Marx-Type solid-state bipolar modulator topologies: Performance comparison

The operation of generalized Marx-type solid-state bipolar modulators is discussed and compared with simplified Marx-derived circuits, to evaluate their capability to deal with various load conditions. A comparative analysis on the number of switches per cell, fiber optic trigger count, losses, and switch hold-off voltages has been made. A circuit topology is obtained as a compromise in terms of operating performance, trigger simplicity, and switching losses. A five-stage laboratory prototype of this circuit has been assembled using 1200 V insulated gate bipolar transistors (IGBTs) and diodes, operating with 1000 V dc input voltage and 1 kHz frequency, giving 5 kV bipolar pulses, with 2.5 mu s pulse width and 5 mu s relaxation time into resistive, capacitive, and inductive loads.

Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Hox genes control the specification of global vertebral domains

Dissertação apresentada para obtenção do grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Analysis of Synaptic Proteins in the Cerebrospinal Fluid as a New Tool in the Study of Inborn Errors of Neurotransmission

Abstract In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samplesfrom 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman’s correlation and Student’s t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism. Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases.

Design of switched-capacitor filters using low gain amplifiers

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Cerebrospinal Fluid Synaptic Proteins as Useful Biomarkers in Tyrosine Hydroxylase Deficiency

Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine biosynthesis and a cause of early parkinsonism. Two clinical phenotypes have been described. Type “B”: early onset severe encephalopathy; type “A”: later onset, less severe and better response to L-dopa. We aimed to study the expression of several key dopaminergic and gabaergic synaptic proteins in the cerebrospinal fluid (CSF) of a series of patients with TH deficiency and their possible relation with the clinical phenotype and response to L-DOPA. Dopamine transporter (DAT), D2-receptor and vesicularmonoamine transporter (VMAT2)weremeasured in the CSF of 10 subjectswith THdeficiency byWestern blot analysis. In 3 patients, data of pre- and post-treatmentwith L-DOPA were available, and in one of them, GABA vesicular transporter was determined. Results were compared to an age-matched control population. The concentration of D2-receptors in CSFwas significantly higher in patients with TH deficiency than in controls. Similarly, DAT and vesicular monoamine transporter type 2 were up-regulated. Studies performed before LDOPA, and on L-DOPA therapy showed a paradoxical response with D2 receptor expression increase as L-Dopa doses and homovanillic concentration gradually raised in a B phenotype patient. The opposite results were found in two patients with A phenotype. However, this is a very small sample, and further studies are needed to conclude robust differences between phenotypes. Synaptic proteins are detectable in the CSF and their quantification can be useful for understanding the pathophysiology of neurotransmitter defects and potentially to adjust and personalize treatments in the future.

Prediction of drug targets in human pathogens

The identification of new and druggable targets in bacteria is a critical endeavour in pharmaceutical research of novel antibiotics to fight infectious agents. The rapid emergence of resistant bacteria makes today's antibiotics more and more ineffective, consequently increasing the need for new pharmacological targets and novel classes of antibacterial drugs. A new model that combines the singular value decomposition technique with biological filters comprised of a set of protein properties associated with bacterial drug targets and similarity to protein-coding essential genes of E. coli has been developed to predict potential drug targets in the Enterobacteriaceae family [1]. This model identified 99 potential target proteins amongst the studied bacterial family, exhibiting eight different functions that suggest that the disruption of the activities of these proteins is critical for cells. Out of these candidates, one was selected for target confirmation. To find target modulators, receptor-based pharmacophore hypotheses were built and used in the screening of a virtual library of compounds. Postscreening filters were based on physicochemical and topological similarity to known Gram-negative antibiotics and applied to the retrieved compounds. Screening hits passing all filters were docked into the proteins catalytic groove and 15 of the most promising compounds were purchased from their chemical vendors to be experimentally tested in vitro. To the best of our knowledge, this is the first attempt to rationalize the search of compounds to probe the relevance of this candidate as a new pharmacological target.

Participación de los glucocorticoides en las secuelas emocionales inducidas por estrés

El proyecto tiene como objetivo estudiar la implicancia funcional de los glucocorticoides liberados durante diferentes situaciones estresantes en las consecuencias comportamentales inducidas por las mismas. Asimismo, se evaluará la influencia de los esteroides en los cambios neuroquímicos (neurotransmisión gabaérgica) y neuroendócrinos (actividad del sistema hipotalámico-hipofisario-adrenal, HHA) que subyacen a las alteraciones comportamentales. El estudio contribuye, además, a la caracterización de los mecanismos neurales implicados en los déficits conductuales inducidos por estrés. Durante este período se abordarán los siguientes objetivos específicos: a) Caracterización de los sistemas de receptores centrales (MR o GR) involucrados en la respuesta ansiogénica inducida por una sesión de inmovilización y de las áreas centrales comprometidas. (...) De este modo se extenderán los hallazgos conductuales a la actividad del complejo supramolecular GABAa. b) Evaluación de los efectos inducidos por la inyección sistémica aguda de dexametasona sobre la funcionalidad complejo-receptor GABAa en corteza prefrontal por medio de la determinación de la captación de cloruro estimulada por GABA. (...) De este modo se extenderán los hallazgos conductuales a la actividad del complejo supramolecular GABAa. c) En el paradigma residente-intruso se evaluará la generalización de la acción de Corticoesterona (CS) sobre el diferentes medidas conductuales de reactividad a un evento aversivo novel. (...) d) Se evaluarán los cambios en la actividad del sistema HHA en relación a las diferentes expresiones conductuales (sumisión, "boxing" o "freezing") asociadas a la confrontación social, para lo cual se determinará el curso temporal de secreción de CS y ACTH, antes y después del encuentro agonístico. (...) e) Se investigarán los efectos de una experiencia previa de derrota sobre la respuesta neuroendócrina inducida por exposición a un evento aversivo novel.

