Il cervello dal di dentro

The functional magnetic resonance imaging (fMRI) techniques are a potent probe able to visualize brain functions, by analyzing modification of blood oxygenation, and see the action in specific brain areas in response to activity or thoughts. fMRI thus promise to be a formidable tool not only to draw a new cartography of brain functional areas, but also a new tool to understand some aspects of brain function’s evolution, as well to get insights and to breach the wall into cognition, morality and consciousness. Nevertheless fMRI is not deprived of pitfalls such as limitation in spatial accuracy, reliable reproducibility of brain scan amongst different individuals or in the same person at different stages of life ( age or health versus disease), the different time scale of fMRI measurements (seconds) and neuron’s action potentials (milliseconds). Thus often caution is required in the appreciation of fMRI results and conclusions, that could lead to incorrect interpretation of brain signals and induce to draw spurious conclusions. New applications combining fMRI and real time visualization of one's own brain activity in healthy volunteers or patients promise to enable individuals to modify brain response and thus therapeutically or with other goals intervene in modifying individual behaviors. Specially this last aspect, as well as the concern about the confidentiality and storage of sensible information or forensic uses of such approaches, raises the problem of mind privacy and new ethical questions

Targeting of noncanonical nucleic acid structures for therapeutic intervention and biological investigation

G-quadruplex (G4) structures fall among the most extensively studied noncanonical DNA/RNA conformations, with the current knowledge suggesting that they might play an integral part in many biological processes, including transcriptional regulation, telomeric maintenance, malignant transformation, and cancer development. In the last decade, mounting attention has been also focused on i-motif (iM) structures. Even though the structural aspects of iM formation, the factors leading to its stabilization, and its putative biological roles have been recently reviewed and discussed by the scientific community, there is still much to know about such noncanonical nucleic acid conformation. This PhD project aimed, on the one hand, at providing a more in-depth knowledge about the i-motif structures and, on the other hand, at identifying and developing new molecular tools able to target alternative nucleic acid secondary structures for either therapeutic intervention or biological investigation. Particularly, the thesis has been subdivided as follow: Chapter 1 provides a general description of nucleic acids and noncanonical DNA/RNA conformations, stressing on their structural features and biological roles. Chapter 2 offers an overview of the main methodologies employed in the herein presented studies. Chapter 3 includes two detailed investigations on iM structures. The first one is a systematic study to simultaneously analyze the effect of pH, cation type, and cation concentration (and their possible interactions) on the formation of an i-motif structure in vitro. The second one uses multivariate data analysis to bring out valuable structural information on i-motif DNA from circular dichroism and thermal difference spectra. Chapter 4 describes new G4-targeting compounds that we identified or rationally designed and synthesized as potential anti-cancer agents. In Chapter 5, new molecules are reported that might be used as tools to clarify the biological roles of G4 and iM structures and their intriguing relationship. The last section of this PhD thesis collects the general conclusions. Finally, the Appendix reports the published scientific articles as cited throughout the thesis. Paper IV is not included since the Manuscript is still in preparation and has not been submitted at the present time

The serotonin receptor 7 and the structural plasticity of brain circuits

Serotonin (5-hydroxytryptamine, 5-HT) modulates numerous physiological processes in the nervous system. Together with its function as neurotransmitter, 5-HT regulates neurite outgrowth, dendritic spine shape and density, growth cone motility and synapse formation during development. In the mammalian brain 5-HT innervation is virtually ubiquitous and the diversity and specificity of its signaling and function arise from at least 20 different receptors, grouped in 7 classes. Here we will focus on the role 5-HT7 receptor (5-HT7R) in the correct establishment of neuronal cytoarchitecture during development, as also suggested by its involvement in several neurodevelopmental disorders. The emerging picture shows that this receptor is a key player contributing not only to shape brain networks during development but also to remodel neuronal wiring in the mature brain, thus controlling cognitive and emotional responses. The activation of 5-HT7R might be one of the mechanisms underlying the ability of the CNS to respond to different stimuli by modulation of its circuit configuration

Dopamine: The Neuromodulator of Long-Term Synaptic Plasticity, Reward and Movement Control

Dopamine (DA) is a key neurotransmitter involved in multiple physiological functions including motor control, modulation of affective and emotional states, reward mechanisms, reinforcement of behavior, and selected higher cognitive functions. Dysfunction in dopaminergic transmission is recognized as a core alteration in several devastating neurological and psychiatric disorders, including Parkinson's disease (PD), schizophrenia, bipolar disorder, attention deficit hyperactivity disorder (ADHD) and addiction. Here we will discuss the current insights on the role of DA in motor control and reward learning mechanisms and its involvement in the modulation of synaptic dynamics through different pathways. In particular, we will consider the role of DA as neuromodulator of two forms of synaptic plasticity, known as long-term potentiation (LTP) and long-term depression (LTD) in several cortical and subcortical areas. Finally, we will delineate how the effect of DA on dendritic spines places this molecule at the interface between the motor and the cognitive systems. Specifically, we will be focusing on PD, vascular dementia, and schizophrenia

A short peptide that preferentially binds c-MYC G-quadruplex DNA

G-quadruplexes (G4s) are non-canonical DNA secondary structures. The identification of selective tools to probe individual G4s over the ~700,000 found in the human genome is key to unravel the biological significance of specific G4s. We took inspiration from a crystal structure of the bovine DHX36 helicase bound to the G4 formed in the promoter region of the oncogene c-MYC to identify a short peptide that preferentially binds MYC G4 with nM affinity over a small panel of parallel and antiparallel G4s tested

Enhancement of Dopaminergic Differentiation in Proliferating Midbrain Neuroblasts by Sonic Hedgehog and Ascorbic Acid

We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction

What dictates the accumulation of desmosterol in the developing brain?

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Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages

Previous gene targeting studies in mice have implicated the nuclear protein Krüppel-like factor 7 (KLF7) in nervous system development while cell culture assays have documented its involvement in cell cycle regulation. By employing short hairpin RNA (shRNA)-mediated gene silencing, here we demonstrate that murine Klf7 gene expression is required for in vitro differentiation of neuroectodermal and mesodermal cells. Specifically, we show a correlation of Klf7 silencing with down-regulation of the neuronal marker microtubule-associated protein 2 (Map2) and the nerve growth factor (NGF) tyrosine kinase receptor A (TrkA) using the PC12 neuronal cell line. Similarly, KLF7 inactivation in Klf7-null mice decreases the expression of the neurogenic marker brain lipid-binding protein/fatty acid-binding protein 7 (BLBP/FABP7) in neural stem cells (NSCs). We also report that Klf7 silencing is detrimental to neuronal and cardiomyocytic differentiation of embryonic stem cells (ESCs), in addition to altering the adipogenic and osteogenic potential of mouse embryonic fibroblasts (MEFs). Finally, our results suggest that genes that are key for self-renewal of undifferentiated ESCs repress Klf7 expression in ESCs. Together with previous findings, these results provide evidence that KLF7 has a broad spectrum of regulatory functions, which reflect the discrete cellular and molecular contexts in which this transcription factor operates. © 2010 Elsevier Inc

GINGER: A feasibility study

GINGER (Gyroscopes IN General Relativity) is a proposal for an Earth-based experiment to measure the Lense-Thirring (LT) and de Sitter effects. GINGER is based on ring lasers, which are the most sensitive inertial sensors to measure the rotation rate of the Earth. We show that two ring lasers, one at maximum signal and the other horizontal, would be the simplest configuration able to retrieve the GR effects. Here, we discuss this configuration in detail showing that it would have the capability to test LT effect at 1%, provided the accuracy of the scale factor of the instrument at the level of 1 part in 1012 is reached. In principle, one single ring laser could do the test, but the combination of the two ring lasers gives the necessary redundancy and the possibility to verify that the systematics of the lasers are sufficiently small. The discussion can be generalised to seismology and geodesy and it is possible to say that signals 10-12 orders of magnitude below the Earth rotation rate can be studied; the proposed array can be seen as the basic element of multi-axial systems, and the generalisation to three dimensions is feasible adding one or two devices and monitoring the relative angles between different ring lasers. This simple array can be used to measure with very high precision the amplitude of angular rotation rate (the length of the day, LOD), its short term variations, and the angle between the angular rotation vector and the horizontal ring laser. Finally this experiment could be useful to probe gravity at fundamental level giving indications on violations of Einstein Equivalence Principle and Lorenz Invariance and possible chiral effects in the gravitational field