Plant viruses have developed a number of strategies that enable them to become obligate intracellular parasites of many agricultural crops. Potato virus X (PVX) belongs to a group of positive-sense, single-stranded plant RNA viruses that replicate on host membranes and form elaborate structures known as viral replication complexes (VRCs) that contain viral RNA (vRNA), proteins and host cellular components. VRCs are the principal sites of viral genome replication, virion assembly and packaging of vRNA for export into neighbouring cells. For many animal viruses, host membrane association is crucial for RNA export. For plant viruses, it is not yet known how vRNA is transported to and through plant plasmodesmata. PVX encodes genetic information required for its movement between cells; three viral triple gene block (TGB) movement proteins and a viral coat protein are essential for viral trafficking. This research project studies the relationship between PVX and its host plants, Nicotiana benthamina and Nicotiana tabacum. A particular focus of this project is exploration of the structural and functional significance of the PVX VRC and how the virus recruits cell host components for its replication and movement between cells. The role of specific viral proteins in establishing the VRC, and the ways in which these interact with host organelles, was investigated. A combination of different approaches was used, including RNA-binding dyes and a Pumilio-based bimolecular fluorescence complementation assay for detection of the vRNA, fluorescent reporters for virusencoded proteins, fluorescent reporters for host organelles involved in viral replication, and also transgenic tobacco plants expressing reporters for specific plant components (endoplasmic reticulum, Golgi, actin, microtubules and plasmodesmata). In addition, mutagenesis was used to study the functions of individual viral proteins in replication and movement. All of these approaches were combined to achieve live-cell imaging of the PVX infection process. The PVX VRC was shown to be a highly compartmentalised structure; (+)-stranded vRNA was concentrated around the viral TGB1 protein, which was localised in discrete circular compartments within the VRC while coat protein was localised to the external edges of the VRC. The vRNA was closely associated with host components (endoplasmic reticulum and actin) shown to be involved in the formation of the VRC. The TGB2/TGB3 viral proteins were shown to colocalise with the host endomembranes (ER) and to exit these compartments in the form of motile granules. vRNA, TGB1, TGB2 and CP localised to plasmodesmata of the infected cells. TGB1 was shown to move cell-to-cell and recruit ER, Golgi and actin in the absence of viral infection. In the presence of virus, TGB1 targeted the VRCs in several neighbouring cells. A model of PVX replication and movement is proposed in which TGB1 functions as a key component for recruitment of host components into the VRC to enable viral replication and spread.

The primary research aims of this thesis were to analyse the mechanism of Ca2+/calmodulin (CaM) signalling during conidial germination and conidial anastomosis tube (CAT)-mediated fusion in Neurospora crassa. Ca2+ is an ubiquitous signalling molecule that regulates many important processes in filamentous fungi including spore germination, hyphal growth, mechanosensing, stress responses, circadian rhythms, and the virulence of pathogens. Transient increases in cytosolic free calcium ([Ca2+]c) act as intracellular signals. As the primary intracellular Ca2+ receptor, calmodulin (CaM) converts these Ca2+ signals into responses by regulating the activities of numerous target proteins. Ca2+-free medium, antagonists of L-type Ca2+ channels, CaM and calcineurin were found to inhibit CAT fusion. In addition, my results showed that CAT chemotropism is dependent on extracellular Ca2+. 65 genes were identified as likely components of the Ca2+ signalling machinery of N. crassa based on a comparative genomic analysis of S. cerevisiae, A. fumigatus and C. albicans. Deletion mutants of 29 of these genes were characterized in relation to their possible roles during colony initiation and development. Four of these mutants (Δcna-1, Δcnb-1, Δcamk-1, Δplc-2, and Δrgs-1), which were homokaryons, exhibited strong morphological phenotypes associated with CAT fusion. To identify the protein machinery involved in Ca2+/CaM signalling during colony initiation, proteins that directly or indirectly interacted with CaM were isolated from germlings by immunoprecipitation and analyzed by mass spectroscopy. A total of 286 putative Ca2+/CaM-interacting proteins were identified in this way and 30 of these proteins contained CaM-binding motifs. This proteomics analysis provided evidence for Ca2+/CaM signalling playing a role in regulating the activity of a wide range of proteins including MAP kinases in the cell integrity pathway, Ras/Rho signalling pathway, and microtubule and actin cytoskeletal proteins. GFP labelled CaM localized as dynamic spots associated with the plasma membrane and cytoplasm in both germ tubes and CATs. Significant CaM accumulation was observed in the tips of CATs growing towards each other, around fusion pores at sites of CAT fusion, and at developing septa in germ tubes. CaM localization was influenced by the actin and microtubule cytoskeleton during the colony initiation. Inhibition of F-actin polymerization with latrunculin-A suppressed the pronounced accumulation of CaM at growing germ tube and CAT tips. The movement of CaM associated with spindle pole bodies was prevented by treatment with the microtubule polymerization inhibitor benomyl. The absence of myo-5 resulted in reduced CAT fusion and the lack recruitment of CaM at growing tips indicating a role for the motor protein, myosin-5, in these processes. Finally, by expressing the genetically encoded Ca2+ sensor GCaMP6s under the control of tef-1 promoter in N. crassa, I have been able to image [Ca2+]c dynamics in this fungus for the first time. Using this I have been able to detect localized [Ca2+]c spikes and waves in conidia, germ tubes and CATs. However, I obtained no clear evidence for localized [Ca2+]c changes being associated with CAT chemotropism or fusion.

