Microglial involvement in neurological disorders is well-established, being microglial activation not only associated with neurotoxic consequences, but also with neuroprotective effects. The studies presented here, based on microglia rat primary cell cultures and mainly on microglial conditioned medium (MCM), show insights into the mechanism of Superoxide dismutase 1 (SOD1) and Apolipoprotein E (ApoE) secretion by microglia as well as their neuroprotective effect towards primary cerebellar granule neurons (CGNs) exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). SOD1 and ApoE are released respectively through non-classical lysosomal or the classical ER/Golgi-mediated secretion pathway. Microglial conditioned medium, in which SOD1 and ApoE accumulated, protected CGNs from degeneration and these effects were replicated when exogenous SOD1 or ApoE was added to a non-conditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. ApoE release is negatively affected by microglia activation, both with lipopolysaccharide (LPS) and Benzoylbenzoyl-ATP (Bz-ATP) but is stimulated by neuronal-conditioned medium as well as in microglia-neurons co-culture conditions. This neuronal-stimulated microglial ApoE release is differently regulated by activation states (i.e. LPS vs ATP) and by 6-hydroxydopamine-induced neurodegeneration. In co-culture conditions, microglial ApoE release is essential for neuroprotection, since microglial ApoE silencing through siRNA abrogated protection of cerebellar granule neurons against 6-OHDA toxicity. Therefore, these molecules could represent a target for manipulation aimed at promoting neuroprotection in brain diseases. Considering a pathological context, and the microglial ability to adopt a neuroprotective or neurotoxic profile, we characterize the microglial M1/M2 phenotype in transgenic rats (McGill-R-Thy1-APP) which reproduce extensively the Alzheimer’s-like amyloid pathology. Here, for the first time, cortical, hippocampal and cerebellar microglia of wild type and transgenic adult rats were compared, at both early and advanced stages of the pathology. In view of possible therapeutic translations, these findings are relevant to test microglial neuroprotection, in animal models of neurodegenerative diseases.

Il presente studio ha indagato e valutato alcune abilità cognitive del cane: la capacità di discriminare quantità e le capacità di apprendimento mediante imitazione; quest’ultima è poi stata messa in relazione con l’attaccamento nei confronti del proprietario. Per l’esecuzione della prima indagine sono stati messi appunto due test: il primo si è basato esclusivamente sulla presentazione di uno stimolo visivo: diversi quantitativi di cibo, differenti tra loro del 50%, sono stati presentati al cane; la scelta effettuata dai soggetti testati è stata premiata con differenti tipi di rinforzo differenziale o non differenziale. Il secondo test è stato diviso in due parti: sono stati presentati al cane diversi quantitativi di cibo sempre differenti tra loro del 50% ma nella prima parte del test l’input sensoriale per il cane è stato esclusivamente uditivo mentre nella seconda parte è stato sia uditivo che visivo. Ove è stato possibile è stato applicato ai cani un cardiofrequenzimetro al fine di eseguire una valutazione delle variazioni della frequenza cardiaca nel corso del test. Lo scopo è stato quello di valutare se i soggetti testati erano in grado di discriminare la quantità maggiore. La seconda indagine ha analizzato le capacità di apprendimento di 36 soggetti che sono stati suddivisi in cani da lavoro e pet. I soggetti protagonisti dello studio hanno eseguito il Mirror Test per la valutazione dell’apprendimento per imitazione. I soggetti presi in considerazione, sono stati sottoposti a scansione termografica all’inizio ed al termine del test ed è stata rilevata la loro frequenza respiratoria nella fase iniziale e finale del test. In 11 soggetti che hanno eseguito il precedente test è stato possibile eseguire anche il Strange Situation Test per la valutazione dell’attaccamento al proprietario; i test in questione sono stati videoregistrati ed analizzati per mezzo di un software preposto (OBSERVER XT 10). / The present study investigated and evaluated some cognitive abilities of the dog: the ability to discriminate quantity and to learn through imitation; the latter kind of learning was related to the dog’s attachment to the owner. In the first analysis two tests were applied: the first is based on the presentation of a visual stimulus: different amounts of food, different from each other by 50% , were presented to the dog; the choice of the dogs was rewarded with different types of differential or non-differential reinforcement. The second test was divided into two parts: different quantities of food (different from each other by 50 %) were submitted to the dogs; in the first part of the test, the sensory input was exclusively auditory, while in the second part both auditory and visual. Where feasible, a heart rate monitor was applied to the dogs, in order to perform an assessment of the heart rate changes during the test. The aim of the test was to evaluate if the subjects were able to discriminate the larger amount of food. The second study analyzed the learning ability of 36 subjects, divided into working dogs and pets. The subjects performed the Mirror Test for the assessment of learning by imitation. In addition the subjects were subjected to thermal scanning at the beginning and at the end of the test and their respiratory rate was detected before and after the test. Finally, in 11 of these subjects the Strange Situation Test for the assessment of attachment to the owner, was performed. All tests were videotaped and then analyzed using a dedicated software (OBSERVER XT 10).

