Stochastic models and dynamic measures for the characterization of bistable circuits

During the last few years, a great deal of interest has risen concerning the applications of stochastic methods to several biochemical and biological phenomena. Phenomena like gene expression, cellular memory, bet-hedging strategy in bacterial growth and many others, cannot be described by continuous stochastic models due to their intrinsic discreteness and randomness. In this thesis I have used the Chemical Master Equation (CME) technique to modelize some feedback cycles and analyzing their properties, including experimental data. In the first part of this work, the effect of stochastic stability is discussed on a toy model of the genetic switch that triggers the cellular division, which malfunctioning is known to be one of the hallmarks of cancer. The second system I have worked on is the so-called futile cycle, a closed cycle of two enzymatic reactions that adds and removes a chemical compound, called phosphate group, to a specific substrate. I have thus investigated how adding noise to the enzyme (that is usually in the order of few hundred molecules) modifies the probability of observing a specific number of phosphorylated substrate molecules, and confirmed theoretical predictions with numerical simulations. In the third part the results of the study of a chain of multiple phosphorylation-dephosphorylation cycles will be presented. We will discuss an approximation method for the exact solution in the bidimensional case and the relationship that this method has with the thermodynamic properties of the system, which is an open system far from equilibrium.In the last section the agreement between the theoretical prediction of the total protein quantity in a mouse cells population and the observed quantity will be shown, measured via fluorescence microscopy

MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure

MLP is a LIM-only protein of terminally differentiated striated muscle cells, where it accumulates at actin-based structures involved in cytoarchitecture organization. To assess its role in muscle differentiation, we disrupted the MLP gene in mice. MLP (-/-) mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Ultrastructural analysis revealed dramatic disruption of cardiomyocyte cytoarchitecture. At birth, these hearts were not hypertrophic, but already abnormally soft, with cell-autonomous and MLP-sensitive alterations in cytoarchitecture. Thus, MLP promotes proper cardiomyocyte cytoarchitecture, whose perturbation can lead to dilated cardiomyopathy. In vivo analysis revealed that MLP-deficient mice reproduce the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans, providing the first model for this condition in a genetically manipulatable organism

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7-/-). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7-/-, but not in mGluR7+/-, mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7-/- mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs

Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1))

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes