Made available in DSpace on 2014-07-29T15:25:24Z (GMT). No. of bitstreams: 1 Cristiana Marinho de Jesus-Franca.pdf: 619773 bytes, checksum: d2bad54175727229ffb76acf89d235f2 (MD5) Previous issue date: 2009-05-15 / Little is known about the sedatives effectiveness for dental treatment in children under 3 years. The efficacy of oral midazolam sedation associated with protective stabilization was evaluated. In this randomized clinical trial, healthy children younger than 36 months were randomly allocated in groups: 1- protective stabilization; 2-protective stabilization associated with midazolam 1.0 mg/kg. The treatment was performed by an operator in a total of 55 sessions. A total of 26 children, 15 boys and 11 girls, were analysed: Group 1 (n = 12) - age (mean ± standard deviation) 27.50 ± 6.87, Group 2 (n = 14) - age (mean ± standard deviation) 26.86 ± 5.32. Child´s behavior was assessed using the Ohio State University Behavior Rating Scale (OSUBRS) and the heart rate record. A trained dentist recorded both behavior and physiological parameters. No difference in behaviors was found between groups on child initial examination carried out without sedation. In treatment sessions, only group 2 showed negative correlation between performance and the number of invasive Abstract procedures (Spearman rho = 0.469, P = 0.049). No significant differences between groups 1 and 2 OSUBRS scores were found in treatment sessions. The boys (77.50 ± 16.69) in group 2 showed higher percentage of negative scores (OSUBRS) than girls (26.67 ± 27.33). Statistically significant differences (Mann Whitney U test) between groups 1 and 2 were observed in heart rate during the forceps or low / high rotation use (1 = 139.67 ± 29.37 beats per minute, 2 = 164.97 ± 25.84; P = 0.003) and when the suture or rubber dam was placed (1=142,94 ± 23,19; 2=164,18 ± 23,69; P=0.005). A global analysis of children behavior during dental care was made overlooking the intervention groups (Friedman test). No associations were observed between the percentage of negative scores (OSUBRS) and several treatment sessions sequences. The percentage of negative scores (OSUBRS) was correlated with child's age (Spearman s rho =- 0.522, P = 0.006) and the percentage of negative scores in treatment the sessions (Spearman s rho = 0.405, P = 0.040). It was concluded that oral midazolam was not effective for sedation in up to 3 years old children for dental treatment purposes, and that this group of children did not change their negative behavior in the next sessions required for finishing the dental treatment planned / Pouco se sabe sobre a eficácia de sedativos em crianças menores de 3 anos, visando o tratamento odontológico. Verificou-se a eficácia, nessa população, do midazolam oral associado à estabilização protetora. Neste ensaio clínico randomizado, crianças saudáveis menores de 36 meses foram aleatoriamente alocadas nos grupos: 1-Estabilização protetora (controle); 2-Estabilização protetora associada ao midazolam oral 1,0 mg/kg. O tratamento foi realizado por um operador e um observador avaliou o comportamento por meio da Escala de Classificação Comportamental da Universidade do Estado de Ohio (OSUBRS) e do registro da frequência cardíaca. Foram analisadas informações do comportamento de 26 crianças, 15 meninos e 11 meninas, atendidas em 55 sessões: Grupo 1 (n=12) idade (média ± desvio padrão) 27,50 ± 6,87 ; Grupo 2 (n=14) idade (média ± desvio padrão) 26,86 ± 5,32. Não houve diferenças estatisticamente significantes entre os grupos na consulta inicial, realizada sem sedação. Nas sessões de tratamento, apenas o grupo 2 evidenciou correlação entre comportamento mais negativo e número de procedimentos invasivos (Spearman rho=0,469, P=0,049). Não houve diferenças estatisticamente significantes entre os grupos 1 e 2 nos escores OSUBRS nas sessões de tratamento. Os meninos (77,50 ± 16,69), no grupo 2, apresentaram maior porcentagem de escores negativos (OSUBRS) do que as meninas (26,67 ± 27,33). Diferenças estatisticamente significantes (teste U de Mann Whitney), Resumo entre os grupos 1 e 2, foram observadas na frequência cardíaca durante o uso de motor ou fórceps (1=139,67 ± 29,37 batimentos por minuto; 2=164.97 ± 25,84; P=0,003) e de isolamento absoluto ou sutura (1=142,94 ± 23,19; 2=164,18 ± 23,69; P=0,005). Desconsiderando os grupos de intervenção, verificou-se (teste de Friedman) que o comportamento das crianças durante o atendimento odontológico não diferia da primeira para a última sessão de tratamento, uma vez que não foram observadas associações entre a porcentagem dos escores negativos da escala OSUBRS e a sequência de várias sessões de tratamento. Observou-se, no entanto que a porcentagem dos escores mais negativos da escala OSUBRS durante o exame odontológico inicial correlacionou-se com a idade da criança (Spearman rho=-0,522, P=0,006) e com a porcentagem de escores negativos nas sessões de tratamento (Spearman rho=0,405, P=0,040). Nas condições deste ensaio clínico, concluiu-se que o midazolam oral não foi eficaz em crianças menores de 3 anos e que o comportamento negativo dessas crianças não modificou com o transcorrer das sessões de tratamento

