Doctor of Philosophy / Department of Grain Science and Industry / Hulya Dogan / Jon Faubion / Bread quality and final crumb grain are reflective of the ability for wheat flour dough to retain and stabilize gas cells during the baking process. The visco-elastic properties of dough allow for the incorporation of air cells and expansion during fermentation and baking. The gluten-starch matrix provides the backbone support. However, following the end of proofing and during the beginning of baking, the structure weakens due to over-extension and expansion and the matrix begins to separate and eventually break down. Native wheat lipids, which are found in small quantities in wheat flour, provide a secondary support for gas cell stabilization because of their amphiphilic characteristics and ability to move to the interface and form condensed monolayers. The objectives of this research were to evaluate the influence of native wheat lipids on the rheological properties of dough and the microstructure of bread. Native wheat lipids were extracted from straight-grade flour and separated into total, free, bound, nonpolar, glycolipids, and phospholipids using solid-phase extraction (SPE) with polar and nonpolar solvents. Defatted flour was reconstituted using each lipid fraction at a range of levels between 0.2% and 2.8%. Dough and bread were made following AACC Method 10-10.03. Rheological testing of the dough and evaluation of the microstructure of the bread was conducted using small and large deformation testing, C-Cell imaging, and x-ray microtomography analysis to determine changes in visco-elastic properties and gas cell structure and distribution. Rheological assessment through small amplitude oscillatory measurements demonstrated that nonpolar, phospholipids, and glycolipid fractions had a greater interaction with both proteins and starch in the matrix, creating weaker dough. Nonpolar, phospholipids, and glycolipids, varied in their ability to stabilize gas cells as determined by strain hardening index. C-Cell imaging and x-ray microtomograpy testing found that treatments containing higher concentrations of polar lipids (glycolipids and phospholipids) had a greater effect on overall loaf volume, cell size, and distribution. This illustrates that level and type of native wheat lipids influence the visco-elastic properties of dough and gas cell size, distribution, cell wall thickness, and cell stability in bread.

Diabetes Mellitus (DM) is a metabolic disorder in which the body fails to achieve glucose homeostasis, due to either insulin resistance or reduced insulin secretion or both. This inadequate glucose control leads to hyperglycemia which, if left unchecked, leads to secondary complications like nephropathy, neuropathy, retinal degeneration and other serious conditions. In non-disease state, normal glucose level in the blood is maintained by pancreatic β-cells, which secrete insulin. However, during diabetes development, there is loss of β-cell mass and function; resulting in decreased insulin secretion which is the ultimate cause of hyperglycemia. The ability to non-invasively monitor changes in the β-cell mass during the development or treatment of diabetes would be a significant advance in diabetes management. However, a primary limitation for analysis of β-cell mass and developing dysfunction is the lack of specificity of β-cell targeting agents. Our novel approach for achieving the required specificity for a usable β-cell targeted contrast agent is to target a set of receptors on the cell surface that, as a combination, are unique to that cell. Through genetic screening, Glucagon Like Peptide-1 Receptor (GLP-1R) and α2Adrenergic Receptor (α2AR) were chosen as a potential molecular barcode for β-cells since their combination expression is relatively unique to the β-cells. GLP-1R and α2AR are both G-protein couple receptors (GPCRs) that, apart from being a β-cell specific combination, play an important role in regulating fundamental downstream signaling pathways in β-cells. To target these receptors effectively, we synthesized a multivalent ligand composed of Yohimbine (Yhb), an α2 adrenergic receptor (α2AR) antagonist, linked to an active Glucagon-like Peptide 1 analog (GLP-1₇₋₃₆). In this manuscript, I describe the synthesis and characterization of binding selectivity and signaling ability of GLP-1/Yhb at the cellular level. Using high throughput binding assays, we observed high affinity binding of GLP-1/Yhb to βTC3 cells, a β-cell mimetic line expressing both receptors, at a Kd of ~3 nM. Using microscopy, we observed significant Cy5-tagged GLP-1/Yhb binding and rapid internalization in cells expressing the complementary receptor pair at low concentrations, as low as 1 nM and 5 nM. When one of the receptors was made inaccessible due to presence of saturating quantities of a single unlabeled monomer, GLP-1/Yhb-Cy5 failed to bind to the cells at low concentrations (<10 nM). Similarly, in cells where either GLP-1R or α2AR were knocked down (using shRNA), binding of GLP-1/Yhb was significantly reduced (≤half of cells with both receptors), indicating strong selectivity of the ligand to cells expressing the combination of receptors. We also observed that GLP-1/Yhb construct modulates downstream signaling inβ TC3 cells resulting in enhanced Glucose Stimulated Insulin Secretion (GSIS). In presence of stimulatory glucose, GLP-1/Yhb significantly potentiated GSIS with a half-maximal effective dose of 2.6 nM. Compared to GLP-1₇₋₃₆ alone or GLP-1₇₋₃₆ and Yhb monomers added together, only GLP-1/Yhb could significantly potentiate GSIS at 1 nM, demonstrating that GLP-1/Yhb could translate high affinity binding to increased efficacy for GSIS potentiation. Unlike for insulin secretion, high affinity divalent binding did not translate to increased cAMP production at low concentrations, with significant increases above baseline seen only at 10 nM and higher. Nevertheless, these data show that GLP-1/Yhb binds selectively to β-cells and affects signaling, demonstrating its potential for targeted β-cell imaging and therapy. Overall, our work indicates that synthetic heterobivalent ligands, such as GLP-1/Yhb can be developed to increase cellular specificity and sensitivity making them a strong candidate for both noninvasive imaging and targeted therapy.

La transcription est une étape fondamentale dans l'expression des gènes. Cependant, elle reste incomplètement caractérisée dans les cellules vivantes. Pour mieux comprendre la dynamique de la transcription, notre laboratoire a amélioré le système de marquage d'ARN en utilisant la séquence codante pour MS2, facilement fusionnée avec le promoteur d'intérêt et inséré copie unique dans deux lignées cellulaires HeLa cellules. Cette construction permet une vue quantitative de la transcription, a l’échelle de la molécule unique, en temps réel. Nous avons trouvé que le VIH-1 est transcrit par des groupes de polymérases nominés convois. La transcription oscille de manière aléatoire avec des périodes actives (ON) et inactives (OFF) et est contrôlée indépendamment.Sur la base de cette découverte, nous avons étudié: (i) comment l'architecture de différents promoteurs de mammifères contrôle la cinétique transcriptionnelle; et (ii) le rôle du transactivateur transcriptionnel (Tat), le régulateur principal de la transcription du VIH-1, dans les cellules vivantes. Pour traiter ces questions, une nouvelle méthode de modélisation a été établi, combinant l'information des fluctuations transcriptionnelles avec différentes résolutions temporelles. Cela a donné une vue complète et précise du processus stochastique, décrit par le modèle de Markov. Cinq des six promoteurs de mammifères pourraient être définis par trois états, probablement contrôlés par des mécanismes différents. Le passage entre ces états est défini par les constantes de vitesse et l'écart entre eux pourrait potentiellement expliquer la différence dans la quantité d'ARN produit. De manière intéressante, nous avons constaté que les taux de passage entre les états inactifs et profondément silencieux sont la marque distinctive de différents promoteurs, suggérant que les événements cruciaux définissant les profils transcriptionnels sont en fait des événements pré-transcriptionnels.Pour étudier le rôle de Tat, des lignées cellulaires contenant un rapporteur du VIH-1 et une quantité différente de Tat ont été produites. Avec cette approche décrite ci-dessus, nous avons montré que Tat, précédemment caractérisé en tant qu'acteur dominant dans la libération de la polymérase en pause, agit longtemps avant que la transcription soit initiée. Ces résultats frappants apportent de nouvelles perspectives concernant la dynamique transcriptionnelle du VIH-1 contrôlée par Tat. / Transcription is a fundamental step in gene expression. However, it is incompletely characterized in single living cells. To address this question, our laboratory developed the improved RNA tagging system using MS2-binding protein that could easily be fused with the promoter of interest inserted in a single copy in HeLa cell lines. This construct allows quantitative, single molecule view of the transcription in a real time. We have found that HIV-1 is transcribed by groups of closely spaced polymerases referred as convoys. The transcription oscillates randomly between active (ON) and inactive (OFF) periods that are controlled independently.On the basis of this discovery, we further investigated: (i) how architecture of different mammalian promoters controls the transcriptional kinetics; and (ii) the role of transcriptional transactivator (Tat), the master regulator of in HIV-1 transcription in living cells. To address this, new pipeline for the quantification was established, combining the information of transcriptional fluctuations with different temporal resolutions. This gave the full and precise view of the stochastic switching, described by the Markov model. Five of six mammalian promoters could be defined by three states, probably controlled by different mechanisms. Switching between them is defined by the rate constants and the discrepancy among them could potentially explain the difference in the amount mRNA produced. Interestingly, we found that switching rates between inactive, deeply silent states are the hallmark of different promoters, suggesting that the crucial events defining the transcriptional profiles are in fact pre-transcriptional events.To address the role of Tat, cell lines containing HIV-1 reporter and different amounts of Tat were produced. With the above described approach, we found that Tat, previously characterized as dominant player in the release of the paused polymerase, actually acts long before the transcription is initiated. These striking results bring new insights of HIV-1 transcriptional dynamics controlled by Tat.

