Thesis (Ph. D.)--University of California, San Diego, 2000. / Vita. Includes bibliographical references (leaves 151-168).

Thesis (Ph.D. in Toxicology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 137-149).

Neural crest cells arise during neurulation, a process that occurs during the third week of embryogenesis. These diverse cells then divide into various subtypes including cranial neural crest cells and cardiac neural crest cells. Each of these subtypes gives rise to a wide range of features throughout the fetus. While these cells are extremely diverse, they are also incredibly sensitive to their surrounding environment. Many maternal conditions affect neural crest cell division and migration, but maternal alcohol consumption and hyperglycemia due to gestational diabetes will be discussed in detail, with special attention paid to tissues that derive from cranial neural crest cells. While the initial mechanisms of the pathology vary for both of these conditions, what is remarkable is that they ultimately cause effects in similar ways. Both mechanisms lead to the creation of reactive oxygen species, which in turn trigger apoptotic pathways. Neural crest cell death causes a variety of congenital anomalies in fetuses, including craniofacial defects and cardiac outflow tract defects. Treatment options that have been researched in both conditions also vary, but are based on similar principles. Antioxidant therapies reduce the production of reactive oxygen species, thus reducing the severity of the anomalies affecting the fetus during development. Both maternal alcohol consumption and gestational diabetes are important public health concerns, and their management is of utmost priority in society. By decreasing the rates of women who consume alcohol during pregnancy, and managing gestational diabetes in those at highest risk, the rates of fetal congenital defects could be decreased.

O objetivo deste estudo foi investigar os efeitos do tratamento musicoterapêutico aplicado ao desenvolvimento da memória não declarativa em crianças com Síndrome de Williams (SW) e com Síndrome do Álcool Fetal (SAF). Foram conduzidos dois experimentos de antes e depois. Um incluindo 10 indivíduos com SW e outro 10 indivíduos com SAF com idade entre 6 a 17 anos, que receberam 13 sessões de tratamento com periodicidade de uma vez por semana em formato individual e duração de 45min cada sessão. As avaliações foram feitas pelas escalas IMTAP (Individualized Music Therapy Assessment Profile) e WISC-III (Escala Wechsler de Inteligência para Crianças) que mensuraram respostas de QI e habilidades cognitivas antes e depois das intervenções com musicoterapia. Os resultados foram mensurados por um avaliador cego, antes e depois das intervenções, através da pontuação das escalas WISC-III e IMTAP. A média calculada nos diferentes tempos do estudo para as crianças com SAF pela escala WISC-III antes do tratamento foi de 70.9 e desvio padrão 2.67 (IC 95% 65.65 a 76.15 p= 0.001) e média do IMTAP de 73.90 e desvio padrão de 1.90 (IC 95% 70.17 a 77.63 p=0.001) e no período após a intervenção a média da escala WISC-III foi de 78.60 e desvio padrão de 2.41 (IC 95% 74.40 a 82.80 p=0.001) e do IMTAP 85.70 e desvio padrão de 2.52 (IC 95% 80.75 a 90.65 p=0.001). Com relação às crianças com síndrome de Williams a média calculada pela escala WISC-III antes do tratamento foi de 52.2 e desvio padrão de 1.26 (IC 95% 49.72 a 54.68 p= 0.001) e média do IMTAP de 70.2 e desvio padrão de 0.77 (IC 95% 68.69 a 71.71 p= 0.001) e no período após a intervenção a média da escala WISC-III foi de 59 e desvio padrão de 1.6 (IC 95%55.86 a 62.14 p=0.001) e IMTAP 83.3 e desvio padrão de 1.68 (IC 95% 80.01 a 86.59 p=0.001). Com este estudo conseguimos verificar que as intervenções com musicoterapia apresentaram um efeito positivo para estas populações, mesmo com pouco tempo de tratamento, com relação ao desenvolvimento de habilidades cognitivas. Os resultados observados na investigação dos efeitos da musicoterapia aplicada ao desenvolvimento da memória não declarativa de crianças com síndrome de Williams e síndrome Alcoólica Fetal são inconclusivos. Sugere-se para as próximas investigações uma amostra maior, grupo controle e mais tempo de tratamento. Estas modificações poderão aumentar a precisão para observar os efeitos do tratamento nestas populações. / This study aimed to investigate the effects of music