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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Made available in DSpace on 2016-06-02T20:30:54Z (GMT). No. of bitstreams: 1 4805.pdf: 574299 bytes, checksum: 5bb78686ed42e85503db237f6aa036cc (MD5) Previous issue date: 2012-12-10 / Financiadora de Estudos e Projetos / Several studies have shown that benzodiazepines (BDZ) in periaqueductal gray (PAG) can produce anxiolytic-like effects in different animal models of anxiety. In addition, BDZ drugs also impair learning and memory performance in rodents. Despite the known role of PAG in modulated defensive behaviors in animal models, little is known about its role in modulated of emotional memory. In this sense, the objective of this study was to investigate the effects of midazolam, injected into the PAG, on the acquisition, consolidation and retrieval of aversive memory. For this, we used male mice of the Swiss-Albino weighing between 25-30g (n=7-11). After stereotactic surgery with implantation of a cannula in the PAG, the animals on the test day were divided into three experiments for later exposed to the test "step-down" (SD), as follows: Experiment 1, intra-PAG with saline and midazolam (MDZ) at doses of 3.0 and 30 nmol/0.1μl, in condition of pre-training to evaluate the acquisition of aversive memory; Experiment 2 was like Experiment 1, except for the condition of the injection pretest to evaluate the retrieval of aversive memory; Experiment 3 was like Experiment 1, except for the condition of injection post training to evaluate the consolidation of aversive memory. The animals were trained in the inhibitory avoidance task that was to distributed the animals into two groups: N/Sh - without exposure to shock, W/Sh - with exposed to shock (0.5 mA) for 10 seconds, to record the latency of descent (L1). Twenty-four hours later, each animal was exposed again on SD to record latency (L2), but without shock. The results were evaluated by analysis of variance (ANOVA) of three factors (Factor 1: condition; Factor 2: pre-treatment Factor 3: treatment) for L1 and L2. The results showed that there was an increase of L2 after exposure of mice to SD without shock, confirming that the aversive stimulus (shock) was strong enough to promote facilitation of aversive memory. The two doses (3.0 and 30 nmol) of MDZ intra-PAG decreased the risk assessment of mice, characterized by the fast descent of the platform in L1. This result suggests that GABAbenzodiazepine agonist impaired the acquisition, consolidation and retrieval of aversive memory in mice. Taken together, these results suggest that GABAA receptors within PAG seem to modulate the response related to aversive memory induced by shock. / Varios estudos tem demonstrado que os benzodiazepinicos (BDZ), administrados na substancia cinzenta periaquedutal (SCP), podem produzir efeito ansiolitico em diferentes modelos animais de ansiedade e tambem prejudicar a aprendizagem e a memoria em roedores. Apesar do ja conhecido papel da SCP em modular comportamentos defensivos em modelos animais, pouco se sabe sobre o seu papel na modulacao da memoria emocional. Neste sentido, o objetivo desde estudo foi investigar os efeitos do midazolam, intra-SCP, sobre a aquisicao, consolidacao e evocacao da memoria aversiva. Para isto, utilizamos camundongos machos da linhagem Suico-Albino, pesando entre 25-30g (n=7-11/grupo). Os animais apos cirurgia estereotaxica com implantacao de canula na SCP, no dia do teste foram distribuidos em tres Experimentos para posteriormente serem expostos ao teste de step-down (SD), a saber: Experimento 1, injecao intra-SCP com salina e midazolam (MDZ) nas doses de 3,0 e 30 nmol/0,1μl, na condicao de pre-treino ao SD para avaliar a aquisicao da memoria aversiva; Experimento 2, conforme Experimento 1, exceto pela condicao de injecao pre-teste ao SD para avaliar