Rett's Syndrome (RTT) is a severe neurodevelopmental disorder, characterized by cognitive disability that appears in the first months/years of life. Recently, mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been detected in RTT patients characterized by early-onset seizures. CDKL5 is highly expressed in the brain starting from early postnatal stages to adulthood, suggesting the importance of this kinase for proper brain maturation and function. However, the role/s of CDKL5 in brain development and the molecular mechanisms whereby CDKL5 exerts its effects are still largely unknown. In order to characterize the role of CDKL5 on brain development, we created a mice carrying a targeted conditional knockout allele of Cdkl5. A first behavioral characterization shows that Cdkl5 knockout mice recapitulate several features that mimic the clinical features described in CDKL5 patients and are a useful tool to investigate phenotypic and functional aspects of Cdkl5 loss. We used the Cdkl5 knockout mouse model to dissect the role of CDKL5 on hippocampal development and to establish the mechanism/s underlying its actions. We found that Cdkl5 knockout mice showed increased precursor cell proliferation in the hippocampal dentate gyrus. Interestingly, this region was also characterized by an increased rate of apoptotic cell death that caused a reduction in the final neuron number in spite of the proliferation increase. Moreover, loss of Cdkl5 led to decreased dendritic development of new generated granule cells. Finally, we identified the Akt/GSK3-beta signaling as a target of Cdkl5 in the regulation of neuronal precursor proliferation, survival and maturation. Overall our findings highlight a critical role of CDKL5/AKT/GSK3-beta signaling in the control of neuron proliferation, survival and differentiation and suggest that CDKL5-related alterations of these processes during brain development underlie the neurological symptoms of the CDKL5 variant of RTT.

Este trabajo identifica a HERC2 como una proteína que se une a p53 y modula su actividad transcripcional mediante la regulación de su oligomerización. En una primera etapa, implementamos un sistema de geles en gradiente Tris-acetato que permitió el análisis de proteínas gigantes como HERC2 y otras de menor tamaño (hasta de 10 kDa) en un solo gel de electroforesis. Después, estudiamos el papel de HERC2 en la regulación de p53, mapeando los dominios involucrados en la interacción, evaluando los niveles de expresión, actividad transcripcional, localización subcelular y oligomerización de p53 en ausencia de HERC2, para demostrar finalmente que la expresión ectópica del dominio CPH de HERC2 es suficiente para promover la oligomerización de p53 y aumentar su actividad. También se demostró que una mutación patológica de HERC2, que origina un cambio de aminoácido en la posición 594 causa la inestabilidad de la proteína, reduciendo casi por completo su expresión. Esta mutación causa una enfermedad con un fenotipo muy similar al síndrome de Angelman. Con los fibroblastos de individuos afectados por la mutación en HERC2, realizamos ensayos para estudiar la actividad transcripcional de p53 encontrándola reducida. Finalmente, demostramos que los ratones knockout para Herc2 son letales en homocigosis, demostrando que Herc2 es un gen esencial en el desarrollo embrionario de los ratones. / This work identifies HERC2 as a protein that binds to p53 and modulates its transcriptional activity by regulating its oligomerization. As a first step, we developed an electrophoretic analysis using Tris-acetate polyacrylamide gels to detect giant proteins such as HERC2 and other smaller proteins (down to 10 kDa) in the same electrophoresis gel. Then, we found that HERC2 interacted with p53 and identified the domains involved in the interaction. Also, we evaluated the levels of expression, transcriptional activity, subcellular localization and oligomerization of p53 when HERC2 was depleted, to finally demonstrate that ectopic expression of HERC2 (CPH domain) was sufficient to promote the oligomerization of p53 and increase its activity. Also, we demonstrated that a HERC2 pathological mutation, which causes an amino acid change at position 594, induced the instability of the protein and reduced its expression. This mutation causes a disease similar to Angelman syndrome. Fibroblasts affected by the mutation in HERC2 have the transcriptional activity of p53 reduced. Finally, we showed that Herc2 knockout mice are lethal in homozygous, demonstrating that Herc2 is an essential gene in the embryonic development of mice.