Comportamiento de preferencia a estímulos feromonales relacionados con la reproducción en la rata

Estudios previos en nuestro laboratorio han demostrado cambios en la actividad de la enzima GAD que sintetiza a GABA, en el bulbo olfatorio (BO) e hipotálamo de ratas durante la tarde del proestro (liberación de gonadotrofinas) cuando son expuestas a distintas feromonas relacionadas con la reproducción. El objetivo de este trabajo es investigar las modificaciones en la actividad de GAD en el BO e hipotálamo y en la conducta preferencial de la rata en la noche del proestro (momento de máxima receptividad hacia el macho) para olores de congéneres en distintos estados hormonales los cuales influyen en la calidad de las feromonas producidas. La participación del sistema GABAérgico y la influencia del serotoninérgico en dicha conducta será también estudiada. (...) En conjunto los estudios conductuales y de la actividad enzimática darán información de la participación GABAérgica y probable modulación por serotonina, en los eventos mencionados. Objetivos generales Investigar la participación GABAérgica y la influencia serotoninérgica en la conducta preferencial de una rata en la noche del proestro, a la presentación de distintas feromonas, estudiando además la respuesta de la enzima que sintetiza GABA en el bulbo olfatorio. Se compararán los resultados obtenidos con estudios de la actividad de GAD ya realizados en el BO en la tarde del proestro.

Efecto de la hiponutrición perinatal sobre distintos parámetros farmacológicos en el animal adulto

Numerosas evidencias, clínicas y experimentales, han demostrado que la hiponutrición temprana, en coincidencia con el período de ontogénesis del SNC (Sistema Nervioso Central), produce una serie de alteraciones morfológicas, neurofisiológicas, neuroquímicas y comportamentales que perduran en el sujeto adulto, aún luego de largos períodos de recuperación nutricional. (...) En los últimos 20 años, el concepto de cómo la malnutrición afecta las funciónes cerebrales superiores ha evolucionado considerablemente. Los hallazgos más recientes, basados en cambios en la reactividad a fármacos y a alteraciones en el funcionalismo de receptores neuronales, sugieren que los comportamientos y procesos cognitivos afectados por la hiponutrición temprana podrían ser consecuencia de la incapacidad de sujetos malnutridos en producir respuestas emocionales adecuadas y cambios adaptativos en los sistemas neuronales ante situaciones estresantes, más que por déficit cognitivo per-se. La hiponutrición infantil representa un enorme impacto económico-social para la humanidad y por lo tanto la mejor comprensión de los mecanismos neuronales involucrados en la regulación de las funciones cerebrales superiores afectadas por la malnutrición temprana puede representar un significativo aporte al conocimiento de la fisiopatología y/o tratamiento de alteraciones que afectan a millones de niños. En el presente proyecto, en ratas adultas sometidas a un esquema de hiponutrición perinatal y sus respectivos controles, se estudiará: 1) El desarrollo de tolerancia y síndrome de abstinencia a la acción ansiolítica de drogas que median sus efectos a través del complejo-receptor GABA-A. Paralelamente a la evaluación conductual luego de tratamientos crónicos, se intentará correlacionar las alteraciones observadas con parámetros neuroquímicos del funcionalismo neuronal. 2) El desarrollo de tolerancia al efecto analgésico de drogas analgésicas narcóticas. Se intentará correlacionar los resultados con la densidad de receptores opiáceos en distintas estructuras cerebrales

Efecto de la abstinencia a Diazepam en el desarrollo de dependencia a etanol: Tratamiento farmacológico