La maurocalcine (MCa) est une toxine de 33 acides aminés issus du venin de scorpion Scorpio maurus palmatus. Ce peptide a initialement été étudié pour son activité pharmacologique en tant qu'activateur du récepteur à la ryanodine (RyR1) des muscles squelettiques. En étudiant comment cette toxine pouvait atteindre le RyR qui est localisé à l'intérieur des cellules, il a été montré que la maurocalcine pouvait être classé dans la liste croissante des peptides de pénétration cellulaire. Depuis la découverte que la maurocalcine peut servir de vecteur à la délivrance intracellulaire de streptavidine fluorescente, les données se sont accumulées pour illustrer l'incroyable valeur biotechnologique de cette toxine. Plusieurs nouveaux analogues ont été produits qui séparent les propriétés pharmacologiques et de pénétration cellulaire du peptide comme une maurocalcine sans ponts disulfulres, synthétisée en remplaçant les résidus cystéine par des acides aminobutyriques, ou en remplaçant tous les acides aminés par leur isomère de conformation D. La maurocalcine s'est avérée efficace pour la délivrance cellulaire de nanoparticules ouvrant ainsi une myriade possible d'applications high-tech. Enfin, la maurocalcine a été couplé à la doxorubicine, un agent anti-tumoral, pour rendre chimio-sensibles des cellules cancéreuses devenues chimio-résistantes. Il semble donc que la maurocalcine débute sa carrière comme outil biotechnologique, mais aussi que cette toxine s'avèrera utile pour déchiffrer finement les détails mécanistiques du fonctionnement du récepteur à la ryanodine. / Maurocalcine (MCa) is a 33 mer toxin initially identified from a tunisian scorpion venom, scorpio maurus palmatus. This peptide initially triggered our interest for its pharmacological activity on Ryanodine Receptor type 1 (RyR1) of skeletal muscles. In studying how this toxin reaches the intracellular RyR1, it has been shown that MCa could be placed in the growing family of cell penetrating peptides. Since the discovery that MCa can act as a transport agent for the intracellular delivery of fluorescent streptavidine, data have accumulated to illustrate the amazing biotechnological properties of this toxin. Several new analogs have been produced that keep cell penetration properties and lose pharmacological activity of the native molecule. This is the case for a linear analog of MCa synthesized by replacing internal cysteine residues by aminobutyric acid, or by the synthesis of a MCa analog with all its amino acid in D conformation. MCa proved efficient for the intracellular delivery of nanoparticles leading to a myriad of hi-tech applications. Finally, MCa has been grafted on an anti-tumor agent, doxorubicin, to made chemo-resistant tumor cells chemo-sensitive. So it seems that MCa begins its career as a biotechnological tool, and that this toxin will be helpful to see the light on the mechanistic aspects of RyR function.

No description available.

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.