Tesi realitzada a l'Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) / La Leucoencefalopatia Megalencefàlica amb quists subcorticals, també anomenada MLC, és un tipus rar de leucodistròfia vacuolitzant. Actualment encara es desconeix el mecanisme fisiopatològic de la malaltia, i per tant ni hi ha cap tractament possible per als pacients. S’han descrit dos gens implicats en la malaltia MLC. El primer gen descobert s’anomena MLC1 i codifica per una proteïna de membrana que porta el mateix nom. El segon gen s’anomena GLIALCAM i codifica per una proteïna transmembrana de tipus I que també porta el mateix nom. S’ha decrit que la proteïna GlialCAM actua com a subunitat ß de MLC1 ja que es capaç de dirigir-la i concentrar-la a les unions cel•lulars. Per altra banda, GlialCAM també s’ha descrit com a subunitat auxiliar del canal de Cl- ClC-2 ja que és capaç de modificar les propietats d’activació i rectificació del canal. En la present tesi s’han generat i estudiat diferents models animals i cel•lulars per a l’estudi de la malaltia. En primer lloc, s’ha generat i s’ha caracteritzat un model de ratolí knock-out per a Mlc1. Gràcies a aquest model s’ha observat que la proteïna MLC1 és únicament astrocitària i que la proteïna GlialCAM no es independent de MLC1, ja que en absència d’aquesta es troba deslocalitzada en el cerebel. També s’ha pogut descriure per primer cop la implicació del canal de Cl- ClC-2 en la fisiopatologia, ja que els seus nivells de proteïna disminuixen en el cerebel i el canal es troba gairebé inactiu en els oligodendròcits de l’animal knock-out. Les característiques fenotípiques que presenta el model de ratolí equivalen a les característiques observades en els pacients en fases inicials de la malaltia, ja que l’animal tot i que mostra presència de vacuoles no presenta deteriorament motor i macrocefàlia aparent. També s’ha generat un model de peix zebra knock-out per a zmlc1. Aquest model presenta avantatges respecte el ratolí, com per exemple el baix cost o l’aplicació de tècniques genètiques a gran escala. Aquest model ha permés observar de nou que realment GlialCAM necessita a MLC1 per a la seva correcta localització. També s’ha observat que l’ortòleg zGlialCAMa conserva la seva funció de entre espécies ja que també es capaç de modificar les corrents de ClC-2. Aquests resultats obtinguts amb els models s’han pogut comparar amb el cervell d’una pacient. Aquest cervell demostra que MLC1 és necessària per a la correcta localització de GlialCAM en la regió del cerebel. Per altra banda, s’han desenvolupat diferents models cel•lulars. Primerament s’han estudiat els astròcits del ratolí knock-out. Aquestes cel•lules mancades de MLC1 també presenten vacuoles per tot el citoplasma, però no mostren canvis en la localització ni en els nivells de proteïna de GlialCAM i ClC-2. Aquest fet juntament amb altres estudis del grup van fer pensar si la condició necessària per a que es veguessin afectades aquestes proteïnes estaria relacionada amb el procés del sifoneig de K+. Estudis realitzats en astròcits de rata demostren que en condicions d’un alt contingut de K+, com per exemple durant una alta activitat neuronal, GlialCAM i ClC-2 és localitzen juntament a les membranes cel•lular i ClC-2 canvia les seves propietat de canal. Paral•lelament, estudis realitzats en oligodendròcits de rata també demostren que aquest fet també succeix en aquest tipus cel•lular. / Megalencefalic leukoencephalopathy with subcortical cysts, also known as MLC, is a rare type of leukodystrophy. Currently still unknown pathophysiological mechanism of the disease, and therefore there is no effective treatment possible for patients. There are two genes involved in the MLC disease. Gene was first discovered was MLC1 and this encodes for a membrane protein with the same name. The second gene is called GLIALCAM and encodes for a transmembrane protein type I that also carries the same name. In our group is has been described that GlialCAM acts as a protein ß subunit of MLC1 because it is able to direct and concentrate in the cellular junctions. Moreover, GlialCAM also act as auxiliary subunit of CLC-2 Cl channel as it is capable of modifying the activation and rectification properties of the channel. In this work we have developed two different models to study the physiopathology. The results show that GlialCAM affected by the absence of MLC1. It has been also demonstrated that ClC-2 is implicated in the disease.These results were compared with a patient brian and has been shown that MLC1 is important for the correct location of GlialCAM in the cerbellum. Have also been developed a different cellular models. The results with this models show that GlialCAM and ClC-2 could have a functional role in the process of potassium siphoning.

Il nucleo accumbens (NAc), il maggior componente del sistema mesocorticolimbico, è coinvolto nella mediazione delle proprietà di rinforzo e nella dipendenza da diverse sostanze d’abuso. Le sinapsi glutammatergiche del NAc possono esprimere plasticità, tra cui una forma di depressione a lungo termine (LTD) dipendente dagli endocannabinoidi (eCB). Recenti studi hanno dimostrato un’interazione tra le vie di segnalazione del sistema eCB e quelle di altri sistemi recettoriali, compreso quello serotoninergico (5-HT); la vasta colocalizzazione di recettori serotoninergici e CB1 nel NAc suggerisce la possibilità di un’interazione tra questi due sistemi. In questo studio abbiamo riscontrato che una stimolazione a 4 Hz per 20 minuti (LFS-4Hz) delle afferenze glutammatergiche in fettine cerebrali di ratto, induce una nuova forma di eCB-LTD nel core del NAc, che richiede l’attivazione dei recettori CB1 e 5-HT2 e l’apertura dei canali del Ca2+ voltaggio-dipendenti di tipo L. Inoltre abbiamo valutato che l’applicazione esogena di 5-HT (5 M, 20 min) induce una LTD analoga (5-HT-LTD) a livello delle stesse sinapsi, che richiede l’attivazione dei medesimi recettori e l’apertura degli stessi canali del Ca2+; LFS-4Hz-LTD e 5-HT-LTD sono reciprocamente saturanti. Questi risultati suggeriscono che la LFS-4Hz induce il rilascio di 5-HT, che si lega ai recettori 5-HT2 a livello postsinaptico incrementando l’influsso di Ca2+ attraverso i canali voltaggio-dipendenti di tipo L e la produzione e il rilascio di 2-arachidonoilglicerolo; l’eCB viaggia a ritroso e si lega al recettore CB1 a livello presinaptico, causando una diminuzione duratura del rilascio di glutammato, che risulta in una LTD. Queste osservazioni possono essere utili per comprendere i meccanismi neurofisiologici che sono alla base della dipendenza da sostanze d’abuso, della depressione maggiore e di altre malattie psichiatriche caratterizzate dalla disfunzione della neurotrasmissione di 5-HT nel NAc. / The nucleus accumbens (NAc), a major component of the mesolimbic system, is involved in the mediation of reinforcing and addictive properties of many dependence-producing drugs. Glutamatergic synapses within the NAc can express plasticity, including a form of endocannabinoid (eCB)-long-term depression (LTD). Recent evidences demonstrate cross-talk between eCB signaling pathways and those of other receptor systems, including serotonin (5-HT); the extensive co-localization of CB1 and 5-HT receptors within the NAc suggests the potential for interplay between them. Here, we found that 20 min low-frequency (4 Hz) stimulation (LFS-4Hz) of glutamatergic afferences in rat brain slices induces a novel form of eCB-LTD in the NAc core, which requires 5-HT2 and CB1 receptors activation and L-type voltage-gated Ca2+ channels opening. Moreover, we found that exogenous 5-HT application (5 μM, 20 min) induces an analogous LTD (5HT-LTD) at the same synapses, requiring the activation of the same receptors and the opening of the same Ca2+ channels; LFS-4Hz-LTD and 5-HT-LTD were mutually occlusive. Present results suggest that LFS-4Hz induces the release of 5-HT, which acts at 5-HT2 postsynaptic receptors increasing Ca2+ influx through L-type voltage-gated channels and 2-arachidonoyl-glycerol production and release; the eCB travels retrogradely and binds to presynaptic CB1 receptors, causing a long-lasting decrease of glutamate release resulting in LTD. These observations might be helpful to understand the neurophysiological mechanisms underlying drug addiction, major depression and other psychiatric disorders characterized by dysfunction of 5-HT neurotransmission in the NAc.

