Biological activity of Pyrrole-Imidazole polyamides in vivo

This thesis focuses on biological activity of pyrrole-imidazole polyamides in vivo. The work presented includes experiments underlining sequence selectivity of these compounds in living cells and potential methods to improve it. A large fraction of this thesis is devoted to activity of Py-Im in murine models of cancer. We investigated the pharmacokinetics and biodistribution of two compounds – targeted to 5'-WGGWCW-3' and 5'-WTWCGW-3' sequences – and characterized their activity by measuring their effects on tumor growth, gene expression in vivo and in tissue culture, and their effects on physiology of tumors. The initial theoretical studies suggested that a large fraction of genomic sites are bound by Py-Im polyamides non-specifically and experimental data shows that the programmed binding sequence is not a sole determinant of the patterns of gene regulation. Despite the likely presence of non-specific effects of Py-Im polyamides in living cells, in vivo administration of Py-Im polyamides resulted in tolerable host toxicity and anti-tumor activity. Py-Im polyamide targeted to Estrogen Receptor Response Element showed downregulation of ER-driven gene expression in tumor cells, while the compound targeted to hypoxia response element reduced vascularization of tumors and their growth rate, induced apoptosis of cells in hypoxic areas and reduced expression of proangiogenic and prometastatic factors. Further studies, showed that polyamides distributed to many of the tested tissues and their FITC-conjugates showed nuclear uptake. The gene expression effects were also present in murine tissues, such as liver and kidneys, indicating a potential for use for Py-Im polyamides in non-cancerous diseases.

Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The α6-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for α6-containing nAChRs. Our goal was to isolate the role of α6-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423’s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.

The Physiology and Computation of Pyramidal Neurons

A variety of neural signals have been measured as correlates to consciousness. In particular, late current sinks in layer 1, distributed activity across the cortex, and feedback processing have all been implicated. What are the physiological underpinnings of these signals? What computational role do they play in the brain? Why do they correlate to consciousness? This thesis begins to answer these questions by focusing on the pyramidal neuron. As the primary communicator of long-range feedforward and feedback signals in the cortex, the pyramidal neuron is set up to play an important role in establishing distributed representations. Additionally, the dendritic extent, reaching layer 1, is well situated to receive feedback inputs and contribute to current sinks in the upper layers. An investigation of pyramidal neuron physiology is therefore necessary to understand how the brain creates, and potentially uses, the neural correlates of consciousness. An important part of this thesis will be in establishing the computational role that dendritic physiology plays. In order to do this, a combined experimental and modeling approach is used.

This thesis beings with single-cell experiments in layer 5 and layer 2/3 pyramidal neurons. In both cases, dendritic nonlinearities are characterized and found to be integral regulators of neural output. Particular attention is paid to calcium spikes and NMDA spikes, which both exist in the apical dendrites, considerable distances from the spike initiation zone. These experiments are then used to create detailed multicompartmental models. These models are used to test hypothesis regarding spatial distribution of membrane channels, to quantify the effects of certain experimental manipulations, and to establish the computational properties of the single cell. We find that the pyramidal neuron physiology can carry out a coincidence detection mechanism. Further abstraction of these models reveals potential mechanisms for spike time control, frequency modulation, and tuning. Finally, a set of experiments are carried out to establish the effect of long-range feedback inputs onto the pyramidal neuron. A final discussion then explores a potential way in which the physiology of pyramidal neurons can establish distributed representations, and contribute to consciousness.