published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

<p>Programmed cell death, or apoptosis, has during the last decade received a lot of attention due to its involvement in a large number of pathological conditions. Since death is always irreversible, it is important for cells to fully control the initiation and execution of this process. One of many apoptosis-regulatory proteins is XIAP, which blocks the action of caspases, a family of proteases that are important during apoptosis. However, apoptosis inhibitors have to be tightly controlled since too little cell death can lead to the development of tumors and other diseases. This thesis is the result of an aspiration to fully understand the function and regulation of XIAP.</p><p>By using the yeast-2-hybrid system, we identified two novel binding partners of XIAP. The first, GPS2, was found to bind XIAP and inhibit its ability to block caspase-activity. In addition, GPS2 induced caspase-mediated cell death in two different tumour cell lines and XIAP inhibited this effect.</p><p>The second binding partner, Nulp1, preferentially bound XIAP in the presence of the apoptosis-inducer staurosporine. Nulp1 induced or sensitized cell lines to cell death when overexpressed, but this was not blocked by caspase-inhibitors or XIAP, suggesting a different reason for binding than apoptosis regulation. With the aim to understand the Nulp1-XIAP interaction, we continued to study Nulp1 <i>in vivo</i> and <i>in vitro</i>. We studied three different splice variants of Nulp1 and found that they were regulated by poly-ubiquitination and nuclear shuttling. Also, Nulp1 was expressed in embryonic mice, especially in the cortical plate, hippocampal neurons and cerebellar granular neurons. Expression of Nulp1 decreased with age but was still present in cerebellar deep nuclei and Purkinje cells of adult mice. </p><p>To summarize, we have identified GPS2 as an apoptosis-inducing factor and an inhibitor of XIAP <i>in vitro</i>, and Nulp1 as a XIAP-interacting protein during staurosporine-induced apoptosis.</p>

Chez les rongeurs, le traitement par le cortex à tonneaux de l'information sensorielle en provenance des vibrisses est mal compris. En effet, malgré l'aide fournie par l'organisation de ce cortex en une reproduction stricte de la topographie de l'appareil sensoriel, il a été difficile jusqu'à présent d'identifier de façon indiscutable le système de filtrage linéaire et non-linéaire qu'utilisent les neurones du cortex à tonneaux durant leur traitement des scènes tactiles auxquelles ils sont exposés. Pour mieux identifier ces traitements corticaux, nous avons développé un système de stimulation vibrissale permettant d'appliquer des déflections sur un grand nombre de vibrisses indépendamment, dans toutes les directions possibles et ce à travers une vaste gamme fréquentielle. En utilisant ce dispositif de stimulation multivibrissale durant des enregistrements extracellulaires de l'activité électrique des neurones du cortex à tonneaux de rats anesthésiés, nous avons pu identifier plus précisément le filtrage linéaire des stimulations vibrissales, qui s'avère similaire pour tous les neurones que nous avons pu enregistrer. Par ailleurs, en explorant les aspects non-linéaires du traitement effectué par ces neurones, nous avons noté qu'ils se séparent en deux familles distinctes : d'un côté des neurones "locaux" qui se sont avérés sensibles à des contrastes locaux dans les déflections multivibrissales. De l'autre, des neurones "globaux" capables au contraire de détecter des situations où les déflections sont similaires pour de nombreuses vibrisses. Enfin, en effectuant d'autres enregistrements dans la couche II/III du cortex à tonneaux, cette fois à l'aide d'un microscope deux-photons, nous avons pu noter que les neurones appartenant aux familles locales et globales étaient séparés en groupes spatialement distincts et que la position spatiale des neurones était plus généralement étroitement liée à l'ensemble de leurs propriétés de filtrage des déflections vibrissales.

This dissertation research explored the capabilities of neuronal networks grown on substrate integrated microelectrode arrays in vitro with emphasis on utilizing such preparations in three specific application domains: pharmacology and drug development, biosensors and neurotoxicology, and the study of burst and synaptic mechanisms. Chapter 1 details the testing of seven novel AChE inhibitors, demonstrating that neuronal networks rapidly detect small molecular differences in closely related compounds, and reveal information about their probable physiological effects that are not attainable through biochemical characterization alone. Chapter 2 shows how neuronal networks may be used to classify and characterize an unknown compound. The compound, trimethylol propane phosphate (TMPP) elicited changes in network activity that resembled those induced by bicuculline, a known epileptogenic. Further work determined that TMPP produces its effects on network activity through a competitive inhibition of the GABAA receptor. This demonstrates that neuronal networks can provide rapid, reliable warning of the presence of toxic substances, and from the manner in which the spontaneous activity changes provide information on the class of compound present and its potential physiological effects. Additional simple pharmacological tests can provide valuable information on primary mechanisms involved in the altered neuronal network responses. Chapter 3 explores the effects produced by a radical simplification of synaptic driving forces. With all synaptic interactions pharmacologically limited to those mediated through the NMDA synapse, spinal cord networks exhibited an extremely regular burst oscillation characterized by a period of 2.9 ± 0.3 s, with mean coefficients of variation of 3.7, 4.7, and 4.9 % for burst rate, burst duration, and inter-burst interval, respectively (16 separate cultures). The reliability of expression of this oscillation suggests that it may represent a fundamental mechanism of importance during periods of NMDA receptor dominated activity, such as embryonic and early postnatal development. NMDA synapse mediated activity produces a precise oscillatory state that allows the study of excitatory-coupled network dynamics, burst mechanisms, emergent network properties, and structure-function relationships.

