Two adaptation mechanisms regulate cellular migration in Dictyostelium discoideum

Dictyostelium discoideum is a simple model widely used to study many cellular functions, including differentiation, gene regulation, cellular trafficking and directional migration. Adaptation mechanisms are essential in the regulation of these cellular processes. The misregulation of adaptation components often results in persistent activation of signaling pathways and aberrant cellular responses. Studying adaptation mechanisms regulating cellular migration will be crucial in the treatment of many pathological conditions in which motility plays a central role, such as tumor metastasis and acute inflammation. I will describe two adaptation mechanisms regulating directional migration in Dictyostelium cells. The Extracellular signal Regulated Kinase 2 (ERK2) plays an essential role in Dictyostelium cellular migration. ERK2 stimulates intracellular cAMP accumulation in chemotaxing cells. Aberrant ERK2 regulation results in aberrant cAMP levels and defective directional migration. The MAP Phosphatase with Leucine-rich repeats (MPL1) is crucial for ERK2 adaptation. Cells lacking, MPL1 (mpl1- cells) displayed higher pre-stimulus and persistent post-stimulus ERK2 phosphorylation, defective cAMP production and reduced cellular migration. Reintroduction of a full length Mpl1 into mpl1- cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild type cells (Wt). These results suggest Mpl1 is essential for proper regulation of ERK2 phosphorylation and optimal motility in Dictyostelium cells. Cellular polarization in Dictyostelium cells in part is regulated by the activation of the AGC-related kinase Protein Kinase Related B1 (PKBR1). The PP2A regulatory subunit, B56, and the Glycogen Synthase Kinase 3 (GSK3) are necessary for PKBR1 adaptation in Dictyostelium cells. Cells lacking B56, psrA-cells, exhibited high basal and post-stimulus persistent phosphorylation of PKBR1, increased phosphorylation of PKBR1 substrates, and aberrant motility. PKBR1 adaptation is also regulated by the GSK3. When the levels of active GSK3 are reduced in Wt and psrA- cells, high basal levels of phosphorylated PKBR1 were observed, in a Ras dependent, but B56 independent mechanism. Altogether, PKBR1 adaptation is regulated by at least two independent mechanisms: one by GSK3 and another by PP2A/B56.

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

Foundational Forensic Techniques for Cellular and Ad Hoc Multi-hop Networks

The Internet has become an integral part of our nation's critical socio-economic infrastructure. With its heightened use and growing complexity however, organizations are at greater risk of cyber crimes. To aid in the investigation of crimes committed on or via the Internet, a network forensics analysis tool pulls together needed digital evidence. It provides a platform for performing deep network analysis by capturing, recording and analyzing network events to find out the source of a security attack or other information security incidents. Existing network forensics work has been mostly focused on the Internet and fixed networks. But the exponential growth and use of wireless technologies, coupled with their unprecedented characteristics, necessitates the development of new network forensic analysis tools. This dissertation fostered the emergence of a new research field in cellular and ad-hoc network forensics. It was one of the first works to identify this problem and offer fundamental techniques and tools that laid the groundwork for future research. In particular, it introduced novel methods to record network incidents and report logged incidents. For recording incidents, location is considered essential to documenting network incidents. However, in network topology spaces, location cannot be measured due to absence of a 'distance metric'. Therefore, a novel solution was proposed to label locations of nodes within network topology spaces, and then to authenticate the identity of nodes in ad hoc environments. For reporting logged incidents, a novel technique based on Distributed Hash Tables (DHT) was adopted. Although the direct use of DHTs for reporting logged incidents would result in an uncontrollably recursive traffic, a new mechanism was introduced that overcome this recursive process. These logging and reporting techniques aided forensics over cellular and ad-hoc networks, which in turn increased their ability to track and trace attacks to their source. These techniques were a starting point for further research and development that would result in equipping future ad hoc networks with forensic components to complement existing security mechanisms.

