Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Lectins and hemagglutinins are carbohydrate binding proteins present in a diversity of organisms including humans, vertebrate and invertebrate animals, plants, fungi, and bacteria. They are usually the abundant storage proteins in leguminous plants. They display a host of biological activities such as antitumor, antifungal, antiviral, insecticidal, and antibacterial activities. / The biological properties of isolated proteins, including hemagglutinating, antifungal, anti-tumor and HIV-1 reverse transcriptase inhibitory activities, were examined. Their biochemical and biological properties were compared with other purified proteins. / The seeds contain an abundance of proteins, some of which are storage proteins but may play a role of protection from pathogenic microbes and phytophagous insects. Antifungal peptides/proteins, antiviral proteins, ribosome-inactivating proteins, proteinase inhibitors, chitinases, proteinases, and defensins, are some examples of the myriad of seed proteins. The aforementioned proteins are collectively called plant defense proteins in view of their antipathogenic activities. These antifungal proteins exhibit a wide range of molecular masses and amino acid sequences. / Two lectins with potentially exploitable activities were purified from Capparis spinosa seeds and Hibiscus mutabilis seeds, respectively. A hemagglutinin was isolated from Phaselous vulgaris , cultivar "French bean 35", and detailed apoptotic pathway in breast cancer cells, MCF-7 cells, was investigated. A novel dimeric beta-lactoglobulin-like antifungal protein and an antifungal amidase were purified from Passiflora edilus seeds and Peltophorum pterocarpum, respectively. / Lam, Sze Kwan. / Adviser: Tsi Bun Ng. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 188-204). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The altered protein expression and activity of receptor tyrosine kinases (TK) are implicated in the progression of various types of cancers. One such dysfunction is the overexpression of the epidermal growth factor receptor (EGFR) that correlates with aggressive tumor progression and poor prognosis. On the other hand, c-Src non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been strongly linked to progression to distant metastases. c-Src-induced phosphorylation of EGFR is required for EGF-mediated mitogenesis, tumorigenesis and tumour invasiveness. Thus we surmised that molecules termed "combi-molecules" designed to block both EGFR and c-Src should not only possess significant growth inhibitory potency but also strong anti-invasive properties. In this thesis, we utilized molecular modeling to design molecules containing two moieties: one that straddles the structure of the known Src inhibitor PP2 and the other that mimics the backbone of Iressa, a potent EGFR inhibitor. Of all the molecules synthesized, only SB163 containing the longest spacer between the two moieties was capable of inducing a dose dependent inhibition of both Src and EGFR. More importantly, SB163 blocked cell motility in the wound healing assay and showed significantly greater anti-invasive activity than a PP2+Iressa combination. The observation that SB163 was a less potent EGFR or Src inhibitor than Iressa and PP2 suggests that its superior potency when compared with the PP2+ Iressa combination may be at least partially attributed to mechanisms other than EGFR or Src blockade. This was also corroborated by the fact that SB163, despite its significant bulkiness (>700) could induce dose dependent inhibition of other kinase such PDFGR and Abl. The results in toto suggest that conferring multiple kinase targeting properties to single molecules can lead to highly anti-proliferative and anti-invasive agents. Traditionally, multi-kinase targeted molecules were discovered serendipitously through multi-kinase testing. Here we initiated a more rational approach to the design of single multi-targeted molecules. Cancer being a complex disease driven by tumours characterized by multiple disordered signaling pathways, this approach may well represent a novel avenue in the therapy of refractory malignancies.

No description available.

Interferon (IFN) and phosphatidylinositol 3-kinase (PI3K) are apoptosis regulators that are targeted by viruses to promote survival of infected cells. Significant crosstalk exists between IFN and PI3K, and this study sought to investigate the relationships between IFN, PI3K and apoptosis during virus infection. Parainfluenza virus 5 (PIV5) and influenza A virus (IAV) are both negative-sense single-stranded RNA viruses that encode multifunctional proteins in order to maximise their genome coding capacity. The PIV5 V and IAV NS1 proteins are well-studied as IFN antagonists and, in addition, both are reported to modulate PI3K signalling. Less well-studied is the role of these proteins in apoptosis regulation; the ability of V and NS1 to inhibit apoptosis was therefore investigated. PIV5/V was found to limit cell death in response to a number of apoptosis inducers in a manner that required its STAT1- degradative activity and also inhibited activation of the PI3K downstream target, Akt. IAV/NS1 binds directly to PI3K to stimulate its activity, and this is reported to mediate anti-apoptotic signalling during IAV infection. However, a virus expressing an NS1 unable to bind PI3K did not induce more apoptosis than wt virus. NS1 expression, either in a stable cell-line or during virus infection, was also unable to protect cells from pro-apoptotic stimuli. NS1-mediated PI3K activation similarly had no effect on IFN production or ISG expression in infected cells. In contrast, other NS1 mutant viruses induced large amounts of apoptosis. These viruses also induced significant levels of IFN and were unable to cause apoptosis in IFN-deficient cells, indicating that NS1 limits apoptosis induction through its IFN antagonist functions. The implications of this work for anti-cancer and anti-viral therapies are discussed.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.

GnRH neurons represent the final common pathway for the regulation of the reproductive axis and they are modulated by multiple signals. It has recently been shown that a potent effector of GnRH neuron function is an afferent network of kisspeptin-producing neurons. Kisspeptin released from these neurons acts upon a specific receptor (gpr-54) expressed on GnRH neurons, and increases the secretion of GnRH from the hypothalamus. The kisspeptin system has since been implicated as a downstream mediator for regulation of the Hypothalamic-Pituitary-Gonadal (HPG) axis by steroid hormones, metabolic signals and photoperiod, potentially placing it at the centre of reproductive physiology. However, the supporting evidence to date has been indirect, relying on interpretation of changes in mRNA levels and immuno-histochemical staining to infer the actions of kisspeptin upon the central control of reproduction. The detailed mechanisms of kisspeptin action are yet to be fully elucidated. The research within this thesis elucidates the effect of kisspeptin on the HPG axis via the development of kisspeptin-10 (kp-10) analogues with antagonistic properties. Functionally important residues within the peptide were delineated. Structure-activity studies of kp-10 analogues indicated that residues Asn2, Trp3, Phe6, Arg9 and Phe10 interact with gpr-54 to facilitate receptor binding. Two other residues, Tyr1 and Leu8 were shown to be critical for receptor activation by kisspeptin. Four synthetic peptide antagonists were selected according to a consensus sequence for good antagonism: X1-N-W-N-X5-F-G-X8-R-F-NH2 where X1 = D-Ala or D-Tyr, X5 = Gly or D-Ser and X8 = D-Trp or D-Leu. One of the antagonists, peptide 234, was used in in vivo studies, where it inhibited the amplitude of GnRH and LH pulses without affecting basal secretion of GnRH or LH. These results indicate for the first time that basal and pulsatile secretion of these factors is regulated by separate pathways. Use of the antagonist also demonstrated the direct involvement of endogenous kisspeptin in steroid hormone negative feedback, positive regulation of the pre-ovulatory LH surge and in regulating the onset of puberty in rodents, as had been suggested via indirect methods. Although a major role of the kisspeptin system is in the regulation of the HPG axis, the system may also be important in the inhibition of cancer cell metastasis and in placental development (trophoblast cell invasion) but little is known about the mechanisms involving kisspeptin in these processes. This thesis describes novel signalling mechanisms for the regulation cell migration by kisspeptin, involving the MAPK and GSK3β signalling pathways. Using a stably transfected CHO cell line, kisspeptin-gpr-54 signalling can activate all members of the MAPK pathway, the β- catenin/GSK3β pathway, NFκB and FAK. These factors are involved in inhibiting the migration of these cells via an ERK1/2-p90rsk-GSK3β-β catenin pathway to potentially up- regulate formation of adherens junctions at the plasma membrane. This pathway was also shown to be involved in the inhibition of migration within an immortalised human first trimester placental trophoblast cell line and in human umbilical vein endothelial cells. Some of these pathways were also active within a mouse GnRH neuronal cell line, where ERK1/2, NFκB and GSK3β were activated by kisspeptin with no effect on migration. However, the role of these pathways in the GnRH neuronal cells requires further investigation. In summary, the research presented within this thesis defines receptor-binding and activating residues within kisspeptin-10, which should enable more details of ligand-receptor binding interactions to be fully elucidated. Novel gpr-54 antagonists have been identified and used in in vivo studies. The thesis demonstrates the direct involvement of endogenous kisspeptin in the regulation of GnRH/LH secretion at the onset of puberty and throughout the reproductive cycle in mature animals. The antagonists developed within this thesis represent useful tools to further delineate mechanisms of kisspeptin action within the HPG axis and peripheral tissues. Other findings describe kisspeptin signalling mechanisms for the inhibition of cell migration, potentially important in a variety of normal and pathological processes, including for the first time a description of the regulation of GSK3β and β-catenin signalling factors by kisspeptin and gpr-54.