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Osteoporosis and depression are prevalent among older postmenopausal women 65 years or older. Bisphosphonates (BPs) and selective serotonin reuptake inhibitors (SSRIs) or serotonin norepinephrine reuptake inhibitors (SNRIs) are commonly used medications to treat these conditions. Inhibitory effects of BPs on osteoclasts are responsible for the reduction in fracture risk. SSRIs, however, are associated with increased fracture risk through decreasing osteoblasts and increasing osteoclastic activity. These effects of SSRIs could attenuate the beneficial effects of BPs. This dissertation describes the concomitant use of BPs and SSRIs among postmeopausa women and reports findings from examining the association between concomitant use of BPs and SSRIs and fracture risk. Separate cross-sectional analyses were performed using data from the 2004-2008 Medical Expenditure Panel Survey (MEPS) and Medicare Part D prescriptions claims data (2008-2010) to examine usage patterns of BPs and SSRIs/SNRIs for women aged ≥45 years and ≥65 years, respectively. For our second objective, a nested-case control was conducted using Medicare claims data (2008-2010). Data from Medicare inpatient claims were linked to Medicare Part D data for all female BP users 65 years or older. We used Cox proportional hazards model to assess the increased risk of osteoporotic-related fractures among propensity score matched (1:1 ratio) cohorts of concomitant users of BPs and SSRIs and BP alone users. Concomitant use of BPs and SSRIs was prevalent and increased with age for each timeframe examined. Findings showed that approximately 12% (using MEPS) and 28% (using Medicare data) of women on BPs were also on SSRIs. For the second objective, 4,214 propensity score matched pairs (average age=80.4 years) of subjects were analyzed. Findings showed that concomitant use of BPs and SSRIs was associated with statistically significant increased risk for any fracture (HR=1.29, 95% CI, 1.07-1.57), but statistically non-significant increased risk for hip (HR=1.16, 95% CI, 0.92-1.47) and vertebral fractures (HR=1.55, 95% CI, 0.97-2.48). Current findings indicate that concomitant use of BPs and SSRIs is not uncommon among postmenopausal women and suggest potential attenuation of antifracture efficacy of BPs by SSRIs. Further studies are needed to understand the clinical impact of concomitant use of these medications among older postmenopausal women.

INTRODUÇÃO: Testes provocativos com drogas serotonérgicas não apresentaram resultados consensuais em pacientes com TOC, sugerindo que a atividade serotonérgica seja heterogênea neste transtorno. É possível que a disfunção serotonérgica se relacione à resistência ao tratamento com inibidores de recaptura de serotonina (IRS) neste transtorno. O objetivo deste estudo foi comparar a atividade serotonérgica em pacientes com TOC resistente e responsivo ao tratamento com IRS e voluntários normais através de teste provocativo com citalopram. MÉTODOS: Foram estudados 30 pacientes com TOC resistente a IRS (RT), 30 pacientes com TOC respondedor (RP) e 30 voluntários sem transtorno mental (CN) pareados por sexo e idade. Cada indivíduo recebeu 20 mg de citalopram intravenoso. Foram dosados: prolactina, cortisol, hormônio de crescimento no plasma e serotonina em plasma rico em plaquetas nos seguintes tempos: -20, 0, 20, 40, 60, 80, 100, 120, 140 e 160 minutos. Como medidas da variação hormonal foram comparadas a variação percentual máxima (delta%) e a área sob a curva da variação. RESULTADOS: O citalopram não induziu sintomas ansiosos ou piora dos sintomas obsessivo-compulsivos nos pacientes. Também não houve mudanças significativas na concentração periférica de serotonina e na concentração de GH. A droga induziu um pico de secreção de prolactina no grupo controle (delta%=65,76 ± 105,1) maior que nos grupos RT e RP (RT delta%=17,41 ± 31,06; RP delta%=15,87 ± 31,71; p<0,05 em relação ao grupo controle). A secreção de cortisol mostrou-se atenuada apenas no grupo RT: (RT delta%=20,98 ± 58,14; RP delta%= 47,69 ± 66,94; CN delta%= 63,58 ± 88,4; p<0,05). CONCLUSÕES: Tanto pacientes resistentes como respondedores ao tratamento com IRS apresentaram resposta atenuada de prolactina ao citalopram em comparação a voluntários saudáveis, mas apenas pacientes resistentes apresentaram também resposta atenuada de cortisol, sugerindo maior disfunção serotonérgica neste grupo. / INTRODUCTION: Serotonergic pharmacological challenge tests have failed to produce consensual results in patients with OCD diagnosis, suggesting a heterogeneous 5-HT activity in this disorder. It is possible that serotonergic dysfunction is