therapy treatment applied to the non declarative memory development in the Williams Syndrome (WS) and the Fetal Alcohol Syndrome children (FAS). A before and after experiment was conducted which included 10 WS individuals and another 10 individuals with FAS aged between 6 to 17, each received 13 treatment sessions, with weekly intervals, individually and 45min sessions. The evaluations were executed by the WISC III and the IMTAP scales measuring the IQ responses and the cognitive abilities before and after the music therapy interventions. A random evaluator measured the before and after results of the interventions using the WISC-III and the IMTAP scales scores. Regarding the FAS children’s analysis different time sequence, the WISC-III scales calculated average before treatment was 70.9 and standard deviation 2.67 (CI 95% 65.65 a 76.15 p= 0.000) the IMTAP average was 73.90 and standard deviation 1.90 (CI 95% 70.17 a 77.63 p=0.000) the post period intervention average for the WISC-III scale was 78.60 and standard deviation 2.41 (CI 95% 74.40 a 82.80 p=0.000) and for the IMTAP was 85.70 and standard deviation 2.52 (CI 95% 80.75 a 90.65 p=0.000). In relation to the Williams Syndrome children the WISC-III calculated average before treatment was 52.2 and standard deviation 1.26 (CI 95% 49.72 a 54.68 p= 0.000) the IMTAP average was 70.2 and standard deviation 0.77 (CI 95% 68.69 a 71.71 p= 0.000) post period intervention average for the WISC-III scale was 59 and standard deviation 1.6 (CI 95% 55.86 a 62.14 p=0.000) the IMTAP scale was 83.3 and standard deviation 1.68 (CI 95% 80.01 a 86.59 p=0.000). This study verifies that intervention with music therapy presents positive effects for these populations, even in short term treatment with relation to the cognitive skills development. The results observed in the investigation of the music therapy effects applied to the non declarative memory development of children with Williams Syndrome and Fetal Alcohol Syndrome is inconclusive. Therefore, for future investigations larger samples, group control, and longer treatment time is recommended. This modification can improve observation accuracy of treatment effects in these populations.

abstract: In the fifteen years between the discovery of fetal alcohol syndrome (FAS) in 1973 and the passage of alcohol beverage warning labels in 1988, FAS transformed from a medical diagnosis between practitioner and pregnant women to a broader societal risk imbued with political and cultural meaning. I examine how scientific, social, moral, and political narratives dynamically interacted to construct the risk of drinking during pregnancy and the public health response of health warning labels on alcohol. To situate such phenomena I first observe the closest regulatory precedents, the public health responses to thalidomide and cigarettes, which established a federal response to fetal risk. I then examine the history of how the US defined and responded to the social problem of alcoholism, paying particular attention to the role of women in that process. Those chapters inform my discussion of how the US reengaged with alcohol control at the federal level in the last quarter of the twentieth century. In the 1970s, FAS allowed federal agencies to carve out disciplinary authority, but robust public health measures were tempered by uncertainty surrounding issues of bureaucratic authority over labeling, and the mechanism and extent of alcohol’s impact on development. A socially conservative presidency, dramatic budgetary cuts, and increased industry funding reshaped the public health approach to alcoholism in the 1980s. The passage of labeling in 1988 required several conditions: a groundswell of other labeling initiatives that normalized the practice; the classification of other high profile, socially unacceptable alcohol-related behaviors such as drunk driving and youth drinking; and the creation of a dual public health population that faced increased medical, social, and political scrutiny, the pregnant woman and her developing fetus. / Dissertation/Thesis / Doctoral Dissertation Biology 2016

