatibilidade foram classificados quanto à severidade da reação inflamatória e quantificados em relação aos eventos presentes nas áreas pré-determinadas. Ao final de 28 dias não houve diferença estatística (p>0,05) entre nenhum grupo, tanto para o pH quanto para a liberação de íons cálcio. MTA, EBP e EBOB apresentaram atividade antimicrobiana quando em contato com todos os microrganismos, e EBOZr e EBOZn apenas quando em contato com Staphylococcus aureus e Candida glabrata. A reação inflamatória observada com os implantes subcultâneos diminuiu no decorrer do período de análise, porém ao final de 42 dias MTA ainda apresentou uma discreta reação inflamatória, situação não encontrada com o EB. Desta forma, concluiu-se que EB apresentou propriedades químicas e biológicas semelhantes ao MTA, além de apresentar menor reação tecidual, sendo biocompatível quando testado em tecido subcutâneo de ratos. / The purpose of this study was to evaluate the pH, calcium ion release, in vitro antimicrobial activity of a calcium aluminate cement (EndoBinder) with different radiopacifiers (bismuth oxide, zinc oxide, or zirconium oxide) and the biocompatibility of calcium aluminate cement with bismuth oxide radiopacifier in comparison with the mineral trioxide aggregate (MTA). For the evaluation of pH and calcium ion release 25 sample (n=5) measuring 2,0 x 10 mm were obtained from EndoBinder Pure (EBP), EndoBinder with bismuth oxide (EBOB), EndoBinder with zinc oxide (EBOZn), EndoBinder with zirconium oxide (EBOZr) and mineral trioxide aggregate (MTA) wich were immersed in a flask containing 10 ml of deionized and distilled water (pH=6,9). After 2, 4, 12, 24, 48 hours and 7, 14, 28 days, meassurements of the pH and calcium ion release from the water where the samples were immersed were assessed. To determine the in vitro antimicrobial activity of the cements (EBP, EBOB, EBOZN, EBOZr and MTA) 15 samples of each material (n=3) measuring 5,0 x 5,0 mm were placed in contact with five types of microorganisms: Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 10231) e a Candida glabrata (ATCC 2001) .The MullerHinton agar (MH) diffusion method was employed and the plates were kept at room temperature for 2 hours for pre diffusion and then incubated at 37 C° for 24 hours. Zones of inhibition around the specimens were measured using a 0.5mm precision millimeter ruler. The biocompatibility of the test materials was evaluated from the implantation of 2 polyethylene tubes in the subcutaneous tissue of 15 male rats (n=5), 1 being scapular (EBOB) and 1 pelvic (MTA). The external tube wall was considered as the control group. After 7, 21 and 42 days animals were sacrificed, obtaining 5 tissue samples per group at each time interval of analysis then histological analysis was performed. The test results for pH, calcium ion release and in vitro antimicrobial activity were submitted to statistical analysis (two-way ANOVA, Bonferroni, p <0.05). The results of biocompatibility were classified according to severity of inflammatory reaction and quantified in relation to present events in pre-determined areas. After 28 days there was no statistical difference (p> 0.05) between any group for both the pH and calcium ions release. MTA, EBP and EBOB showed antimicrobial activity against all microorganisms and EBOZr and EBOZn only against Staphylococcus aureus and Candida glabrata. The inflammatory reaction observed with subcutaneous implants decreased during the period of analysis however, after 42 days MTA still had a discrete inflammatory reaction, this situation was not found with EBOB. Thus, it was concluded that EB has been shown to have similar biological and chemical properties compared with MTA, it also showed less tissue reaction and biocompatibility when tested in rat subcutaneous tissue.