While mechanical loading is essential for articular cartilage homeostasis, it also plays a central role in the etiology of osteoarthritis. The mechanotransduction events underlying these dual effects, however, remain unclear. Previously, we have shown that lethal amounts of reactive oxygen species (ROS) were liberated from mitochondrial complex 1 in response to a mechanical insult. The sensitivity of this response to an actin polymerase inhibitor, cytochalasin B, indicated a link between ROS release and cytoskeletal distortion caused by excessive compressive strain. It did not, however, rule out the possibility that ROS may also mediate the beneficial effects of normal stresses that induce lower tissue strains required for proper homeostasis. If this possibility is true, one would expect the amount of ROS released in loaded cartilage to be positively correlated with the level of strain, and ROS should only reach lethal levels under super-physiological deformations. To test this hypothesis, full cartilage tissue strains were measured in cartilage explants subjected to static normal stresses of 0, 0.1, 0.25, 0.5, and1.0 MPa. After compression, the percentage of ROS-producing cells was measured using the oxidation-sensitive fluorescent probe, dihydroethidium, and confocal microscopy. In support of our theory, the percentage of fluorescing cells increased linearly with increasing strains (0-75%, r2 = 0.8, p < 0.05). Additionally, hydrostatic stress, which causes minimal tissue strain, induced minimal ROS release. In terms of cell viability, cartilage explants compressed with strains >40% experienced substantial cell death, while explants with strains

The neuropeptides orexin-A and orexin-B were discovered in 1998 and were first described as regulators of feeding behaviour. Later research has shown that they have an important role in the regulation of sleep. Two G protein-coupled receptors, OX1 and OX2 orexin receptors, mediate the cellular responses to orexins. The overall aim of this thesis was to investigate the OX1 orexin receptors signalling to phospholipases. Previous investigations have determined that orexin receptors induce Ca2+ elevations through both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). In this thesis we investigated the importance of these influxpathways on orexin-mediated phospholipase (PLC) activation. The results demonstrate that ROC influx is enough to fully support orexin-stimulated PLC activation but that SOC influx has a further amplifying role. We also investigated the metabolites generated after PLC activation, inositolphosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities one of them leading to DAG production without co-occurring IP3 production at low orexin receptor stimulation. The results also suggest that at even lower orexin receptor stimulation DAG is produced via the activation of phospholipase D. In this thesis we also investigated if the ubiquitous phospholipase A2 (PLA2) signalling system is involved in orexin receptor signalling. The results demonstrate that stimulation of the OX1 orexin receptors leads to arachidonic acid (AA) release. This release is fully dependent on Ca2+ influx, probably through ROC, and at the same time the studies demonstrate that ROC influx is partly dependent on PLA2 activation. At low orexin receptor activation the AA release seemed to in part rely on extracellular signal-regulated kinase. We also devised two methods to aid in these investigations. The first method enabled studies of the receptor-operated Ca2+ influx without interference of the co-occurring store-operated Ca2+ influx. This was done by the expression of IP3-metabolising enzymes IP3-3-kinase-A and IP3-5-phosphatase-I. The second method enables quantification of DAG and IP3 signalling in fixed cells using GFP-fused indicators, leading to a semi-quantitative but easily applicable pharmacological assay.