O objetivo deste estudo foi investigar os efeitos do tratamento musicoterapêutico aplicado ao desenvolvimento da memória não declarativa em crianças com Síndrome de Williams (SW) e com Síndrome do Álcool Fetal (SAF). Foram conduzidos dois experimentos de antes e depois. Um incluindo 10 indivíduos com SW e outro 10 indivíduos com SAF com idade entre 6 a 17 anos, que receberam 13 sessões de tratamento com periodicidade de uma vez por semana em formato individual e duração de 45min cada sessão. As avaliações foram feitas pelas escalas IMTAP (Individualized Music Therapy Assessment Profile) e WISC-III (Escala Wechsler de Inteligência para Crianças) que mensuraram respostas de QI e habilidades cognitivas antes e depois das intervenções com musicoterapia. Os resultados foram mensurados por um avaliador cego, antes e depois das intervenções, através da pontuação das escalas WISC-III e IMTAP. A média calculada nos diferentes tempos do estudo para as crianças com SAF pela escala WISC-III antes do tratamento foi de 70.9 e desvio padrão 2.67 (IC 95% 65.65 a 76.15 p= 0.001) e média do IMTAP de 73.90 e desvio padrão de 1.90 (IC 95% 70.17 a 77.63 p=0.001) e no período após a intervenção a média da escala WISC-III foi de 78.60 e desvio padrão de 2.41 (IC 95% 74.40 a 82.80 p=0.001) e do IMTAP 85.70 e desvio padrão de 2.52 (IC 95% 80.75 a 90.65 p=0.001). Com relação às crianças com síndrome de Williams a média calculada pela escala WISC-III antes do tratamento foi de 52.2 e desvio padrão de 1.26 (IC 95% 49.72 a 54.68 p= 0.001) e média do IMTAP de 70.2 e desvio padrão de 0.77 (IC 95% 68.69 a 71.71 p= 0.001) e no período após a intervenção a média da escala WISC-III foi de 59 e desvio padrão de 1.6 (IC 95%55.86 a 62.14 p=0.001) e IMTAP 83.3 e desvio padrão de 1.68 (IC 95% 80.01 a 86.59 p=0.001). Com este estudo conseguimos verificar que as intervenções com musicoterapia apresentaram um efeito positivo para estas populações, mesmo com pouco tempo de tratamento, com relação ao desenvolvimento de habilidades cognitivas. Os resultados observados na investigação dos efeitos da musicoterapia aplicada ao desenvolvimento da memória não declarativa de crianças com síndrome de Williams e síndrome Alcoólica Fetal são inconclusivos. Sugere-se para as próximas investigações uma amostra maior, grupo controle e mais tempo de tratamento. Estas modificações poderão aumentar a precisão para observar os efeitos do tratamento nestas populações. / This study aimed to investigate the effects of music therapy treatment applied to the non declarative memory development in the Williams Syndrome (WS) and the Fetal Alcohol Syndrome children (FAS). A before and after experiment was conducted which included 10 WS individuals and another 10 individuals with FAS aged between 6 to 17, each received 13 treatment sessions, with weekly intervals, individually and 45min sessions. The evaluations were executed by the WISC III and the IMTAP scales measuring the IQ responses and the cognitive abilities before and after the music therapy interventions. A random evaluator measured the before and after results of the interventions using the WISC-III and the IMTAP scales scores. Regarding the FAS children’s analysis different time sequence, the WISC-III scales calculated average before treatment was 70.9 and standard deviation 2.67 (CI 95% 65.65 a 76.15 p= 0.000) the IMTAP average was 73.90 and standard deviation 1.90 (CI 95% 70.17 a 77.63 p=0.000) the post period intervention average for the WISC-III scale was 78.60 and standard deviation 2.41 (CI 95% 74.40 a 82.80 p=0.000) and for the IMTAP was 85.70 and standard deviation 2.52 (CI 95% 80.75 a 90.65 p=0.000). In relation to the Williams Syndrome children the WISC-III calculated average before treatment was 52.2 and standard deviation 1.26 (CI 95% 49.72 a 54.68 p= 0.000) the IMTAP average was 70.2 and standard deviation 0.77 (CI 95% 68.69 a 71.71 p= 0.000) post period intervention average for the WISC-III scale was 59 and standard deviation 1.6 (CI 95% 55.86 a 62.14 p=0.000) the IMTAP scale was 83.3 and standard deviation 1.68 (CI 95% 80.01 a 86.59 p=0.000). This study verifies that intervention with music therapy presents positive effects for these populations, even in short term treatment with relation to the cognitive skills development. The results observed in the investigation of the music therapy effects applied to the non declarative memory development of children with Williams Syndrome and Fetal Alcohol Syndrome is inconclusive. Therefore, for future investigations larger samples, group control, and longer treatment time is recommended. This modification can improve observation accuracy of treatment effects in these populations.