Orientador: Celso Aparecido Bertran, Italo Odone Mazali / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-15T20:33:27Z (GMT). No. of bitstreams: 1 Lopes_JoaoHenrique_M.pdf: 3741277 bytes, checksum: 839c748f4fd68d1140366429af1efbc5 (MD5) Previous issue date: 2010 / Mestrado / Físico-Química / Mestre em Química

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19229号 / 医博第4028号 / 新制||医||1011(附属図書館) / 32228 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 妻木 範行, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The fundamental attribute of neurons is their cellular electrical excitability, which is based on the expression of a plethora of ligand- and voltage-gated membrane channels that give rise to prominent membrane currents and membrane potential variations that represent the biophysical substrate underlying the transfer and integration of information at the cellular level. Dendrites have both an electrical and a biochemical character, which are closely linked. In contrast, glial cells are non-electrically excitable but nevertheless display a form of excitability that is based on variations of the Ca2+ concentration in the cytosol rather than electrical changes in the membrane. Cytoplasmic Ca2+ serves as an intracellular signal that is responsible for controlling a multitude of cellular processes. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca2+]i rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. Ca2+ handling in the cell is maintained by operation of multiple mechanisms of Ca2+ influx, internal release, diffusion, buffering and extrusion. Ca2+ tends to be a rather parochial operator with a small radius of action from its point of entry at the cytoplasm resulting in the concept of microdomains. Dendritic Ca2+ signaling have been shown to be highly compartmentalized and astrocytic processes have been reported to be constituted by hundreds of microdomains that represent the elementary units of the astrocyte Ca2+ signal, from where it can eventually propagate to other regions of the cell. The astrocyte Ca2+ elevation may thus act as intra and intercellular signal that can propagate within and between astrocytes, signaling to different regions of the cell and to different cells. The spatio-temporal features of neuron-to-astrocyte communication, results from diverse neurotransmitters and signaling pathways that converge and cooperate to shape the Ca2+ signal in astrocytes. Alterations in Ca2+ homeostasis have been shown to be associated with major pathological conditions of the brain such as epilepsy, ischemia and neurodegenerative diseases. Although there are evidences of Ca2+ rise in hippocampal neurons in in vitro models of epilepsy (Pal et al., 1999; Limbrick et al., 2001), there is no information on the Ca2+ regulatory mechanisms operating in discrete compartments of the epileptic neuron following Ca2+ influx through voltage gated calcium channels (VGCCs). In the first part of the work, the spatial and temporal profiles of depolarization induced changes in the intracellular Ca2+ concentration in the dendrites of cultured autaptic hippocampal pyramidal neurons rendered epileptic experimentally have been addressed. Our in vitro epilepsy model consisted of hippocampal neurons in autaptic culture that were grown in the presence of kynurenate and high Mg2+, and subsequently washing the preparation free of the blockers. To understand the differences in Ca2+ handling mechanisms in different compartments of a control neuron and the kynurenate treated neuron, a combination of whole-cell patch-clamp recording and fast Ca2+ imaging methods using the Ca2+ indicator Oregon Green 488 BAPTA-1 was applied. All our analysis was focused on localized regions in the dendrite that showed pronounced Ca2+ transients upon activation of high voltage activated (HVA) Ca2+ channels. The spatial extent of Ca2+ signals suggested the presence of distinct dendritic compartments that respond to the depolarizing stimulus. Further, the local Ca2+ transients were observed even in the presence of NMDA and AMPA receptor antagonists, suggesting that the opening of VGCCs primarily triggered the local Ca2+ changes. The prominent changes in intracellular Ca2+ observed in these dendritic regions appear to be sites where Ca2+ evoked dendritic exocytosis (CEDE) takes place. Since cellular Ca2+ buffers determine the amplitude and diffusional spread of neuronal Ca2+ signals, quantitative estimates of the time-dependent spread of intracellular Ca2+ in the dendritic compartments in the control and treated neurons were done using image processing techniques. Physiological changes in Ca2+ channel functioning were also induced by kynurenate treatment and one such noticeable difference was the observation of Ca2+ dependent inactivation in the treated neurons. We provide evidences of localized Ca2+ changes in the dendrites of hippocampal neurons that are rendered epileptic by kynurenate treatment, suggesting that these sites are more vulnerable (Padmashri et al., 2006). This might contribute to the epileptiform activity by local changes in cellular and membrane properties in complex ways that remains to be clearly understood. Status Epilepticus (SE), stroke and traumatic brain injury are all associated with large increases in extracellular glutamate concentrations. The concentration of glutamate in the extracellular fluid is around 3-4 µM and astrocytes are primarily responsible for the uptake of glutamate at the synapses. The extracellular levels of glutamate has been shown to increase dramatically (16 fold) in human SE suggesting an important role of glutamate in