related with inadequate response to treatment with serotonin reuptake inhibitors (SRI). The aim of this study was to compare the neuroendocrine response to a serotonergic challenge in serotonin reuptake inhibitors treatment resistant and responders OCD patients and healthy volunteers. Methods: Thirty OCD resistant patients, 30 responders patients and 30 healthy volunteers, age and sex matched, were included. Each subject received 20 mg of intravenous citalopram. Prolactin, cortisol, growth hormone and serotonin were determined at the following times after the onset of citalopram infusion: -20, 0, 20, 40, 60, 80, 100, 120, 140 and 160 minutes. The maximal percentage variation (delta%) and area under the curve were compared as measures of hormonal variation. RESULTS: Citalopram has not induced any worsening of anxious or obsessive-compulsive symptoms among patients. No significant changes were observed either at platelet rich plasma serotonin concentration or at plasma growth hormone concentration. Citalopram has induced an increase in prolactin secretion in the control group (delta%=65.76 ± 105.1) while RT and RP groups showed blunted prolactin response (RT delta%=17.41 ± 31.06; RP delta%=15.87 ± 31.71; p<0.05). The cortisol response to citalopram was attenuated only in the RT group: (RT delta%=20.98 ± 58.14; RP delta%= 47.69 ± 66.94; CN delta%= 63.58 ±88.4; p<0.05). CONCLUSIONS: We concluded that either treatment resistant as responders patients have blunted prolactin response to citalopram, but only resistant patients also show an attenuated cortisol response, suggesting a more disrupted central serotonergic transmission in this group.

INTRODUÇÃO: Testes provocativos com drogas serotonérgicas não apresentaram resultados consensuais em pacientes com TOC, sugerindo que a atividade serotonérgica seja heterogênea neste transtorno. É possível que a disfunção serotonérgica se relacione à resistência ao tratamento com inibidores de recaptura de serotonina (IRS) neste transtorno. O objetivo deste estudo foi comparar a atividade serotonérgica em pacientes com TOC resistente e responsivo ao tratamento com IRS e voluntários normais através de teste provocativo com citalopram. MÉTODOS: Foram estudados 30 pacientes com TOC resistente a IRS (RT), 30 pacientes com TOC respondedor (RP) e 30 voluntários sem transtorno mental (CN) pareados por sexo e idade. Cada indivíduo recebeu 20 mg de citalopram intravenoso. Foram dosados: prolactina, cortisol, hormônio de crescimento no plasma e serotonina em plasma rico em plaquetas nos seguintes tempos: -20, 0, 20, 40, 60, 80, 100, 120, 140 e 160 minutos. Como medidas da variação hormonal foram comparadas a variação percentual máxima (delta%) e a área sob a curva da variação. RESULTADOS: O citalopram não induziu sintomas ansiosos ou piora dos sintomas obsessivo-compulsivos nos pacientes. Também não houve mudanças significativas na concentração periférica de serotonina e na concentração de GH. A droga induziu um pico de secreção de prolactina no grupo controle (delta%=65,76 ± 105,1) maior que nos grupos RT e RP (RT delta%=17,41 ± 31,06; RP delta%=15,87 ± 31,71; p<0,05 em relação ao grupo controle). A secreção de cortisol mostrou-se atenuada apenas no grupo RT: (RT delta%=20,98 ± 58,14; RP delta%= 47,69 ± 66,94; CN delta%= 63,58 ± 88,4; p<0,05). CONCLUSÕES: Tanto pacientes resistentes como respondedores ao tratamento com IRS apresentaram resposta atenuada de prolactina ao citalopram em comparação a voluntários saudáveis, mas apenas pacientes resistentes apresentaram também resposta atenuada de cortisol, sugerindo maior disfunção serotonérgica neste grupo. / INTRODUCTION: Serotonergic pharmacological challenge tests have failed to produce consensual results in patients with OCD diagnosis, suggesting a heterogeneous 5-HT activity in this disorder. It is possible that serotonergic dysfunction is related with inadequate response to treatment with serotonin reuptake inhibitors (SRI). The aim of this study was to compare the neuroendocrine response to a serotonergic challenge in serotonin reuptake inhibitors treatment resistant and responders OCD patients and healthy volunteers. Methods: Thirty OCD resistant patients, 30 responders patients and 30 healthy volunteers, age and sex matched, were included. Each subject received 20 mg of intravenous citalopram. Prolactin, cortisol, growth hormone and serotonin were determined at the following times after the onset of citalopram infusion: -20, 0, 20, 40, 60, 80, 100, 120, 140 and 160 minutes. The maximal percentage variation (delta%) and area under the curve were compared as measures of hormonal variation. RESULTS: Citalopram has not induced any worsening of