El 17β-estradiol, una hormona clásicamente asociada a funciones reproductivas, ha adquirido trascendencia por sus efectos en varios tejidos no asociados a la reproducción. Asimismo, se ha propuesto que el 17β-estradiol contribuiría a la supervivencia celular en varios tejidos. En músculo esquelético ha sido descripta la presencia de receptores de estrógenos (ER) aunque el mecanismo molecular activado por la hormona y el rol fisiológico de la misma en este tejido no han sido totalmente dilucidados. En este laboratorio, utilizando la línea celular de músculo esquelético murino C2C12 se demostró que, sumado a la localización citosólica-nuclear, los ER se encuentran en mitocondria y fracción microsomal. En esta tesis, se investigó el rol del 17β-estradiol sobre las cascadas de señalización que se activan durante la apoptosis en mioblastos esqueléticos, caracterizando los mecanismos moleculares involucrados y enfatizando sobre la modulación de funciones mitocondriales. Los estudios realizados sugieren que la inducción de apoptosis con H2O2 en células C2C12 activa diferentes vías de señalización que incluyen la fosforilación temprana de PKD, y posteriormente las de JNK, PKCδ, PKCθ, p53 y p66Shc, efectos que son revertidos con el pretratamiento con 17β-estradiol. En particular, PKCδ actúa upstream JNK que, a su vez, conduce a la fosforilación y translocación mitocondrial de p66Shc en respuesta al H2O2. Este efecto también es contrarrestado por 17β-estradiol, resultando en la protección del potencial de membrana mitocondrial y la prevención de la apertura prolongada del poro de permeabilidad transitoria mitocondrial. Esta acción protectora del 17β-estradiol sobre la fisiología mitocondrial también incluye la inhibición de la translocación de Bax hacia mitocondria. Asimismo, se observó que uno de los sucesos en el inicio de la apoptosis disparados por H2O2 es el aumento en la formación de estructuras tipo-TNT (tunneling nanotubes) entre las células para promover la transferencia de mitocondrias sanas desde las células no estresadas a las estresadas, mientras que el pretratamiento con la hormona disminuye su formación, efecto probablemente relacionado con la reducción en la proporción de células estresadas que necesitarían revertir la disfunción mitocondrial. Estas mitocondrias disfuncionales producen mayor cantidad de especies reactivas del oxígeno y cuando sus niveles aumentan, las enzimas antioxidantes son fundamentales para contrarrestar sus efectos negativos. En esta tesis se observó que la disminución de la capacidad antioxidante inducida por H2O2 es revertida por el pretratamiento con 17β-estradiol mediante la regulación de las actividades enzimáticas y/o expresión proteica de MnSOD, GPx y CAT a través de ERs. Por otra parte, se evidenció la regulación de FoxOs por 17β-estradiol que induciría la inhibición de la transcripción de genes proapoptóticos, y además que el inductor de apoptosis regula negativamente a nivel transcripcional a Bcl-2, y positivamente a p66Shc, PUMA y PERP, acciones que son revertidas por 17β- estradiol. Por otro lado, la regulación transcripcional de MDM2 por el H2O2, que involucra a ERK, se relacionaría con la actividad de p53 y FoxO3a inactivo. Esta tesis aporta conocimientos básicos sobre los mecanismos moleculares activados por 17β-estradiol frente a la apoptosis inducida por H2O2 en células musculares esqueléticas que resultan en supervivencia celular. / 17β-estradiol, a hormone typically associated with reproductive functions, has gained significance for its effects on several tissues not associated with reproduction. Furthermore, it has been proposed that 17β-estradiol contributes to cell survival in various tissues. In skeletal muscle it has been described the presence of estrogen receptors (ER) but the molecular mechanism activated by the hormone and its physiological role over this tissue have not been fully elucidated. In this laboratory, using the murine skeletal muscle cell line C2C12 it was shown that, in addition to the cytosolic-nuclear localization, the ER was found in mitochondria and microsomal fraction. In this thesis, it was investigated the role of 17β-estradiol on the signaling cascades that are activated during apoptosis in skeletal myoblasts, characterizing the molecular mechanisms involved and emphasizing on modulation of mitochondrial functions. The studies suggest that induction of apoptosis with H2O2 in C2C12 cells activates different signaling pathways including early phosphorylation of PKD, and then the phosphorylation of JNK, PKCδ, PKCθ, p53 and p66Shc, effects that are reversed by pretreatment with 17β-estradiol. In particular, PKCδ acts upstream JNK which, in turn, leads to phosphorylation and mitochondrial translocation of p66Shc in response to H2O2. This effect is counteracted by 17β-estradiol, resulting in the protection of mitochondrial membrane potential and prevention of sustained opening of the mitochondrial permeability transition pore. This protective effect of 17β-estradiol on mitochondrial physiology also includes the inhibition of Bax translocation to mitochondria. It was also observed that one of the events in the beginning of apoptosis triggered by H2O2 is an augmented formation of TNT-like structures (tunneling nanotubes) between cells that promotes the transfer of healthy mitochondria from unstressed to stressed cells, whereas pretreatment with the hormone decreases its formation, effects that would probably be related to the reduction in the proportion of stressed cells that needs to reverse mitochondrial dysfunction. Since dysfunctional mitochondria produce more reactive oxygen species, when their levels are increased, antioxidant enzymes are essential to counteract their negative effects. In this thesis, it was observed that the decrease in H2O2-induced antioxidant capacity is reversed by pretreatment with 17β-estradiol by regulating enzyme activities and/or protein expression of MnSOD, GPx and CAT via ERs. Additionally, it was observed that the regulation of FoxOs mediated by 17β- estradiol would be related to the inhibition of transcription of proapoptotic genes, and moreover that the apoptotic inductor negatively regulates the transcriptional level of Bcl-2, and positively the transcriptional levels of p66Shc, PUMA and PERP. These actions are reversed by pretreatment with 17β-estradiol. Also, it was suggested that the transcriptional regulation of MDM2 by H2O2 involved ERK and that it could be related to p53 activity and inactive FoxO3a. This thesis provides basic information on the 17β-estradiol-activated mechanisms against H2O2-induced apoptosis in skeletal muscle cells resulting in cell survival molecular mechanisms.

Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postis- chemic and diabetic retinas. Progesterone, testosterone, estriol, and 17ß-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 lM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y1, and adenosine A1 receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.

Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postis- chemic and diabetic retinas. Progesterone, testosterone, estriol, and 17ß-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 lM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y1, and adenosine A1 receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.

Organic contaminants from industrial and/or domestic effluents may be harmful to humans directly or indirectly by degrading the quality of the aquatic environment. Consequently these contaminants must be reduced to levels that are not harmful to humans and the environment before disposal. Chemical, physical and biological methods exist for the removal of these pollutants from effluents. Among the available chemical methods, heterogeneous photocatalytic oxidation has been found particularly effective in removing a large number of persistent organics in water. In this study, photocatalytic degradation was explored for the removal of reactive azo-dye (textile dye), triclocarban (disinfectant), clopyralid (herbicide) and three endocrine disrupting compounds (EDCs) (estrone, 17ß-estradiol and 17α-ethinylestradiol) from synthetic effluents. The major factors affecting the photocatalytic processes including the initial concentration of the target compounds, the amount of catalyst, the light intensity, the type of catalyst, the electron acceptor, the irradiation time and the pH were studied. Other oxidation techniques including (O3, H2O2, UV) were also studied. Generally UV light is used in combination with titanium dioxide, as photocatalyst, to generate photoinduced charge separation leading to the creation of electron-hole pairs. The holes act as electron acceptors hence the oxidation of organics occur at these sites. These holes can also lead to the formation of hydroxyl radicals which are also effective oxidants capable of degrading the organics. The results obtained in this study indicated that photolysis (i.e. UV only) was found to have no effect on the degradation of reactive azo-dye (RO16). However, complete photocatalytic degradation of 20 mg/L (3.24×10-2 mM) RO16 was achieved in 20 minutes in the presence of 1g/L TiO2 Degussa P25 at pH 5.5. Comparison between various types of catalysts (i.e. Degussa P25, VP Aeroperl, Hombifine N) gave varied results but Degussa P25 was the most effective photocatalyst hence it was selected for this study. For RO16 the optimum catalyst concentration was 0.5 g/L TiO2 with initial concentration of 20 mg/L RO16. It was found that the disappearance of RO16 satisfactorily followed the pseudo first-order kinetics according to Langmuir-Hinshelwood (L-H) model. The rate constant was k= 0.0928 mol/min. Photodegradation of TCC was studied in 70%v acetonitrile: 30%v water solutions. UV light degraded TCC effectively and the reaction rates increased with decreasing initial concentration of TCC. UV/TiO2 gave unsatisfactory degradation of triclocarban (TCC) since only 36% were removed in 60 minutes with initial concentration of TCC 20 mg/L. The degradation of clopyralid and the EDCs was studied using three oxidation systems UV/TiO2, UV/H2O2 and O3. Complete degradation of clopyralid (3,6-DCP) was achieved with UV/TiO2 in about 90 minutes at an optimum catalyst concentration of 1g/L. Zero-order kinetics was found to describe the first stage of the photocatalytic reaction in the concentration range 0.078-0.521 mM. At pH 5 the rate constant was 2.09×10-6-4.32×10-7 M.s-1.Complete degradation of all the three EDCs was achieved with UV/H2O2 in 60 minutes at catalyst concentration of (2.94×10-2 M). On the other hand complete degradation of the EDCs was achieved in just 2 minutes with ozonation. For high concentration EDCs, TiO2/UV gave low efficiency of degradation as compared with ozone and H2O2/UV. First-order kinetics was found to describe the photocatalytic reaction of the EDCs. / Education Service Department of the Libyan Government

One of the defining characteristics of the brain is its plasticity, which is the ability to alter and reorganize neuronal circuits. The brain is constantly being shaped and moulded by the external world through endogenous factors like neurotransmitters, growth factors and circulating hormones. 