290 research outputs found
Analisi funzionale del C-terminale di Xotx2 e Xotx5 nello sviluppo precoce di Xenopus laevis
I geni <i style='mso-bidi-font-style:
normal'>Otx dei Vertebrati, omologhi di <i
style='mso-bidi-font-style:normal'>orthodenticle (<i style='mso-bidi-font-style:
normal'>otd) di Drosophila,
svolgono un ruolo di primaria importanza nella specificazione della regione
rostrale del sistema nervoso centrale (SNC) dei Vertebrati. Studi su questi
geni sono stati condotti dapprima in topo dove sono stati inizialmente
caratterizzati Otx1 e <i
style='mso-bidi-font-style:normal'>Otx2. Successivamente
sono stati isolati Otx3 in Zebrafish
e Xotx1, Xotx2, <i style='mso-bidi-font-style:
normal'>Xotx4 ed Xotx5 in <i
style='mso-bidi-font-style:normal'>Xenopus laevis. Nei mammiferi <i
style='mso-bidi-font-style:normal'>otx5 non è presente, ma si trova un
altro gene della famiglia otx detto <i
style='mso-bidi-font-style:normal'>crx (cone rod homeobox), la cui
relazione con otx5 non è
definitivamente chiarita. Crx è
implicato nello sviluppo dellâocchio e mutazioni di <i style='mso-bidi-font-style:
normal'>Crx provocano nellâuomo diverse malattie della retina, che
comprendono lâamaurosi congenita di Leber (una grave degenerazione dei coni e
dei bastoncelli nellâinfanzia), la distrofia dei coni e dei bastoncelli
nellâadulto e la retinite pigmentosa.
In questo lavoro di tesi <span
class=GramE>abbiamo concentrato la nostra attenzione sui geni <i
style='mso-bidi-font-style:normal'>Xotx2 e <i style='mso-bidi-font-style:
normal'>Xotx5 di Xenopus,
nellâambito di un progetto di ricerca rivolto a chiarire il loro ruolo
nello sviluppo dellâocchio. Come tutti i geni della classe <i
style='mso-bidi-font-style:normal'>otd/otx, <i style='mso-bidi-font-style:
normal'>Xotx2 e Xotx5 contengono
un omeodominio della classe bicoid, caratterizzato da uno specifico residuo di
lisina in posizione 50 (corrispondente alla posizione 9 dellâelica di
riconoscimento) che conferisce loro la specificitĂ di legame al DNA.
Esperimenti condotti in topo hanno mostrato che <i style='mso-bidi-font-style:
normal'>Otx2 è espresso nellâintero epiblasto di embrioni
precoci. Successivamente la sua espressione diviene sempre
piĂš ristretta alle regioni piĂš anteriori che includono il neuroectoderma
corrispondente alle regioni telencefaliche, diencefaliche e mesencefaliche. A <span
class=GramE>sottolineare lâimportanza di tale gene vi è il fatto che
topi Otx2-/- muoiono
precocemente nellâembriogenesi per la totale assenza di tali strutture e per
malformazioni generalizzate del piano corporeo (dovuti a movimenti anomali
durante la gastrulazione). Lâespressione di Xotx2
nellâembriogenesi di Xenopus è simile
a quella osservata nel topo, ed interessa inizialmente
la regione dellâorganizzatore di Spemann, e successivamente le regioni rostrali
del SNC, incluso lâocchio.
Dal canto suo <i
style='mso-bidi-font-style:normal'>Xotx5 ha unâespressione nellâembrione
precoce simile a quella di Xotx2. Si
trova infatti nella regione dellâorganizzatore di
Spemann allo stadio di gastrula precoce e, poco dopo, anche nel neurectoderma
anteriore. Successivamente alla chiusura del tubo
neurale però, si ritrova esclusivamente espresso nellâocchio e nella ghiandola
pineale, entrambe strutture deputate alla fotorecezione. Esperimenti di
sovraespressione in embrioni precoci hanno mostrato effetti simili alla
sovraespressione di Xotx2, producendo
riduzioni delle strutture posteriori e lâinduzione di strutture anteriori e
tessuto neurale nellâintero embrione.
Lâespressione di questi geni durante lo sviluppo dellâocchio
ha spinto a ulteriori approfondimenti che hanno
mostrato il ruolo chiave dei due geni nel differenziamento di tale struttura. <span
class=GramE>Infatti lâespressione di Xotx5,
durante lo sviluppo dellâocchio si restringe progressivamente sia alle cellule
bipolari che ai fotorecettori. Invece lâespressione di <i style='mso-bidi-font-style:
normal'>Xotx2 viene progressivamente ristretta
alle sole cellule bipolari. Esperimenti di lipotrasfezione di precursori
retinici con costrutti per i due geni mostrano che <i style='mso-bidi-font-style:
normal'>Xotx2 spinge i precursori verso un destino bipolare, mentre <i
style='mso-bidi-font-style:normal'>Xotx5 spinge le cellule verso un destino
di fotorecettori. Scopo del presente lavoro di tesi è stato quello di
analizzare lâimportanza delle varie porzioni del C-terminale nellâazione <span
class=GramE>di transattivazione di questi fattori di trascrizione. A
questo scopo, si è provveduto alla creazione di vari costrutti <i
style='mso-bidi-font-style:normal'>Xotx2 e <i style='mso-bidi-font-style:
normal'>Xotx5, deleti al C-terminale, tramite PCR. Successivamente,
il loro mRNA è stato trascritto âin vitroâ e microiniettato in embrioni di <i
style='mso-bidi-font-style:normal'>Xenopus allo stadio di 4 blastomeri. Gli
embrioni sono stati poi caratterizzati in base al fenotipo osservato e
sottoposti ad analisi con opportuni markers molecolari allo scopo di
evidenziare strutture ectopiche indotte in seguito allâiniezione. Lâanalisi degli
effetti di tali costrutti è preliminare a successivi esperimenti di
lipotrasfezione di precursori cellulari retinici; lâintento è di identificare
quali domini funzionali di Xotx2 e Xotx5 siano essenziali per la loro azione di
specificazione di tipi cellulari in retina.</p
Relationship of Oligomerization to DNA Binding of Wheat Dwarf Virus RepA and Rep Proteins
AbstractMembers of the genus Mastrevirus (family Geminiviridae) produce a complementary-sense (c-sense) transcription unit with the potential to encode two proteins, RepA and Rep. In the present work, we have studied the DNAâprotein complexes formed by the Wheat dwarf virus (WDV) RepA protein within the WDV large intergenic region. WDV RepA forms large nucleoprotein complexes near the TATA boxes of the viral complementary-sense and virion-sense (v-sense) promoters (the RepA C- and V-complexes, respectively), a location similar to those of WDV RepâDNA complexes but with distinct DNase I footprints. We have also studied the relationship of oligomerization of WDV RepA and Rep proteins to DNAâprotein complex formation. Using chemical cross-linking, we have determined that both WDV proteins can form oligomers in solution. Interestingly, the pH is critical for the monomerâoligomer equilibrium and small changes produce a displacement in such a way that at pH ⤠7.0, the predominant species is an octamer while at pH ⼠7.4 it is a monomer. Complex formation is also strongly affected by pH and occurs more efficiently at pH 7.0â7.4. We found that preformed oligomers interact very poorly with DNA. Thus, our data are consistent with a stepwise model for proteinâDNA complex assembly in which monomers interact with DNA and then with other monomers to assemble an oligomeric structure on the DNA. These results may be relevant for studies on the DNA binding, replication, and transcription properties of geminivirus proteins
Neuroinflammation: A Signature or a Cause of Epilepsy?
: Epilepsy can be both a primary pathology and a secondary effect of many neurological conditions. Many papers show that neuroinflammation is a product of epilepsy, and that in pathological conditions characterized by neuroinflammation, there is a higher probability to develop epilepsy. However, the bidirectional mechanism of the reciprocal interaction between epilepsy and neuroinflammation remains to be fully understood. Here, we attempt to explore and discuss the relationship between epilepsy and inflammation in some paradigmatic neurological and systemic disorders associated with epilepsy. In particular, we have chosen one representative form of epilepsy for each one of its actual known etiologies. A better understanding of the mechanistic link between neuroinflammation and epilepsy would be important to improve subject-based therapies, both for prophylaxis and for the treatment of epilepsy
Analysis of immunization time, amplitude, and adverse events of seven different vaccines against SARS-CoV-2 across four different countries
BackgroundScarce information exists in relation to the comparison of seroconversion and adverse events following immunization (AEFI) with different SARS-CoV-2 vaccines. Our aim was to correlate the magnitude of the antibody response to vaccination with previous clinical conditions and AEFI.MethodsA multicentric comparative study where SARS-CoV-2 spike 1-2 IgG antibodies IgG titers were measured at baseline, 21-28 days after the first and second dose (when applicable) of the following vaccines: BNT162b2 mRNA, mRNA-1273, Gam-COVID-Vac, Coronavac, ChAdOx1-S, Ad5-nCoV and Ad26.COV2. Mixed model and Poisson generalized linear models were performed.ResultsWe recruited 1867 individuals [52 (SD 16.8) years old, 52% men]. All vaccines enhanced anti-S1 and anti-S2 IgG antibodies over time (p<0.01). The highest increase after the first and second dose was observed in mRNA-1273 (p<0.001). There was an effect of previous SARS-CoV-2 infection; and an interaction of age with previous SARS-CoV-2 infection, Gam-COVID-Vac and ChAdOx1-S (p<0.01). There was a negative correlation of Severe or Systemic AEFI (AEs) of naĂŻve SARS-CoV-2 subjects with age and sex (p<0.001); a positive interaction between the delta of antibodies with Gam-COVID-Vac (p=0.002). Coronavac, Gam-COVID-Vac and ChAdOx1-S had less AEs compared to BNT162b (p<0.01). mRNA-1273 had the highest number of AEFIs. The delta of the antibodies showed an association with AEFIs in previously infected individuals (p<0.001).ConclusionsThe magnitude of seroconversion is predicted by age, vaccine type and SARS-CoV-2 exposure. AEs are correlated with age, sex, and vaccine type. The delta of the antibody response only correlates with AEs in patients previously exposed to SARS-CoV-2.Registration numberClinicalTrials.gov, identifier NCT05228912
Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo
Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.Peer reviewe
Guidance on aneugenicity assessment
The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo. A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo. If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health-based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health-based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment
The N-Terminal Domain of ERK1 Accounts for the Functional Differences with ERK2
The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output
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