Control of circadian rhythm on cortical excitability and synaptic plasticity

Living organisms navigate through a cyclic world: activity, feeding, social interactions are all organized along the periodic succession of night and day. At the cellular level, periodic activity is controlled by the molecular machinery driving the circadian regulation of cellular homeostasis. This mechanism adapts cell function to the external environment and its crucial importance is underlined by its robustness and redundancy. The cell autonomous clock regulates cell function by the circadian modulation of mTOR, a master controller of protein synthesis. Importantly, mTOR integrates the circadian modulation with synaptic activity and extracellular signals through a complex signaling network that includes the RAS-ERK pathway. The relationship between mTOR and the circadian clock is bidirectional, since mTOR can feedback on the cellular clock to shift the cycle to maintain the alignment with the environmental conditions. The mTOR and ERK pathways are crucial determinants of synaptic plasticity and function and thus it is not surprising that alterations of the circadian clock cause defective responses to environmental challenges, as witnessed by the bi-directional relationship between brain disorders and impaired circadian regulation. In physiological conditions, the feedback between the intrinsic clock and the mTOR pathway suggests that also synaptic plasticity should undergo circadian regulation

Influenza dei pathway coinvolti nella riparazione delle rotture a doppio filamento del DNA nell'instabilita' genomica indotta da BRCA1 in Saccharomyces cerevisiae

Nell’uomo, il gene BRCA1 codifica per una proteina facente parte di un complesso multiproteico implicato nella riparazione del DNA. Tali pathway di riparazione si ritrovano nell'organismo modello Saccharomyces cerevisae e questo rende il lievito un ottimo candidato per questo tipo di studi. Poichè, il lievito non ha l'omologo umano di BRCA1, l'espressione viene mediata attraverso un vettore plasmidico tramite crescita in terreno contenente l’induttore specifico che in questo caso è il galattosio. Studi precedenti effettuati dal gruppo dove ho svolto la tesi, hanno dimostrato che, in lievito, l'espressione di varianti missenso di BRCA1 associate a tumore causano un aumento della ricombinazione e che l’espressione di BRCA1 wild type determina un difetto nella crescita. Lo scopo di questo studio è di determinare se questi pathway specifici di riparazione possano influenzare la ricombinazione e mutazione genica e che quindi potrebbero avere un ruolo nella tumorigenesi dovuta a BRCA1. In particolare, i geni omologhi a quelli di lievito che influenzano il fenotipo indotto dalle varianti BRCA1, potrebbero avere un ruolo nella tumorigenesi come “modificatori” del rischio in pazienti che portano queste varianti. I ceppi aploidi di Saccharomyces cerevisiae difettivi per i suddetti pathway di riparazione presi in esame sono: RSY6rad51Δ difettivo nella riparazione per ricombinazione omologa, RSY6rad50Δ e RSY6mre11Δ difettivi nella riparazione per ricombinazione omologa e non. Poiché è noto che il mismatch repair influenza la ricombinazione in lievito, abbiamo costruito per gene targeting un nuovo ceppo difettivo per il mismatch repair chiamato RSY6msh6Δ per poter determinare anche l’effetto di questo gene sul fenotipo indotto da BRCA1. Questi ceppi sono stati trasformati con plasmidi contenenti BRCA1 wt o le varianti BRCA1 scelte secondo la loro classificazione. Prima di determinare l’effetto di BRCA1 in questi ceppi, abbiamo determinato il livello della proteina BRCA1 wild type o mutata in tutti i ceppi utilizzati. I risultati di Western blot mostrano che sia la proteina wt che le varianti scelte vengono espresse in tutti ceppi di lievito. Questo risultato è considerato molto importante, quando si vuole validare un saggio funzionale. Poiché i ceppi derivati da RSY6 contengono un sistema di ricombinazione intracromosomica che permette di misurare la frequenza di tale evento direttamente in piastra, abbiamo determinato se l’espressione di BRCA1 wt e delle varianti influenzasse la ricombinazione. Dai risultati ottenuti sembra che questi pathway non influiscano in modo significativo nel processo di ricombinazione genica nel ceppo aploide, eccetto che nel ceppo RSY6rad50Δ dove l’espressione di alcune varianti diminuisce della frequenza di ricombinazione. Al contrario, in tutti i ceppi studiati alcune varianti sembrano determinare un aumento della mutazione. In particolare, nel ceppo RSY6rad50Δ dove tutte le varianti inducono un forte aumento della frequenza di mutazione indicando che questo pathway ha una forte relazione con BRCA1 e quindi può essere un forte modficatore del rischio in pazienti che portano mutazione sul gene BRCA1. L’espressione di BRCA1 wt inibisce la crescita in lievito. Poiché, il ruolo dei pathway della riparazione del DNA su questo fenotipo non è completamente chiaro, abbiamo determinato se l’effetto sulla crescita determinato dall’espressione di varianti BRCA1 associate a tumore possa essere in qualche modo modulato da questi pathway che, come ampiamente riportato, influenzano la sopravvivenza e la crescita cellulare. I dati ottenuti mostrano chiaramente che le diverse varianti inducono diversi livelli di inibizione della crescita nei vari ceppi deleti, indicando quindi che questi pathway modulano l'effetto inibitorio indotto da BRCA1. Ancora una volta, si dimostra con questa tesi che il lievito Saccharomyces cerevisiae può essere un sistema genetico importante per determinare quali interazioni sono rilevanti per la tumorigenesi dovuta a mutazioni del gene BRCA1

Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3\u2032 single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer

Conservación de secciones corporales y órganos de gatuzo, mustelus schmitti (Pisces, chondrichthyes), por inyección de silicona a temperatura ambiente para ser utilizados en el aprendizaje de anatomía comparada

Comparative Anatomy deals with the study of the ontogenetic and phylogenetic changes of the vertebrates, requiring complementing the theoretical aspects with the observation of structures in specimens belonging to different taxonomic groups. The aim of the present study was to test the injection of silicone at room temperature in organs and trunk sections of Mustelus schmitti as an alternative to the plastination technique. Samples consisted in brain, eyes, heart, proximal end of the ventral aorta, digestive tract, spleen, pancreas, kidneys, testis and cross body section at a pre-caudal level. Material was fixed with formalin (10-5 %), dehydrated with growing concentrations of isopropyl (30 % - 50 % - 70 % - 90 % - 100 % - 100 %), impregnated with diluted commercial silicone and cured at room temperature. The whole process took 66 days. The brain was the unique organ that could not undergo the complete procedure because it did not resist the injection of silicone. The other pieces resulted in materials that characterised by being off-colour, dry, semi-flexible, lightweight, odourless, and non-toxic. They showed no signs of fungal colonization or bacterial degradation after two years of being obtained. Shrinkage was observed, which ranged among 2-25 % for total length, and from 5-26 % for maximum width (mean values: 14 and 15 %, respectively), being testicle the organ that suffered greater shrinkage in both dimensions. The degree of contraction in length and width for each of the samples was generally similar (difference £ 3 %), indicating that not striking deformation occurred. Deformation was observed only for the trunk section, eye, stomach, pancreas and valvular intestine. The technique did not affect the morphology of the structures, allowing the correct visualization of all the basic features required to recognise them. We conclude that this simple and economic method is an adequate alternative to be implemented for the conservation of small-size materials with educational purposes in Comparative Anatomy courses.Fil: Popp, Albertina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Basso, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Lodovichi, Mariela Victoria. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sidorkewicj, Nora Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentin

Adaptation and transformation of existing space into a plastination laboratory: Experience at the Universidad Nacional del Sur, Argentina