Embryonic murine neuronal networks cultured on microelectrode arrays were used to quantify acute electrophysiological effects of fluoxetine and ethanol. Spontaneously active frontal cortex cultures showed highly repeatable, dose-dependent sensitivities to both compounds. Cultures began to respond to fluoxetine at 3 µM and were shut off at 10-16 µM. EC50s mean ± S.D. for spike and burst rates were 4.1 ± 1.5 µM and 4.5 ± 1.1 µM (n=14). The fluoxetine inhibition was reversible and without effect on action potential wave shapes. Ethanol showed initial inhibition at 20 mM, with spike and burst rate EC50s at 52.0 ± 17.4 mM and 56.0 ± 17.0 mM (n=15). Ethanol concentrations above 100 -140 mM led to cessation of activity. Although ethanol did not change the shape and amplitude of action potentials, unit specific effects were found. The combined application of ethanol and fluoxetine was additive. Ethanol did not potentiate the effect of fluoxetine.

Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic network failure to develop in a concentration-dependent time window between 50% and 90% activity loss. Investigation of entry routes suggested the L-type but not N-type calcium channels to be the main entry pathway for zinc. Data are presented implicating the chloride channel to be an additional entry route.

The Dorsal Raphe Nucleus (DRN) is a complex brain region that has been implicated in disorders such as anxiety and depression. The DRN is divided into subregions through its rostrocaudal and mediolateral axis. It has been reported that after a single restraint session there is differential spatial activation of nitric oxide synthase (NOS) across the DRN. The temporal profile of NOS activity during acute stress is not known but it is important because duration of acute stress is associated with different general responses. In this report rats were restrained for 1, 3, or 6 hours and nicotinamide adenine phosphate diaphorase (NADPH-d) was stained as an index to NOS activity to determine the spatio-temporal profile of NOS throughout a 6 hour restraint. Astrocyte reactivity was also measured to determine whether NOS activation correlated with GFAP expression since astrocytes react to neural activity and store and release l-arginine, the precursor for nitric oxide production. The results showed that the DRN had a dynamic response to acute restraint stress, most notably in the caudal lateral wings where activation increased after 3 hours of restraint (p = > 0.001) but neuron count decreased after 6 hours (p = 0.040). Astrocytes did not correlate with NOS activation but they showed spatio-temporal differences as well whereas they were more active in the rostral half of the DRN. In conclusion, the present study suggests that NOS produced in the DRN may have a role in prolonged exposure to acute stress and that subregions show differential NOS activation.

Humans spend a third of their lives sleeping but very little is known about the physiological and genetic mechanisms controlling sleep. Increased data from sleep phenotyping studies in mouse and other species, genetic crosses, and gene expression databases can all help improve our understanding of the process. Here, we present analysis of our own sleep data from the large-scale phenotyping program at The Jackson Laboratory (JAX), to identify the best gene candidates and phenotype predictors for influencing sleep traits. The original knockout mouse project (KOMP) was a worldwide collaborative effort to produce embryonic stem (ES) cell lines with one of mouse’s 21,000 protein coding genes knocked out. The objective of KOMP2 is to phenotype as many as of these lines as feasible, with each mouse studied over a ten-week period (www.mousephenotype.org). The phenotyping for sleep behavior is done using our non-invasive Piezo system for mouse activity monitoring. Thus far, sleep behavior has been recorded in more than 6000 mice representing 343 knockout lines and nearly 2000 control mice. Control and KO mice have been compared using multivariate statistical approaches to identify genes that exhibit significant effects on sleep variables from Piezo data. Using these statistical approaches, significant genes affecting sleep have been identified. Genes affecting sleep in a specific sex and that specifically affect sleep during daytime and/or night have also been identified and reported. The KOMP2 consists of a broad-based phenotyping pipeline that consists of collection of physiological and biochemical parameters through a variety of assays. Mice enter the pipeline at 4 weeks of age and leave at 18 weeks. Currently, the IMPC (International Mouse Phenotyping Consortium) database consists of more than 33 million observations. Our final dataset prepared by extracting biological sample data for whom sleep recordings are available consists of nearly 1.5 million observations from multitude of phenotyping assays. Through big data analytics and sophisticated machine learning approaches, we have been able to identify predictor phenotypes that affect sleep in mice. The phenotypes thus identified can play a key role in developing our understanding of mechanism of sleep regulation.