a evocacao da memoria aversiva; Experimento 3, conforme Experimento 1, exceto pela condicao de injecao pos-treino ao SD para avaliar a consolidacao da memoria aversiva. Os animais foram treinados na tarefa de esquiva inibitoria que consistiu em distribuir os animais em dois grupos: S/Ch - sem exposicao ao choque; C/Ch com exposicao ao choque (0,5mA) por 10s, para registro da latencia de descida (L1). Vinte e quatro horas apos, cada animal foi exposto novamente ao SD para registro da latencia (L2), mas sem o choque. Os resultados foram avaliados pela analise de variancia (ANOVA) de tres fatores (Fator 1: condicao; Fator 2: pre-tratamento; Fator 3: tratamento), durante L1 e L2. Os resultados mostraram que ocorreu aumento de L2, apos a exposicao de camundongos ao SD sem apresentacao de choque, confirmando que o estimulo aversivo (choque) foi forte o suficiente para promover facilitacao da memoria aversiva. As duas doses (3,0 e 30 nmol) de MDZ intra-SCP diminuiram a avaliacao de risco dos camundongos, caracterizada pela rapida descida da plataforma em L1. Este resultado sugere que este agonista GABABenzodiazepinico, prejudicou a aquisicao, evocacao e consolidacao da memoria aversiva em camundongos. Em conjunto, esses resultados sugerem que os receptores GABAA localizados na SCP participam da modulacao das respostas relacionadas a memoria aversiva induzida pelo choque.

O objetivo do trabalho foi avaliar comparativamente o efeito do midazolam 7,5 mg administrado por via oral e da sedação consciente empregando óxido nitroso associado ao oxigênio a 50% em pacientes submetidos à extração de terceiros molares inferiores na alteração do nível de ansiedade do paciente por meio da dosagem de cortisol salivar, no nível de saturação de oxigênio, na frequência cardíaca e na pressão arterial, na produção de amnésia anterógrada, além de avaliar as Escalas de Ansiedade Dental de Corah (DAS), Escala Visual Análoga para Ansiedade (VAS), Escala Verbal de Ansiedade e Inventário de Spielberger (STAI) na detecção de ansiedade dental. Realizou-se estudo split-mouth, no qual vinte e oito pacientes do gênero masculino foram submetidos à extração de terceiros molares inferiores sob anestesia local e sedação com midazolam e óxido nitroso associado ao oxigênio. Foram obtidos dados objetivos (dosagem de cortisol salivar, saturação de oxigênio, frequência cardíaca e pressão arterial) e subjetivos (Escala de Ansiedade Odontológica de Corah, Inventário de Ansiedade Traço-Estado, Escala Visual Análoga de Ansiedade e Escala Verbal de Ansiedade). Os resultados evidenciaram que os dois métodos de sedação empregados produziram efeitos benéficos e foram seguros na redução da ansiedade pré-operatória sem apresentar efeitos cardiovasculares ou respiratórios siginificantes, e que o midazolam 7,5 mg administrado por via oral foi mais eficaz na redução do cortisol salivar. Considerando as escalas de avaliação de ansiedade empregadas, constatou-se que a Escala de Ansiedade Dental de Corah mostrou ser a mais indicada para se avaliar ansiedade dental. / The study objective was to comparatively assess the effect of midazolam 7.5mg P.O. and conscious sedation with Nitrous oxide associated with oxygen at 50% in patients undergoing lower third molar extractions on the change in the anxiety level of patients by means of salivary cortisol dosage, on oxygen saturation level, on heart rate, on blood pressure, and on production of anterograde amnesia, as well as to assess the Corah\'s Dental Anxiety Scale (DAS), Anxiety Visual Analogue Scale (VAS), Anxiety Verbal Scale and Spilberger State-trait Anxiety Inventory (STAI) for the detection of dental anxiety. It was carried out a split-mouth study, in which twenty-eight male patients underwent lower third molar extraction under local anesthesia and sedation with midazolam and nitrous oxide associated with oxygen. Objective (salivary cortisol dosage, oxygen saturation, heart rate and blood pressure) and subjective (Corah\'s Dental Anxiety Scale, Spilberger State-trait Anxiety Inventory, Anxiety Visual Analogue Scale, and Verbal Anxiety Scale) data have been obtained. The results showed that both sedation methods used produced beneficial effects and were safe in reducing pre-operatory anxiety, showing no significant cardiovascular or respiratory effects, and midazolam 7.5 mg P.O. was more effective in reducing the salivary cortisol. Regarding the anxiety scales employed, it was found that the Corah\'s Dental Anxiety Scale was more indicated to assess dental anxiety.

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No. of bitstreams: 2 Dissertação - Heloisa de Sousa Gomes - 2013.pdf: 3199526 bytes, checksum: fedf466f42b9c4b911e2e0c9629d72de (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-02-26 / The fear and anxiety represent a barrier for dental care and can cause behavioral and physiological changes. These changes can be evaluated through behavioral scales and the measurement of salivary cortisol levels. The aim of this study was to evaluate the level of salivary cortisol in children during restorative dental treatment under moderate sedation with midazolam and taken a placebo and verify the correlation between this physiological measure and the assessment of the behavior conducted through the scale Ohio State University Behavioral Rating Scale (OSUBRS). A randomized controlled crossover triple-blind clinical trial was conducted with 18 healthy children from 2 to 5-year olds with necessity of at least two sessions of restorative dental treatment. Each child was undergo treatment under sedation with 1mg/kg oral midazolam in one session and the other with a placebo and in both sessions protective stabilization was associated. The assessment of child behavior was conducted from videos of clinical sessions using the scale of OSUBRS and the salivary cortisol level was evaluated in 4 moments on the two sessions (waking up, on arrival at the Dental School (DS), 25 minutes after the anesthesia and 25 minutes after finishing the procedure). The saliva samples were analyzed by the Enzyme Immunoassay test to get the mean salivary cortisol level. The results showed that the salivary cortisol level was lower when the children had received midazolam than when they had received the placebo at the moment of anesthesia (p=0.004). It was! Observed greater variation in cortisol level when the children received placebo than when they received midazolam. However, there were no differences between salivary cortisol levels observed in the four moments during treatment with sedation (p = 0.319) or placebo (p = 0.080). Regarding the child behavior it was not observed improvement during the treatment with sedation compared with placebo. The salivary cortisol levels showed no statistically significant correlation with the child’s behavior assessed by the scale OSUBRS during dental treatment under sedation or placebo. Therefore it was concluded that the oral midazolam dose of 1.0 mg/kg is effective in reducing the levels of salivary cortisol of children from 2 to 5-year olds during restorative dental treatment. However, this reduction in the level of cortisol in saliva did not reflect in better clinical behavior of these children. / O medo e ansiedade representam uma barreira durante o atendimento odontológico e podem causar alterações comportamentais e fisiológicas. Essas alterações podem ser avaliadas através de escalas comportamentais e da mensuração do nível de cortisol salivar. O objetivo do presente estudo foi avaliar o nível de cortisol salivar de crianças durante o tratamento odontológico restaurador sob sedação moderada com midazolam e com placebo, e verificar a correlação entre esta medida fisiológica e a avaliação do comportamento realizada através da escala Ohio State University Behavioral Rating Scale (OSUBRS). Realizou-se um ensaio clínico randomizado controlado cruzado triplo-cego com 18 crianças saudáveis de 2 a 5 anos de idade com necessidade de pelo menos duas sessões de tratamento odontológico restaurador. Cada criança foi submetida a tratamento sob sedação com midazolam oral (1 mg/kg) e outra sessão com placebo, sendo que em ambas as sessões realizou-se estabilização protetora. A avaliação do comportamento infantil foi realizada a partir de vídeos das sessões clínicas utilizando-se a escala OSUBRS e o nível de cortisol salivar foi avaliado em 4 momentos nas duas sessões (ao acordar, ao chegar na Faculdade de Odontologia (FO), 25 minutos após a anestesia e 25 minutos após o término do procedimento). As amostras de saliva foram analisadas por um ensaio imunoenzimático para obter a média do nível de cortisol salivar. Os resultados demonstraram que o nível de cortisol salivar foi menor quando as crianças receberam o midazolam do que quando receberam o placebo no momento da anestesia (p=0,004). Observou-se maior variação no nível de cortisol salivar quando as crianças receberam o placebo do que quando receberam o midazolam. No entanto, não foi demonstrada diferença entre os níveis de cortisol salivar verificados nos quatro momentos de coleta durante o tratamento com sedação (p=0,319) e com o placebo (p=0,080). Em relação ao comportamento infantil, não foi observado melhora durante o atendimento com sedação comparado com o placebo. Os níveis de cortisol salivar não apresentaram correlação com o comportamento infantil avaliado através da escala OSUBRS, tanto durante o tratamento sob sedação quanto com o placebo. Portanto, concluiu-se que o midazolam oral na dose de 1,0 mg/kg é eficaz na redução do nível de cortisol salivar de crianças de 2 a 5 anos de idade durante o tratamento odontológico restaurador. Entretanto, essa redução do nível de cortisol na saliva não refletiu em um melhor comportamento clínico dessas crianças.

