Gilda Stefanelli

Gilda Stefanelli
University of Toronto | U of T · Department of Psychology

24.54
 · 
PhD Neurobiology
About
36
Research items
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Introduction
Gilda Stefanelli currently works at the Department of Psychology, University of Toronto. Gilda does research in molecular and epigenetic basis of memory formation.
Research Experience
Research
Research items (36)
Article
Full-text available
MeCP2 is a fundamental protein associated with several neurological disorders, including Rett syndrome. It is considered a multifunctional factor with a prominent role in regulating chromatin structure; however, a full comprehension of the consequences of its deficiency is still lacking. Here, we characterize a novel mouse model of Mecp2 bearing th...
Article
Memory formation is a protracted process that initially involves the hippocampus and becomes increasingly dependent on the cortex over time, but the mechanisms of this transfer are unclear. We recently showed that hippocampal depletion of the histone variant H2A.Z enhances both recent and remote memories, but the use of virally mediated depletion r...
Article
Full-text available
Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to...
Article
Full-text available
Histone variants were recently discovered to regulate neural plasticity, with H2A.Z emerging as a memory suppressor. Using whole-genome sequencing of the mouse hippocampus, we show that basal H2A.Z occupancy is positively associated with steady-state transcription, whereas learning-induced H2A.Z removal is associated with learning-induced gene expr...
Article
Full-text available
MeCP2 binds to methylated DNA in a chromatin context and has an important role in cancer and brain development and function. Histone deacetylase (HDAC) inhibitors are currently being used to palliate many cancer and neurological disorders. Yet, the molecular mechanisms involved are not well known for the most part and, in particular, the relationsh...
Article
Full-text available
MeCP2 is a transcriptional regulator whose functional alterations are responsible for several autism spectrum and mental disorders. Post-translational modifications (PTMs), and particularly differential phosphorylation, modulate MeCP2 function in response to diverse stimuli. Understanding the detailed role of MeCP2 phosphorylation is thus instrumen...
Answer
thank you all for your suggestions...this is the last experiment for my paper and, of course, everything that could go wrong is going wrong...including my attention...
Question
Hi all, 
Hope someone will answer me ad not everybody is on vacations...
I was isolating my RNA using trizol from neurons but I accidentally forgot to perform the ET-OH wash after precipitating with isopropanol. 
I resuspended in 10 ul and measured concentration and got low yields, when I realised my mistake I added 10 ul more and measured again the yields were much higher I don't know why, and so were the ratios 260/280.
Any suggestion? can I proceed with retrotranscription? just through everything away and start from neurons again?
Thank you all in advance
Gilda
Question
Hi all,
this is another of my mysterious questions. I am working with brain chromatin during development, in particular with P4 and P60 mouse brains, and I am always getting a weird result. whether I am preparing chromatin for ChIP or just isolating it after Mnase digestion, I always get a huge amount of DNA from P4 brains but very small amount from P30. Before you ask I treat every sample with almost 200ug of PK for 2 hours. My first thought was that DNA could get stuck to proteins and so I could lose it during my phenol chloroform extraction. I always start from same amount of nuclei so DNA amount should be same. Anybody can help me solve this?
Answer
thank you all for your valuable suggestions, I think I will just transfect my neurons with GFP for the moment and then figure out something to fix it...this will drive me crazy!!!!
Question
Hi everybody!
I have to do an EMSA to see if my mutations in my protein of interest abolished binding to DNA. Foe lack of time and of permissions of using radioactive labels my boss suggested to try with ethidium bromide staining. Anyone having any luck so far? Or anyone who can suggest me a protocol?
thank you in advance
Gilda
Answer
Hi Nicolas,
thank you very much for your answer. I can't believe these things can happen even though I have seen them happening many times now...science is weird too many times!!! It is very strange as one of my colleague was using this lentivirus before me and she was having green neurons...maybe now they decided they had enough...
I will give a look at that paper but the problem is that we do not have the backbone constructs...
thank you so much anyway you were extremely helpful
Gilda
Question
Hi all,
I have another mistery to solve. I am trying to infect primary hyppocampal neurons with lentiviruses expressing my protein and then iresGFP. construct work good and are green when transfected. I produced my lentivirus and used to infect 293T cells to obtain titer and the 293T cells are green after infection. When I infect my neurons however, they are not green, I tryied to perform immunofluorescence and western blot and in both cases I see my protein overexpressed but no GFP.
Can anybody help me?
Gilda
Question
I am doing ChIP for MeCP2 from total brain Chromatin. I start with 25ug of Chromatin. Assuming my antibody works good, how much DNA do you usually recover from your ChIPs in terms of ng/ul?
thanks
Gilda
Article
Mutations in MECP2 cause a broad spectrum of neuropsychiatric disorders of which Rett syndrome (RTT) represents the best-defined condition; both neuronal and non-neuronal functions of the methyl-binding protein underlie the related pathologies. Nowadays, MeCP2 is recognized as a multi-functional protein that modulates its activity depending on its...
Article
Full-text available
Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regula...
Answer
thank you Menagro, this is what I already do and my westerns were just perfect...I hope I can solve this problem soon...
Answer
can the sample buffer be a problem if the formulation is wrong? i mean can it leave the total protein run good but make me lose the signal for the phosphorylated?
because now I see some bands but the background is too high, if I wash more I lose the signal, if I put the secondary again no signal as the primary has been washed away too. So can it be my sample buffer that makes me lose the phosphorylation?
Answer
I stained both memebrane with ponceau, the proteins are there and after staining for my antibody and getting no signal I stain for H4 and get a perfect signal. there is no dedgradation going on as the total protein is perfect on the other membrane. by the way i stain the whole membrane and do not cut the strip. I do not understand I just do everything as I always did and it always worked...
Answer
There are also protease inhibitors and plus the total protein is not degraded...
Question
I am having serious troubles with my sds pages and hope that somebody can help me. I study a phosphorylated isoform of a protein and my antibody has always detected the protein enough was to put inhibitors of phosphatases (phosstop roche). I recently ran a gel and something strange happened: I always load two gels to see the total protein and the phosphorylated and I got a nice signal for the total but zero signal for the phosphorylated. Checked the protein with ponceau and nigrosin and were there as well as H4, my normalizer. to check if the antibody was dead I run another gel with my samples and a positive control and I had a good staining for my positive control but zero for my samples. I am dying as I put as usual tons of phosstop and when I used to forget to put it I still had some signal even though very weak while now I am getting nothing at all.
Please, if you have any ideas or suggestions please comment. 2 months, zero results is very frustrating.
Thank you.
Article
Full-text available
Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes...
Article
Rett syndrome (RTT) is a neurodevelopmental disorder with no efficient treatment that is caused in the majority of cases by mutations in the gene methyl-CpG binding-protein 2 (MECP2). RTT becomes manifest after a period of apparently normal development and causes growth deceleration, severe psychomotor impairment and mental retardation. Effective a...