Live cell imaging can be used to study dynamic cellular systems at single cell resolution. In heterogeneous cell populations, analyzing cell properties at the single cell level reduces the generalization of results caused by population-based assays. This thesis details the implementation of live cell imaging and single cell tracking to characterize heterogeneous cell systems undergoing dynamic processes over multiple generations. This approach enables the consideration of both spatial and temporal variables as well as the mapping of cell phenotype trajectories along their generational lineages. Cell-, lineage-, and colony-level properties are used as descriptors of the underlying molecular mechanisms that they are produced by. These may be unexpected, emergent properties that can not be predicted or completely characterized at the molecular level. Analysis of these properties can reveal and characterize the properties and processes of dynamic, heterogeneous cell systems. Live cell imaging culture strategies were developed to enable characterization of both two- and three-dimensional cell systems. Computational modeling was performed to evaluate the conditions imposed by a confined imaging chamber that enables single cell resolution imaging of monolayer and multilayer cell systems. Imaging chamber dimensions and cell colony/aggregate sizes were calculated that would prevent the introduction of metabolite transport limitations and allow for stable, long term imaging. Methods for single cell tracking and analysis were also developed, which produces a database detailing the tracked, observed and extracted properties of every cell and colony, while maintaining the lineage structure of the data. Visualizations such as lineages, histograms and scatter plots were implemented to enable interactive data analysis and querying. These methods were used to characterize heterogeneity in two separate cell systems: human islet of Langerhans-derived progenitor cells, and human embryonic stem cells. Islet-derived progenitors are an expandable source of cells with potential for treatment of diabetes. Here, it was shown that there is an unequal contribution of islets to the progenitor derivation process. Islet-derived progenitors consist of two distinct sub-populations of cells that were distinguished by morphological identification during live cell image analysis. These sub-populations possess unique proliferation profiles and appear to exist in a dynamic state with each other. Three-dimensional tracking of islet progenitor derivation was implemented, but suffered from a lack of resolution to capture the dynamic nature of the transformation process. However, entire islets were imaged and tracked successfully under maintenance conditions, suggesting that this system may be useful for other cell types. These results highlight that live cell imaging and cell tracking may not be suitable for all cell systems and that inclusion of other analytical information, such as immunocytochemistry, would improve the power of cell tracking analysis. Human embryonic stem cell cultures were studied using live cell imaging to identify the mechanisms by which they differentiate to produce supportive niche cells. Cell tracking, morphology scoring and lineage analysis revealed a previously unappreciated level of heterogeneity within human embryonic stem cell colonies. The results show that a sub-population of human embryonic stem cells exist that are precursors to niche cell differentiation. However, these cells exist in a dynamic equilibrium with self-renewing stem cells, which is dependant on the presence of existing local niche cells. Sub-optimal niche conditions leads to the production of niche differentiation-competent cells and, significantly, considerable cell death. The effect of cell death is the clonal selection of self-renewing cells that contribute to colony expansion. Overall, these results highlight the importance of the co-transfer of existing niche cells and the dynamic balance that regulates human embryonic stem cell self-renewal and differentiation. This thesis displays the utility of live cell imaging, cell tracking and cell, colony and lineage analysis for studying dynamic heterogeneous systems. Furthermore, it highlight the fact that cell-, lineage- and colony-level analysis can uncover previously unappreciated heterogeneity and unknown sub-populations of cells. The system does not rely on characterization at the molecular level, but uses higher order measures to generalize them. However, future incorporation of cell, lineage and colony information with molecular-level information may results in analytical power not possible from either level alone. Such systems will be valuable tools in the growing fields of stem cell biology and systems biology.