17β-estradiol, which is the most potent estrogen among the group of ovarian steroid hormones, has widespread effects throughout the central nervous system. Apart from its actions on regions of the brain concerned with reproduction, estradiol has profound effects on brain areas not classically associated with reproductive function like cerebral cortex, midbrain, brainstem, hippocampus and spinal cord. This enables the hormone to influence learning and memory, emotions, affective state, cognition, motor coordination and pain sensitivity. Estradiol exerts these effects by regulating gene expression via intracellular estrogen receptors. In addition to this, the hormone interacts with receptors at the cell membrane to rapidly alter the electrical activity of neurons and astrocytes, and regulate second messenger systems. The aim of this study was to investigate the cellular and functional effects of estradiol on neuronal networks and on signaling between neurons and astrocytes in primary mixed hippocampal cultures. Estradiol is proconvulsant; it increases neuronal excitability and decreases the threshold for seizures. This property of estradiol is instrumental in precipitating catamenial seizures in women with epilepsy. These are epileptic seizures influenced by cyclical hormone changes and occur in over one-third to half of women with epilepsy. In the first part of the work, the effects of 24-hour estradiol treatment on hippocampal neurons were investigated using fluorescence imaging and electrophysiological techniques. Further, the ability of gabapentin, an antiepileptic drug sometimes used to treat hormone sensitive seizures, to counteract the effects of estradiol was studied. Synaptic vesicles were labeled by uptake of FM 1-43, and high K+- triggered exocytotic release was monitored by fluorescence imaging. The reduction in intensity of FM 1-43 fluorescence, which is a measure of vesicular release, was enhanced by estradiol, suggesting that estradiol upregulates the exocytotic machinery. The high K+-evoked intracellular Ca2+ rise in neurons, studied by loading the neurons with the Ca2+ indicator dye fluo-3 AM, was potentiated following estradiol treatment. Electrophysiological recordings from neurons following estradiol treatment showed an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a larger number of mEPSC events with a predominant NMDA component. Many of the estradiol-induced excitatory effects on the neuronal network were abolished by incubating the cultures with a combination of estradiol and gabapentin suggesting a mechanism of action for the drug in the treatment of hormone sensitive seizures. Glial cells were once regarded as passive, supportive elements in the nervous system. This view of glial cells has drastically changed over the past decade and it is now known that glial cells are dynamic signaling elements in the brain. In view of the emerging importance of glia in the physiology of the nervous system and accumulating evidence of direct effects of steroid hormones on these cells, the subsequent part of the work delves into the consequences of 24-hour estradiol treatment on astrocytes and neuron-to-astrocyte signaling. Estrogen receptors have been described on both neurons and astrocytes in the hippocampus suggesting a complex interplay between the two cell types in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration, ([Ca2+]i). Astrocyte ([Ca2+]i) transients can modulate neuronal activity, indicating a bi-directional form of communication between neurons and astrocytes. Using simultaneous electrophysiology and calcium imaging techniques, neuronal activity-evoked ([Ca2+]i) changes in fluo-3 AM loaded astrocytes were monitored. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca2+]i) elevations in adjacent astrocytes which could be blocked by 200 µM MCPG and also 1 µM TTX. Comparison of astrocytic ([Ca2+]i) transients in control and estradiol treated cultures revealed that the amplitude of the ([Ca2+]i) transient, the number of responsive astrocytes and the ([Ca2+]i) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca2+]i) rise in astrocytes in response to local application of the metabotropic glutamate receptor agonist t-ACPD was attenuated in estradiol treated cultures suggesting functional changes in the astrocyte metabotropic glutamate receptor following 24-hour treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca2+]i) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling. The acute effects of estradiol on astrocyte-to-astrocyte and astrocyte-to-neuron signaling have been addressed in the next part of the study. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes, and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. In addition to its rapid actions on neuronal electrical activity, estradiol can rapidly alter astrocyte ([Ca2+]i) levels through a plasma membrane-associated estrogen receptor. The functional consequences of acute estradiol treatment (5 min) on astrocyte-astrocyte and astrocyte-neuron communication were investigated using calcium imaging and electrophysiological techniques. Mechanical stimulation of an astrocyte evoked a ([Ca2+]i) rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a ([Ca2+]i) wave. Following acute treatment with estradiol, the amplitude of the ([Ca2+]i) elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the ([Ca2+]i) rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, t-ACPD. Mechanical stimulation of astrocytes induced ([Ca2+]i) elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated ([Ca2+]i) rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol mediated hormonal control.