Numerosos estudios sugieren que los efectos conductuales y neuroquímicos del etanol y las benzodiazepinas (BDZs) están estrechamente relacionados. La administración aguda de BDZs y etanol potencia el efecto del GABA, sugiriendo que ambas drogas producirían efectos depresores del sistema nervioso central por incremento de la neurotransmisión GABAérgica. La administración prolongada de etanol y BDZS produce tolerancia a alguno de los efectos agudos tanto de la droga primaria, como de un agente relacionado (tolerancia cruzada). Este hecho se ha observado tanto en humanos como en animales. La interrupción del tratamiento crónico con dichas drogas provoca la aparición del síndrome de abstinencia. (...) Considerando que la ansiedad es un factor crítico tanto en la abstinencia a BDZs como en la génesis y mantenimiento del alcoholismo y que la abstinencia a diazepam podría provocar alteraciones a largo plazo en la capacidad de adaptación al estrés, se podría hipotetizar que esos efectos podrían estar implicados en la recurrencia al consumo de BDZs, etanol u otras drogas de abuso, luego de la interrupción de un tratamiento crónico. De lo anteriormente expuesto se deduce que fármacos que normalicen potenciales alteraciones en los mecanismos adaptativos frente al estrés podrían ser de utilidad para revertir las alteraciones observadas durante la expresión del sindrome de abstinencia inducido por etanol y BDZs. En relación a este tema, hemos demostrado recientemente que carbamazepina (CBZ), droga efectiva para el tratamiento del sindrome de abstinencia inducido por BDZs y etanol, revierte las alteraciones conductuales y neuroquímicas descriptas en animales con abstinencia a BDZs. Estos resultados sugieren que el tratamiento con dicho fármaco podría ser de utilidad terapéutica para la normalización de las alteraciones inducidas por la interrupción del consumo. Objetivos El objetivo general del presente proyecto es investigar una posible facilitación del desarrollo de dependencia al etanol en animales previamente sometidos a la abstinencia de benzodiazepinas. Este objetivo se extiende al estudio de nuevas alternativas terapéuticas que tengan capacidad potencial para revertir las alteraciones producidas por la abstinencia a etanol.

Einfluss frühkindlicher Sozialerfahrung auf die funktionelle Reifung des anterioren cingulären Cortex der Strauchratte (Octodon degus)

Maternal separation, rodent, anterior cingulate, prefrontal cortex, spines, synapses, GABA, calbindin, parvalbumin, calretinin, CRH, CRF

Neural Mechanisms and Delayed Gastric Emptying of Liquid Induced Through Acute Myocardial Infarction in Rats

Background: In pathological situations, such as acute myocardial infarction, disorders of motility of the proximal gut can trigger symptoms like nausea and vomiting. Acute myocardial infarction delays gastric emptying (GE) of liquid in rats. Objective: Investigate the involvement of the vagus nerve, α 1-adrenoceptors, central nervous system GABAB receptors and also participation of paraventricular nucleus (PVN) of the hypothalamus in GE and gastric compliance (GC) in infarcted rats. Methods: Wistar rats, N = 8-15 in each group, were divided as INF group and sham (SH) group and subdivided. The infarction was performed through ligation of the left anterior descending coronary artery. GC was estimated with pressure-volume curves. Vagotomy was performed by sectioning the dorsal and ventral branches. To verify the action of GABAB receptors, baclofen was injected via icv (intracerebroventricular). Intravenous prazosin was used to produce chemical sympathectomy. The lesion in the PVN of the hypothalamus was performed using a 1mA/10s electrical current and GE was determined by measuring the percentage of gastric retention (% GR) of a saline meal. Results: No significant differences were observed regarding GC between groups; vagotomy significantly reduced % GR in INF group; icv treatment with baclofen significantly reduced %GR. GABAB receptors were not conclusively involved in delaying GE; intravenous treatment with prazosin significantly reduced GR% in INF group. PVN lesion abolished the effect of myocardial infarction on GE. Conclusion: Gastric emptying of liquids induced through acute myocardial infarction in rats showed the involvement of the vagus nerve, alpha1- adrenergic receptors and PVN.

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Clinical benefit of fulvestrant in postmenopausal women with advanced breast cancer and primary or acquired resistance to aromatase inhibitors: final results of phase II Swiss Group for Clinical Cancer Research Trial (SAKK 21/00).

BACKGROUND: The aim of this study was to evaluate the efficacy and tolerability of fulvestrant, an estrogen receptor antagonist, in postmenopausal women with hormone-responsive tumors progressing after aromatase inhibitor (AI) treatment. PATIENTS AND METHODS: This is a phase II, open, multicenter, noncomparative study. Two patient groups were prospectively considered: group A (n=70) with AI-responsive disease and group B (n=20) with AI-resistant disease. Fulvestrant 250 mg was administered as intramuscular injection every 28 (+/-3) days. RESULTS: All patients were pretreated with AI and 84% also with tamoxifen or toremifene; 67% had bone metastases and 45% liver metastases. Fulvestrant administration was well tolerated and yielded a clinical benefit (CB; defined as objective response or stable disease [SD] for >or=24 weeks) in 28% (90% confidence interval [CI] 19% to 39%) of patients in group A and 37% (90% CI 19% to 58%) of patients in group B. Median time to progression (TTP) was 3.6 (95% CI 3.0 to 4.8) months in group A and 3.4 (95% CI 2.5 to 6.7) months in group B. CONCLUSIONS: Overall, 30% of patients who had progressed following prior AI treatment gained CB with fulvestrant, thereby delaying indication to start chemotherapy. Prior response to an AI did not appear to be predictive for benefit with fulvestrant.