Tesis presentada a la Universidad de Chile para optar al grado de Doctor en Farmacología / No autorizada por el autor para ser publicada a texto completo en el Portal de Tesis Electrónicas / La polimerización y agregación del péptido Aβ1-42 son procesos poco dilucidados hasta el momento, sin embargo, están fuertemente relacionados al desarrollo de la Enfermedad de Alzheimer, trastorno neurodegenerativo, causante principal de demencia senil para el cual no existe cura o prevención efectiva hasta nuestros días. Una de las estrategias estudiadas con el fin de prevenir y buscar la cura de esta enfermedad es la de buscar compuestos que inhiban la agregación de Aβ1-42 y consecuentemente la formación de especies tóxicas que causan la muerte neuronal y el desarrollo de la enfermedad. En esta tesis se evaluó la acción inhibidora de la agregación de una serie de 6 derivados dihidropiridínicos (fenil-DHP, 3-OH-DHP, 4-OH-DHP, 3,4-OH-DHP, 3,5-OH- DHP y 3,4,5-OH-DHP) que difieren en el número y posición de hidroxilos en la molécula que por semejanzas estructurales con otros compuestos encontrados en la literatura se constituyeron en buenos candidatos para este fin. Estos compuestos poseen además las propiedades de ser inhibidores de la generación de radicales libres, inhibidores de canales de calcio y posibles antihipertensivos, constituyéndose en compuestos aún más atractivos, ya que el estrés oxidativo, el desbalance en la homeostasis de calcio y la hipertensión, son también factores desencadenantes de la EA. Mediante las técnicas de Fluorescencia por Interacción con tioflavina-T, Electroforesis en Geles y TEM, los resultados demostraron que los compuestos con uno o ningún grupo hidroxilo (fenil-DHP, 3-OH-DHP y 4-OH-DHP) no son inhibidores de la agregación de Aβ1-42. Sin embargo, los compuestos , 3,4-OH-DHP, 3,5-OH-DHP y 3,4,5-OH-DHP son inhibidores de la agregación siendo 3,4-OH-DHP el compuesto más eficaz con una inhibición máxima de 88±5% y una IC50 de 5.6±0.1µM siendo significativamente más potente que el resveratrol (IC50=9.8±1.3 µM), compuesto polifenólico usado como control en esta tesis. Los efectos sobre la toxicidad celular inducida por fibras amiloides fueron evaluados, para los compuestos activos, en cultivo primario de hipocampo de ratas E18; observándose que una inhibición en la agregación de Aβ1-42 está relacionada con una inhibición de la muerte celular inducida por fibras amiloides. Por otra parte, en esta tesis se desarrolló un nuevo método electroquímico que permite evaluar el proceso de agregación y desagregación de Aβ1-42 con el fin de complementar, con nueva información, la metodología existente, ya que los métodos convencionales poseen diversas limitaciones y no pueden ser constituidos como un único método válido. Se generaron electrodos de carbón vítreo modificados con nanotubos de carbono y fue posible seguir el proceso de agregación mediante la señal de oxidación del residuo de Tyr en estadios tempranos cuando oligómeros pequeños son formados, revelando información conformacional del extremo N-terminal de la molécula. Además, este nuevo método fue útil para evaluar la desagregación del péptido LPFFD-NH2, conocido desagregante. / Polymerization and aggregation of the Aβ1-42 peptide are not complete elucidated processes until now, however, they are strongly related to the development of Alzheimer's disease, a neurodegenerative disorder, the primary cause of dementia for which no cure or effective prevention exist so far. One of the strategies studied in order to prevent and to find a cure for this disease is to find compounds that inhibit the aggregation of Aβ1-42 and consequently the formation of toxic species that cause neuronal death and disease development. In this thesis, the aggregation inhibitory activity of a series of 6 dihydropyridine derivatives (phenyl-DHP, 3-OH-DHP, 4-OH-DHP, 3,4-OH-DHP, 3,5-OH-DHP and 3,4,5- OH-DHP) was evaluated. These compounds differ to each other in the number and position of hydroxyl groups in the molecule and since they have structural similarities with other compounds found in the literature they are constituted in good candidates for this purpose. These compounds also have other characteristics like inhibitors of free radical generation, calcium channel blockers and potential antihypertensive activity that make them more attractive since oxidative stress, the imbalance in calcium homeostasis and hypertension, are also triggers factors of Alzheimer's disease. By mean of Thioflavin-T Fluorescence Assay, Gel Electrophoresis and TEM the results demonstrated that the compounds with one or no hydroxyl group (phenyl-DHP, 3-OH-DHP and 4-OH-DHP) are not inhibitors of Aβ1-42 aggregation. However, the compounds, 3,4-OH-DHP, 3,5-OH-DHP and 3,4,5-OH-DHP are inhibitors of