DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.<p>Previous research in our laboratory found that among DNA repair mutants of <i>Saccharomyces cerevisiae</i>, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in <i>E. coli</i> cells.<p>Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.<p>The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

Many cells in the inferior colliculus (IC) are excited by contralateral and inhibited by ipsilateral stimulation and are thought to be important for sound localization. These excitatory-inhibitory (EI) cells comprise a diverse group, even though they exhibit a common binaural response property. Previous extracellular studies proposed specific excitatory and/or inhibitory events that should be evoked by each ear and thereby generate each of the EI discharge properties. The proposals were inferences based on the well established response features of neurons in lower nuclei, the projections of those nuclei, their excitatory or inhibitory neurochemistry, and the changes in response features that occurred when inhibition was blocked. Here we recorded the inputs, the postsynaptic potentials, discharges evoked by monaural and binaural signals in EI cells with in vivo whole cell recordings from the inferior colliculus (IC) of awake bats. We also computed the excitatory and inhibitory synaptic conductances from the recorded sound evoked responses. First, we showed that a minority of EI cells either inherited their binaural property from a lower binaural nucleus or the EI property was created in the IC via inhibitory projections from the ipsilateral ear, features consistent with those observed in extracellular studies. Second, we showed that in a majority of EI cells ipsilateral signals evoked subthreshold EPSPs that behaved paradoxically in that EPSP amplitudes increased with intensity, even though binaural signals with the same ipsilateral intensities generated progressively greater spike suppressions. These ipsilateral EPSPs were unexpected since they could not have been detected with extracellular recordings. These additional responses suggested that the circuitry underlying EI cells was more complex than previously suggested. We also proposed the functional significance of ipsilaterally evoked EPSPs in responding to moving sound sources or multiple sounds. Third, by computing synaptic conductances, we showed the circuitry of the EI cells was even more complicated than those suggested by PSPs, and we also evaluated how the binaural property was produced by the contralateral and ipsilateral synaptic events. / text

In the central nervous system, most of the processes ranging from ion channels to neuronal networks occur in a closed loop, where the input to the system depends on its output. In contrast, most experimental preparations and protocols operate autonomously in an open loop and do not depend on the output of the system. Real-time software technology can be an essential tool for understanding the dynamics of many biological processes by providing the ability to precisely control the spatiotemporal aspects of a stimulus and to build activity-dependent stimulus-response closed loops. So far, application of this technology in biological experiments has been limited primarily to the dynamic clamp, an increasingly popular electrophysiology technique for introducing artificial conductances into living cells. Since the dynamic clamp combines mathematical modeling with electrophysiology experiments, it inherits the limitations of both, as well as issues concerning accuracy and stability that are determined by the chosen software and hardware. In addition, most dynamic clamp systems to date are designed for specific experimental paradigms and are not easily extensible to general real-time protocols and analyses. The long-term goal of this research is to develop a suite of real-time tools to evaluate the performance, improve the efficacy, and extend the capabilities of the dynamic clamp technique and real-time neural electrophysiology. We demonstrate a combined dynamic clamp and modeling approach for studying synaptic integration, a software platform for implementing flexible real-time closed-loop protocols, and the potential and limitations of Kalman filter-based techniques for online state and parameter estimation of neuron models.

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In the inner and outer hair cells (OHCs) of the inner ear, an unconventional myosin 15a localizes at the tips of mechanosensory stereocilia and plays an important role in forming and maintaining their normal structure. A missense mutation makes the motor domain of myosin 15a dysfunctional and is responsible for the congenital deafness DFNB3 in humans and deafness and vestibular defects in Shaker-2 (Sh2) mouse model. All hair cells of homozygous Shaker-2 mice (Myo15sh2/sh2) have abnormally short stereocilia, but, only stereocilia of Myo15sh2/sh2OHCs start to degenerate after the first few days of postnatal development and lose filamentous tip links between stereocilia that are crucial for mechanotransduction. The exact mechanisms of this degeneration are unknown even though they may underlie DFNB3 deafness in humans. We hypothesize that structural abnormalities in Myo15sh2/sh2 OHCs may alter the mechanical forces applied to the mechano-electrical transduction (MET) channels resulting in abnormal ionic homeostasis, which may lead to eventual degeneration of Myo15sh2/sh2 OHCs. Therefore, we investigated the ionic conductances and integrity of mechanotransduction apparatus in Myo15sh2/sh2 OHCs. Surprisingly, we found that myosin 15a-deficiency is associated not only with structural abnormalities of OHC stereocilia but also with alterations of voltage-gated ion conductances.

Two classes of ion-channels comprising 22 members were prepared. Three members were linear oligo-esters with terephthalate core designed to span both leaflets of the bilayer; these were prepared in a modular synthesis in three linear steps. 19 half-channels based on cyclodextrins with functionalized primary-rims were prepared by the Huisgen Cu+-catalyzed [3+2]-cyclization; three distinct synthetic protocols were established to be applicable to these substrates. The voltage-clamp experiment was used to characterize the ion transport properties of these 22 compounds as well as 5 oligo-esters previously prepared by solid-phase synthesis. All but two were active in bilayers, with the majority of these compounds showing highly complex conductance activities. Exponentially voltage-dependent currents were observed for two compounds (both terephthalate-derived); exclusive “square-top” activities were observed for one solid-phase–derived compound and one cyclodextrin-based channels; fractal openings were observed for at least two cyclodextrin-based channels. An “activity grid” notation was proposed as an empirical, coarse, but model-free method of treating the complex data. Through an exhaustive analysis of previously published synthetic ion channels, disparate compounds were found to share modes of activity. Supporting software were developed to facilitate the preparation of activity grids from current traces acquired for the aforementioned 27 compounds. Resulting activity grids for individual experiments were collated to generate an activity profile for each compound, from which a structure–activity map was established and could be compared to the literature data. Four core findings emerged. First, the activity grid notation is sufficiently expressive to denote highly complex mixture of activities. Second, systematic application of the notation reduces selection bias in data analysis. Third, many synthetic ion channels share highly sim- ilar activities and suggests the participation of the lipids, water, and ions in pore-formation. Lastly, the cyclodextrin half-channels are generally membrane active, and their activities are clearly modulated by structural variations. / Graduate

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 108-118). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 148-158.