Hepatocyte growth factor (HGF) is a multifunctional mitogen, stimulating cell proliferation, motility, angiogenesis and morphogenesis via activating its receptor, c-Met tyrosine kinase. Overexpression of HGF and c-Met has been shown as a characteristic of cancer transformation and metastasis. Inhibition of HGF/c-Met signaling may abrogate the malignant and metastatic states of cancer cells and offer a useful therapeutic approach for treating cancers. Thus with the aim of creating inhibitors to HGF-cMet signaling, we constructed plasmids containing truncated N-terminus of HGF, NK1, NK2, NK3 and NK4, respectively, and transfected into MDA MB 435S cells by electroporation. After selection with antibiotics, stable transfectants were obtained. Proliferation assay showed that the truncated NKs significantly inhibited the growth of the cells. Moreover, wound healing assay showed that migration of the NK-transfected cells were also significantly inhibited in comparison with GFP-transfected and nontransfected cells. These results therefore suggest that truncated NKs may be good inhibitors to HGF/c-Met signaling in the proliferation and migration of human breast cancer cells in vitro. In the future, the in vivo animal model is needed to be carried out to further clarify the clinical values of truncated NKs for application to cancer therapy.

The ability of plants to not only detect but also adjust to their environment is crucial for their survival. The genes involved in photomorphogenesis – developmental changes in response to light – and their regulation have long been of interest to researchers. While the phytochrome and cryptochrome photoreceptors have been isolated and partially characterized, the downstream components of the light signaling pathway which transmit the perceived light signals and regulate gene expression are still being discovered. A negative regulator of photomorphogenesis, DET1 (de-etiolated 1), was discovered in a mutant screen for plants that develop a light grown phenotype in the dark. DET1 is nuclear localized, but its exact function remains unknown. Two contrasting mechanisms for the role of DET1 in the regulation of gene expression have been proposed based on studies of the tomato and human orthologs of DET1. In order to reveal the mechanism and molecular context of DET1 action, suppressor mutant screens were employed to discover additional genes acting in conjunction with DET1 (designated as TED genes). In this research, new genomic methods were developed and employed to identify the genes underlying the ted1-1SD and ted2-1D suppressor mutations. A long hypocotyl QTL and suppression of the det1-1 dark grown phenotype by the Bensheim (Be-0) ecotype of Arabidopsis mapped to the HAT4 gene, a homeoboxdomain leucine-zipper transcription factor involved in shade-avoidance responses. Sequence analysis uncovered two functionally distinct alleles of HAT4 in the Be-0 alleles of HAT4 compared to the genomic standard Columbia (Col-0) ecotype. Expression analysis showed that in addition to negative autoregulation by itself, HAT4 is also negatively regulated by DET1. The ted2-1D mutation was mapped to a 57 Kbp interval on chromosome I containing three likely candidate genes. Suppression of the det1-1 phenotype by ted2-1D is overdominant which is highly unusual and typically associated with hybrid vigor or heterosis traits. The discovery of the genes underlying the ted1-1SD and ted2-1D suppressor mutations have furthered the understanding of the role for DET1 in regulation of photomorphogenesis as well as mechanisms involved in overall gene regulation during light signaling.

The issue of preventive health policy has become increasingly important topic. A point worth emphasizing here for the managers, market scholars, or policy makers is that whether the health policy would change the consumer behavior. For considerable efforts to encouraging consumer taking the physical examination, here, marketing signaling and costumers perceived trust might be considered as marketing strategies in this dissertation.

Hedgehog signaling is essential during embryogenesis and in the maintenance of adult