Live cell imaging can be used to study dynamic cellular systems at single cell resolution. In heterogeneous cell populations, analyzing cell properties at the single cell level reduces the generalization of results caused by population-based assays. This thesis details the implementation of live cell imaging and single cell tracking to characterize heterogeneous cell systems undergoing dynamic processes over multiple generations. This approach enables the consideration of both spatial and temporal variables as well as the mapping of cell phenotype trajectories along their generational lineages. Cell-, lineage-, and colony-level properties are used as descriptors of the underlying molecular mechanisms that they are produced by. These may be unexpected, emergent properties that can not be predicted or completely characterized at the molecular level. Analysis of these properties can reveal and characterize the properties and processes of dynamic, heterogeneous cell systems. Live cell imaging culture strategies were developed to enable characterization of both two- and three-dimensional cell systems. Computational modeling was performed to evaluate the conditions imposed by a confined imaging chamber that enables single cell resolution imaging of monolayer and multilayer cell systems. Imaging chamber dimensions and cell colony/aggregate sizes were calculated that would prevent the introduction of metabolite transport limitations and allow for stable, long term imaging. Methods for single cell tracking and analysis were also developed, which produces a database detailing the tracked, observed and extracted properties of every cell and colony, while maintaining the lineage structure of the data. Visualizations such as lineages, histograms and scatter plots were implemented to enable interactive data analysis and querying. These methods were used to characterize heterogeneity in two separate cell systems: human islet of Langerhans-derived progenitor cells, and human embryonic stem cells. Islet-derived progenitors are an expandable source of cells with potential for treatment of diabetes. Here, it was shown that there is an unequal contribution of islets to the progenitor derivation process. Islet-derived progenitors consist of two distinct sub-populations of cells that were distinguished by morphological identification during live cell image analysis. These sub-populations possess unique proliferation profiles and appear to exist in a dynamic state with each other. Three-dimensional tracking of islet progenitor derivation was implemented, but suffered from a lack of resolution to capture the dynamic nature of the transformation process. However, entire islets were imaged and tracked successfully under maintenance conditions, suggesting that this system may be useful for other cell types. These results highlight that live cell imaging and cell tracking may not be suitable for all cell systems and that inclusion of other analytical information, such as immunocytochemistry, would improve the power of cell tracking analysis. Human embryonic stem cell cultures were studied using live cell imaging to identify the mechanisms by which they differentiate to produce supportive niche cells. Cell tracking, morphology scoring and lineage analysis revealed a previously unappreciated level of heterogeneity within human embryonic stem cell colonies. The results show that a sub-population of human embryonic stem cells exist that are precursors to niche cell differentiation. However, these cells exist in a dynamic equilibrium with self-renewing stem cells, which is dependant on the presence of existing local niche cells. Sub-optimal niche conditions leads to the production of niche differentiation-competent cells and, significantly, considerable cell death. The effect of cell death is the clonal selection of self-renewing cells that contribute to colony expansion. Overall, these results highlight the importance of the co-transfer of existing niche cells and the dynamic balance that regulates human embryonic stem cell self-renewal and differentiation. This thesis displays the utility of live cell imaging, cell tracking and cell, colony and lineage analysis for studying dynamic heterogeneous systems. Furthermore, it highlight the fact that cell-, lineage- and colony-level analysis can uncover previously unappreciated heterogeneity and unknown sub-populations of cells. The system does not rely on characterization at the molecular level, but uses higher order measures to generalize them. However, future incorporation of cell, lineage and colony information with molecular-level information may results in analytical power not possible from either level alone. Such systems will be valuable tools in the growing fields of stem cell biology and systems biology.

Gene expression is a fundamental process in the cell and is made up of two parts – the information flow from DNA to RNA, and from RNA to protein. Here, we examined specific sub-processes in Escherichia coli gene expression using newly available tools that permit genome-wide analysis. We begin our studies measuring mRNA and protein abundances in single cells by single-molecule fluorescence microscopy, and then focus our attention to studying RNA generation and degradation by high throughput sequencing. The details of the dynamics of gene expression can be observed from fluctuations in mRNA and protein copy numbers in a cell over time, or the variations in copy numbers in an isogenic cell population. We constructed a yellow fluorescent fusion protein library in E. coli and measured protein and mRNA abundances in single cells. At below ten proteins per cell, a simple model of gene expression is sufficient to explain the observed distributions. At higher expression levels, the distributions are dominated by extrinsic noise, which is the systematic heterogeneity between cells. Unlike proteins which can be stable over many hours, mRNA is made and degraded on the order of minutes in E. coli. To measure the dynamics of RNA generation and degradation, we developed a protocol using high throughput sequencing to measure steady-state RNA abundances, RNA polymerase elongation rates and RNA degradation rates simultaneously with high nucleotide-resolution genome-wide. Our data shows that RNA has similar lifetime at all positions throughout the length of the transcript. We also find that our polymerase elongation rates measured in vivo on a chromosome are generally slower than rates measured on plasmids by other groups. Studying nascent RNA will allow further understanding of RNA generation and degradation. To this end, we have developed a labeling protocol with a nucleoside analog that is compatible with high throughput sequencing.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by over expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Using serial live cell immunofluorescence imaging of human fibroblasts undergoing reprogramming, we traced the emergence of nascent iPS cell colonies among heterogeneous cell populations and defined the kinetics of marker expression. We identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters, and differentiation into teratomas, we determined that only one colony type represented bona fide iPS cells, whereas the others represented reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B, and REX1 distinguished the fully reprogrammed state, whereas Alkaline Phosphatase, SSEA-4, GDF3, hTERT and NANOG proved insufficient as markers. Reprogramming in chemically defined medium favored formation of bona fide iPS cell colonies relative to partially reprogrammed colonies. These data highlight the need for rigorous characterization and standardization of putative iPS cells.