O objetivo deste estudo foi investigar os efeitos do tratamento musicoterapêutico aplicado ao desenvolvimento da memória não declarativa em crianças com Síndrome de Williams (SW) e com Síndrome do Álcool Fetal (SAF). Foram conduzidos dois experimentos de antes e depois. Um incluindo 10 indivíduos com SW e outro 10 indivíduos com SAF com idade entre 6 a 17 anos, que receberam 13 sessões de tratamento com periodicidade de uma vez por semana em formato individual e duração de 45min cada sessão. As avaliações foram feitas pelas escalas IMTAP (Individualized Music Therapy Assessment Profile) e WISC-III (Escala Wechsler de Inteligência para Crianças) que mensuraram respostas de QI e habilidades cognitivas antes e depois das intervenções com musicoterapia. Os resultados foram mensurados por um avaliador cego, antes e depois das intervenções, através da pontuação das escalas WISC-III e IMTAP. A média calculada nos diferentes tempos do estudo para as crianças com SAF pela escala WISC-III antes do tratamento foi de 70.9 e desvio padrão 2.67 (IC 95% 65.65 a 76.15 p= 0.001) e média do IMTAP de 73.90 e desvio padrão de 1.90 (IC 95% 70.17 a 77.63 p=0.001) e no período após a intervenção a média da escala WISC-III foi de 78.60 e desvio padrão de 2.41 (IC 95% 74.40 a 82.80 p=0.001) e do IMTAP 85.70 e desvio padrão de 2.52 (IC 95% 80.75 a 90.65 p=0.001). Com relação às crianças com síndrome de Williams a média calculada pela escala WISC-III antes do tratamento foi de 52.2 e desvio padrão de 1.26 (IC 95% 49.72 a 54.68 p= 0.001) e média do IMTAP de 70.2 e desvio padrão de 0.77 (IC 95% 68.69 a 71.71 p= 0.001) e no período após a intervenção a média da escala WISC-III foi de 59 e desvio padrão de 1.6 (IC 95%55.86 a 62.14 p=0.001) e IMTAP 83.3 e desvio padrão de 1.68 (IC 95% 80.01 a 86.59 p=0.001). Com este estudo conseguimos verificar que as intervenções com musicoterapia apresentaram um efeito positivo para estas populações, mesmo com pouco tempo de tratamento, com relação ao desenvolvimento de habilidades cognitivas. Os resultados observados na investigação dos efeitos da musicoterapia aplicada ao desenvolvimento da memória não declarativa de crianças com síndrome de Williams e síndrome Alcoólica Fetal são inconclusivos. Sugere-se para as próximas investigações uma amostra maior, grupo controle e mais tempo de tratamento. Estas modificações poderão aumentar a precisão para observar os efeitos do tratamento nestas populações. / This study aimed to investigate the effects of music therapy treatment applied to the non declarative memory development in the Williams Syndrome (WS) and the Fetal Alcohol Syndrome children (FAS). A before and after experiment was conducted which included 10 WS individuals and another 10 individuals with FAS aged between 6 to 17, each received 13 treatment sessions, with weekly intervals, individually and 45min sessions. The evaluations were executed by the WISC III and the IMTAP scales measuring the IQ responses and the cognitive abilities before and after the music therapy interventions. A random evaluator measured the before and after results of the interventions using the WISC-III and the IMTAP scales scores. Regarding the FAS children’s analysis different time sequence, the WISC-III scales calculated average before treatment was 70.9 and standard deviation 2.67 (CI 95% 65.65 a 76.15 p= 0.000) the IMTAP average was 73.90 and standard deviation 1.90 (CI 95% 70.17 a 77.63 p=0.000) the post period intervention average for the WISC-III scale was 78.60 and standard deviation 2.41 (CI 95% 74.40 a 82.80 p=0.000) and for the IMTAP was 85.70 and standard deviation 2.52 (CI 95% 80.75 a 90.65 p=0.000). In relation to the Williams Syndrome children the WISC-III calculated average before treatment was 52.2 and standard deviation 1.26 (CI 95% 49.72 a 54.68 p= 0.000) the IMTAP average was 70.2 and standard deviation 0.77 (CI 95% 68.69 a 71.71 p= 0.000) post period intervention average for the WISC-III scale was 59 and standard deviation 1.6 (CI 95% 55.86 a 62.14 p=0.000) the IMTAP scale was 83.3 and standard deviation 1.68 (CI 95% 80.01 a 86.59 p=0.000). This study verifies that intervention with music therapy presents positive effects for these populations, even in short term treatment with relation to the cognitive skills development. The results observed in the investigation of the music therapy effects applied to the non declarative memory development of children with Williams Syndrome and Fetal Alcohol Syndrome is inconclusive. Therefore, for future investigations larger samples, group control, and longer treatment time is recommended. This modification can improve observation accuracy of treatment effects in these populations.