The canonical PI3K/Akt pathway is activated downstream of numerous receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors, and is a crucial regulator of growth and survival in metazoans. The deregulation of Akt is implicated in the pathogenesis of numerous diseases including cancer, making the identification of modifiers of its activity of high chemotherapeutic interest. In a transheterozygous genetic screen for modifiers of embryonic Akt function in Drosophila, in which the PI3K/Akt signaling pathway is conserved, we identified the A subunit of the vacuolar ATPase (Vha68-2) as a positive regulator of Dakt function. Our characterization of this genetic interaction in the larval stage of development revealed that Vha68-2 mutant phenotypes stereotypically mimicked the growth defects observed in mutants of the Drosophila insulin signaling pathway (ISP). The loss of Vha68-2 function, like Dakt-deficiency, was found to result in organismal and cell-autonomous growth defects, and consistent with its putative role as a positive regulator of Dakt function, both the mutational and pharmacological inhibition of its activity were found to downregulate Akt iv activation. Genetic epistasis experiments in somatic clones of Vha68-2/dPTEN double mutants demonstrated that the loss of Vha68-2 function suppressed the growth defects associated with dPTEN-deficiency, placing Vha68-2 activity downstream of dPTEN in the ISP, while the examination of PI3K activity and PH domain-dependent membrane recruitment in pharmacologically inhibited larval tissues further placed Vha68-2 function downstream of PI3K. These findings were recapitulated in cultured NIH-3T3 cells, whose treatment with bafilomycin A1, a potent and specific inhibitor of V-ATPase, resulted in the downregulation of Akt phosphorylation, particularly in non-cytoplasmic intracellular compartments. Furthermore, cellular subfractionation of bafilomycin-treated NIH-3T3 cells demonstrated a decrease in the localization of Akt to early endocytic structures, and a downregulation in the localization and activation of Akt in the nuclei of both Drosophila and mammalian cells. Finally, the pharmacotherapeutic relevance of V-ATPase inhibition was addressed in two tumor models – multiple myeloma and glioblastoma – and our preliminary findings in these cancers, which are often associated with ectopic PI3K/Akt signaling, showed significant cytotoxic efficacy in vitro, warranting its consideration as a tractable pharmacological option in the treatment of cancer.

Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Pharmacology. Includes bibliographical references.

Zugl.: Bayreuth, Univ., Diss., 2006

Universiẗat, Diss., 1999--Kiel.

Doctor of Philosophy / Department of Biology / Kristin Michel / Malaria remains a major human vector-borne disease, greatly contributing to global human morbidity and mortality. Control of mosquito vectors that transmit malaria continues to be dependent on the widespread application of chemical insecticides through indoor residual spraying and insecticide treated bed nets. However, resistance to these insecticides is spreading within many mosquito populations, adding an ever-increasing urgency to the development of alternative vector control measures. The mosquito immune system is a potential novel target for such alternative measures, as the immune response initiated in these insects during infection with vector-borne disease agents is a key determinant of vector competence and, thus, contributes to a species’ vectorial capacity. These immune responses, additionally, interact with and respond to parasitic or symbiotic biocontrol agents employed to kill or manipulate infection outcome with vector-borne disease agents. Entomopathogenic fungi, including Beauveria bassiana, have been considered as an alternative vector control measure, functioning as biopesticides. The Toll pathway is a key antifungal immune pathway in insects that impacts an insect’s ability to survive fungal infections. A better understanding of Toll signaling immune function and evolution in anophelines, both vector and nonvector, can thus help to improve future biocontrol methods of important vector mosquitoes like Anopheles gambiae. In this dissertation, I report the use of B. bassiana strain I93-825 in An. gambiae to analyze the impact of Toll pathway modulation on mosquito survival. Mosquito survivorship was strongly affected by B. bassiana exposure dose by several measured parameters including median survival, longevity, and hazard. Modulation of Toll signaling, by way of knockdown by RNA interference, revealed a dose-dependent trade-off between immune activation state and survivorship in An. gambiae. To better determine the full Toll immune signaling repertoire in mosquitoes, I annotated and describe the evolutionary history of intracellular Toll pathway members and Toll-like receptors (TLRs) within 21 mosquito genomes. The intracellular signaling pathway is conserved with 1:1 orthology, and evolutionary rates across different intracellular pathway members vary widely as compared to the conserved protein core of these mosquito species. In contrast, TLRs evolved largely by duplication events within certain anopheline lineages, most dramatically in the An. gambiae complex, where six TOLL1/5 paralogs likely possess different ligand binding specificities. Thus, these TLRs should be prioritized for experimental analyses of TLR immune function in An. gambiae. Taken together, the work in this dissertation identifies Toll pathway modulation as a potential resistance mechanism that could impact malaria biocontrol strategies and provides a foundation for future detailed studies of Toll pathway function in important mosquito vector species.