For centuries tissue conservation has been sought. Now alternative techniques that minimize the risk of toxicity have emerged. Plastination, developed by Prof. Gunther von Hagens in 1977 is such. The principle of this technique is replacement of the fluid and lipid present in biological tissues by polymers, obtaining odorless, dry, durable and non-toxic specimens. Given these widely recognized benefits, it is an alternative already used by more than 300 scientific-educational institutions around the world. However, the technique requires a laboratory that meets certain structural characteristics, related to biosafety. Construction and start-up involve significant costs. The main objective of this work is to report our experience at Universidad Nacional del Sur (Argentina), where a pre-existing space was modified to transform it into a plastination lab, with substantially lower costs than designing and building from scratch would have required.Fil: Popp, Albertina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Lodovichi, Mariela Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Castillo, Diego Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Casanave, Emma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Sidorkewicj, Nora Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Terapia manipulativa ortopédica na dor vertebral crônica: uma revisão sistemática

Objective: To examine the effectiveness of Manipulative Orthopedic Therapy (MOT) in cases of Chronic Vertebral Pain. Methods: A systematic search was performed in PEDro, Medline, and Science Direct databases in June 2010, retrieving recent randomized clinical trials that documented the effects of MOT in Chronic Vertebral Pain. Results: Eight articles about chronic cervical pain and eleven articles about chronic low back pain were reviewed. On chronic cervical pain, six papers showed that MOT associated with exercises was effective, maintaining the improvement of symptoms for up to 24 months. One study showed that MOT (with no exercises) was better than massage and another study showed that MOT provided immediate pain relief. In chronic low back pain, five clinical trials showed that MOT associated with exercises was effective in the short and long term; two papers showed that MOT was more effective than analgesics and anti-inflammatory drugs. Three studies showed that MOT is effective as a single treatment and one study showed that MOT is no more effective than stretching exercises. Conclusion: The MOT techniques (except osteopathy) are effective remedies in cases of chronic spinal pain. However, its effects in reduction of pain occur only in the short term and the combination of therapeutic exercises is needed for an effective long term result. More studies should be made to compare different MOT techniques and different exercise techniques with each other in cases of chronic spinal pain.Objetivo: Analisar a eficácia da Terapia Manipulativa Ortopédica (TMO) nos casos de dores vertebrais crônicas. Métodos: Uma busca sistematizada foi realizada nas bases de dados PEDro, Medline e Science Direct no mês de junho de 2010, reunindo ensaios clínicos randomizados recentes que documentassem os efeitos da TMO na Dor Vertebral Crônica. Resultados: Foram revisados oito artigos sobre dor cervical crônica e onze sobre dor lombar crônica. Quanto a dor cervical crônica, seis estudos mostraram que a TMO associada a exercícios são eficazes, mantendo a melhora dos sintomas por até 24 meses. Um estudo mostrou que a TMO (sem exercícios) é superior à massagem e outro estudo mostrou que a TMO proporciona alívio imediato da dor. Quanto a dor lombar crônica, cinco ensaios clínicos mostraram que a TMO associada a exercícios são eficazes a curto e longo prazo; dois trabalhos mostraram que a TMO é mais eficaz do que analgésicos e anti-inflamatórios. Três estudos mostram que a TMO como único tratamento é eficaz, um estudo mostra que a manipulação não é mais eficaz que exercícios de extensão. Conclusão: As técnicas de TMO (exceto osteopatia) são recursos eficazes nos casos de dores crônicas da coluna. Entretanto, seus efeitos de redução da dor ocorrem somente a curto prazo, sendo necessário a associação de exercícios terapêuticos para um resultado eficaz a longo prazo. Mais estudos devem ser feitos para comparar diferentes técnicas de TMO e diferentes técnicas de exercícios entre si nos casos de dores crônicas da coluna

Variación ontogenética de la forma de la mandíbula de Chaetophractus villosus (Mammalia, Xenarthra, Cingulata)