Made available in DSpace on 2016-03-15T19:40:27Z (GMT). No. of bitstreams: 1 Stella Maria Coda Pinto Alves.pdf: 2917004 bytes, checksum: 4023390b83759490fdd7f6eb4340a61f (MD5) Previous issue date: 2007-09-13 / Alcohol consumption during pregnancy is associated with a wide range of effects on birth, from which fetal alcohol syndrome is considered to lead to the most severe phenotypes. The teratogenic effects of alcohol on the human growth represent a spectrum of features on facial anomalies, growth deficiency at intrauterine into postnatal period, attention deficits and poor academic achievements. The aim of the present investigation was to study cephalometry as a diagnostic tool concerning craniofacial disorders in children and adolescents with confirmed mothers' alcohol intake history during the pre natal period. The specific objective was to analyse McNamara and Björk-Jarabak craniofacial parameters of ten children and adolescents who were exposed to alcohol during their mothers' pregnancy. The age of the subjects ranged from 11 to 18 years old, 5 were female. It was measured linear and angular variables compared to previous standards as descrived by McNamara and Björk-Jarabak. For this purpose, five linear measures and four angular parameters were taken into account in order to better describe craniofacial growth changes: maxilla length, mandibular length, anterior facial height, anterior cranial base, posterior cranial base, saddle angle, articular angle, gonial angle, upper gonial angle. Posterior cranial base, anterior facial height, mandibular size and tegumentary profile parameters were analysed as a whole in order to undertake global craniofacial development. The study showed that ten children and adolescents who were exposed to alcohol during their mothers' pregnancy were associated with linear measurements changes relative to posterior cranial base (p=0.01), mandibular size (p= 0.0009), face height (p= 0.01) and upper gonial angle (p= 0.01). / A exposição ao álcool durante a gestação é responsável pelo amplo espectro de alterações ocasionadas ao feto, cuja manifestação mais grave é a síndrome fetal alcoólica. O efeito teratogênico do álcool no homem apresenta evidências clínicas caracterizadas por anomalias faciais, atraso no crescimento intrauterino ou após o nascimento, e deficiências relacionadas ao aprendizado e à atenção. O objetivo geral deste estudo foi testar a análise cefalométrica como auxílio diagnóstico dos efeitos fetais do álcool em sujeitos com história de exposição ao álcool durante a gestação. O objetivo específico foi estudar as análises cefalométricas de McNamara e Björk-Jarabak nestes indivíduos. Foram selecionados dez sujeitos com história de ingestão de bebidas alcoólicas pelas respectivas mães no período gestacional. As idades variam de 11 a 18 anos, sendo 5 de cada sexo. Foram mensuradas grandezas lineares e angulares, e comparadas com a norma destas medidas de acordo com padrões cefalométricos das análises de McNamara e Björk-Jarabak. Foram analisados os dados obtidos a partir de cinco medidas lineares (comprimento efetivo da maxila, comprimento efetivo da mandíbula, altura facial anterior e inferior, base anterior do crânio, base posterior do crânio) e quatro medidas angulares (ângulo da sela, ângulo articular, ângulo goníaco, plano superior do ângulo goníaco). As grandezas relacionadas à base do crânio, à altura facial, à mandíbula e ao perfil tegumentar foram avaliadas em conjunto para permitir análise global do crescimento craniofacial. Os dez sujeitos com história de exposição ao álcool no período gestacional mostraram as seguintes medidas com diferença estatística significante em relação à norma: grandezas lineares relacionadas à base posterior do crânio (p= 0,01), comprimento efetivo da mandíbula (p= 0,0009); altura facial antero inferior (p= 0,01) e a grandeza angular plano superior do ângulo-goníaco (p= 0,01).

