Avaliação in vitro de genótipos de citros a Phytophthora parasitica.

Este trabalho teve como objetivo avaliar in vitro a reação de porta-enxertos de citros (Citrus spp.) a Phytophthora parasitica. As plântulas foram cultivadas em meio MS por 40 dias sendo, submetidas a fotoperíodo de 18 h, à temperatura de 25 ºC. A inoculação foi realizada através da inserção de agulha infestada com micélio de P. parasitica. A avaliação foi realizada aos 15 dias após a inoculação, medindo-se o comprimento das lesões em centímetros. O delineamento experimental foi inteiramente casualizado, com 15 repetições. Os resultados estão de acordo com as reações de campo dos genótipos e podem ser de grande utilidade em trabalhos envolvendo resistência varietal e seleção precoce de plantas

Caracterização agronômica e molecular de acessos de Citrus sunki do banco de germoplasma de citros do Centro APTA Citros Sylvio Moreira.

Este trabalho objetivou caracterizar e avaliar acessos de tangerina Sunki (Citrus sunki Hort. ex Tan.) e assemelhados, pertencentes ao Banco Ativo de Germoplasma de Citros do Centro APTA Citros Sylvio Moreira/IAC, Cordeirópolis, SP. Foram avaliadas características agronômicas: altura (A), diâmetro (D), relação A/D, massa e número de gomos dos frutos, número de sementes viáveis e abortadas, comprimento e diâmetro das sementes, número de embriões e características de suco dos frutos: rendimento de suco, sólidos solúveis, acidez total, ratio e sólidos solúveis por caixa (40,8 kg de frutos). Para as análises moleculares foram utilizados DNA genômico total extraído de folhas frescas, acessando-se polimorfismo genético mediante emprego de 11 pares de iniciadores microssatélites. Os acessos de tangerina Sunki CV200 e CV200 (BMS) apresentaram 100% de similaridade genética com os iniciadores microssatélites utilizados e se mostraram semelhantes em relação às características agronômicas. Os acessos Suen Kat CV201 e CV202 apresentaram diferenças entre si, tanto em relação a polimorfismo genético molecular, quanto morfológico e também não se agruparam com os acessos de tangerina Sunki

QTL mapping associated with rooting stem cuttings from Citrus sunki vs. Poncirus trifoliata hybrids.

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When multidisciplinary surgical trans-orbital approaches should be considered to reach the skull base

SUMMARY The transorbital approaches are a group of surgical procedures performed passing through the orbital spaces and aimed to reach deeper areas. This kind of surgery has been proved to be safe and effective in the management of selected lesions of the anterior, middle and infratemporal fossa. The aim of the present study is to perform a review of the literature, in order to draw the reader’s attention on the main features of this kind of surgery, focusing on the anatomical background and the surgical setting; we will also summary the current indications and contraindications to this approach and find out the related complications and the possible alternatives. Even if we consider the transorbital approach as a promising route to the skull base, we underline that there is no better approach over another and the choice must always consider several elements. Furthermore, as for every skull base procedure, a multidisciplinary management is strongly advisable

Trehalose prodegradative role on AR aggregates in a muscle model of Spinal and Bulbar Muscular Atrophy.

Spinal and Bulbar Muscular Atrophy (SBMA) is a motor neuronal disease whose onset and progression have been recently linked also to a muscular defect. SBMA is caused by a polyglutammine tract in the exon 1 of the androgen receptor protein (ARpolyQ). When AR is activated by testosterone a fraction of the protein misfolds and become toxic to cells. Moreover if ARpolyQ is not correctly removed from cellular environment it also forms aggregates that could damage many cellular process. In this work we have studied the protein quality control system (composed of a chaperone network and two main degradative pathways: proteasome and autophagy) in a cellular muscular model of SBMA. We use C2C12 stably transfected with ARwt or ARpolyQ bearing an elongation of 100 glutammine. Initially we performed a filter trap assay (FTA) on both cell line treated with testosterone. We observed that testosterone triggers the aggregation of ARpolyQ but not of ARwt. We also observed that testosterone treatment caused mortality in C2C12 with ARpolyQ. By real time PCR we found that there was not activation of the PQC system in presence of ARpolyQ but that the expression of AchR was significantly lower than in control cell. These data suggest that ARpolyQ led to muscular atrophy. We investigate degradative systems that degrade AR and found that autophagy is highly involved in AR degradation. We facilitate autophagy towards the overexpression of HspB8. We observed that HspB8 counteracted testosterone dependent aggregation of ARpolyQ. We know that trehalose in motorneuronal model of SBMA induce the expression of HspB8. We then enhanced autophagy with trehalose and found that ARpolyQ aggregation was almost completely reverted. We co-treated cells with trehalose and bafilomycin and found that this condition abolished trehalose effect. We demonstrated that trehalose effect depend upon an efficient autophagic flux. By rtPCR we observed that trehalose enhance the expression of a wide range of genes related to autophagy. Interestingly we also found VCP overexpression in presence of trehalose. The valosin containing protein VCP is a multi-functional protein involved also in the ERAD pathway. So we inhibit VCP with DBEQ a specific inhibitor of the ATPase activity of VCP and found that testosterone dependent aggregation was significantly increased. Interestingly we found that trehalose treatment counteracted this DBEQ associated aggregation. In conclusion we characterized C2C12 as a reliable muscle model of SBMA. we also found that autophagy is highly involved in ARpolyQ degradation and consequently we demonstrated that autophagy activation rescues ARpolyQ aggregation in muscle cells. We finally observed that also the ERAD pathway plays an important role in ARpolyQ degradation. AFM-TELETHON; FONDAZIONE TELETHON; FONDAZIONE CARIPLO; FONDAZIONE ARISLA; Ministero della Sanità; Joint Programme Neurodegenerative Disease (JPND)

Multiple Roles of Transforming Growth Factor Beta in Amyotrophic Lateral Sclerosis

Transforming growth factor beta (TGFB) is a pleiotropic cytokine, known to be dysregulated in many neurodegenerative disorders and particularly in amyotrophic lateral sclerosis (ALS). This motor neuronal disease is non-cell autonomous, as it affects not only motor neurons, but also their surrounding glial cells, and their target skeletal muscle fibers. Here, we analyze the multiple roles of TGFB in these cell types, and how TGFB signaling is altered in ALS tissues. Data reported support a crucial involvement of TGFB in the etiology and progression of ALS, leading us to hypothesize that an imbalance of TGFB signaling, diminished at the pre-symptomatic stage and then increased with time, could be linked to ALS progression. A reduced stimulation of the TGFB pathway at the beginning of disease blocks its neuroprotective effects and promotes glutamate excitotoxicity. At later disease stages, the persistent activation of the TGFB pathway promotes an excessive microglial activation and strengthens muscular dysfunction. The therapeutic potential of TGFB is discussed here, in order to foster new approaches to treat ALS

Mutations in VCP induce lysosomal alterations and autophagy activation in ALS neuronal models

Valosin Containing Protein (VCP) is an ATPase protein member of the AAA+ protein family. VCP is involved in various pathways that concur in maintaining cellular homeostasis. VCP mutations have been correlated different proteinopathies including neurodegenerative diseases as ALS. VCP-mutants are associated associated with the presence of alteration of the Protein Quality Control System: ubiquitin inclusions, TDP- 43 mis-localization and aggregation, and abnormal vacuoles. To date, the mechanisms correlated to VCP- mutants that lead to cell toxicity and death are still not defined. In this study, we identify VCP-mutants pathological mechanisms in an ALS-model. We overexpress VCP WT, VCP R155H and VCP R191Q in NSC-34, a motor neuron mouse immortalized cell line. In first instance, we found that both VCP mutants from insoluble aggregates and induce lysosomal alteration in size, morphology, activity and membrane breakage. Lysosomal damage is known to lead to cell toxicity and death, so cells activate different mechanisms to remove damaged lysosome as the activation of autophagy. Therefore, we studied variance in the autophagic flux in presence of VCP-mutants by analysing LC3 conversion and p62 accumulation. We could determine that VCP-mutants are correlated with an activation of the autophagic flux. Moreover, by analysing transcription factors that regulate autophagy we determined that VCP-mutants positively regulates autophagic flux by specifically activating the transcription factor TFE3. Results also determined that TFE3 activation triggered by VCP-mutants presence is mediated by calcineurin, a Ca2+ dependent phosphatase. In parallel, we excluded the involvement of TFEB in this pathway. Overall, these data suggest that lysosomal damage and leakage induced by VCP-mutants activate calcineurin which in turn mediates TFE3 dephosphorylation and nuclear translocation inducing autophagy. In support to this we found that VCP mutants enhances insoluble protein-aggregates with a specific dependency from the autophagic pathway

VCP mutants cause lysosomal alterations and autophagy induction in ALS-neuronal model

Valosin Containing Protein (VCP) is an ATPase protein that has a key role in various pathways critical for the maintenance of cellular homeostasis and vitality. In particular, VCP is involved in the Protein Quality Control System. Indeed, VCP- mutants have been correlated to different proteinopathies as IBMPFD and ALS. The presence of VCP mutations has been associated with ubiquitin inclusions, TDP-43 mislocalization and aggregation, and abnormal vacuoles. To date VCP-mutants pathological mechanisms are still controversial. Thus, we decided to better define VPC-mutants pathological mechanisms in an ALS-model overexpressing VCP WT, VCP R155H, and VCP R191Q in NSC-34, a motor neuron mouse immortalized cell line. Firstly, we determined that VCP-mutants form insoluble aggregates in this neuronal model. In addition, we observed that the presence of VCP-mutants triggers significant lysosomal alterations in morphology, size, activity, and membrane breakage. Lysosomal alterations have been described to induce cell toxicity and death. To remove damaged lysosomes and therefore to maintain cell vitality, cells activate different mechanisms like autophagy induction. Thus, we analysed LC3 conversion and p62 accumulation, markers of autophagic flux, to determine if the presence of VCP-mutants triggered activation of the autophagic flux. Data showed that VCP- mutants were correlated with an activation of the autophagic flux. Moreover, we determined that the activation of the autophagic flux was specifically regulated by TFE3 calcineurin-dependent dephosphorylation and activation. Calcineurin is a calcium-dependent phosphatase that could be activated by lysosomal leakage supporting a correlation between VCP-mutants lysosomal damage and autophagy activation. In addition, we excluded the involvement of TFEB in this pathway. Together these data suggest that lysosomal damage and leakage induced by VCP- mutants activate calcineurin which in turn mediates TFE3 dephosphorylation and nuclear translocation inducing autophagy. In support of this, we found that VCP- mutants enhanced insoluble protein-aggregates with a specific dependency on the autophagic pathway