the aggregation being 3.4-OH-DHP the inhibitor compound with the highest maximum inhibition of 88 ± 5% and an IC50 of 5.6 ± 0.1μM being significantly more potent than resveratrol (IC50 = 9.8 ± 1.3 µM), a polyphenolic compound used as a control in this thesis. The effects on cellular toxicity induced by amyloid fibrils were evaluated for these compounds in primary cultures of E18 rat hippocampus. It was observed that the inhibition on the aggregation of Aβ1-42 is related to the inhibition of cell death induced by amyloid fibers. On the other hand, in this thesis was developed a new electrochemical method to assess the process of aggregation and disaggregation of Aβ1-42 in order to supplement, with new information, the existing methodology, since conventional methods have several limitations and cannot be constituted as a single valid method. Glassy carbon electrodes modified with carbon nanotube were generated and it was possible to follow up the aggregation process by mean of the oxidation signal of Tyr residue in the early stages when small oligomers are formed, revealing conformational information of the N-terminal part of the molecule. In addition, this new methodology was useful for assessing the disaggregation activity of LPFFD-NH2 peptide, a known disaggregating compound. / Fondecyt

Tesis presentada a la Universidad de Chile para optar al grado académico de Magíster en Bioquímica, área de especialización en bioquímica clínica y Memoria para optar al título profesional de Bioquímico / La neurotransmisión noradrenérgica está relacionada con procesos como el aprendizaje y la memoria. Se ha observado en el cuadro depresivo una disminución en la neurotransmisión de noradrenalina (NA), alteraciones de la arquitectura dendrítica y la disminución de los niveles de factores tróficos como el factor neurotrófico derivado del cerebro (BDNF). El hecho de que una variación de los niveles de NA pueda activar diferentes receptores adrenérgicos en el espacio sináptico, podría explicar en parte los mecanismos de plasticidad sináptica involucrados en cambios morfofuncionales, desde acciones sobre la complejidad dendrítica hasta la modulación sobre procesos complejos como aprendizaje y memoria. Esta noción es apoyada por el hecho de que los fármacos que incrementan los niveles de NA en el espacio sináptico revierten la atrofia dendrítica observada en modelos animales de depresión, y recuperan el deterioro cognitivo. En base a estos antecedentes hemos propuesto en esta Tesis que: “La activación de receptores β‐adrenérgicos promueve un aumento en la complejidad dendrítica en cultivos primarios de neuronas hipocampales”. Se obtuvieron cultivos hipocampales de ratas en estado fetal de 18 días (E18), y se cultivaron durante 7 días in vitro (7 DIV). En estos cultivos se evaluó a través de Western Blot la respuesta frente a la estimulación de receptores β‐adrenérgicos, midiendo la fosforilación de la Proteína de Unión al Elemento de Respuesta al AMP cíclico (CREB) frente a estímulos cortos (5‐ 15 minutos) con noradrenalina (100 nM) en ausencia y presencia del β‐antagonista propranolol (10 μM). Junto con ello, se observó la distribución de CREB fosforilado a través de inmunocitoquímica luego de estímulos de 2 horas con los mismos fármacos. No fue posible a través de estas técnicas observar modificaciones en la intensidad de la señal o la distribución de p‐CREB. Sin embargo, la adición de NA fue capaz de promover un incremento en el calcio citosólico, como se demostró a través del uso de sondas fluorescentes, y una reducción en la actividad neuronal evaluada por registros whole-cell. Los cultivos a los 7 DIV presentan receptores β2‐adrenérgicos, visualizados a través de Western Blot. Más aún, estudios de inmunocitoquímica muestran que el receptor β2 presenta una distribución principalmente somatodendrítica. A través de la estimulación farmacológica de los cultivos durante 24 horas con NA 100 nM o clenbuterol 25 nM, en ausencia y presencia de propranolol 10 μM, se estudió si la activación de receptores adrenérgicos modula la morfología dendrítica. Ésta fue evaluada a través de la medición de puntos de intersección (Análisis de Sholl), puntos de ramificación, largo y número de dendritas. La estimulación con clenbuterol 25 nM no produjo efecto sobre ninguno de estos parámetros, descartando la participación de los receptores β2‐ adrenérgicos en la arquitectura dendrítica. Tampoco produjo efectos en la morfología de las dendritas la administración por separado de noradrenalina o propranolol. Sin embargo, los cultivos estimulados con NA posterior a la incubación con propranolol mostraron una reducción en el número de intersecciones del análisis de Sholl en el rango de los 30‐80 μm en comparación con el control. Estos cambios están