Purpose: Compare moderate oral sedation of pediatric patients using Hydroxyzine and Meperidine with either Diazepam or Midazolam in management of pediatric dental patients. Methods: Randomized, double-blind, crossover pilot study of patients 3 to 7 years of age requiring two sedation visits. Frankl and Houpt behavior scores recorded at injection time, initiation of treatment and 100% oxygen at end of treatment. Postoperative phone call surveys conducted within eight hours and within 24 hours of discharge. Wilcoxon Signed-Rank tests, Fisher’s Exact Chi-squared test and 0.10 significance level. Results: 25 subjects completed 35 sedations. Eight participants completed both treatments and demonstrated significantly higher total Houpt Scores with Diazepam at all treatment stages. Frankl scores favored Diazepam at injection time. More abnormal behavior was found with Midazolam, less memory of the visit with Diazepam, but longer sleep time with Diazepam. Conclusions: Sedation with the Hydroxyzine, Meperidine and Diazepam regimen may allow for a better overall sedation experience. Postoperative monitoring is essential. The results are promising and demonstrate the value of a larger study on sedation with Diazepam.

O Objetivo deste estudo controlado e duplo-cego foi avaliar os efeitos cardiovasculares induzidos pelo bloqueio pterigomandibular com o anestésico local cloridrato de lidocaína 2%, associado à epinefrina, seguido da administração de ansiolítico (midazolam 15mg) ou placebo, para realização de cirurgia de colocação de implantes dentários inferiores bilaterais, em 22 pacientes (13 mulheres 9 homens). Os parâmetros cardiovasculares Pressão Arterial Sistólica (PAS), Diastólica (PAD), Média (PAM) e Freqüência Cardíaca (FC) foram monitorados pelos métodos oscilométrico e fotopletismográfico. Os valores médios foram registrados a cada minuto e de forma contínua durante as etapas do experimento da seguinte forma: Fase 0 Período basal; Fase 1 Anestesia local; Fase 2 incisão; Fase 3 perfuração; Fase 4 colocação dos implantes; Fase 5 sutura; Fase 6 período final. Os indivíduos que receberam midazolam não apresentaram alterações de PAS, PAD, PAM e FC significativas (p>0,01) comparada ao placebo. Ocorreram alterações significantes dos valores para o grupo que recebeu placebo e dos valores médios do grupo que receberam midazolam na freqüência cardíaca / The purpose of this controlled and double-blind trial was to evaluate cardiovascular effects induced by pterigomandibular block of local anesthetic (LA) 2 % lidocaína hydrochloride with epinephrine, followed administration of benzodiazepine (midazolam 15mg on hour prior) or placebo during the surgical phase of placement of the lower bilateral dental implant in 22 normotensive outpatients (13 female and 9 male). The cardiovascular parameters systolic (SP), diastolic (DP) and mean (MP) pressures and heart rate (HR) were monitored by oscillometric and photopletismographic methods in 06 clinical phases during the procedure. The mean values were recorded every minute and in a continuous way during the phases of the experiment following the sequence: phase 0 basal period; phase 1 - anesthesia local; phase 2 mucoperiostal flap; phase 3 perforation; phase 4 placement of dental implants; phase 5 suture; phase 6 the end period. The group with received midazolam didnt induce significant SP, DP, MP and HR changes (p>0.01) compared to placebo. Significantly higher values in placebo group and mean values in midazolam group in heart-frequency were observed.