La exposición a estímulos estresantes de tipo emocional induce activación neuronal en muchas regiones del SNC que cursa en paralelo con una estimulación del eje hipotálamo-hipofisario-adrenal (HPA). La activación neuronal suele valorarse mediante la inducción de genes de expre-sión temprana como c-fos, mientas que la activación del eje HPA se basa en la expresión del factor liberador de corticotropina (CRF) en el núcleo paraventricular del hipotálamo (PVN) y en la liberación de hormonas como la ACTH y los glucocorticoides. Numerosos estudios sugieren que la expresión de c-fos y CRF alcanza un máximo alrededor de los 15-30 min de iniciada la exposición al estrés, para disminuir posteriormente a pesar de la persistencia del estímulo estre-sante. Esta disminución de la respuesta podría obedecer a cambios en las señales estimuladoras y/o inhibidoras que llegan a la neurona como consecuencia de la exposición prolongada al mis-mo estímulo estresante, o bien a mecanismos de represión intracelular. Razonamos que si la hipótesis de los cambios en las señales es correcta, la expresión de dichos genes se restablecería en gran medida en respuesta a un nuevo estímulo estresante, en tanto que si la segunda hipótesis es la válida, la expresión debería estar bloqueada. Utilizando la rata como modelo, hemos estudiado en un primer experimento mediante hibrida-ción in situ (ISH), los cambios en la expresión de c-fos y CRF, y los cambios en los niveles plas-máticos de ACTH y corticosterona tras la exposición prolongada (unas 4 h) a una inmoviliza-ción en plancha (IMO) y cómo esta exposición a la IMO afecta a la respuesta a un nuevo estímu-lo estresante (nado forzado). Los resultados de expresión de c-fos indicaron que en la corteza prefrontal medial (mPFC), el septum lateral ventral (LSv) y la amígdala medial (MeA) la res-puesta al nado tras la IMO prolongada es capaz de restablecerse en gran medida, dando validez a la hipótesis de una reducción de las señales que llegan a las neuronas. Sin embargo, en el PVN la expresión de c-fos en respuesta al nado tras la IMO prolongada se encuentra en su mayor parte bloqueada, sugiriendo un bloqueo intracelular. En consonancia con los resultados de expresión de c-fos en el PVN, tanto la expresión de CRF como la secreción de ACTH en respuesta al nado se encuentran totalmente bloqueadas tras la IMO prolongada. Una interpretación correcta de estos primeros resultados, precisa conocer si las poblaciones neuronales activadas durante la IMO prolongada coinciden o no con las que responden al nado tras esta IMO. Al objetivo de identificar estas neuronas hemos realizado un nuevo experimento similar al anterior pero incluyendo un grupo de animales que fue liberado de la IMO prolonga-da y re-expuesto a una nueva IMO. La finalidad de este grupo era diferenciar la activación debi-da exclusivamente al nado de la que pudiera producirse por otros procesos como la simple ma-nipulación del animal o la propia liberación de la IMO. Para identificar las poblaciones neuro-nales que responden a la IMO prolongada y al nado, hemos realizado un doble marcaje de la proteína c-Fos y el mRNA que codifica para esta proteína mediante inmunofluorescencia e ISH fluorescente (IF-FISH). Basándonos en la dinámica de ambos marcadores, las neuronas que responden a la IMO prolongada mostrarían fundamentalmente proteína y poco o nulo mRNA, mientras que las que responden al nado aplicado tras esta IMO mostrarían fundamentalmente mRNA y nula proteína. Tras este análisis hemos observado que en regiones como el mPFC, el LSV y la MeA, la exposición al nado tras la IMO prolongada provocó la activación de nuevas neuronas sugiriendo cierta especificidad en la respuesta a ambos estímulos estresantes. Sin em-bargo, en el PVN con la exposición al nado tras la IMO prolongada no provocó activación de nuevas neuronas, confirmando la existencia de una sola población neuronal que responde indis- tintamente frente a estímulos estresantes de distinta naturaleza que