the mechanism of seizure activity and seizure related brain damage (Carlson et al., 1992). Several other studies have also shown a persistent increase in extracellular glutamate concentration to potentially neurotoxic concentrations in the epileptogenic hippocampus (During and Spencer, 1993; Sherwin, 1999; Cavus et al., 2005). We addressed the problem related to the effects of prolonged glutamate pretreatment on Ca2+ signaling in an individual astrocyte and its adjoining astrocyte (astrocyte pair), rather than on a syncytium of astrocytes in culture. Individual astrocytes may have functional domains that respond to an agonist through distinct receptor signaling systems. These are difficult to observe in studies that are done on glial syncytium because of spatial limits of image capture. This was examined with simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca2+ imaging using the Ca2+ indicator Oregon Green 488 BAPTA-1 to identify the Ca2+ microdomains. Transient Ca2+ changes locked to the depolarization were observed in certain compartments in the astrocyte processes of the depolarized astrocyte and the responses were more pronounced in the adjoining astrocyte of the astrocyte pair. The Ca2+ transient amplitudes were enhanced on pretreatment of cells with glutamate (500 µM for 20 minutes). Estimation of local Ca2+ diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. In order to understand the underlying mechanisms, we performed the experiments in the presence of different blockers for the metabotropic glutamate receptor, inositol 1,4,5 triphosphate (IP3) receptors and gap junctions. Ca2+ transients recorded on pretreatment of cells with glutamate showed attenuated responses in the presence of the metabotropic glutamate receptor (mGluR) antagonist α-Methyl(4-Carboxy-Phenyl) Glycine (MCPG). Intracellular heparin (an antagonist of IP3 receptor) introduced in the depolarized astrocyte did not affect the Ca2+ transients in the heparin loaded astrocyte, but attenuated the [Ca2+]i responses in the adjoining astrocyte suggesting that IP3 may be the transfer signal. The uncoupling agent 1-Octanol attenuated the [Ca2+]i responses in the adjoining cell of the astrocyte pair in both the control and glutamate pretreated astrocytes indicating the role of gap junctional communication. The findings of [Ca2+]i responses within discrete regions of astrocytic processes suggest that astrocytes may be comprised of microdomains whose properties are altered by glutamate pretreatment. The data also indicates that glutamate induced alterations in Ca2+ signaling in the astrocyte pair may be mediated through phospholipase C (PLC), IP3, internal Ca2+ stores, VGCCs and gap junction channels (Padmashri and Sikdar, 2006). Neuronal (EAAC-1) and glial (GLT-1 and GLAST) glutamate transporters facilitate glutamate reuptake after synaptic release. Transgenic mice with GLT-1 knockout display spontaneous epileptic activity (Tanaka et al., 1997) and loss of glial glutamate transporters using chronic antisense nucleotide administration was reported to result in elevated extracellular glutamate levels and neurodegeneration characteristic of excitotoxity (Rothstein et al., 1996). Dysfunction of glutamate transporters and the resulting increase of glutamate have been speculated to play an important role in infantile epilepsies (Demarque et al., 2004). We examined the effects of pretreatment with glutamate in the presence of the glutamate transport inhibitor threo-β-hydroxy-aspartate (TBHA) and in Na+-free extracellular medium to understand whether this resulted in any alteration in the astrocytic intracellular Ca2+ dynamics following activation of voltage gated calcium channels. The Ca2+ responses were found to be attenuated in both the cases indicating that the elevated levels of extracellular glutamate due to blockade of glutamate transporters may influence the responses mediated by the astrocytic glutamate receptors. Our studies indicate that the heightened extracellular glutamate concentration is not gliotoxic in our experimental system, although it may have a profound effect on altering the activity of surrounding neurons which was not addressed in the present work. Several studies have indicated that neurons control the level of gap junction mediated communication between astrocytes (Giaume and McCarthy, 1996; Rouach et al, 2000). All our earlier studies were done on process bearing astrocytes that were co-cultured with neurons. We have addressed the question as to whether the spatio-temporal changes in [Ca2+]i in astrocyte pairs differ if the astrocytes are cultured in the absence of neurons. The results indicate that there is indeed a significant reduction in the responses that are evoked in response to the depolarization pulse in the adjoining cell of the astrocyte pair. These experiments demonstrate that neurons in the cocultures may selectively enhance the Ca2+ responses possibly by increasing the coupling between the two cells.