O citocromo P450 (CYP450) e suas isoformas podem ser reguladas por compostos exogenos, endogenos e patologias. O Diabetes Mellitus (DM) e uma sindrome de etiologia multipla, decorrente da incapacidade da insulina em exercer adequadamente sua acao, sendo capaz de alterar a atividade do CYP450. Este estudo avalia a influencia do DM na atividade das isoformas CYP2D6 e CYP3A4 utilizando como marcadores de atividade o metoprolol (MET) e o midazolam (MDZ) respectivamente. Ratos machos Wistar, 200-250 g, foram tratados com dose unica, por via oral 20 mg/Kg para MET e 15 mg/Kg para MDZ. O DM foi induzido por estreptozotocina (50 mg/Kg) por via intravenosa. Midazolam, metoprolol e alfa-hidroximetoprolol foram analisados em plasma por cromatografia liquida de ultraperformance (coluna XR-ODS 100 mm x 3,0 mm) acoplada a espectrometro de massas. O preparo das amostras foi realizado por extracao liquido-liquido. A analise farmacocinetica do MDZ em ratos mostrou reducao significativa (54,01 vs 12,24 L/h/kg) no clearance dos animais diabeticos em relacao ao controle e aumento significativo (402,66 vs 1742,6 h.ng/mL) do AUC0- ‡ no grupo diabetico quando comparado com o controle. O DM modificou o AUC0- ‡ do MET no grupo diabetico em relacao ao controle (998,09 vs 348,45 h.ng/mL) acompanhado de reducao significativa do clearance no grupo diabetico (46,85 vs 17,80 L/h/kg). Desde que nao foi observada qualquer alteracao na farmacocinetica do alfa-hidroximetoprolol, o aumento do AUC0- ‡ do MET pode ser devido a inibicao de outra via de metabolizacao pelo DM, como comprovado pela alteracao na via do CYP3A. A analise dos dados sugere que o DM inibe a atividade do CYP3A4 e nao exerce influencia sobre a atividade do CYP2D6 / Cytochrome P450 (CYP450) and its isoforms can be regulated by exogenous and endogenous compounds and also diseases. Diabetes Mellitus (DM) is a multiple etiology syndrome resulting from a lack or inability of insulin to properly exercise its action, that can modify the CYP450 activity. This research evaluates the influence of DM on CYP2D6 and CYP3A4 isoforms activity employing metoprolol (MET) and midazolam (MDZ) as probe drugs. Male Wistar rats 200-250g, were treated with single oral dose of 20mg/kg for MET and 15 mg/kg for MDZ. Diabetes mellitus was induced by streptozotocin (50mg/kg) intravenously administered. Midazolam, metoprolol and alpha-hydroxymetoprolol were analyzed in plasma by ultraperformance liquid chromatography (column XR-ODS) coupled to a mass spectrometer. The sample preparation was perfomed using liquid-liquid extraction. Pharmacokinetics of MDZ in rats showed a significant decrease (54,01 vs 12,24 L/h/kg) of total clearance in the diabetic animals compared to control group and a significant increase (402.66 vs 1742.6 h.ng/mL) AUC0- ‡ in the diabetic group compared with the control. The DM altered the AUC0- ‡ of MET in the diabetic group compared to control (998.09 vs 348.45 h.ng/mL) followed by a significant decrease in clearance in the diabetic group (46.85 vs 17.80 L/h/kg). Since no changes in alpha- hydroxymetoprolol pharmacokinetics was observed, the increase in MET AUC0- ‡ can be explained by DM inhibition of other MET metabolization pathways. Data analysis suggests that DM inhibits CYP3A4 activity and had no effect on CYP2D6 activity / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq

by Szeto Yuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 257-283). / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.vi / Abstract --- p.ix / Contents --- p.xi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoies --- p.is / Chapter 1.1.1 --- Ontogeny of the hematopoietic system --- p.1 / Chapter 1.1.2 --- Hierarchy of hematopoietic cells --- p.3 / Chapter 1.1.3 --- Characteristics of a functional blood system and the need for regulation --- p.11 / Chapter 1.1.4 --- Interrupted hematopoiesis -- Leukemia --- p.13 / Chapter 1.2 --- Regulation of myeloid cell differentiation / Chapter 1.2.1 --- Regulation of hematopoiesis --- p.16 / Chapter 1.2.2 --- Models of hematopoiesis --- p.18 / Chapter 1.2.3 --- Genes regulation of myeloid cell differentiation and its study --- p.21 / Chapter 1.2.4 --- Genes differentially expressed and involved in myeloid cell differentiation --- p.24 / Chapter 1.3 --- Induced myeloid cell differentiation / Chapter 1.3.1 --- Induced myeloid cell differentiation --- p.46 / Chapter 1.3.2 --- WEHI-3B JCS cells --- p.48 / Chapter 1.3.3 --- Chemical inducers -- Flavonoids and benzodiazepines --- p.51 / Chapter 1.4 --- The aim of study --- p.59 / Chapter Chapter Two --- Cytokine Expression in Biochanin A- and Midazolam-treated JCS cells / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cytokine and myeloid differentiation --- p.62 / Chapter 2.1.2 --- Phenotypic studies biochanin A- and midazolam-treated JCS cells --- p.65 / Chapter 2.1.3 --- Cytokine regulation at transcriptional level --- p.68 / Chapter 2.1.4 --- Cytokine mRNA phenotyping by a semi-quantitative approach --- p.69 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Cell line --- p.72 / Chapter 2.2.2 --- Chemicals and buffers --- p.72 / Chapter 2.2.3 --- DIG system --- p.73 / Chapter 2.2.4 --- Enzymes and nucleic acids --- p.73 / Chapter 2.2.5 --- Solutions --- p.74 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of total RNA by guanidinium thiocyanate/cesium chloride isopycnic gradient --- p.75 / Chapter 2.3.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.76 / Chapter 2.3.3 --- Southern blotting --- p.79 / Chapter 2.3.4 --- Cycle titration and dot blotting --- p.79 / Chapter 2.3.5 --- DIG 3' end labeling of probes --- p.81 / Chapter 2.3.6 --- Hybridization and stringency wash --- p.81 / Chapter 2.3.7 --- Chemiluminescent detection --- p.82 / Chapter 2.3.8 --- Quantitation by densitometry --- p.82 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Analysis of total RNA --- p.83 / Chapter 2.4.2 --- mRNA phenotyping --- p.85 / Chapter 2.4.3 --- Summary of mRNA phenotyping results --- p.98 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- mRNA phenotyping --- p.100 / Chapter 2.5.2 --- Cytokine gene regulation --- p.106 / Chapter 2.5.3 --- mRNA quantitation using the current method --- p.108 / Chapter Chapter Three --- Identification and Isolation of Genes that are Differentially Expressed during Midazolam-induced JCS Cell Differentiation / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Methods for studying differentially expressed genes --- p.110 / Chapter 3.1.2 --- RNA fingerprinting by arbitrarily-primed PCR (RAP-PCR) and differential display (DDRT-PCR) --- p.113 / Chapter 3.1.3 --- Re-amplification of PCR products by touchdown PCR --- p.118 / Chapter 3.1.4 --- Strategies to avoid false positives --- p.119 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Cell line and bacterial culture --- p.121 / Chapter 3.2.2 --- Chemicals --- p.121 / Chapter 3.2.3 --- Enzymes and nucleic acids --- p.122 / Chapter 3.2.4 --- Kits --- p.122 / Chapter 3.2.5 --- Solutions --- p.122 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Isolation of total RNA --- p.124 / Chapter 3.3.2 --- First strand cDNA synthesis --- p.124 / Chapter 3.3.3 --- RNA fingerprinting by arbitrarily-primed PCR --- p.124 / Chapter 3.3.4 --- First round cDNA probe screening --- p.126 / Chapter 3.3.5 --- Subcloning of differentially amplified fragments --- p.129 / Chapter 3.3.6 --- Second round cDNA probe screening --- p.133 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Spectrophotometric analysis of total RNA --- p.134 / Chapter 3.4.2 --- Normalization of samples --- p.135 / Chapter 3.4.3 --- RNA fingerprinting of arbitrarily-primed PCR --- p.136 / Chapter 3.4.4 --- Re-amplification of PCR products --- p.138 / Chapter 3.4.5 --- First round cDNA probe screening --- p.139 / Chapter 3.4.6 --- Subcloning of the differentially amplified fragments --- p.143 / Chapter 3.4.7 --- Second round cDNA probe screening --- p.145 / Chapter 3.4.8 --- A comparison of the first and second screening --- p.149 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Towards the steps to isolate differentially expressed genes --- p.151 / Chapter 3.5.2 --- Expression profiles predicted at different stage of the procedures --- p.156 / Chapter 3.5.3 --- Representation of the total mRNA in the cell --- p.158 / Chapter 3.3.4 --- Comparison of the original and modified protocol of RAP-PCR --- p.159 / Chapter 3.3.5 --- Advantages of the modified protocol and further refinements --- p.163 / Chapter Chapter Four --- Characterization of the Putative Differentially Expressed Genesin Midazolam-induced JCS cells / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- DNA sequencing --- p.165 / Chapter 4.1.2 --- Automated DNA sequencing and analysis --- p.168 / Chapter 4.1.3 --- Genbank and BLAST homology search --- p.171 / Chapter 4.1.4 --- Internal primer design for RT-PCR --- p.174 / Chapter 4.1.5 --- Genes involved in both myeloid cell differentiation and embryonic development --- p.177 / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Selected recombinant plasmids --- p.180 / Chapter 4.4.2 --- Total RNAs --- p.180 / Chapter 4.2.3 --- Chemicals --- p.180 / Chapter 4.2.4 --- Enzymes and nucleic acids --- p.181 / Chapter 4.2.5 --- Kits --- p.181 / Chapter 4.2.6 --- Solutions --- p.181 / Chapter 4.3 --- Methods / Chapter 4.3.1 --- Preparation of selected recombinant plasmid DNA --- p.182 / Chapter 4.3.2 --- Sequencing --- p.182 / Chapter 4.3.3 --- Data analysis and assessment by ALF manager and DNAsis --- p.184 / Chapter 4.3.4 --- Sequence search by BLASTN program --- p.185 / Chapter 4.3.5 --- Primer design by Oligo´ёØ ver. 34 --- p.186 / Chapter 4.3.6 --- Differential expression confirmed by RT-PCR --- p.186 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Analysis of selected recombinant plasmid DNA --- p.187 / Chapter 4.4.2 --- Sequencing results --- p.191 / Chapter 4.4.3 --- BLASTN search results --- p.212 / Chapter 4.4.4 --- Primer design of the sequenced fragments --- p.222 / Chapter 4.4.5 --- "Expression profile of the isolated genes in midazolam-, biochanin A- induced JCS cells and mouse embryos" --- p.223 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Sequence analysis of the isolated gene fragments --- p.233 / Chapter 4.5.2 --- Expression profiles of the isolated genes --- p.236 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Studies on leukemic cell differentiation / Chapter 5.1.1 --- Differentiation pathways revealed by different inducers --- p.241 / Chapter 5.1.2 --- Lineage preference during differentiation --- p.243 / Chapter 5.2 --- Differentiation program triggered by midazolam / Chapter 5.2.1 --- Signaling pathways initiated by biochanin A and midazolam --- p.245 / Chapter 5.2.2 --- Differentially expressed genes during midazolam-induced differentiation --- p.247 / Chapter 5.2.3 --- Expression patterns of the isolated differentially expressed genesin midazolam and biochanin A-induced JCS cells --- p.248 / Chapter 5.2.4 --- Myeloid genes in embryonic development --- p.250 / Chapter 5.3 --- Future studies of the isolated fragments --- p.252 / Chapter 5.4 --- Conclusion --- p.256 / Reference --- p.257 / Append --- p.ix / Chapter A1. --- Ambiguity codes for sequencing --- p.i / Chapter A2. --- Myeloid cell lines --- p.ii / Chapter A3. --- Details of manufacturer's products --- p.iii / Chapter A4. --- List of machine and equipment --- p.v