es bloqueada tras una esti-mulación prolongada. Una gran parte de la activación neuronal observada en términos de expresión de c-fos en res-puesta a los distintos estímulos estresantes podría deberse a procesos de activación generalizada o de arousal que enmascararía aquellas neuronas que son realmente importantes en la respuesta al estrés. En un último experimento hemos pretendido disminuir la aportación del arousal, ad-ministrando a nivel sistémico DSP-4, una neurotoxina altamente selectiva para las terminales noradrenérgicas provenientes del LC. Una semana después de la administración de DSP-4, ex-pusimos a los animales a estímulos estresantes de diferente intensidad (ambiente nuevo, olor al depredador e IMO) para posteriormente analizar la expresión de c-fos mediante FISH en el mPFC, el LSv, la MeA y el PVN. El alcance de la lesión sobre las terminales noradrenérgicas del LC se valoró mediante inmunoflorescencia de la dopamina beta-hidroxilasa (DβH). En concor-dancia con datos de la literatura, sólo en el mPFC y la MeA se pudo apreciar una disminución en las terminales noradrenérgicas. La lesión con el DSP-4 no tuvo un impacto sobre la expresión de c-fos en respuesta a los distintos estímulos estresantes, salvo en la respuesta del LSv a la IMO donde se observó una disminución. En conjunto, los resultados sugieren que en la reducción de la expresión de c-fos observada tras la exposición prolongada a un estímulo emocional pueden intervenir tanto la familiarización con el estímulo y la consiguiente reducción de las señales estimuladoras como la represión in-tracelular de la expresión del gen. La contribución de cada uno de los mecanismos es marcada-mente dependiente del área estudiada. Por otro lado, parecen existir poblaciones de neuronas que responden específicamente a la IMO y al nado, aunque muchas de ellas pueden ser comunes a ambos. En la respuesta del eje HPA podrían añadirse bloqueos adicionales que probablemente contribuyen a reducir el posible impacto negativo de una liberación excesiva de glucocorticoi-des. El escaso impacto de la administración de DSP-4 podría explicarse por la existencia de me-canismos compensatorios y por lo tanto sería necesario valorar otras alternativas para disminuir la aportación del arousal a la respuesta de estrés. / Exposition to emotional stressful stimuli leads to neuronal activation of several regions of the Central Nervous System (CNS) that converge into the stimulation of the hypothalamic-pituitary-adrenal axis (HPA). This neuronal activation can be measured by the induction of early expression genes (IEG) such as c-fos, while the activation of the HPA axis can be measured by the expression of the corticotrophin-releasing-factor (CRF) in the paraventricular nucleus of the hypothalamus (PVN) and also by the release of hormones such as ACTH and glucocorti-coids. There are several studies that suggest the expression of c-fos and CRF to reach its maxi-mum around 15-30 min after initiation of the exposition to stress. A decline in these parameters is observed even with the persistence of the stressful stimuli. This decline could be due to chang-es in the stimulatory/inhibitory signals arriving to the neurons as a consequence of a prolonged exposition to the same stressful stimuli, or it could also be due to intracellular repression mech-anisms. If the former hypothesis is correct, the expression of such genes would be mostly reestablished in response to new stressful stimuli, however, if the second hypothesis is valid, the expression should be blocked. In a first experiment we studied changes in the expression of c-fos and CRF in the rat CNS and changes in plasma levels of ACTH and corticosterone after a prolonged exposure (4 h approx.) to immobilization (IMO). We then assessed how this exposure could affect the response to a new stressor (forced swim). The results of expression of c-fos indicated that in the medial pre-frontal cortex (mPFC), the ventral lateral septum (LSv) and the medial