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-06-09T17:05:06Z No. of bitstreams: 2 Tese - Letícia Mendes de Castro - 2016.pdf: 2261024 bytes, checksum: 4cfaccebd66c7e3ea4eda9b31d53d16d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-10T11:19:37Z (GMT) No. of bitstreams: 2 Tese - Letícia Mendes de Castro - 2016.pdf: 2261024 bytes, checksum: 4cfaccebd66c7e3ea4eda9b31d53d16d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-06-10T11:19:37Z (GMT). No. of bitstreams: 2 Tese - Letícia Mendes de Castro - 2016.pdf: 2261024 bytes, checksum: 4cfaccebd66c7e3ea4eda9b31d53d16d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2016-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Brazil has one of the largest commercial cattle herds worldwide, but its meat quality is highly variable. The national herd is largely composed of Bos indicus breeds, which in general have less tender meat than Bos taurus cattle, decreasing the product value. This study was carried out to identify relevant regions and biological pathways associated with meat tenderness in Polled Nellore cattle. It was also aimed to evaluate the effect of different quality control protocols in GWAS for meat tenderness in Polled Nellore cattle. The data consisted of Warner-Bratzler shear force (WBSF) values of Longissimus muscle after 7 days of ageing, from 427 Polled Nellore animals. The animals were genotyped using either the Illumina BovineHD Beadchip (777k) or the GGP-Indicus Chip (77k). SNPs were excluded when Call Rate < 90%, then the imputation from the GGP to the HD Chip was performed using the FImput’s software. To study the different quality control protocols and their influence in GWAS, 590,915 markers were used. The effect of different QCs were verified using 16 protocols with three thresholds for MAF (MAF < 0.01; < 0.05 and < 0.10) and HWP (p < 0.01; < 0.0001 and < 0.00001) and their possible combinations. GWASs were performed using the PD3/EMMAx method with the remaining markers of each QC. For GWAS performed for pathway analysis, 369,007 markers were used after SNPs were excluded when Call rate < 90%, HWP p < 0.01 and MAF < 0.01. Group of slaughter and sex were included as fixed effects. Significant markers (p < 0.0001) were found in all analysis, in which the chromosomes with more significant SNPs of the different QCs were 3, 17, 20, 21, 25 and 27, and in the pathway study were located on chromosomes 3, 13, 17, 20, 21 and 25 explaining great proportion of variation, indicating possible QTLs associated with meat tenderness in those genomic regions. The analyses of different QCs showed that there is an effect of quality control over GWAS, and the filter for MAF influenced the results more broadly. A pathway enrichment analysis based on SNPs from GWAS was performed using FatiGO’s procedure. 22,365 annotated genes, including 1,010 significant genes were used. Thus, 22 GO terms and two IP entries were deemed enriched. Several of these functional categories, such as protein tyrosine and serine/threonine kinase activity, calcium ion binding and growth factors can be related to WBSF in Polled Nellore cattle. These results help to elucidate the metabolic pathways related to this trait, which is of extreme economic and social importance to Brazil as Nellore is the dominant beef cattle breed in the country. / O Brasil tem um dos maiores rebanhos bovinos comerciais do mundo, mas a qualidade da carne é altamente variável. O rebanho nacional é em grande parte composto de raças Bos indicus, que em geral têm carne menos macia do que o gado Bos taurus, diminuindo o valor do produto. Objetivou-se nesse estudo identificar regiões genômicas e vias biológicas relevantes associadas com a maciez da carne em bovinos da raça Nelore Mocho. Além disso, objetivou-se também avaliar diferentes protocolos de controle de qualidade dos SNPs e as possíveis influências nos resultados de GWAS. Os dados consistiram em valores de WBSF do músculo Longissimus dorsi, após maturação de sete dias, de 427 animais Nelore Mocho. Os animais foram genotipados em marcadores SNP Illumina BovineHD Beadchip (777k) ou Chip GGP-Indicus (77k). Todos os SNPs passaram por um Call Rate de 90% para posterior imputação utilizando o software FImput. Para averiguar os diferentes protocolos de qualidade e suas influências no GWAS foram utilizados 590.915 marcadores. Os efeitos dos diferentes QCs foram verificados utilizando 16 protocolos com três limiares para MAF (MAF < 0,01;< 0,05 e < 0,10) e HWP (p < 0,01; < 0,0001 e < 0,00001) e suas possíveis combinações. Os GWASs foram realizados utilizando método P3D/EMMAx com os marcadores restantes de cada QC. No GWAS realizado para posterior análise das vias utilizou-se 369.007 marcadores após a exclusão de SNPs baseada nos filtros Call Rate < 90%, HWP p < 0,01 e MAF < 0,01. Grupo de abate e sexo foram incluídos no modelo como efeitos fixos. Marcadores significativos (p < 0,0001) foram localizados em todas as análises, e os cromossomos com maior quantidade de SNPs significativos dos diferentes QCs foram 3, 17, 20, 21, 25 e 27. No estudo de vias foram localizados SNPs significativos nos cromossomos 3, 13, 17, 20, 21 e 25, que explicaram maior proporção da variação, indicando que existem QTLs associados à maciez da carne nessas regiões do genoma. As análises dos diferentes QCs evidenciaram efeito do controle de qualidade dos SNPs sobre o GWAS e o filtro para MAF influenciou de maneira mais ampla os resultados. Foi realizada uma análise de enriquecimento de vias baseando-se nos SNPs do GWAS, utilizando o procedimento FatiGO. Apenas os genes com no mínimo um SNP significativo (p < 0,01) foram considerados. Foram utilizados 22.365 genes anotados, incluindo 1.010 genes significativos. Um total de 22 termos GO e duas entradas IP foram consideradas enriquecidas com genes significamente associados com a maciez da carne. Várias dessas categorias funcionais como atividade da proteína tirosina quinase e serina/treonina quinase, ligantes ao íon cálcio e fatores de crescimento, podem estar relacionadas com WBFS em bovinos da raça Nelore Mocho. Estes resultados ajudam elucidar as vias relacionadas com essa característica de extrema importância econômica para o Brasil, já que o Nelore é a raça de gado de corte dominante no país.