The cognitive and/or behavioural problems associated with Fetal Alcohol Spectrum Disorder (FASD) place this population at increased risk of involvement in the justice system. Although FASD poses challenges at each stage of the justice system, legal discussion and commentary have pinpointed the sentencing stage as the phase in which the issue of FASD is most commonly raised and considered. The purpose of this study is to examine if (and how) FASD is being taking into consideration at sentencing. To this end, a comparative analysis of 87 sentencing judgments (42 FASD offenders and 45 non-FASD offenders) reported in Quicklaw was conducted. Cases were matched on most serious offence (assault, robbery and sexual assault) and jurisdiction (Yukon, British Columbia and Ontario). Descriptions of FASD and non-FASD offenders as reported by judges were found to differ in a number of significant ways. Similarly, sentencing purposes applied to each offender group emerged as distinct. Despite these distinctions, no differences were found in the type and length of sentence handed down (even after controlling for criminal record and breaches). These findings indicate a need for further research and possible policy changes.

Fetal alcohol exposure is the most common known cause of preventable mental retardation, yet we know little about how microglia respond to, or are affected by, alcohol in vivo. Using an acute (single day) model of moderate (3 g/kg) to severe (5 g/kg) alcohol exposure in postnatal day (P) 7 or P8 mice we have found that alcohol-induced cortical neuroapoptosis is closely correlated in space and time with the appearance of activated microglia near dead cells. Microglia found in close proximity to dying neurons selectively engulfed those that were in later stages of apoptosis. Remarkably, most dead cells were cleared and microglia began to deactivate within 1-2 days of the initial insult. Coincident with microglial activation and deactivation, in the 5 g/kg alcohol model, there was a transient but substantial increase in pro-inflammatory factor (PIFs) expression. Work in BAX-null mice demonstrated that microglial activation and PIF expression were linked to BAX-dependent neuroapoptosis. As such, the level of microglial activation scaled with alcohol-induced cell death. Therefore, acute alcohol exposure in the developing cortex causes transient microglial activation and mobilization, promoting clearance of dead cells and tissue recovery. Moreover, cortical microglia show a remarkable capacity to rapidly deactivate following even severe neurodegenerative insults in the developing brain. Given that alcohol exposure on either P7 or P8 induced comparable levels of neuroapoptosis and microglial activation, we hypothesized that alcohol exposure on two sequential days (P7 and P8) would exacerbate neuroapoptosis and extend microglial activation. Instead, we found that the period of neuroapoptosis and microglial activation was similar after one day of alcohol exposure on P7 or after two days of exposure on P7 and P8. This was true for both the moderate and severe alcohol paradigms. Potentially, the low levels of cell death produced by the second day of injection may be due to neuroprotective mechanisms elicited by the first day of alcohol injection. In support of this idea, a preliminary microarray analysis of cortical gene expression 12 and 24 h after 5 g/kg alcohol exposure shows a decrease in expression of several pro-apoptotic factors and an increase in the expression of pro-survival factors, including neurotrophins. Of particular interest, BDNF, which has previously been shown to inhibit alcohol-induced neuroapoptosis, showed an eight-fold increase in expression at 24 h following 5 g/kg alcohol exposure and in situ hybridization showed strong BDNF expression near cortical regions with high levels of cell death. Future studies will be needed to extend the analysis of microglial activation states in this two-day injection model and to further explore the possibility that BDNF expression by microglia enacts neuroprotective mechanisms against a second insult. Finally, work in cell culture has suggested that chronic alcohol exposure may potentiate or inhibit microglial phagocytosis of dead cells. These studies raise the possibility that alcohol may directly affect microglial mobility which is important for their surveillance and synaptic remodeling functions. Therefore, we measured the effect of increasing doses of alcohol (0, 0.25, 0.5 and 1%) on microglial migration, branch motility, and morphology in dissociated BV-2 cell cultures and in acutely isolated neonatal (P5-6) brain slices. The results indicate that alcohol dose-dependently inhibits microglial migration and ruffling in cell culture, but in brain slices even high alcohol concentrations (0.5%) only reduce microglial branch motility by ~2%. When combined with our evidence for efficient microglial phagocytic clearance of dead cells in the neonatal cortex, these data suggest that while there is a measurable effect of acute alcohol exposure on microglial mobility, it does not impede microglia from performing their surveillance and phagocytic functions in vivo.