http://hdl.handle.net/1721.1/7891 Tue, 21 May 2013 23:13:03 GMT 2013-05-21T23:13:03Z http://hdl.handle.net/1721.1/78153 Sodium channel diversity in the vestibular ganglion : evidence for Nav̳1.5-like and Nav̳1.8-like currents Liu, Xiao-Ping, Ph. D. Massachusetts Institute of Technology Voltage-gated sodium (Nav) channels are diverse, comprising nine known mammalian subunits, which are classified pharmacologically into tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-insensitive (TTX-1) categories. The pattern of Nav channel expression shapes response properties of neurons, while changes in these expression patterns are related to many pathological conditions. Previous RT-PCR results indicated the expression of a variety of Nav channel subunits in the vestibular ganglion, the sensory ganglion that conveys information about motion and orientation. The expressed subunits included several TTX-1 subunits with unique biophysical properties that have been extensively characterized in somatosensory neurons and the heart, but never reported in published electrophysiological studies of the vestibular ganglion. Using whole-cell patch clamp, we show the presence of two types of TTX-l Nav currents in acutely dissociated rat vestibular ganglion neurons (VGNs) from the first postnatal week: a fast and negatively-inactivating current (midpoint of inactivation: ~-100 mV) that resembled current previously described for the Nav1.5 subunit, and a slower current with a depolarized voltage range of inactivation (midpoint ~-30 mV) which had properties consistent with Nav1.8 channels. All neurons also expressed TTX-S Nav currents with similar properties to those previously described in VGN (midpoint of inactivation: ~-75 mV). The Nav1.5-like current contributed about 10% of the total Nav current, was expressed in most VGNs on the first postnatal day (P1), and gradually decreased in prevalence throughout the first week. The Nav1.8-like current was present in ~25% of cells and was correlated with broader action potentials, higher voltage thresholds, and minimal spike height accommodation. We confirmed the expression of Nav1.8 using a reporter mouse in which fluorescence is restricted to Nav1.8- expressing cells; intense fluorescent signal was seen in many VGN cell bodies and peripheral processes. These results suggest that Nav1.8 may be expressed in non-somatosensory peripheral neurons. Nav channel expression in immature VGNs may contribute to development, while differential expression in adulthood may underlie diversity of mature response properties. Thesis (Ph. D. in Health Sciences and Technology)--Harvard-MIT Division of Health Sciences and Technology, 2012.; Cataloged from PDF version of thesis. In title on title page, double underscored "v̳" appears as subscript.; Includes bibliographical references (p. 73-83). Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/1721.1/78153 2012-01-01T00:00:00Z http://hdl.handle.net/1721.1/78152 Establishment of the epigenetic landscape in mammalian embryonic stem cells Koche, Richard Patrick Temporal and spatial variation of histone methylation is an important factor in mammalian development. Deciphering the details of such epigenetic phenomena has the potential to enrich both stem cell biology and therapeutics, as well as offer insight into various pathologies. While the enzymatic machinery responsible for these transitions is well known, it is their localization to specific genomic regions that controls cell fate, and this has largely remained a mystery. The goal of this thesis was to use an integrative genomics approach to elucidate the role of cis elements in the establishment of repressive chromatin domains. To this effect, we determined the genetic basis for localization of Polycomb repressive complexes (PRCs) in mammalian embryonic stem (ES) cells. First, by generating genomewide chromatin state maps in mouse and human by high throughput sequencing, we utilized a comparative and motif dictionary approach to computationally identify potential Polycomb recruitment elements. Surprisingly, we found that PRC recruitment is best explained by localization to clusters of unmethylated CpG dinucleotides, elements originally associated with gene activation. Next, in a series of transgenic assays involving human and E. coli sequence, we were able to reconstitute the chromatin state of an epigenetic memory element in mouse ES cells. Finally, we found that as somatic identity is reset during induced pluripotent stem (iPS) cell reprogramming, these same elements are central to a coordinated response in which active chromatin domains are established prior to and independently of transcription. Taken together, these studies highlight the role of a particular cis element in the establishment of both active and repressive chromatin domains. Furthermore, this dynamic underscores how a static genetic element can be utilized to enable the chromatin-based plasticity required of stem cell differentiation and lineage specification. Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2012.; Cataloged from PDF version of thesis.; Includes bibliographical references. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/1721.1/78152 2012-01-01T00:00:00Z http://hdl.handle.net/1721.1/78151 Identifying genes that are required for the maintenance of pancreatic ductal adenocarcinoma Jenq, Harry We searched for genes that are potentially important for the maintenance of Pancreatic Ductal Adenocarcinoma (PDAC). PDAC is the 4th leading cause for cancer-related deaths and exhibits a 5-year survival rate of less than 5%. Since PDAC is a Kras-driven cancer in that greater than 90% of PDACs contain a Kras mutation, we tested genes that are downstream of Kras. We used RNAi technology to inhibit approximately 30 genes in the canonical Kras effector pathways, Mapk, Pi3k, and Ral. These genes were tested in the context of mouse cell lines derived from a genetically engineered mouse model of PDAC with conditional mutations in Kras G12D and p53. An individual gene-by-gene approach and a pooled high-throughput screening strategy were taken to identify important genes. We identified mTOR, and to a lesser extent, Raptor and Rictor, as genes that are important for the maintenance of PDAC both in vitro and in vivo. In addition, we show that inhibition of mTOR and Raptor are synthetic lethal in that PDAC lines are sensitive to their inhibition, while non-tumorigenic cell lines are not as sensitive. Moreover, inhibition of mTOR results in downregulation of mTORCl and mTORC2 targets, while inhibition of Raptor induces downregulation of only mTORC 1 targets. As combination therapies are likely to be more effective, we looked for a drug that could combine effectively with mTOR and Raptor. From screening several small molecule drugs that target the Mapk and Pi3k pathways, we found PDAC lines to be particularly sensitive to AZD6244, while normal cell lines are significantly less sensitive. The combination or either an mTOR or Raptor hairpin and AZD6244 was found to be additive in that the effect on viability is significantly greater than that of each intervention alone. Another approach to combinations is to combine two drugs. AZD6244 and BEZ235, an inhibitor of Pi3k and mTOR, were tested in combination on both PDAC and normal cell lines. The combination was synergistic in PDAC lines but not in normal lines, suggesting that the combination may be effective with low toxicity. In summary, through a screen, we have identified mTOR, Raptor, and Rictor, as being critical components for the maintenance of PDAC. Thesis (Ph. D. in Biomedical Engineering)--Harvard-MIT Division of Health Sciences and Technology, 2012.; Cataloged from PDF version of thesis. Vita; Includes bibliographical references. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/1721.1/78151 2012-01-01T00:00:00Z http://hdl.handle.net/1721.1/78150 Structural and functional adaptations of the Auditory-Motor System : insights from expertise & disorder Halwani, Gus F. (Gus Fizt) While evidence from clinical and functional neuroimaging domains converges on a notion that auditory-motor networks can be remodeled functionally and structurally in response to experiences, studies that seek to evaluate these hypotheses by combining behavioral, functional, and structural measures are rare. Given relatively recent advances in neuroimaging, e.g. diffusion-tensor imaging (DTI) and functional neuroimaging methods (fMRI), it is now possible to structurally and functionally analyze these networks, as well as make inferences about them in situations where the networks are either functionally compromised by an auditory-motor feedback disorder, or structurally enhanced by an intense long-term auditory-motor training regimen. To this end, a three-fold course of study has been undertaken: (1) a between-group comparison of the structural aspects of the arcuate fasciculus (a prominent white-matter fiber tract that reciprocally connects the temporal and inferior frontal lobes and is thought to be important for auditory-motor interactions) of singers and those of matched nonsinging musicians, in order to evaluate the hypothesis that singers will exhibit structural differences specifically for aspects of vocal output that require rapid temporal processing and precise sound-motor matching. (2) a within-subject fMRI comparison of responses of young adults (non-musicians) to auditory feedback that is either unperturbed or shifted in pitch while they perform a pitch-matching task, to ascertain a functional network related to perceiving and perhaps compensating for mismatched auditory feedback. (3) a within-subject pilot study of the network ascertained in (2), now in a smaller group of young adults with an auditory-motor disorder/disconnection syndrome commonly referred to as tonedeafness (TD) or congential amusia (a conditioned marked by a high pitch discrimination threshold as well as readily apparent difficulty in matching pitches), in order to provide insight into how this network might behave in a state of long-term disorder. While this work corroborates previous work in clinical, behavioral, and neuroimaging domains, and sheds light on the organization of these auditory-motor networks (structurally and functionally) in the normal population, it also aids in understanding how these networks may be remodeled and optimized (structurally) in response to intense long-term training, how they adapt to an acutely compromised state (i.e. when input to the network is compromised or perturbed), as well as how they may adapt functionally in a chronically compromised state (i.e. tonedeafness). Taken together, these observations help to explain the functioning of the auditory-motor network in normal individuals and those with communication disorders, as well as well as shedding light on possible mechanisms of recovery as they participate in an intensive long-term auditory-motor therapy program. Thesis (Ph. D. in Speech and Hearing Bioscience and Technology)--Harvard-MIT Division of Health Sciences and Technology, 2012.; Cataloged from PDF version of thesis. Vita.; Includes bibliographical references (p. 78-85). Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/1721.1/78150 2012-01-01T00:00:00Z