It is impossible to remove methylmercury (MeHg) from biological systems because MeHg is found throughout our environment in many fresh and salt water fish. The consumption of fish is important to human nutrition and health. The mechanism of MeHg neurotoxicity must be understood to minimize adverse exposure consequences. The dissertation objective was to: 1) compare mechanisms of MeHg neurotoxicity between animals exposed as adults and those exposed during gestation, and 2) develop an in vitro test model of in vivo MeHg exposure. Total mercury (Hg) levels in tissue / cells were determined by combustion / trapping / atomic absorption. Cell death was determined by Fluoro-Jade histochemical staining and activated caspase 3 immunohistochemistry for in vivo studies, and Trypan blue exclusion, lactate dehydrogenase activity, and cytotoxicity assays for in vitro studies. Mitochondrial membrane potential (MMP), intracellular calcium ion concentration ([Ca2+]i), and production of reactive oxygen species (ROS) were determined using fluorescence microscopy or microplate reader assays. Young adult C57Bl/6 mice were exposed to a total dose of 0, 1.0, or 5.0 mg/kg body weight MeHg divided over postnatal days (P)35 to 39. Pregnant female mice were exposed to a total does of 0, 0.1, or 1.0 mg/kg body weight MeHg divided over gestational days (G)8 to 18. SY5Y cells were exposed to 0, 0.01, 0.1, or 1.0 ?M MeHg or HgCl2 for 24, 48, or 72 hours. Total Hg in brains of young adult mice, mouse pups, and SY5Y cells accumulated in a dose-dependent manner. Cell death increased in SY5Y cells exposed to the highest concentrations of MeHg and HgCl2 used in this study. Cell death increased in the molecular and granule cerebellar cell layers of young adult mice exposed to the highest doses of MeHg used in this study. P0 mouse pups showed no increase in cell death within the cerebellum following MeHg exposure. Cerebella of mice at P10 exhibited decreased dying cells only in the external germinal layer. Low concentrations of MeHg affected MMP in both in vivo and in vitro studies, but did not result in decreased MMP typically associated with higher MeHg concentrations. [Ca2+]i was increased throughout the in vivo experiments in an age- , sexand brain region-dependent manner. Generation of ROS was decreased in both in vivo and in vitro studies with both the MeHg and HgCl2 (in vitro) treatments. In summary, low and moderate MeHg exposure, both in vivo and in vitro, altered mitochondrial function, Ca2+ homeostasis, and ROS differently than what is reported in the literature for higher MeHg exposure concentrations. SY5Y cells were sensitive to low-levels of MeHg and HgCl2 and responded similarly to cells in the whole animal studies, thus making SY5Y cells realistic candidates for mechanistic MeHg studies. Cell culture and whole animal neuronal functional studies at chronic low-level MeHg exposure are limited. These data suggest that low-levels of MeHg may affect neuronal function. Therefore, further chronic low-level MeHg neuronal functional studies are warranted.

This thesis deals with the analysis of micromotion of a single charged calcium ion trapped inside the linear Paul's ion trap and the influence of residual micromotion on the systematic frequency shifts of the clock transition of calcium ion. The fundamental properties of the motion of an ion confined within linear Paul's ion trap are shown in general using a theoretical description. The micromotion component of the overall motion is especially emphasized. A model expressing micromotion in the axial direction of the trap is introduced on the basis of the results of the numerical calculation of electric fields inside the trap. The model is compared to the reality experimentally. Then, the photon-correlation method of detection of micromotion is introduced and subsequently used to minimize and to estimate a measure of residual micromotion in all spacial directions. According to the achievable measure of residual micromotion, the systematic frequency shifts caused by this micromotion are estimated. It can be seen that we are able to reach uncertainties of the relative frequency shifts due to micromotion below 10^20. We expect that uncertainty of total motional systematic frequency shift is in our case limited by thermal motion.