Katecholamine (Dopamin, Adrenalin, Noradrenalin) und Serotonin sind wichtige Monoamin-Neurotransmitter im menschlichen zentralen Nervensystem, deren quantitative Bestimmung von großem medizinischen Interesse ist, weil damit Aussagen zum Verlauf von Nervenkrankheiten und zur Tumorgefährdung des sympathoadrenalen bzw. neuroendokrinen Systems möglich sind. Ascorbinsäure und Harnsäure finden sich in vielen Körperflüssigkeiten. Ihre Bestimmung ist klinisch ebenfalls bedeutend, da deren Konzentration als Indikatoren bekannter Krankheitsbilder dienen. Etablierte Standardmethoden, wie die Hochleistungsflüssigkeitschromatographie (HPLC) und immunologische Nachweisverfahren (ELISA) werden im klinischen Bereich zur Bestimmung der Neurotransmitter genutzt. Diese sind kostenintensiv und zeitaufwändig und daher für die Anwendung in den Arztpraxen, vor allem in Entwicklungsländern nicht geeignet. Elektrochemische Verfahren, insbesondere voltammetrische Messmethode haben den Vorteil, solche Bestimmungen in einfacher Weise zu ermöglichen. In der Literatur finden sich Angaben zu eingesetzten Elektroden auf Kohlenstoffbasis mit hoher Sensitivität für die Katecholamine. Allerdings wurden diese Elektroden meist einzeln hergestellt. Der kommerzielle Durchbruch ist deshalb bisher, hauptsächlich infolge der mangelnden Reproduzierbarkeit der Elektrodeneigenschaften und der Verfügbarkeit einfacher elektronischer Geräte ausgeblieben. Es war daher Ziel dieser Arbeit, durch industrienahe Herstellungsverfahren Graphitelektroden mit reproduzierbaren Eigenschaften zu entwickeln und diese auf ihre Eignung für den quantitativen Nachweis bioaktiver Stoffe zu erproben. Dazu waren Verfahrensschritte zu optimieren, die es erlauben, diese siebgedruckten Graphitelektroden reproduzierbar und kostengünstig zu fertigen und sie auf verschiedene Weise, z.B. durch halbleitende Polymere und nanoskalige Metalle zu modifizieren. Neben den Neurotransmittern enthalten Körperflüssigkeiten unter anderem Ascorbinsäure und Harnsäure in hohen Konzentrationen. Daher waren zunächst Modellanalyten unter Verwendung dieser Stoffe herzustellen. Die voltammetrischen Methoden, wie die zyklische Voltammetrie (CV), die Differentielle Puls-Voltammetrie (DPV) und die Square-Wave-Voltammetrie (SWV) sollten auf ihre Eignung zum Nachweis der bioaktiven Substanzen erprobt werden. Schließlich waren die Elektroden in realen Analyten zu testen. Insgesamt konnte in der vorliegenden Arbeit gezeigt werden, dass ausgewählte Neurotransmitter, Ascorbinsäure und Harnsäure sich mit differentiellen voltammetrischen Verfahren an industrienah hergestellten modifizierten Dickschichtelektroden bestimmen lassen. Es ist erstmalig gelungen, eine modifizierte Dickschichtelektrode zu entwickeln, mit der es möglich ist, Katecholamine unabhängig von Ascorbinsäure (3 mM) und Harnsäure (2 mM) quantitativ nachzuweisen. Damit eröffnen sich neue Wege für den Einsatz von elektrochemischen Sensoren für die einfache Bestimmung der Neurotransmitter vor Ort. Die beschriebenen modifizierten Dickschichtelektroden sind ohne Verlust an elektrochemischer Aktivität an der Luft oder im Grundelektrolyten monatelang lagerfähig. Die Elektroden lassen sich im Gegensatz zu den in der Literatur beschriebenen Elektroden mit Einzelfertigung kostengünstig in großer Stückzahl mit hoher Reproduzierbarkeit herstellen.