acompañados por un descenso en el largo total de las dendritas primarias, y en particular de las dendritas primarias mayores a 40 μm, sin que el número de dendritas primarias por neurona se viera modificado. Un efecto similar se observó en las dendritas secundarias. Estos resultados sugieren que los cambios en el largo dendrítico observados probablemente no se asocian a la activación de receptores β2‐adrenérgicos. Proponemos que la activación de receptores α‐ y β1‐adrenérgicos regularían la morfología neuronal, probablemente con efectos opuestos, controlando el largo dendrítico de las dendritas primarias y secundarias de mayor tamaño. El estudio realizado en esta Tesis por primera vez describe efectos de la activación de receptores adrenérgicos sobre la morfología neuronal, pudiendo explicar en parte los cambios morfológicos y mejoría cognitiva tras el tratamiento de fármacos que incrementan los niveles de NA en el espacio sináptico / Noradrenergic neurotransmission is related with processes as learning and memory. In depressive behavior, it has been observed a decrease in noradrenaline (NA) neurotransmission, alterations on dendritic arborization and a decrease in levels of trophic factors, such as brain derived neurotrophic factor (BDNF). The fact that different levels of NA can activate different adrenergic receptors at synaptic cleft, could explain part of synaptic neuroplasticity mechanisms involved in morphofunctional alterations, from actions on dendritic complexity to modulation of complex processes as learning and memory. This notion is supported by the fact that drugs that increase levels of NA at synaptic cleft reverse dendritic atrophy observed in animal models of depression, and recover cognitive impairment. Considering these antecedents, this Thesis proposed that: “Activation of β‐adrenergic receptors promotes an increase in dendritic complexity in primary cultures of hippocampal neurons”. Hippocampal primary cultures were obtained from embryonic day 18 (E18) Sprague‐Dawley rat fetuses, and cultured for 7 days in vitro (7DIV). In these cultures, changes in phosphorylation of cAMP‐response element binding protein (CREB) were evaluated by Western Blot, in response to short stimulus (5‐15 min) with 100 nM NA in absence and presence of 10 μM propanolol, a β‐adrenergic receptor antagonist. In addition, levels of phosphorylated CREB (p‐CREB) were measured by immunocytochemistry, after stimulation of 2 hours with the same drugs. It was not possible to observe modifications on signal intensity of p‐CREB through these techniques. However, the addition of NA promoted an increase in cytosolic calcium, measured by fluorescent probes for calcium, and a reduction of neuronal activity evaluated by whole‐cell recording. The 7 DIV cultures showed β2‐adrenergic receptors, which were visualized by Western Blot. Moreover, immunocytochemistry studies showed that β2‐adrenergic receptor was distributed in neurons mainly in the somato‐dendritic compartment. Through pharmacological stimulation of cultures for 24 hours with 100 nM NA or 25 nM clenbuterol, with 10 μM propranolol absent or present, the activation of adrenergic receptors and their role in dendritic morphology has been measured. Morphology was evaluated through the measurement of intersection points (Sholl Analysis), branching points, and dendritic length and number. Stimulation with 25 nM clenbuterol did not have effect on any of these parameters, thus any role of β2‐adrenergic receptors in dendritic architecture was discarded. Neither administration of NA nor propranolol alone produced effects on dendrite morphology. However, NA‐stimulated cultures after incubation with propranolol showed a reduction in the number of intersections in Sholl analysis compared with controls, in the 30‐80 μm range. Those changes were accompanied by a reduction in primary dendrites greater than 40 μm, without any modifications in primary dendrite number per neuron. A similar effect was shown in secondary dendrites. These results suggest that changes observed in dendritic length were probably not associated with the activation of β2‐adrenergic receptors. We propose that the activation of α‐ and β1‐adrenergic receptors may regulate neuron morphology, probably with opposite effects, controlling dendritic length of longer primary and secondary dendrites. The study carried out in this Thesis describes for first time the effects of adrenergic receptors activation in dendrite morphology, and may explain in part the morphological changes and cognitive improvement after treatment of drugs that increase levels of NA in the synaptic cleft