La mandíbula de Chaetophractus villosus se caracteriza por un dentario sólido, cuyarama es más ancha que el cuerpo. El proceso coronoides y el cóndilo articular, separadospor una profunda muesca, tienen casi igual altura, y el proceso angular es redondeadoy poco prominente. Algunos estudios han reportado la existencia de dimorfismo sexual,pero la información relacionada con la morfología mandibular en función de la edad sedesconoce. Se evaluaron diferencias de forma mandibular de adultos (N=63) y crías de1-22 días de vida (N=12), utilizando morfometría geométrica. Se digitalizaron landmarks(L) y semilandmarks (SM) sobre fotografías del dentario en vista interna (L=10, SM=7) yexterna (L=9, SM=7). En cada caso, se efectuó ajuste de Procrustes con obtención deconsensos, análisis discriminante, componentes principales, canónico, y análisis deregresión entre coordenadas de Procrustes y logaritmo del centroid size para evaluaralometría. Para ambas vistas, las distancias de Mahalanobis fueron significativas (p <=0,0001), con 100% de clasificación correcta. La varianza acumulada por las dos primerascomponentes fue de 64,48% (vista interna) y 62,20% (externa), con una separaciónevidente de los grupos. En el análisis canónico, las crías se separaron de adultos segúnCV1, con mandíbulas caracterizadas por un proceso coronoides más robusto y menordesarrollo del proceso angular. Además, la rama es más baja y robusta que en adultos.El porcentaje de variación explicado por alometría fue alto (36.99% vista interna, 30.28%vista externa). Se concluye que la forma del dentario sufre modificaciones durante eldesarrollo de los individuos. A mayor desarrollo de procesos coronoides y angular, lafuerza de oclusión mandibular se incrementa, pudiéndose relacionar con diferencias enlos hábitos alimenticios durante las diferentes etapas. Sería importante aumentar lacantidad de crías y agregar neonatos y juveniles a fin de evaluar el tipo de cambioalométrico durante la ontogenia.Fil: Popp, Albertina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Lodovichi, Mariela Victoria. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Sidorkewicj, Nora Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Casanave, Emma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaXXXII Jornadas Argentinas de MastozoologíaPuerto MadrynArgentinaSociedad Argentina para el Estudio de los MamíferosUniversidad Nacional de la Patagonia San Juan Bosc

Axonal Odorant Receptors Mediate Axon Targeting

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine- binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons.Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting

Functional Interaction Between BRCA1 and DNA Repair in Yeast May Uncover a Role of RAD50, RAD51, MRE11A, and MSH6 Somatic Variants in Cancer Development

In this study, we determined if BRCA1 partners involved in DNA double-strand break (DSB) and mismatch repair (MMR) may contribute to breast and ovarian cancer development. Taking advantage the functional conservation of DNA repair pathways between yeast and human, we expressed several BRCA1 missense variants in DNA repair yeast mutants to identify functional interaction between BRCA1 and DNA repair in BRCA1-induced genome instability. The pathogenic p.C61G, pA1708E, p.M775R, and p.I1766S, and the neutral pS1512I BRCA1 variants increased intra-chromosomal recombination in the DNA-repair proficient strain RSY6. In the mre11, rad50, rad51, and msh6 deletion strains, the BRCA1 variants p.C61G, pA1708E, p.M775R, p.I1766S, and pS1215I did not increase intra-chromosomal recombination suggesting that a functional DNA repair pathway is necessary for BRCA1 variants to determine genome instability. The pathogenic p.C61G and p.I1766S and the neutral p.N132K, p.Y179C, and p.N550H variants induced a significant increase of reversion in the msh2Δ strain; the neutral p.Y179C and the pathogenic p.I1766S variant induced gene reversion also, in the msh6Δ strain. These results imply a functional interaction between MMR and BRCA1 in modulating genome instability. We also performed a somatic mutational screening of MSH6, RAD50, MRE11A, and RAD51 genes in tumor samples from 34 patients and identified eight pathogenic or predicted pathogenic rare missense variants: four in MSH6, one in RAD50, one in MRE11A, and two in RAD51. Although we found no correlation between BRCA1 status and these somatic DNA repair variants, this study suggests that somatic missense variants in DNA repair genes may contribute to breast and ovarian tumor development