Analysis of the lateral diffuse metal-oxide semiconductor (LDMOS) power amplifier for cellular base stations

The primary purpose of this thesis was to present a theoretical large-signal analysis to study the power gain and efficiency of a microwave power amplifier for LS-band communications using software simulation. Power gain, efficiency, reliability, and stability are important characteristics in the power amplifier design process. These characteristics affect advance wireless systems, which require low-cost device amplification without sacrificing system performance. Large-signal modeling and input and output matching components are used for this thesis. Motorola's Electro Thermal LDMOS model is a new transistor model that includes self-heating affects and is capable of small-large signal simulations. It allows for most of the design considerations to be on stability, power gain, bandwidth, and DC requirements. The matching technique allows for the gain to be maximized at a specific target frequency. Calculations and simulations for the microwave power amplifier design were performed using Matlab and Microwave Office respectively. Microwave Office is the simulation software used in this thesis. The study demonstrated that Motorola's Electro Thermal LDMOS transistor in microwave power amplifier design process is a viable solution for common-source amplifier applications in high power base stations. The MET-LDMOS met the stability requirements for the specified frequency range without a stability-improvement model. The power gain of the amplifier circuit was improved through proper microwave matching design using input/output-matching techniques. The gain and efficiency of the amplifier improve approximately 4dB and 7.27% respectively. The gain value is roughly .89 dB higher than the maximum gain specified by the MRF21010 data sheet specifications. This work can lead to efficient modeling and development of high power LDMOS transistor implementations in commercial and industry applications.

Community control in cellular protein production : consequences for amino acid starvation

The authors thank M. C. Romano, I. Stansfield, L. Ciandrini, A. Kort, and M. Rehberg for helpful discussions. This work was funded by BBSRC grants BB/F00513/X1 and BB/G010722, and the Scottish Universities Life Science Alliance (SULSA).

Community control in cellular protein production : consequences for amino acid starvation

The authors thank M. C. Romano, I. Stansfield, L. Ciandrini, A. Kort, and M. Rehberg for helpful discussions. This work was funded by BBSRC grants BB/F00513/X1 and BB/G010722, and the Scottish Universities Life Science Alliance (SULSA).

Ionic, cellular and molecular mechanisms underlying the QT prolongation and arrhythmias in diabetic cardiocomplications

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Modulation of 1HERG/1K by cellular metabolites : implication in the arrhythmogenesis during myocardial ischemia

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

The cellular lipids of Romboutsia.

We have examined the lipids of three isolates, Romboutsia lituseburensis, Romboutsia ilealis, and Romboutsia sp. strain FRIFI, of the newly described genus Romboutsia by two-dimensional thin-layer chromatography (2D-TLC) and by liquid chromatography/mass spectrometry (LC/MS). We have found three phospholipids, phosphatidylglycerol (PG), cardiolipin and phosphatidic acid in all three species. A fourth phospholipid, lysyl-PG, was found in R. lituseburensis and strain FRIFI. Polyprenyl-phosphates were identified in the lipid extracts of all three species. Three glycolipids, mono-, di- and tri-hexosyldiacylglycerol, were common to all three species. An additional glycolipid, tetrahexosyl-diacylglycerol was identified in strain FRIFI. Acylated trihexosyldiacylglycerol and acyl-tetrahexosydiacylglycerol were also found in R. ilealis and strain FRIFI. Remarkably, no alk-1-enyl ether lipids (plasmalogens) were present in Romboutsia as distinct from bacteria of the related genus Clostridium in which these ether lipids are common. We have compared the lipidome of Romboutsia with that recently described for Clostridium difficile, which has plasmalogens, no lysyl-PG, and no tetrahexosyl-diacylglycerol. According to 16S rRNA gene sequencing, Romboutsia spp. and C. difficile are closely related (>95% sequence identity).

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

TRPV4 is necessary for trigeminal irritant pain and functions as a cellular formalin receptor.

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4's role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4(-/-) mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca(2+). Using TRPA1-blocker and Trpa1(-/-) mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.

A small molecule activator of p300/CBP histone acetyltransferase promotes survival and neurite growth in a cellular model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.

Ionic, cellular and molecular mechanisms underlying the QT prolongation and arrhythmias in diabetic cardiocomplications

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.