Orientador: Marcelo Damy de Souza Santos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-07-16T10:06:58Z (GMT). No. of bitstreams: 1 Suarez_AchillesA_D.pdf: 1909949 bytes, checksum: bbc422978d4b9da1415add1eba4071d4 (MD5) Previous issue date: 1970 / Resumo: Não informado / Abstract: Not informed. / Doutorado / Física / Doutor em Ciências

AIM: Lysyl oxidase-like 2 (LOXL2) has emerged as a biomarker for oral squamous cell carcinoma (OSCC). Its overexpression is associated with poor prognosis in patients, however, the reason behind it is not well understood. The aim of this study is to determine the target for LOXL2 on human gingival fibroblasts (HGF) treated with cancer-conditioned media. MATERIAL AND METHODS: 30 large (150 x 20mm) plates of human gingival fibroblast cells (HGF) were cultured. For serum depletion, HGF or cancer cells were washed two times with PBS and treated with serum-free DMEM for 24 h. Condition media (CM) was produced by growing cancer cells till they were confluent. Then, 10 plates of HSC3 cells were washed twice with PBS, and were treated with serum-free DMEM for 24 h and the conditioned media was collected. Three (A – CMI, B - CM, C- SF) groups of 10 plates each respectively were made. After incubation for 6 hours, HGFs were washed with ice cold PBS and scraped in about and equal volume of glass beads were added and were subjected to dialysis. Proteins from human fibroblasts were extracted and bioyinylated with biotin hydrazide followed by sodium cyanoborohydride reduction. For affinity binding to Neutravidin, 200 microliters of suspended Neutravidin-agarose was then added to a Neutravidin column at room temperature and equilibrated with 1 ml binding buffer. . The bound proteins were collected and subjected to SDS Page western blot and probed for PDGF Beta, Integrin Alpha V and Beta Actin. RESULTS: PDGFR beta in fibroblasts displayed marked reduction in oxidation when PSX-S1C inhibitor of LOXL2 was added to cancer cell condition media. CONCLUSION: The reduction of affinity of PDGF-BB for PDGFR in both the samples indicates the priming role of LOXL2 on PDGFR, which was evident when the inhibitor was added to the samples.

No description available.

Mitochondrial dynamics includes the processes of fusion, fission, and motility. These processes form interdependent adaptive mechanisms that, together with autophagy, maintain mitochondrial function to meet cellular needs. Mitochondrial dynamics control function directly by inducing bioenergetic remodeling or indirectly by promoting turnover of mitochondria via autophagy. Importantly, mitochondrial dysfunction has been implicated in beta-cell failure during type 2 diabetes. This thesis will investigate the role of dynamics and autophagy in regulating mitochondrial and pancreatic beta-cell function during chronic exposure to excess glucose and fatty acids, termed glucolipotoxicity (GLT). It remains ill-defined what role fusion and motility play in determining mitochondrial turnover, as current methodologies to assess turnover lack subcellular resolution. To address this need we developed the use of MitoTimer, a mitochondrial fluorescent probe that undergoes a time-dependent green-to-red transition. Turnover was revealed by the integrated proportions of young (green) and old (red) MitoTimer protein. The results demonstrate that mitochondrial fusion and motility regulate turnover by promoting the distribution of newer protein to subsets of mitochondria in the network. GLT inhibits mitochondrial fusion and networking in pancreatic beta-cells. Since fusion is dependent on motility we tested the hypothesis that GLT impairs fusion by affecting motility. We determined that GLT arrests motility, which may contribute to mitochondrial and beta-cell dysfunction. We show that excess nutrients increase O-linked β-N-acetyl glucosamine (O-GlcNAc) modification of mitochondrial motor adaptor Milton1, which decreases its activity and results in arrest of motility and increased fission. Thus Milton1 O-GlcNAc modification acts as a nutrient-sensor linking fusion, fission, and motility to nutrient supply in the beta-cell. Finally, GLT inhibits autophagic flux with concurrent lysosomal pH increase in beta-cells. To address the hypothesis that impaired lysosomal acidification is a causative event inhibiting autophagic flux and beta-cell function, we developed lysosome-localizing nanoparticles that expand and acidify upon UV photo-activation. Increasing lysosomal acidity with the nanoparticles increased autophagic flux and restored beta-cell function under GLT, establishing lysosomal pH as a key mediator of nutrient-induced beta-cell dysfunction. In summary the work elucidates the interdependence and specific roles of mitochondrial fusion, fission, motility, and autophagy in dictating beta-cell responses to excess nutrient environment. / 2017-06-15T00:00:00Z

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