A cell's ability to sense and respond to mechanical signals highlights the significance of physical forces in biology; however, to date most biomedical research has focused on genetics and biochemical signaling. We sought to further understand the physical mechanisms that guide the cellular migrations that occur in a number of biological processes, such as tissue development and regeneration, bacterial infections and cancer metastasis. We investigated the migration of single cells and determined whether the biomechanics of these cells could be used to elucidate multi-cellular mechanisms. We first studied Borrelia burgdorferi (Bb), the bacterium that causes Lyme disease. We created a mathematical model based on the mechanical interactions between the flagella and cell body that explained the rotation and undulation of the cell body that occurs as the bacterium swims. This model further predicts how the swimming dynamics could be affected by alterations in flagellar or cell wall stiffnesses. Fitting the model to experimental data allowed us to calculate the flagellar torque and drag for Bb, and showed that Treponema pallidum (Tp), the syphilis pathogen, is biomechanically similar to Bb. Next, we used experimentally-determined parameters of Bb's motility to develop a population-level model that accounts for the morphology and spreading of the "bulls-eye" rash that is typically the first indicator of Lyme disease. This work supported clinical findings on the efficacy of antibiotic treatment regimes. Finally, we investigated the dynamics of epithelial monolayers. We found that intracellular contractile stress is the primary driving force behind collective dynamics in epithelial layers, a result previously predicted from a biophysical model. Taken together, these findings identify the relevance of physics in cellular migration and a role of mechanical signaling in biomedical science.

The myelin basic protein (MBP) family arises from different transcription start sites of the Golli (gene of oligodendrocyte lineage) gene, with further variety generated by differential splicing. The “classic” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath, but also have multiple protein interactions. As an intrinsically disordered protein, MBP has proven to have complex structural and functional relationships with proteins in vitro including actin, tubulin, Ca2+-calmodulin, and multiple protein kinases. The investigations reported in this thesis were to further examine the multifunctionality, and protein:protein interactions of MBP with cytoskeletal and SRC homology 3 domain (SH3) proteins in cells using an oligodendrocyte (OLG) model system to better understand MBP’s contributions to membrane structure, formation, and elaboration in the developing OLG. A new function of MBP has been described showing that classic MBPs can modulate voltage operated calcium channels (VOCCs) by direct or indirect protein-protein interactions at the OLG cytoplasmic leaflet. These interactions contribute to global calcium homeostasis, and may play a complex developmental and spatiotemporal role in the regulation of oligodendrocyte precursor cell (OPC) migration and OLG differentiation. The importance of MBPs SH3 ligand binding domain within its central amino acid region was investigated with the protein-tyrosine kinase Fyn. Co-expression of MBP with a constitutively-active form of Fyn in OLGs resulted in membrane process elaboration, a phenomenon that was abolished by amino acid substitutions within MBP’s SH3-ligand domain. These results suggest that MBP’s SH3-ligand domain plays a key role, and may be required for proper membrane elaboration of developing OLGs. Lastly, interactions of MBP with the OLG cytoskeleton were investigated in OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it had increased co-localization with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. The results presented here advance our understanding regarding protein:protein interactions of MBP, and its multifunctionality in OLGs with regards to membrane formation and elaboration. / This work was supported by the Canadian Institutes of Health Research (MOP #86483, J.M.B. and G.H.), and Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, G.H., RG121541). G.S.T.S. was a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada