v-Rel is the acutely oncogenic member of the NF-[kappa]B family of transcription factors and transforms cells through the altered regulation of pathways normally controlled by cellular NF-[kappa]B. Initial studies revealed that expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAP kinases. This induction is critical for the v-Rel transformed phenotype, as suppression of MAPK activity with chemical inhibitors or siRNA severely limited colony formation of v-Rel transformed cell lines of hematopoietic origin. However, signaling must be maintained within a certain range in these cells, as strong additional activation of either pathway through expression of constitutively active MKK mutants also attenuated the transformed phenotype. Studies in primary spleen cells revealed that MAPK signaling is also required for the early stages of v-Rel-mediated transformation. However, constitutive MAPK activity further enhanced the transformation efficiency of v-Rel in primary cells. These studies, as well as analogous experiments in DT40 cells, indicate distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation. The proto-oncoprotein, c-Rel, only weakly activates ERK and JNK signaling compared to v-Rel. Importantly, elevated MAPK activity enhanced transformation by c-Rel, indicating that the ability of v-Rel to induce MAPK signaling is a major contributor to its oncogenic potential. Taken together, this work demonstrates an important role for ERK and JNK activity in transformation by v-Rel. Additional studies examined mechanisms through which MAPK activity is regulated in v-Rel transformed cells. Feedback regulation of the ERK activator, MKK1, at T292 was shown to limit ERK activation in v-Rel transformed cells, preventing the detrimental effects of constitutive activity. This result is the first indication that this regulation may have a role in the maintenance of transformation. Further, several v-Rel induced cytokines were identified that activate ERK and JNK signaling in v-Rel transformed cells, revealing one means by which v-Rel-dependent transcriptional changes lead to MAPK activation. These studies demonstrate the integration of multiple mechanisms in achieving the optimal levels of MAPK activity that are essential for v-Rel-mediated transformation. / text

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- α. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that β-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- α. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that β-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

Local anesthetics (LAs) are necessary for the regional anesthesia, spinal anesthesia, and pain management. However, the application of LAs may cause neurotoxicity and result in postoperative neurological complications. Lithium is a mood stabilizer for the treatment of bipolar disorder and may exert neuroprotective effects. In this study, we evaluated the effects of lithium on bupivacaine (a frequently used LAs)-induced injury in mouse neuroblastoma neuro 2a (N2a) cells. N2a cells were treated with bupivacaine in the presence or absence of lithium. After treatment, the cell injury was evaluated by examination of viability, morphology changes, and nuclear condensation. The levels of mitochondrial transmembrane potential (δψm) and activation of phosphatidylinositol-3-kinase (PI3K)/ threonine-serine protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also examined. In a separate experiment, we investigated the effect of Akt and ERK inhibition on cell injury after bupivacaine and lithium treatment. Pretreatment of N2a cells with lithium significantly attenuated bupivacaine-induced cell injury. Lithium pretreatment completely reversed the suppression of PI3K/Akt and ERK signalings and significantly prevented the decline of δψ m in N2a cells after bupivacaine treatment. More importantly, pharmacological inhibition of Akt and ERK diminished the protective effect of lithium against bupivacaine-induced neuronal death. Our data suggest that lithium pretreatment provides a protective effect on bupivacaine-induced neuronal cell injury. This action of lithium is mediated through, at least in part, the activating of PI3K/Akt- and ERK-dependent mechanisms. Because lithium is a clinically proved safety drug for neurons, it is worthwhile to identify whether coadministration of LAs with lithium will decrease the risks of LAs-induced postoperative neurological complications in clinic practice.

Thesis advisor: Jennifer Erickson / Policymakers are faced with filtering, understanding, and assessing an overwhelming, and often conflicting, amount of information on a constant basis. States signal resolve over issues, such as during a crisis, or to demonstrate intentions by sending reassurance signals of benign or defensive intentions. But states also have incentives to keep some information private or manipulate the information it sends. Whether or not policymakers believe an adversary’s signals influences, and often determines, the prospect of cooperation or competition. This dissertation examines how policymakers believe the reassurance signals of an adversary. Costly signaling theory argues states can cut through these issues by attaching costs to their signals. Only a sincere state would attach and accept these costs, thus demonstrating the sender is sincere and credible. I argue costly signaling theory is unable to explain variation in why policymakers believe signals in certain situations and not others, despite having costs attached. In this dissertation, I argue policymakers look to see whether sender policymakers risk their own political position to send signals. To risk political vulnerability, sender policymakers must demonstrate they have reduced their control over domestic political processes to send reassurance signals. This is done by sending signals which go against the interests of important domestic constituencies, such as the military or members of the elite. In doing so, sender policymakers demonstrate they are committed to the success of the signal, and will not deflect the costs imposed by signaling failure onto the population or state itself. When sender policymakers demonstrate political vulnerability, target policymakers will believe the signal is genuine. If sender policymakers do not demonstrate political vulnerability, target policymakers will not believe the signal is genuine. I test the domestic political vulnerability thesis by examining how U.S. policymakers believed Soviet reassurance signals during the Cold War. Studying cases of reassurance signaling also allows me to examine for the ability, or inability, of U.S. policymakers to update assessments of Soviet intentions. I select nine cases of Soviet reassurance signaling across three signaling strategies identified by costly signaling theory: strategic arms control (tying hands); conventional troop reductions (sinking costs); and de-escalation signaling. The cases were chosen to test the explanatory power of my theory against the alternative explanations. I use extensive archival research and process tracing to study these cases and find support for the theory of domestic political vulnerability. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Political Science.

Using a wearable tactile display three experiments were conducted in which tactile messages were created emulating five standard US Army and Marine arm and hand signals for the military commands, namely: "Attention", "Halt", "Rally", "Move Out", and "Nuclear Biological or Chemical event (NBC)". Response times and accuracy rates were collected for novices responding to visual and tactile representations of these messages, which were displayed either alone or together in congruent or incongruent combinations. Results indicated synergistic effects for concurrent, congruent message presentations showing superior response times when compared to individual presentations in either modality alone. This effect was mediated by participant strategy. Accuracy similarly improved when both the tactile and visual presentation were concurrently displayed as opposed to separately. In a low workload condition, participants could largely attend to a particular modality, with little interference from competing signals. If participants were not given instructions as to which modality to attend to, participants chose that modality which was received first. Lastly, initial learning and subsequent training of intuitive tactile signals occurred rapidly with large gains in performance in short training periods. These results confirm the promise for tactile messages to augment visual messaging in challenging and stressful environments particularly when visual messaging is maybe preferred but is not always feasible or possible.

No description available.

A cell that senses signals from its environment uses proteins for signal transduction via post translational modifications (PTMs) and protein- protein interactions (PPIs) from cell membrane into the nucleus where genes controlling cell proliferation, differentiation and apoptosis can be turned on or off, i.e. changing the phenotype or fate of the cell. Aberrations within such proteins are prone to cause diseases, such as cancer. Therefore, it is important so study aberrant signaling to be able to understand and treat diseases. In this thesis, signaling aberrations of PTMs and PPIs were analyzed with the use of the in situ proximity ligation assay (in situ PLA), and the thesis also contain method development of rolling circle amplification (RCA), which is the method used for signal amplification of in situ PLA reaction products. Paper I considers the integrity of RCA products. Here, the aim was to generate a smaller and more compact RCA product, for more accurate either visual or automated analysis. This was achieved with the use of an additional so called compaction oligonucleotide that during RCA was able to bind and pull segments of RCA products closer together. The compaction oligonucleotide served to increase the signal to noise ratio and decrease the number of false positive signals. The crosstalk between the Hippo and TGFβ signaling pathways were studied in paper II. Activity of the Hippo signaling pathway is regulated by cell density sensing and tissue control. We found differences in amounts and localization of interactions between the effector proteins of the two pathways depending on cell density and TGFβ stimulation. In paper III the NF-кB signaling pathway constitutively activated in chronic lymphocytic leukemia (CLL) was studied. A 4 base-pair frameshift deletion within the NFKBIE gene, which encodes the negative regulator IкBε, was found among 13 of a total 315 cases by the use of targeted deep sequencing. We found reduced levels of IкBε protein, decreased p65 inhibition, and increased phosphorylation, along with increased nuclear localization of p65 in NFKBIE deleted cases compared to healthy cases. Crosstalk between the Hippo and Wnt signaling pathway are studied within paper IV. Here, we found differences in cellular localization of TAZ/β-catenin interactions depending on colon cancer tumor stage and by further investigate Hippo/WNT crosstalk in cell line model systems we found an increase of complex formations involved in the crosstalk in sparse growing HEK293 cells compared to dense growing cells. Also, active WNT3a signaling was affected by cell density. Since cell density showed to have a big effect on Hippo/WNT crosstalk we continued to investigated the effect of E-cadherin, which has a function in cell junctions and maintenance of epithelial integrity on Hippo/WNT crosstalk. Interestingly, we found that E-cadherin is likely to regulate Hippo/WNT crosstalk.

Gastrulation is a key early developmental event that generates the three primary germ layers (ectoderm, mesoderm, and endoderm) from which organ systems subsequently develop. The physical mechanisms of germ layer formation differ significantly in amniotes (reptiles, birds, and mammals) and anamniotes (e.g. frog and fish), as amniote gastrulation includes an epithelial-mesenchymal transition (EMT) that is absent from anamniote gastrulation. Despite this striking difference, much of our knowledge regarding the mechanisms underlying gastrulation is derived from frog and fish studies. To better understand amniote gastrulation, the work herein investigates three signaling pathways that regulate amniote gastrulation with distinct and overlapping functions. The central hypothesis is that multiple signaling pathways function cooperatively to precisely modulate cell migration through the primitive streak during avian gastrulation.First, I describe a novel function of Fibroblast Growth Factor (FGF) signaling in the preingression epiblast adjacent to the avian primitive streak, where it governs the expression of molecules from diverse signaling pathways and transcription factor families, and which is mediated largely through the Ras/MAPK pathway. Importantly, FGF signaling also regulates cell migration during avian gastrulation.Next, I report the isolation of a novel chicken non-canonical Wnt ligand (Wnt11b) that is specifically expressed in the primitive streak and adjacent preingression epiblast during gastrula stages. In gain and loss of function studies, Wnt11b and Wnt5a/b participate in regulating cell migration through the streak in a largely redundant fashion. Signaling specifically targets the non-canonical pathway, as similar cell migration defects are observed with a non-canonical mutant of Dishevelled, and activating the canonical pathway has no effect on cell migration.Finally, I investigate the function of A-class Eph-ephrin signaling during avian gastrulation, and describe that Eph receptor forward signaling negatively regulates the migration of cells through the primitive streak. This modulation of cell migration occurs independently of the EMT that accompanies avian gastrulation, as cells are able to undergo the normal cadherin transition and the basal lamina is unaffected.Altogether, the work presented herein provides a significant contribution to our understanding of signaling pathways that modulate gene expression and ongoing cell migration during germ layer formation in amniote gastrulation.

Many obese individuals and type 2 diabetes mellitus (T2DM) patients have elevated levels of insulin. Hyperinsulinemia is a major cancer risk factor in T2DM individuals and activated insulin receptor (IR) has been linked to many types of cancer and poor survival. However, the mechanisms that account for the link between the hyper-active insulin signaling and cancer risk is not well understood. PTEN plays an antagonistic role in the canonical insulin signaling pathway, and is the second most commonly mutated tumour suppressor (after p53) found in human cancers. In many cancers the PTEN gene is not deleted, but instead the protein is lost. Therefore the regulation of PTEN protein in humans is of great importance. Here we hypothesized that the activated insulin signaling down-regulates PTEN. Considering that insulin signaling is highly conserved from C. elegans to human, I used C. elegans as a model and showed that DAF-2, the worm homolog of IR, is a negative regulator of DAF-18, the worm homolog of PTEN. In addition, I showed that DAF-28, the worm homolog of insulin, also negatively regulates DAF-18/PTEN. I used western blot and immunostaining to show that the protein level of DAF-18/PTEN is increased in the daf-2/IR and daf-28/insulin mutants. I further showed that daf-18/Pten is genetically epistatic to daf-2/IR in regulating neuronal development. I then employed human cell culture experiments and reported that this negative regulation is conserved in human cancer cell lines. I showed that knocking-down IR through siRNA up-regulates PTEN, and over-expressing a gain-of-function IR down-regulates PTEN. I also showed that insulin stimulation dramatically decreased PTEN and this decrease is dependent on IR. I further confirmed a physical association between IR and PTEN in both human and C. elegans, and reported that IR could phosphorylate PTEN. To provide mechanistic insight to DAF-18/PTEN regulation, I identified another protein, which is a ubiquitin ligase, that functions in insulin signaling to down-regulate DAF-18/PTEN. Additionally, I also provided evidence that insulin signaling cross talks with Eph receptor signaling. In summary, my findings will be informative for cancer biologists to study the roles of these genes in carcinogenesis. / Thesis (Ph.D, Biology) -- Queen's University, 2013-02-04 14:37:29.376