GTPases are integral components of virtually every known signal transduction pathway, and mutations in GTPases frequently cause disease. A genomic analysis identified and annotated 174 GTPases in the sea urchin genome (with 90% expressed in the embryo), covering five classes of GTP-binding proteins: the Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR GTPases, and the translational GTPases. The sea urchin genome was found to contain large lineage-specific expansions within the Ras superfamily. For the Rho, Rab, Arf and Ras subfamilies, the number of sea urchin genes relative to vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increased overall numbers, such that total sea urchin gene numbers of these GTPase families approximate vertebrate gene numbers. This suggests lineage-specific expansions as an important component of genomic evolution in signal transduction. A focused analysis on RhoA, a monomeric GTPase, shows it contributes to multiple signal transduction pathways during sea urchin development. The data reveal that RhoA inhibition in the sea urchin results in a failure to invaginate during gastrulation. Conversely, activated RhoA induces precocious archenteron invagination, complete with the associated actin rearrangements and extracellular matrix secretion. Although RhoA regulates convergent extension movements in vertebrates, our experiments show RhoA activity does not regulate convergent extension in the sea urchin. Instead, the results suggest RhoA serves as a trigger to initiate invagination, and once initiation occurs RhoA activity is no longer involved in subsequent gastrulation movements. RhoA signaling was also observed during endomesodermal specification in the sea urchin. Data show that LvRhoA activity is required, downstream of a partially characterized Early Signal, for SoxB1 clearance from endomesodermal nuclei (and subsequent expression of GataE and Endo16 genes). Investigations also suggest that within the endomesoderm, RhoA clears SoxB1 as part of Wnt8 signaling, as activated RhoA is sufficient to rescue Wnt8-inhibited embryos. These data provide evidence of the first molecular components involved in SoxB1 clearance, as well as highlight a previously unrecognized role for RhoA during endomesodermal specification. These analyses suggest RhoA signaling is integral to the proper specification and morphogenesis of the sea urchin endomesoderm. / dissertation

Environmental experiences play a critical role in an individualʼs risk of becoming addicted. Positive experiences may mitigate addiction vulnerability, whereas adverse experiences, particularly during adolescence, have been shown to increase addiction risk. Social isolation in rodents is a model system used to study the effects of such experiences, yet its impact on the learning and memory processes that underlie addiction remains elusive. Although social isolation is known to alter the functioning of the dopaminergic system, as well as reward processing and learning, its effect on dopamine (DA) neurons of the ventral tegmental area (VTA) is unknown. The data presented in this dissertation demonstrate that social isolation of rats during a critical period in adolescence (postnatal days 21-42) enhances long-term potentiation (LTP) of N-methyl D-aspartate receptor (NMDAR)-mediated glutamatergic transmission in the VTA. Activation of NMDARs is critical to the generation of DA neuron bursts that encode rewards and reward-predictive cues, and NMDARs are necessary for associative reward learning. The isolation-induced enhancement of NMDAR LTP results from augmentation of metabotropic glutamate receptor (mGluR)-dependent calcium (Ca²⁺) signaling via an increase in inositol 1,4,5-trisphosphate(IP3) sensitivity. Isolation-mediated effects on Ca²⁺ signaling and NMDAR plasticity were not reversed by a subsequent period of resocialization. Furthermore, social isolation during this critical period occludes the effect of repeated amphetamine exposure on mGluR/IP₃-mediated Ca²⁺ signaling and synaptic plasticity. Although corticotropin releasing factor (CRF) further facilitates mGluR/IP3-mediated Ca²⁺ signaling in DA neurons, alterations in CRF receptors are not responsible for the effects of isolation on Ca²⁺ signaling and synaptic plasticity. In addition, the learning of associations between environmental stimuli and drug rewards is acquired more quickly and is more resistant to extinction in isolated animals. Data presented in this dissertation lend support to the theory that enhanced mGluR/IP₃-mediated Ca²⁺ signaling and NMDAR plasticity facilitate the learning and memory of drug-associated stimuli. This dissertation provides the first demonstration of a cellular basis for the critical time window of social isolation during adolescence. NMDAR plasticity in the VTA may thus represent a neural substrate by which early life experiences regulate addiction vulnerability. (Note: Behavioral data were acquired by Mickael Degoulet) / text

Over the past several decades, our understanding of the immune system has advanced considerably. With it, an appreciation for its role in a number of diseases, such as cancer and infection has significantly grown. While our increased understanding of the immunological mechanisms underlying these diseases has improved treatment, considerable morbidity and mortality from these illnesses still exists signifying the need for more effective and innovative therapies. Dendritic cell (DC) therapy has been shown to be a promising approach to induce strong immune responses for immunotherapy, and biomaterial-based strategies have been developed to target DCs in vivo to facilitate this purpose. Given the importance of calcium in DC function and activation, we hypothesized that we could develop a biomaterial-based approach to locally and specifically control calcium signaling in DCs in vivo as a novel strategy for immunotherapy. Our first sub-hypothesis was that the calcium used to crosslink alginate gels, a commonly used biomaterial, could activate DCs in vitro; our second sub-hypothesis was that calcium ionophore A23187 could be delivered from biomaterials to activate DCs in vitro; and our third sub-hypothesis was that calcium used to crosslink alginate gels and/or controlled delivery of A23187 could increase local inflammation in vivo. We found that both the calcium released from calcium alginate gels and A23187 matured DCs and enhanced TLR-induced inflammatory cytokine secretion in vitro. Although we were unable to effectively deliver A23187 in vivo, calcium alginate gels injected subcutaneously were able to upregulate a number of inflammatory cytokines and chemokines relative to barium alginate gels. Likewise, when LPS was delivered from calcium alginate gels, the inflammatory effects of LPS on surrounding tissue were enhanced compared to when it was delivered from barium alginate gels. Thus, we confirmed that the calcium crosslinker in alginate gels could activate DCs, and provided a proof-of-principle that calcium signaling could be harnessed in vivo to enhance the immune response. Not only does this work impact the future of biomaterial design, but it may also enhance our understanding of DC biology. This thesis lays the groundwork for a novel and potentially effective strategy for enhancing DC activation in vivo, and suggests that ion signaling pathways in other cell types (both immune and non-immune) could also be targeted using biomaterials. / Engineering and Applied Sciences

No description available.

Heart failure represents an important cause of death in Western Countries. The pathophysiology of heart failure is mainly associated with abnormalities in intracellular calcium control. We previously showed that Egr-1 negatively regulates expression of sodium-calcium exchanger (NCX) in vivo and in vitro. Here we tested the hypothesis that Egr-1 regulates expression of calcium storage proteins in the sarco-endoplasmic reticulum (SER), calsequestrin (CSQ) and/or ER, calreticulin (CRT) directly or indirectly via Egr-1:NFAT (nuclear factor of activated T-cells) formation. Secondarily, we hypothesized that this will reduce calcium mobilization. We found that undifferentiated 1293F cells, overexpressing Egr-1, have reduced CSQ compared to control H9c2 cells. We demonstrated that Egr-1 negatively regulates CSQ but not CRT expression. The Egr-1 mediated decrease in CSQ is linked to decreased calcium availability. Repression is by a novel NAB-independent (NGFI-A binding protein) activity localized to a.a. region 1-307. We conclude that Egr-1-mediated reductions in calcium storage protein expression alter calcium availability for cardiac contraction/relaxation.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 180-204). Also available online.