amygdala (MeA) the response after prolonged IMO can be greatly restored, validating the hypothesis of a reduction of signals arriving to the neurons. However, in the PVN, the c-fos expression in response to forced swimming after prolonged IMO is largely blocked, suggesting an intracellular repression. Corroborating the results of c-fos expression in the PVN, the expression of CRF and ACTH secretion in response to forced swim are completely blocked after prolonged IMO. A correct interpretation of these first results requires knowing whether the neuronal popula-tions activated during prolonged IMO are the same as those responding to the forced swim ap-plied after the prolonged IMO. In order to identify these neurons we performed a new experi-ment which was similar to the previous one but including a new group of animals that was re-exposed to a second IMO instead of the forced swim. The purpose of this group was to differen-tiate activation due exclusively to forced swim from the one that could be produced by other processes such as the simple manipulation of the animal or the release of IMO. To identify the neuronal populations that respond to prolonged IMO and forced swim, we have performed a double labeling of c-Fos protein and its mRNA. Given the dynamics of both markers, the neu-rons which respond to the prolonged IMO would show essentially protein and little or no mRNA, while those responding to forced swim applied after this IMO would show essentially mRNA and no protein. After this analysis we found that in regions such as the mPFC, LSV and MeA, exposure to forced swim after prolonged IMO triggered activation of new neurons sug-gesting some specificity in the response to both stressors. However, in the PVN exposure to forced swim after the prolonged IMO did not cause activation of new neurons, confirming the existence of a single neuronal population that responds similarly to different stressful stimuli, by which activation is inhibited after prolonged stimulation. A great part of the neuronal activation observed in terms of expression of c-fos in response to various stressful stimuli could be due to generalized activation or arousal processes. This activa- tion could mask those neurons that are really important in the stress response. In a final experi-ment we tried to decrease the contribution of arousal administrating DSP-4, a neurotoxin that is highly selective for LC noradrenergic terminals. One week after the administration of DSP-4, we exposed the animals to stressful stimuli differing in intensity (new enviroment, predator odor and IMO) and then we analyzed the expression of c-fos in the mPFC, LSV, MeA and PVN . The extent of the lesion on the noradrenergic terminals of LC was assessed by immunofluorescence of dopamine beta-hydroxylase (DβH). We observed only a decrease in noradrenergic terminals in the mPFC and MeA supporting data found in the bibliography. The DSP-4 lesion had no effect on the expression of c-fos in response to various stressors, except in the LSV in animals exposed to IMO where a decrease was observed. The results of this work suggest that the reduction of c-fos expression observed after prolonged exposure to an emotional stimulus may involve both familiarization with the stimulus, with the consequent reduction of stimulatory signals, as well as an intracellular repression of this gene. The contribution of each mechanism seems to be markedly dependent of each region con-cerned. On the other hand, it seems to exist neuronal populations that respond specifically to IMO and forced swim, although many of them may be common to both. The HPA axis response could probably include additional blockages that may help to reduce the negative impact of an excessive glucocorticoid release. The limited impact of the DSP-4 administration could be ex-plained by the existence of compensatory mechanisms, therefore other alternatives to reduce the contribution of arousal to stress response should be considered.

Obiettivo della tesi è stato quello di studiare il ruolo svolto dall’ipotalamo laterale (LH) nella regolazione dei processi di integrazione dell’attività autonomica e termoregolatoria con quella degli stati di veglia e sonno. A questo scopo, l’attività dell’LH è stata inibita per 6 ore (Esperimento A) mediante microiniezioni locali dell’agonista GABAA muscimolo nel ratto libero di muoversi, nel quale sono stati monitorati in continuo l’elelttroencefalogramma, l’elettromiogramma nucale, la pressione arteriosa (PA) e la temperatura ipotalamica (Thy) e cutanea. Gli animali sono stati studiati a temperatura ambientale (Ta) di 24°C e 10°C. I risultati hanno mostrato che l’inibizione acuta dell’LH riduce l’attività di veglia e sopprime la comparsa del sonno REM. Ciò avviene attraverso l’induzione di uno stato di sonno NREM caratterizzato da ipersincronizzazione corticale, con scomparsa degli stati transizionali al sonno REM. Quando l’animale è esposto a bassa Ta, tali alterazioni si associano a un ampio calo della Thy, che viene compensato da meccanismi vicarianti solo dopo un paio d’ore dall’iniezione. Sulla base di tali risultati, si è proceduto ad un ulteriore studio (Esperimento B) volto ad indagare il ruolo del neuropeptide ipocretina (prodotto in modo esclusivo a livello dell’LH) nei processi termoregolatori, mediante microiniezioni del medesimo nel bulbo rostrale ventromediale (RVMM), stazione cruciale della rete nervosa preposta all’attivazione dei processi termogenetici. La somministrazione di ipocretina è stata in grado di attivare la termogenesi e di potenziare la comparsa della veglia, con concomitante lieve incremento della PA e della frequenza cardiaca, quando effettuata alle Ta di 24°C o di 10°C, ma non alla Ta di 32°C. In conclusione, i risultati indicano che l’LH svolge un ruolo cruciale nella promozione degli stati di veglia e di sonno REM e, per tramite dell’ipocretina, interviene in modo coplesso a livello del RVMM nella regolazione dei processi di coordinamento dell'attività di veglia con quella termoregolatoria. / Aim of my thesis was to investigate the lateral hypothalamus (LH) role in the regulation of autonomic and thermoregulatory integration processes with wakefulness and sleep states. For this purpose, LH was inhibited for up to 6 hours ( Experiment A) by local microinjections of the GABAA agonist muscimol in free behaving rats, implanted with electrodes for chronic electroencephalogram, nuchal electromyogram, arterial pressure (PA ), hypothalamic (Thy ) and skin temperature. The animals were exposed to 24 ° C and 10 ° C ambient temperature ( Ta ). The results showed that the acute inhibition of LH reduces wakefulness and suppresses the onset of REM sleep. This occurs through the induction of NREM sleep state, characterized by high cortical synchronization levels, with REM sleep transitional states disappearance. At low Ta, these results were associated with a large Thy decrease, reversed by compensatory systems a couple of hours after the injections. Starting from these results, we proceeded to investigate (Experiment B) the role of the hypocretin neuropeptide (produced exclusively in the LH ) in thermoregulatory processes, microinjecting hypocretin in the Rostral Ventromedial Medulla ( RVMM ), a crucial center of the neural network responsible for the thermogenetic processes activation. The hypocretin administration was able to activate thermogenesis and enhance wakefulness, with concomitant slight blood pressure and heart rate increase, at Ta of 24 ° C and 10 ° C, but not at Ta of 32 ° C. In conclusion, the results indicate that the LH plays a crucial role in wakefulness and REM sleep promotion and, through hypocretin, it intervenes in a complex way in regulating the wakefulness coordination processes, at the RVMM level.

No description available.

Fundamental biological processes such as morphogenesis, tissue regeneration, and cancer invasion, depend on the collective migration of cell groups. The mechanisms that result in collective migration are not well understood, partially because the physical forces that initiate and maintain collective cell migration remain largely unknown. These forces include the traction forces, exerted by the cells on the extracellular matrix, and the cell-cell forces, transmitted between adjacent cells through cell-cell junctions. While the former have been studied, the latter have never been measured in the context of collective cell migration. The objective of this thesis has been to study these forces and integrate them in order to define the biomechanical mechanisms involved in the expansion of an epithelial monolayer. The thesis is presented as a compilation of two articles. In the first article, a new method for measuring intra-and intercellular forces in a cell monolayer was reported. It was shown that cells tend to align and migrate in the direction of maximal principal stress, demonstrating that intercellular forces act as a guidance mechanism during collective cell migration. In the second article, the expansion of an epithelial monolayer was studied. A new experimental model based on a barrier migration assay using polydimethylsiloxane membranes was implemented, allowing the study of epithelial expansion in a controlled and systematic manner. Structural and morphological changes at the cell level were observed during the expansion of the cellular monolayer. Furthermore, a mechanical wave propagates slowly spanning the entire monolayer, traversing intercellular junctions in a cooperative manner and building up differentials of mechanical stress. A minimal model based on sequential fronts of cytoskeletal reinforcement and fluidization captured essential features of this wave generation and propagation. These findings established a mechanism of long-range cell guidance, symmetry breaking and pattern formation during monolayer expansion.