Acetylcholine is a neurotransmitter that regulates neurite branching, induces neurite outgrowth, and synapse formation. Because of its various roles in neuronal activities, acetylcholine-based materials may also be useful in nerve repair. We present a series of biodegradable polymers with varying concentrations of acetylcholine-like motifs. We hypothesize that neurite sprouting and extension can be enhanced by using materials to present biochemical and physical cues. Acetylcholine-like motifs were incorporated by the polycondensation of diglycidyl sebacate, aminoethyl acetate, and leucine ethyl ester, which permitted control over acetylcholine motif concentration. Interactions between the polymers and neurons were characterized using rat dorsal root ganglia explants (DRG). We screened the potential application of these materials in nerve tissue engineering using the following criteria: 1) neurite sprouting, 2) neurite length, and 3) distribution of the neurite lengths. The ability of DRG to sprout neurites was influenced by the concentration of acetylcholine motifs of the polymer. Addition of acetylcholine receptor antagonists to DRG cultured on the polymers significantly decreased neurite sprouting, suggesting acetylcholine receptors mediate sprouting on the polymers. Future studies may examine how neurons on acetylcholine-based polymers exhibit changes in downstream signaling events and cell excitability that are associated with receptor activation. In preparation for testing the acetylcholine-based polymers in vivo, porous scaffolds with longitudinally oriented channels were fabricated using fiber templating and salt leaching. Micro computed tomography, scanning electron microscopy, and cryo-sectioning revealed the presence of longitudinally oriented channels. Channel volume and average pore size of the scaffolds were controlled by the number of fibers and salt fusion time. Future studies may involve testing the effect of acetylcholine-motifs by coating polymers onto such scaffolds or assessing the effect of the scaffold's dimensional properties on nerve regeneration.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 6 uppsatser.

Thesis (Ph.D. (Physiology))--Mahidol University, 1984.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [114]-130).

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [64]-71).

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 165 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Montana, 2007. / Title from title screen Description based on contents viewed Oct. 12, 2007. Includes bibliographical references (p. 151-177).

Made available in DSpace on 2015-07-22T20:49:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-26. Added 1 bitstream(s) on 2015-08-11T03:25:31Z : No. of bitstreams: 1 Publico-00387.pdf: 2025016 bytes, checksum: 1d807dcd8112ef82a43a87d394716763 (MD5) / Objetivos: 1- Investigar o efeito imediato (01 sessão) e mediato (10 sessões) da acupuntura no tratamento da síndrome de apnéia-hipopnéia obstrutiva do sono (SAHOS) de grau moderado. 2- Avaliar a especificidade da técnica, comparando acupuntura manual, eletroacupuntura 2 Hz e eletroacupuntura 10 Hz. 3- Avaliar o envolvimento da serotonina neste mecanismo de ação da acupuntura. Método: Um ensaio clínico, randomizado, controlado, simples cego, foi conduzido no Departamento de Fisiologia, no Setor de Distúrbios do Sono, no Setor de Medicina Chinesa-Acupuntura e no Laboratório de Rim e Hormônios da Universidade Federal de São Paulo - Escola Paulista de Medicina, no período de Janeiro de 2004 a outubro de 2006. Foram recrutados 43 pacientes previamente diagnosticados clínica e polissonograficamente como portadores de SAHOS moderada {índice de apnéia-hipopnéia (IAH) > 15/hora < 30/hora}. Os pacientes foram randomizados em quatro grupos: grupo controle (CTRL), grupo acupuntura manual, grupo eletroacupuntura 2 Hz e grupo eletroacupuntura 10 Hz. O grupo controle não recebeu qualquer tipo de intervenção. Os grupos acupuntura manual e eletroacupuntura receberam tratamento por 05 semanas (2 aplicações por semana). Todos os pacientes foram avaliados pré e pós-tratamento através de exame polissonográfico e da dosagem de serotonina. A dosagem de serotonina foi feita por HPLC-ED (Cromatografia Líquida acoplada a Detecção Eletroquímica). Resultados: Em relação ao efeito imediato (01 sessão) da acupuntura, houve diminuição significativa do IAH (P=0,006; P=0,009), dos eventos respiratórios (P=0,012; P=0,005), dos microdespertares (P=0,046; P=0,05) e da saturação de O2 (P=0,006; P=0,035) nos grupos acupuntura manual e eletroacupuntura 10 Hz, antes e depois de uma aplicação de acupuntura. O índice de apnéia (P=0,028) e o índice de hipopnéia (P=0,046) tiveram diminuição significativa apenas no grupo eletroacupuntura 10 Hz. Não houve alterações significativas nos grupos eletroacupuntura 2 Hz e grupo