anxious or obsessive-compulsive symptoms among patients. No significant changes were observed either at platelet rich plasma serotonin concentration or at plasma growth hormone concentration. Citalopram has induced an increase in prolactin secretion in the control group (delta%=65.76 ± 105.1) while RT and RP groups showed blunted prolactin response (RT delta%=17.41 ± 31.06; RP delta%=15.87 ± 31.71; p<0.05). The cortisol response to citalopram was attenuated only in the RT group: (RT delta%=20.98 ± 58.14; RP delta%= 47.69 ± 66.94; CN delta%= 63.58 ±88.4; p<0.05). CONCLUSIONS: We concluded that either treatment resistant as responders patients have blunted prolactin response to citalopram, but only resistant patients also show an attenuated cortisol response, suggesting a more disrupted central serotonergic transmission in this group.

A epilepsia é a condição crônica mais prevalente dentre as doenças neurológicas graves, associada com taxas significativas de morbidade e mortalidade. Pacientes com epilepsia farmacorresistente possuem uma taxa de mortalidade 2 a 3 vezes maior que indivíduos sem epilepsia. A epilepsia do lobo temporal (ELT) é a principal causa de epilepsia farmacorresistente nos adultos e também o protótipo da epilepsia cirurgicamente tratável, portanto, com maior acessibilidade para estudo de possíveis mecanismos epileptogênicos. Esclerose hipocampal (EH) é o achado neuropatológico mais comum em pacientes com ELT. Evidências, baseadas em experimentos animais e estudos em humanos, sugerem que as vias serotoninérgicas desempenham um importante papel na epileptogênese. Pacientes com ELT, causadas pela EH, apresentam maior prevalência de transtornos do humor e psicose contribuindo para uma pior qualidade de vida, com consequente impacto negativo nas respostas terapêuticas farmacológicas e cirúrgicas. Além disso, alguns estudos sugerem a existência de um mecanismo patogênico operante comum entre estas condições. Portanto, esta é um importante variável para análise. O objetivo desse estudo foi correlacionar as variáveis clínicas da epilepsia e a presença de transtornos psiquiátricos coexistentes com a concentração de serotonina (5-HT), a densidade dos receptores serotoninérgicos e a densidade do transportador serotoninérgico (5-HTT) no hipocampo dos pacientes com ELT-EH, que foram submetidos à cirurgia devido à presença de epilepsia farmacorresistente. Foram avaliadas amostras de 44 hipocampos de pacientes cirurgicamente tratados para ELT-EH. A concentração de 5-HT foi avaliada por cromatografia líquida de alta eficiência (HPLC) com detecção por fluorescência. 5-HTT e os receptores serotoninérgicos 5-HT1A, 5-HT2A, 5-HT6 e 5-HT7 foram avaliados por Western Blot. Níveis mais baixos de concentração de 5-HT estiveram associados com a presença de crises TCG (Wilcoxon-Mann-Whitney; p = 0.019). A densidade aumentada do receptor 5-HT1A esteve associada com maior duração da epilepsia (coeficiente de correlação de Spearman: p = 0.040) e a densidade diminuída do receptor 5-HT6 esteve associado com presença de EME (Wilcoxon-Mann-Whitney: p = 0.0027). A densidade dos receptores 5-HT2A e 5-HT7 e do 5-HTT não estiveram associadas com variáveis clínicas da epilepsia. A concentração de 5-HT, a densidade dos receptores e a densidade de 5-HTT não estiveram associadas à presença dos transtornos psiquiátricos neste grupo de pacientes. Nossos achados sugerem que as vias serotoninérgicas estão associadas com mecanismos de epileptogênese. Não foi evidenciado associações entre as vias serotoninérgicas e a presença de comorbidades psiquiátricas neste grupo de pacientes com ELT-EH / Epilepsy is the most prevalent neurological condition and it is associated with significative morbidities and mortalities rates. Patients with refractory epilepsy have a 2- to 3-fold higher mortality rate than people without epilepsy. Temporal lobe epilepsy (TLE) is the most common form of adult drug-resistant epilepsy. It is also the prototype of a surgically treatable epilepsy and because of that it is the most accessible for studies focused in epileptogenesis. Hippocampal sclerosis (HS) is the most common neuropathological finding in patients with TLE. Evidences from experimental, clinical and image studies suggest that the serotonergic system play an important role in epileptogenesis. Patients with TLE-HS have a higher prevalence of mood disorder and psychosis contributing for a worse quality of life with and consequent negative impact in pharmacological and surgical responses. Furthermore, studies suggest a common pathogenic mechanism operant in both conditions. Therefore, this is an important analytical variable. The objective of this study was to correlate clinical variables of epilepsy and the presence of psychiatric disease with serotonin (5-HT) concentration, serotonergic receptor density and serotonergic transporter (5-HTT) density in the hippocampus of TLE-HS patients submitted for surgery due to refractory epilepsy. It was analyzed 44 hippocampal tissue samples from surgical treated TLE-HS patients. 5-HT concentration was assessed by high pressure liquid chromatography (HPLC) with fluorescence detection. 5-HTT and serotonergic receptors 5-HT1A, 5-HT2A, 5-HT6 and 5- HT7 were assessed by Western Blotting. Lower levels of 5-HT concentration were associated with the presence of generalized tonic-clonic seizures (Wilcoxon-Mann-Whitney; p = 0.019). A higher 5-HT1A receptor density was associated with longer epilepsy duration (Spearman correlation coefficient: p = 0.040). Lower 5-HT6 receptor density was associated with the presence of status epilepticus (Wilcoxon-Mann-Whitney: p = 0.0027). 5-HT2A receptor, 5- HT7 receptor and 5-HTT densities were not associated with clinical variables of epilepsy. 5-HT concentration, serotonergic receptors and transporter densities were not associated with the presence of psychiatric disease in this group of patients. Our findings suggest that the serotonergic pathways are associated with epileptogenesis mechanisms. It was not evidenced any association between serotonergic pathways and psychiatric comorbidities in this group of TLE-HS patients

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke. / Li, Jin. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts in English. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [in Chinese] --- p.v / TABLE OF CONTENT --- p.vi / LISTS OF FLOWCHARTS --- p.xii / LISTS OF FIGURES --- p.xiii / LIST OF TABLES --- p.xvi / LIST OF EQUIPMENTS --- p.xvii / LIST OF ABBREVATIONS --- p.xvii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- Central nervous system disorder --- p.1 / Chapter 1.1.1 --- Stroke --- p.1 / Chapter 1.1.2 --- Spinal cord injuries --- p.4 / Chapter 1.1.3 --- Parkinson's disease --- p.6 / Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8 / Chapter 1.2 --- Stem cell therapy --- p.10 / Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11 / Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11 / Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15 / Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16 / Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18 / Chapter 1.3 --- Stem cells --- p.20 / Chapter 1.3.1 --- Embryonic stem cells --- p.21 / Chapter 1.3.1.1 --- Derivation and characterization --- p.21 / Chapter 1.3.1.2 --- Biology of ES cells --- p.21 / Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21 / Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24 / Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25 / Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27 / Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28 / Chapter 1.3.2 --- Neural stem cells --- p.30 / Chapter 1.3.2.1 --- Derivation and characterization --- p.30 / Chapter 1.3.2.2 --- Biology of NS cells --- p.32 / Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34 / Chapter 1.3.4 --- Mesenchymal stem cells --- p.35 / Chapter 1.4 --- Serotonin (5-HT) --- p.36 / Chapter 1.4.1 --- Distribution --- p.37 / Chapter 1.4.2 --- Metabolism --- p.37 / Chapter 1.4.3 --- Biological effects of 5-HT --- p.38 / Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40 / Chapter Chapter2 --- Aim --- p.43 / Chapter 2.1 --- Hypothesis and study objectives --- p.43 / Chapter Chapter3 --- Materials and Methods --- p.49 / Chapter 3.1 --- Chemicals and Reagents --- p.49 / Chapter 3.1.1 --- Cell culture --- p.49 / Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51 / Chapter 3.1.3 --- Cell proliferation assay --- p.52 / Chapter 3.1.4 --- Cell apoptosis assay --- p.52 / Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52 / Chapter 3.1.6 --- Western blotting --- p.55 / Chapter 3.1.7 --- Molecular biology --- p.56 / Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58 / Chapter 3.1.9 --- MAO activity assay --- p.58 / Chapter 3.1.10 --- Endogenous ROS production assay --- p.58 / Chapter 3.2 --- Consumable --- p.58 / Chapter 3.3 --- Cells --- p.60 / Chapter 3.3.1 --- Feeder cell --- p.60 / Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60 / Chapter 3.3.2 --- ES cells --- p.61 / Chapter 3.3.2.1 --- ES cell D3 --- p.61 / Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61 / Chapter 3.3.3 --- NS cells --- p.61 / Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61 / Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61 / Chapter 3.4 --- In-house prepared solutions --- p.62 / Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63 / Chapter 3.4.2 --- Gelatin solution 01% --- p.62 / Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62 / Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63 / Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63 / Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63 / Chapter 3.4.7 --- Laminin solution --- p.64 / Chapter 3.4.7 --- MEF Maintenance medium --- p.64 / Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64 / Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65 / Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65 / Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65 / Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66 / Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66 / Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67 / Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67 / Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67 / Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68 / Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68 / Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68 / Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69 / Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69 / Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69 / Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70 / Chapter 3.4.22 --- RIPA --- p.70 / Chapter 3.4.23 --- Resolving buffer (8X) --- p.70 / Chapter 3.4.24 --- Stacking buffer (4X) --- p.71 / Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71 / Chapter 3.4.26 --- Transfer buffer (10X) --- p.72 / Chapter 3.4.27 --- Transfer buffer (IX) --- p.72 / Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72 / Chapter 3.4.29 --- TBS (10X) --- p.73 / Chapter 3.4.30 --- TBS-T (IX) --- p.73 / Chapter 3.4.31 --- Stacking gel --- p.73 / Chapter 3.4.32 --- Resolving gel --- p.74 / Chapter 3.5 --- Methods --- p.75 / Chapter 3.5.1 --- Cell culture --- p.75 / Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75 / Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75 / Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76 / Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76 / Chapter 3.5.1.5 --- Passage of culture --- p.77 / Chapter 3.5.1.5 --- 6 Cell count --- p.78 / Chapter 3.5.1.7 --- Cytospin --- p.78 / Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78 / Chapter 3.5.1.9 --- Cryopreservation --- p.79 / Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79 / Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81 / Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83 / Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84 / Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85 / Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85 / Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86 / Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87 / Chapter 3.5.2 --- Cell proliferation assay --- p.87 / Chapter 3.5.2.1 --- Cell morphology --- p.87 / Chapter 3.5.2.2 --- WST-1 assay --- p.88 / Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88 / Chapter 3.5.2.4 --- NCFC assay --- p.89 / Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89 / Chapter 3.5.3.1 --- RNA extraction --- p.89 / Chapter 3.5.3.2 --- RNA quantitation --- p.90 / Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90 / Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91 / Chapter 3.5.3.5 --- RNA Integrity Check --- p.91 / Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91 / Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92 / Chapter 3.5.4 --- Microarray --- p.94 / Chapter 3.5.5 --- Immunofluoresent staining --- p.94 / Chapter 3.5.6 --- Western blot --- p.95 / Chapter 3.5.6.1 --- Harvesting samples --- p.95 / Chapter 3.5.6.2 --- Protein extraction --- p.96 / Chapter 3.5.6.3 --- Protein quantification --- p.96 / Chapter 3.5.6.4 --- SDS-PAGE --- p.97 / Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97 / Chapter 3.5.6.6 --- Blocking the membrane --- p.97 / Chapter 3.5.6.7 --- Immunoblotting --- p.97 / Chapter 3.5.6.8 --- Signal detection --- p.98 / Chapter 3.5.7 --- Cell apoptosis assay --- p.98 / Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98 / Chapter 3.5.7.2 --- TUNEL --- p.99 / Chapter 3.5.8 --- Endogenous ROS assay --- p.100 / Chapter 3.5.9 --- In vivo studies --- p.101 / Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101 / Chapter 3.5.9.2 --- Transplantation --- p.101 / Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102 / Chapter 3.5.10 --- Histological analysis --- p.103 / Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103 / Chapter 3.5.10.2 --- Cryosectioning --- p.103 / Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104 / Chapter 3.6 --- Data analysis --- p.104 / Chapter Chapter4 --- Results --- p.113 / Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113 / Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113 / Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115 / Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115 / Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115 / Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117 / Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119 / Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119 / Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119 / Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120 / Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121 / Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122 / Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122 / Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123 / Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123 / Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127 / Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130 / Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130 / Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130 / Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131 / Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132 / Chapter 4.7.2 --- Cell labelling --- p.132 / Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133 / Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135 / Chapter Chapter5 --- Discussion --- p.180 / Chapter Chapter6 --- Conclusions --- p.192 / References --- p.195

Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe <i>Periplaneta americana</i> durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt.<br> Mikrofluorometrische Ca²⁺-, Na⁺- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. <br><br> Wesentliche Ergebnisse dieser Arbeit waren:<br><br> <ul> <li>Messungen des intrazellulären pH (pH_i) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pH_i-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na⁺-K⁺-2Cl^--Symporter und der Cl^--HCO₃^--Antiporter, gekoppelt mit dem Na⁺ H⁺-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO₂/HCO₃^--Verfügbarkeit ab und war Ca²⁺-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H⁺-ATPase), aber von der Aktivität der basolateralen Na⁺-K⁺-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H⁺-ATPase und Na⁺-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH₄Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pH_i in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na⁺]_i und, zeitlich verzögert, auch der [Ca²⁺]_i. Der [Na⁺]_i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca²⁺]_i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na⁺]_i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na⁺-Ca²⁺-Antiporter in den Ca²⁺-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca²⁺]_i-Erhöhung könnte an der Regulation der Na⁺-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na⁺-K⁺-ATPase noch die V-H⁺-ATPase waren an der Resorption beteiligt.</li> </ul> <br> Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich. / The acinar salivary glands in the cockroach <i>Periplaneta americana</i> are innervated by dopaminergic and serotonergic fibers and secrete a NaCl-rich primary saliva upon stimulation with the biogenic amines dopamine (DA) or serotonin (5-HT). The ducts downstream of the acini are thought to modify the primary saliva by Na⁺reabsorption and K⁺ secretion. The electrolyte and fluid transport processes activated by DA and 5-HT as well as the second messenger pathways mediating between the biogenic amine receptors and the effector transport mechanisms are poorly understood.In this sudy, microfluorometrical Ca²⁺, Na⁺ and pH measurements were performed in combination with pharmacological experiments. Furthermore, ATPase activity assays and microscopical analyses of transepithelial fluid transport were done. The aim of this work has been the characterisation of the DA-induced transport mechanisms in the cockroach salivary glands in order to improve our understanding of the cellular actions of biogenic amines in insects. <br><br> Intracellular pH measurements in duct cells of isolated small lobes of salivary glands consiting of several acini and ducts showed a strong intracellular acidification upon DA or 5-HT stimulation. On the other hand, only a small intracellular acidification could be recognised in isolated ducts without acini. The acini are innervated by dopaminergic and serotonergic fibers, whereas the ducts are innervated only by dopaminergic fibers. Thus, this result demonstrates, that the DA- or 5-HT-induced production of primary saliva in the acini causes the intracellular pH changes in the ducts. Consequently, intracellular pH measurements in ducts are also useful to characterise transport processes in the acini.<br><br> The Na⁺-K⁺-2Cl^- cotransport and/or the Cl^--HCO₃^- exchange combined with the Na⁺ H⁺ exchange (NHE) were responsible for the NaCl uptake at the basolateral membrane in the peripheral cells of the acini during production of primary saliva. The activity of these transporters was regulated by the CO₂/HCO₃^--availability and was Ca²⁺-dependent. The activity of the basolateral Na⁺-K⁺-ATPase, but not of the apical vacuolar-type proton pump (V-H⁺-ATPase) in the duct cells was necessary for the strong intracellular acidification in the ducts with acini. Thus, the Na⁺-K⁺-ATPase seems to energise the saliva modification in the ducts. In ducts with acini, the V-H⁺-ATPase and Na⁺-dependent transporters (e.g. NHE) were responsible for the pH-recovery after a DA- or NH₄Cl-induced intracellular acidification in the duct cells. In the regulation of the intracellular resting pH these transporters played a minor role. In addition, DA induced an increase in the intracellular Na⁺ concentration, followed by an increase in the intracellular Ca²⁺ concentration in duct cells with acini, but never in duct cells without acini. The Na⁺ elevation was probably the result of the activity of apical Na⁺ channels. The DA-induced Na⁺ elevation and a depolarisation of the basolateral membrane of the duct cells reversed a Na⁺-Ca²⁺ exchange activity into the reverse mode causing a graded Ca²⁺ elevation in duct cells. The Ca²⁺ elevation is probably involved in the regulation of the Na⁺ reabsorption during saliva modification. Transepithelial fluid transport in isolated ducts was detected with a fluorescent microscopical method. Already unstimulated isolated ducts reabsorbed fluid from the duct lumen to the bath side. Perhaps unstimulated acini possess a basic secretion rate and this primary saliva is than reabsorbed in the ducts. The fluid reabsorption was ATP-dependent, but the ATP-consuming transport mechanism could not be identified. Neither the basolateral Na⁺-K⁺-ATPase, nor the apical V-H⁺-ATPase were involved in fluid reabsorption. This work extends our knowledge about the complex function of insect salivary glands and about the cellular action of biogenic amines in insects. Additionally, it indicates lots of similarities between the functions of salivary glands in vertebrates and invertebrates.

Within the field of depression the “monoamine hypothesis” has been the leading theory to explain the biological basis of depression. This theory proposes that the biological basis of depression is due to a deficiency in one or more of three key neurotransmitter systems, namely noradrenaline, dopamine and serotonin which are thought to mediate the therapeutic actions of virtually every known antidepressant agent. Citalopram is a selective serotonin-reuptake inhibitor (SSRI) used for the treatment of depression and anxiety disorders. Citalopram is a racemic compound, in other words composed of a 50:50 mixture of two enantiomers (S-(+)-citalopram and R-(-)-citalopram) and with one of the enantiomers (S-(+)-citalopram) accounting for the inhibitory effect. At the time of introduction of citalopram the physician needed a therapeutic drug monitoring service to identify patients with interactions, compliance problems and for handling questions concerning polymorphic enzymes and drug metabolism. An achiral analytical separation method based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for routine therapeutic drug monitoring (TDM) of citalopram and its two main demethylated metabolites. As the data available on citalopram were from achiral concentration determinations and to be able to further investigate citalopram enantiomers effects and distribution, a chiral method for separation of the enantiomers of citalopram and its demethylated metabolites was established. The advances within chiral separation techniques have made measurement of the concentrations of the individual enantiomers in biological fluids possible. The process behind enantioselective separation is however not fully understood and the mechanism behind the separation can be further scrutinized by the use of multivariate methods. A study of the optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram and didesmethylcitalopram on an acetylated ß-cyclodextrin column, by use of two different chemometric programs - response surface modelling and sequential optimization was performed. Sequential optimization can be a quicker mean of optimizing a chromatographic separation; response surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for learning more about the separation mechanism. Studies of the antidepressant effect and pharmacokinetics of citalopram have been performed in adults, but the effects on children and adolescents have only been studied to a minor extent, despite the increasing use of citalopram in these age groups. A study was initiated to investigate adolescents treated for depression, with respect to the steady-state plasma concentrations of the enantiomers of citalopram and its demethylated metabolites. The ratios between the S- and R-enantiomers of citalopram and didesmethylcitalopram were in agreement with studies involving older patients. The concentrations of the S-(+)- and R-(-) enantiomers of citalopram and desmethylcitalopram were also in agreement with values from earlier studies. The results indicate that the use of oral contraceptives may have some influence on the metabolism of citalopram. This might be because of an interaction of the contraceptive hormones with the polymorphic CYP2C19 enzyme. Even though the SSRIs are considered less toxic compared with older monoamine-active drugs like the tricyclic/tetracyclic antidepressants, the risk of developing serious side effects such as ECG abnormalities and convulsions has been seen for citalopram, when larger doses have been ingested. Furthermore, fatal overdoses have been reported where citalopram alone was the cause of death. Data on the toxicity of each of the enantiomers in humans have not been reported and no data on blood levels of the enantiomers in cases of intoxication have been presented. An investigation was initiated on forensic autopsy cases where citalopram had been found at the routine screening and these cases were further analysed with enantioselective analysis to determine the blood concentrations of the enantiomers of citalopram and metabolites. Furthermore the genotyping regarding the polymorphic enzymes CYP2D6 and CYP2C19 were performed. In 53 autopsy cases, we found increasing S/R ratios with increasing concentrations of citalopram. We found also that high citalopram S/R ratio were associated with high parent drug to metabolite ratio and may be an indicator of recent intake. Only 3.8 % were found to be poor metabolizers regarding CYP2D6 and for CYP2C19 no poor metabolizer was found. Enantioselective analysis of citalopram and its metabolites can provide valuable information about the time that has elapsed between intake and death. Genotyping can be of help in specific cases but the possibility of pharmacokinetic interactions is apparently a far greater problem than genetic enzyme deficiency. / On the day of the public defence the status of article IV was: Submitted.

Generalized anxiety disorder (GAD) is associated with painful physical symptoms (PPS). These post hoc analyses of previous trial data assessed PPS and their response to duloxetine treatment in GAD patients. Studies 1 and 2 (n = 840) were 9- to 10-week efficacy trials; study 3 (n = 887) was a relapse prevention trial comprising a 26-week open-label treatment phase and a 26-week double-blind, placebo-controlled treatment continuation phase. Mean baseline visual analog scale scores (VAS, 0–100; n = 1727) ranged from 26 to 37 for overall pain, headache, back pain, shoulder pain, interference with daily activities, and time in pain while awake. In studies 1 and 2, improvement on all VAS scores was greater in duloxetine-treated than in placebo-treated patients (p ≤ 0.01). In study 3, pain symptoms worsened in responders switched to placebo compared with those maintained on duloxetine (p ≤ 0.02). In conclusion, duloxetine was efficacious in the short- and long-term treatment of PPS, which are common in GAD patients.

The objective was to examine duloxetine 60–120mg/day treatment for relapse prevention in adults with generalized anxiety disorder (GAD). Adult patients (N=887; mean age=43.3 years; 61.0% female) with DSM-IV-TR-defined GAD diagnosis were treated with duloxetine for 26 weeks. Patients who completed open-label phase and were treatment responders (≥50% reduction in Hamilton Anxiety Rating Scale total score to ≤11 and “much”/“very much improved” ratings for the last 2 visits of open-label phase) were randomly assigned to receive duloxetine or placebo for a 26-week double-blind continuation phase. Relapse was defined as ≥2-point increase in illness severity ratings or by discontinuation due to lack of efficacy. During the double-blind phase, placebo-treated patients (N=201) relapsed more frequently (41.8%) than duloxetine-treated patients (13.7%, N=204, P≤0.001) and worsened on each outcome measure (P≤0.001, all comparisons). Duloxetine 60–120 mg/day treatment was efficacious and reduced risk of relapse in patients with GAD.