Abstract Adrenomedullin (AM) is a 52-amino acid peptide which is produced in many tissues, including adrenal medulla, lung, kidney and heart. Intravenous administration of AM causes a long-lasting hypotensive effect, accompanied with an increase in the cardiac output in experimental animals. This study was aimed to examine whether AM has any direct effects on myocardial function. In addition to the myocardial contractility, the effects of AM on coronary vascular tone and A-type natriuretic peptide (ANP) release from atria and B-type natriuretic peptide (BNP) gene expression in the ventricles were studied in the perfused rat heart preparation. In spontaneously beating hearts, AM had no effects on the heart rate, but dose-dependently increased the developed tension (DT) with an EC50 of 7 x 10-11 nmol/l, reflecting a potent positive inotropic effect. The lower the initial resting tension, the higher was the elevation in DT. In paced hearts, a protein kinase A inhibitor, H-89, had no effect on AM-induced inotropic effect, and AM did not increase the cAMP content of the ventricular myocardium. In contrast, the inhibitors of sarcoplasmic reticulum Ca2+ stores, ryanodine and thapsigargin, as well as a protein kinase C inhibitor, staurosporine, significantly attenuated the inotropic response to AM. L-type Ca2+ channel blocker, diltiazem, also suppressed the AM-induced elevation in DT. Moreover, AM increased the duration of myocyte action potentials between 10 mV and - 50 mV in isolated rat atria, consistent with an increase in L-type Ca2+ channel current during the plateau. Inotropic effect of endothelin-1 (ET-1), another locally acting peptide, was enhanced by inhibiting the myocardial nitric oxide (NO) synthesis by Nω-nitro-L-arginine methyl ester (L-NAME) in perfused rat heart. The AM-induced inotropic action was unaltered by L-NAME treatment. When AM and ET-1 were administrated in combined infusion, the inotropic response was significantly smaller than that following the infusion of the peptides alone. This attenuated response was more than overcome by infusion of L-NAME, although the individual responses to AM and ET-1 were not modulated by L-NAME at the doses used in the combination. Consistent with its vasodilator action, AM dose-dependently dilated the coronary arteries of the perfused heart. The effect of AM was not dependent on NO under basal conditions or in coronary arteries constricted with ET-1. Furthermore, AM enhanced the stretch-induced release of ANP from the right atrium, but did not affect the ventricular BNP expression induced by ET-1. In conclusion, AM exerts regulatory actions on the heart by increasing cardiac contractility, dilating coronary arteries and modulating stretch-induced ANP release. The inotropic effect of AM was independent of cyclic AMP, but may involve activation of protein kinase C, Ca2+ influx through L-type Ca2+ channels and the release of Ca2+ from the sarcoplasmic reticulum. Endogenous NO production did not modulate the inotropic effect of AM, although the effect of ET-1 was suppressed. Combined administration of AM and ET-1 produced a weak inotropic response most likely because of a potentiated synthesis of NO. Finally, AM had a coronary vasodilator effect and augmented the stretch-induced ANP release in the right atrium.

Abstract How cell and tissue interactions lead to complex structures and differentiated cell types during organogenesis is still one of the most fundermental questions in modern molecular biology. Laminin appears to have a role in branching morphogenesis during organ development. Laminin5 (α3β3γ2) is an epithelium-specific isoform of laminin and previous report has shown that two alternative transcripts for the γ2 chain, the longer γ2 and the shorter γ2*, result from alternative use of the last exon in the human LAMC2 gene. But the transcription of murine laminin γ2 and γ2* and their biological significance have remained unclear. Type XVIII collagen is a newly identified member of the collagen family. It may be involved in the Wnt signaling pathway, since its longest N-terminal variant contains a frizzled domain, which is part of the Wnt receptor and could antagonize Wnt signaling when secreted. Wnt2b is a new member of the Wnt family. Also its function in organogenesis is unknown. In this study, we have investigated the expressions of laminin γ2 and γ2*, type XVIII collagen and Wnt2b during mouse organogenesis. The function of type XVIII collagen in developing lung, kidney and a recombination of ureteric bud and lung mesenchyme tissue and the function of the Wnt-2b gene during kidney organogenesis were studied by using the combined methods of traditional experimental embryology and modern molecular biology. Two alternative laminin γ2 transcripts were demonstrated in mouse. In the developing kidney, the shorter γ2* form was localized in the mesenchyme, whereas the longer γ2 form was only present in the epithelium of the Wolffian duct and in the ureteric bud, indicating different functions for the γ2 variants. Type XVIII collagen was expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomed localized to the epithelial tips in the early-stage lung, while it was confined to the epithelial stalk region and was absent from the nearly formed ureteric tips in the kidney. In recombinants of ureteric bud and lung mesenchyme, the type XVIII collagen expression pattern in the ureteric bud shifted from the kidney to the lung type, accompanied by a shift in epithelial Sonic Hedgehog expression. The lung mesenchyme was also sufficient to induce ectopic lung Surfactant Protein C expression in the ureteric bud. A blocking antibody for the type XVIII collagen reduced the number of epithelial tips in the lung and completely blocked ureteric development with lung mesenchyme, which was associated with a notable reduction in the expression of Wnt2. The shift in type XVIII collagen expression in ureteric bud and lung mesenchyme tissue recombinant was also accompanied by the significant morphological changes in the branching pattern in ureteric bud development. Wnt2b was expressed in numerous developing organs in the mouse embryo, but it was typically localized in the perinephric mesenchymal cells in the region that partly overlaps the presumptive renal stroma at E11.5. Functional studies of the kidney demonstrated that 3T3 cells expressing Wnt2b were not capable of inducing tubule formation but rather stimulated ureteric development. Recombination of ureteric bud treated with cells expressing Wnt2b and isolated kidney mesenchyme resulted in recovery of the expression of epithelial marker genes and better reconstituted organogenesis. Lithium, a known activator of Wnt signaling, was also sufficient to promote ureteric branching in reconstituted kidney in a manner comparable to Wnt2b signaling. Our data suggest that different organ morphogenesis is regulated by an intraorgan patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen. In the mouse kidney, Wnt2b may act as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and the Wnt pathway may regulate ureteric branching directly.

Mutual cell communication in the human body ensures the proper functioning of the essential mechanisms necessary for the life of the individual and preserving the homeostasis of the whole organism. Such communication is established by various types of signal transmission from the recipient cell to the donor cell, depending on the location and type of communicating cells. One such type is signalization through receptor molecules found on the surface or within the cell receiving the signal. These receptors receive the signal molecule in the form of a ligand and bind it to themselves, while activating the receptor and then triggering the intracellular signaling pathways. The most widely represented receptors in the eukaryotic organism include G-protein-coupled receptors, which represent signaling ensured by activation of the intracellular G-protein complex, and one of the main mechanisms occurring in neuronal signaling and signal transmission in the form of a neurotransmitter. Regulation of the amount of receptors on the surface of the cell and transport of the signal molecule into the intracellular spaces of the cell is ensured by the mechanism of endocytosis, whereby internalization of the ligand- bound receptor in the cytoplasm occurs. One of the most researched mechanisms is clatharin-mediated...

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The 5-hydroxytryptamine (5-HT) system modulates numerous physiological and behavioural processes, and dysfunction within this system underlies many behavioural disorders, such as major depression. The 5-HT1A receptor is the primary somatodendritic autoreceptor that controls the firing rate of 5-HT neurons, but is also coupled to numerous signalling pathways. An understanding of 5-HT1A receptor signalling may lead to the development of antidepressant drugs that selectively target therapeutic pathways in treating depression. The 5-HT1A receptor is coupled to inhibitory G-proteins via its intracellular loops 2 and 3. Point mutations within these loops selectively uncouple receptor signalling pathways. In this thesis, I addressed whether mutant receptors’ uncoupling from signalling pathways is associated with alteration in G-protein interaction and coupling. Using bioluminescence resonance energy transfer (BRET) to monitor receptor-G-protein interactions, we show that both wild-type and mutant receptors demonstrate a saturable interaction with Gαi protein in unstimulated conditions. Addition of 5-HT increased the BRET signal for the wild-type 5-HT1A receptor, and this increase was blocked by a 5-HT1A receptor antagonist and G-protein blocker (pertussis toxin). Mutant receptors that were deficient in Gαi signalling, but not those that still signalled to Gαi, failed to respond to receptor activation with increased receptor-Gαi interaction. Pull down studies verified the basal and agonist-induced interaction of 5-HT1A receptors with Gαi proteins. In conclusion, we have shown that the 5-HT1A receptor interacts with Gαi consistent with a pre-coupled model and that 5-HT-induced activation enhances this interaction and requires specific residues in the intracellular loops.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

The gulf toadfish (Opsanus beta) is a member of a group of teleosts that have retained their ornithine urea cycle (OUC) allowing them to excrete nitrogenous waste in the form of urea (ureotely). Urea-­N for the entire day is excreted in 1-­2 quick pulsing events (1-­3 h). This study evaluated the hypothesis that urea-­N pulsing events in gulf toadfish can be triggered by social signals from conspecifics via a specific waterborne messenger. Using a crowding protocol, we found that pre-­conditioned seawater induced a secondary urea pulsing event in naïve conspecifics. Furthermore, it was revealed that other factors such as signal concentration and donor body mass relay information to recipients as well. Fractionation of pre-­conditioned seawater was carried out to narrow possible signal candidates and the aqueous portion was found to contain the active molecule. Ammonia was found to be an important factor controlling the response of toadfish to pre-conditioned seawater.

CC2D1A (coiled-coil and C2 domain containing protein 1A), also known as Freud-1, has been identified as a transcriptional repressor of the serotonin receptor 5-HT1A, a regulator of endosomal budding and an activator of NF-KB signaling. It also acts as a scaffold that promotes activity of the PI3K/Akt pathway upon stimulation by the epidermal growth factor (EGF). Moreover, several studies highlight naturally occurring mutations of CC2D1A in humans that produce varying degrees of intellectual disorder and autism. Use of the Cre-LoxP system to conditionally knockout CC2D1A in mice has provided promising results regarding its effect on 5-HT1A expression and behaviour. This thesis aims to extend the use of this knockout model by studying cell signaling activity in mouse embryonic fibroblasts (MEFs), derived from the CC2D1Aflx/flx transgenic line, that have been treated with a commercially available Cre recombinase to completely knock out CC2D1A. I hypothesize that CC2D1A directly regulates EGF receptor activity and that its Cre-mediated knock down in vitro will entirely block cell signaling pathways activated by the EGF receptor. Western blot analysis demonstrated that, after Cre-mediated CC2D1A knockout, Akt and Erk1/2 phosphorylation were still maintained upon EGF treatment. In addition, overexpressing Freud-1 via transfection had no effect on cell signaling compared to the wild-type control. Analysis of recombinant Freud-1 constructs reveal that a C-terminal truncation enhances its ability to bind to PIP2 and PIP3 – phospholipids essential to the Akt pathway. In addition, immunocytochemistry analysis demonstrates a responsiveness of CC2D1A to EGF treatment. Altogether, these data highlight a unique and effective way in carrying out gene knockout in vitro while also emphasizing the need to further investigate CC2D1A’s importance in regulating cell signaling pathways and functional compensation by other homologous proteins

D.Ing. (Electrical And Electronic Engineering) / Please refer to full text to view abstract