controle quando foram comparados polissonograficamente antes e após a sessão de acupuntura. Quando todos os grupos foram comparados ao grupo controle, apenas o grupo eletroacupuntura 10 Hz apresentou diminuição significativa do IAH. Em relação ao efeito mediato (10 sessões) da acupuntura houve diminuição significativa do tempo de início do sono (P=0,011; P=0,005), do IAH (P=0,011; P=0,006), do índice de apnéia (P=0,042; P=0,005) dos eventos respiratórios (P=0,011; P=0,009) e dos microdespertares (P=0,011; P=0,021) nos grupos acupuntura manual e eletroacupuntura 10 Hz, antes e depois do tratamento. O índice de hipopneia (P=0,021) teve diminuição significativa apenas no grupo eletroacupuntura 10 Hz. O tempo de inicio do sono (P=0,005; P=0,005) dimimuiu significativamente tanto no grupo controle quanto no grupo eletroacupuntura 2 Hz. A exceção deste dado, nenhum outro evento se modificou significativamente nestes grupos. Quando todos os grupos foram comparados ao grupo controle, apenas o grupo eletroacupuntura 10 Hz apresentou diminuição significativa do IAH e dos microdespertares (P=0,004; P=0,008). Em relação ao envolvimento da serotonina no mecanismo de ação imediato da acupuntura na SAHOS Moderada, não observamos significância estatística entre grupos quando comparados ao Controle: Acupuntura Manual (P=0,7674), Eletroacupuntura 2 Hz (P=0,9849) e Eletroacupuntura 10 Hz (P=0,1042). Em relação ao envolvimento da serotonina no mecanismo de ação mediato da acupuntura na SAHOS Moderada o grupo Acupuntura Manual apresentou significância estatística quando comparado ao grupo Controle (P=0,0067) e o grupo Eletroacupuntura 10 Hz, apresentou tendência à significância estatística quando comparado ao grupo Controle (P=0,0984). A não significância estatística destes resultados parece estar relacionada à variabilidade inicial da 5HT e ao pequeno tamanho da amostra. Conclusões: A acupuntura manual e a eletroacupuntura 10 Hz são mais eficazes que a eletroacupuntura 2 Hz em melhorar o IAH e outros eventos respiratórios do sono de pacientes portadores de SAHOS Moderada. Esta eficácia foi observada tanto no efeito imediato (01 sessão de acupuntura), como no efeito mediato (10 sessões de acupuntura). Os resultados preliminares relacionados à serotonina, já apontam para uma direção neurofisiológica que possa justificar a eficácia da acupuntura no tratamento de pacientes portadores da SAHOS. / STUDY OBJECTIVES: 1 - To investigate the immediate (single session) and mediate (ten sessions) effect of acupuncture over the sleep pattern of patients with moderate obstructive sleep hypopnea apnea (OSHA). 2 - To compare manual acupuncture (MA) and electroacupuncture (EA) with different stimulation frequencies (2 Hz and 10 Hz). 3 - To determine the role of endogenous serotonin in this mechanism. SETTING: Department of Physiology, Sleep Division of Department of Psychobiology, Chinese Medicine Division and Department of Nephrology, Federal University of São Paulo, Brazil. INTERVENTIONS: Acupuncture applied in true acupoints accompanied by needle manipulations or electroacupunture 2 hz and 10 Hz for 30 minutes in all patients, except control group pacients. DESIGN: In a randomized, placebo-controlled and single blind study, fourty-three patients with an apnea-hypopnea index (AHI) of 15 to 30 per hour were randomly assigned to 4 groups: control group, MA group, EA 2Hz group and EA 10Hz group. The control group received no intervention. The MA group, EA 2Hz group and EA 10Hz group received treatment for five weeks (02 acupuncture aplications per week). All the patients were evaluated pre-treatment and pos-treatment by polissomnography recordings and assessment of serotonin levels. The 5HT measurement was made with a HPLC-ED (Liquid Chromatografy coupled with Electrochemical Detection). MEASUREMENTS AND RESULTS: 1 – Immediate effect: when compared before and after treatment, the AHI (P=0,006; P=0,009), respiratory events (P=0,012; P=0,005), microarousals (P=0,046; P=0,05) and mean O2 saturation (P=0,006; P=0,035) showed significant improvement in both the manual and electroacupuncture 10 Hz respectively. The apnea index (P=0,028) and the hipopneia index (P=0,046) showed significant improvement only in EA 10 Hz group. There were no significant changes in the electroacupuncture 2 Hz as well as in the control group when compared before and after (first and second for controls) PSG recordings. When compared each group and control group, AHI significantly decreased only in the EA 10 Hz. 2 – Mediate effect: When compared before and after treatment, sleep onset (P=0,011;P=0,005), AHI (P=0,011;P=0,006), apnea index (P=0,042;P=0,005), respiratory events (P=0,011;P=0,009) and microarousals (P=0,011;P=0,021 ) significantly improved in MA and EA 10 Hz respectively. Hipopnea-index (P=0,021) decreased only in the EA 10Hz. Latency for sleep onset (P=0,005;P=0,005) decreased in both EA 2Hz and control group. Except for this change, there were no other significant changes in EA 2 Hz, or in control group after treatment. When compared to the control group the only significant differences were AHI and microarousals that were decreased in EA 10 Hz group (P=0,004;P=0,008). As concerned to the levels of seric serotonin we could not evidence an involvement of this neurotransmitter (as measured in blood) with the immediate effects of acupuncture for OSHA (MA vs. controls [P=0,7674], EA 2 Hz vs controls [P=0,9849]) despite a tendency for a difference for EA 10 Hz as compared to controls (P=0,1042). With regard to an effect of mediate acupuncture in moderate OSHA we found that seric serotonin levels were signficantly different form controls for MA (P=0,0067) with a tendency for group EA 10 Hz (P=0,0984). The lack of more robust differences for seric serotonin levels might have been influcenced by a smaller numeber of subjects as that required for this sort of evaluations. Conclusions: MA and EA 10 Hz have greater efficacy as compared to EA 2 Hz in improving AHI and other respiratory events in patients with moderate OHSA. This effect was shown to hold for both in the immediate (1 acupuncture session) as in the mediate (10 sessions) acupuncture application. The preliminary serotonin results indicate a possible mechanistic link between acupuncture treatment and its beneficial effects for the treatment of patients with moderate OHSA. / TEDE / BV UNIFESP: Teses e dissertações

We employed imaging, electrophysiological, and molecular techniques with the genetically tractable model organism Drosophila melanogaster to unravel fundamental biological and genetic underpinnings regulating synaptic function and plasticity. Using a forward genetic screen, we identified mutations in the Drosophila ortholog of a human WD40 repeat-containing protein termed DDB1 and CUL4 associated factor 12 (DCAF12). We show that DCAF12 likely serves as an adaptor protein for the DDB1-Cul4 E3 ubiquitin ligase complex by recruiting specific target proteins for ubiquitination. DCAF12 is expressed in neurons, muscles, and glia. In mitotically active cells such as muscles, DCAF12 is localized to nuclei and co-localizes in distinct foci with CUL4, suggesting that DCAF12 mediates a nuclear role for the CUL4 E3 ubiquitin ligase complex. In neurons, DCAF12 is localized to both cytoplasmic and nuclear compartments of motor neuron cell bodies, where it colocalizes with Cul4 in nuclei. DCAF12 is also expressed at the periactive zone of presynaptic terminals, but does not distinctly associate with DDB1 or Cul4 at this region. Evoked neurotransmitter release at larval NMJs is significantly reduced in DCAF12 mutants. These defects are rescued by presynaptic expression of wild-type DCAF12, demonstrating that DCAF12 is required presynaptically and serves as an important component of the machinery that facilitates evoked release. In addition, our studies show that DCAF12 is required for the differential expression of glutamate receptor subunits at the larval NMJ through transcriptional and post-translational mechanisms. GluRIID subunit mRNA levels and GluRIIA/C/D subunit protein levels are increased at DCAF12 mutant NMJs. Normal GluRIIA subunit levels can be restored by postsynaptic expression of wild-type DCAF12, but not with a truncated DCAF12 protein lacking a nuclear localization signal (∆NLS-DCAF12). Furthermore, DCAF12 overexpression in muscle nuclei reduces synaptic GluRIIA levels, an effect that can be suppressed by removing a copy of Cul4. These data strongly indicate that DCAF12 in muscle nuclei is required for GluRIIA expression and/or function in a Cul4-dependent manner. Moreover, homozygous DCAF12-GluRIIA double mutants show a strong synthetic lethality phenotype, providing further support for the hypothesis that GluRIIA directly or indirectly requires DCAF12. Mutations in glutamate receptors at larval NMJs trigger a retrograde trans-synaptic signal that leads to a compensatory increase in presynaptic release, which precisely restores the normal efficacy of synaptic transmission and muscle excitation. Reducing the gene dosage of DCAF12 by one gene copy suppresses the initiation and maintenance of GluRIIA-mediated synaptic homeostatic potentiation. This block of synaptic homeostatic potentiation can be rescued by presynaptic expression of DCAF12. In our studies, we determined that DCAF12 is critical for 3 distinct synaptic mechanisms: evoked neurotransmitter release, neurotransmitter reception by regulation of GluR subunit composition, and retrograde synaptic homeostatic signaling. Future research will strive to identify presynaptic and postsynaptic protein targets of DCAF12 and the Cul4 E3 ubiquitin ligase complex and the role of ubiquitination in regulating these synaptic processes.