This research explored the role of extra-therapeutic variables contributing to recovery from chronic Post Traumatic Stress Disorder (PTSD). Within the context of dramatherapy treatment, those variables were identified as three crucial concepts: 'Client, Post Traumatic' (C.PT), ‘Imaginary Existence Zone’ (IEZ) and 'Time Adjusted Encounters' (TAE). Together they created the notion of a Curative Zone (CZ). Establishing and understanding the significance of these new concepts helped the researcher to explain the PTSD recuperation phenomenon. The research was conducted within the qualitative–naturalistic paradigm, and based on real-life dramatherapeutic occurrences. The choice of an inductive case study approach and design was possible due to the fact that a single individual was willing to participate in this research as an active partner by contributing her reflections on the therapy, four years after its termination. Iris, the client and the collaborating respondent was a childless woman aged 43, who suffered from chronic PTSD for three years following stillbirth of her baby daughter and the repetitive failure of fertility treatments. The findings reveal a direct linkage between: the neurological system and its activation, and the cardinal role of the C.PT during TAE, working through prolonged engagements in the IEZ facilitated by dramatherapy. These processes gradually integrated and synthesized to create the CZ, a development which explains this instance of recovery from chronic PTSD. The conclusions are: the chronic PTSD recovery was a holistic body-mind cure phenomenon. It resulted from the interaction between the extra-therapeutic variables, combined with the curative characteristics of the dramatherapeutic nonverbal imaginative language and activities, which compounded a new synergetic constellation. The research findings contribute to the theory and practice of dramatherapy as a discipline; additionally, the model developed by this research can be potentially applied as an appropriate treatment of PTSD. These conclusions challenge valid psychotherapy knowledge regarding effective therapeutic factors that contribute to successful outcomes. However, in this case they verified credible, dependable and transferable attributes features this naturalistic research. Therefore, they make a contribution to knowledge in the dramatherapy field.

Highly structured for efficient communication, natural languages are complex systems. Unlike in their computational cousins, functions and meanings in natural languages are relative, frequently prescribed to symbols through unexpected social processes. Despite grammar and definition, the presence of metaphor can leave unwitting language users "in the dark," so to speak. This is not problematic, but rather an important operational feature of languages, since the lifting of meaning onto higher-order structures allows individuals to compress descriptions of regularly-conveyed information. This compressed terminology, often only appropriate when taken locally (in context), is beneficial in an enormous world of novel experience. However, what is natural for a human to process can be tremendously difficult for a computer. When a sequence of words (a phrase) is to be taken as a unit, suppose the choice of words in the phrase is subordinate to the choice of the phrase, i.e., there exists an inter-word dependence owed to membership within a common phrase. This word selection process is not one of independent selection, and so is capable of generating word-frequency distributions that are not accessible via independent selection processes. We have shown in Ch. 2 through analysis of thousands of English texts that empirical word-frequency distributions possess these word-dependence anomalies, while phrase-frequency distributions do not. In doing so, this study has also led to the development of a novel, general, and mathematical framework for the generation of frequency data for phrases, opening up the field of mass-preserving mesoscopic lexical analyses. A common oversight in many studies of the generation and interpretation of language is the assumption that separate discourses are independent. However, even when separate texts are each produced by means of independent word selection, it is possible for their composite distribution of words to exhibit dependence. Succinctly, different texts may use a common word or phrase for different meanings, and so exhibit disproportionate usages when juxtaposed. To support this theory, we have shown in Ch. 3 that the act of combining distinct texts to form large 'corpora' results in word-dependence irregularities. This not only settles a 15-year discussion, challenging the current major theory, but also highlights an important practice necessary for successful computational analysis---the retention of meaningful separations in language. We must also consider how language speakers and listeners navigate such a combinatorially vast space for meaning. Dictionaries (or, the collective editorial communities behind them) are smart. They know all about the lexical objects they define, but we ask about the latent information they hold, or should hold, about related, undefined objects. Based solely on the text as data, in Ch. 4 we build on our result in Ch. 2 and develop a model of context defined by the structural similarities of phrases. We then apply this model to define measures of meaning in a corpus-guided experiment, computationally detecting entries missing from a massive, collaborative online dictionary known as the Wiktionary.