The thesis consists of four papers that address likelihood-based unit root tests for panel data with cross-sectional dependence arising from common factors. In the first three papers, we derive Lagrange multiplier (LM)-type tests for common and idiosyncratic unit roots in the exact factor models based on the likelihood function of the differenced data. Also derived are the asymptotic distributions of these test statistics. The finite sample properties of these tests are compared by simulation with other commonly used unit root tests. The results show that our LM-type tests have better size and local power properties. In the fourth paper, we estimate the spaces spanned by the common factors and the spaces spanned by the idiosyncratic components of the static factor model by using the quasi-maximum likelihood (ML) method and compare it with the widely used method of principal components (PC). Next, by simulation, we compare the size and power properties of established tests for idiosyncratic unit roots, using both the ML and PC methods. Simulation results show that the idiosyncratic unit root tests based on the likelihood-based residuals generally have better size and higher size-adjusted power, especially when the cross-sectional dimension is small and the time series dimension is large.

Aβ oligomer-induced neurotoxicity has become an important area of therapeutic development in treating Alzheimer’s disease. Platelet-derived growth factor (PDGF) has been shown to be able to protect neurons against several neuronal insults such as ischemia and HIV1 toxin induced cytotoxicity. These neuroprotective effects correlate well with our previous results that demonstrate the neuroprotective effects of PDGF-BB, one of the PDGF receptor ligand subtypes, against NR2B containing NMDA receptor induced excitotoxicity, a possible underlying cause of Aβ oligomer induced synaptic dysfunction and neuronal death. This project examines the neuroprotective effect of PDGF-BB against Aβ1-42 oligomer induced cytotoxicity in both SH-SY5Y cells and primary hippocampal neurons. Cell viability was monitored by MTT assay and the affected signaling pathways were examined using pharmacological methods and Western blotting. The results demonstrated that Aβ1-42 oligomer elicited a dose-dependent toxicity with a sign of saturation at higher dosages, PDGF-BB failed to protect neurons against Aβ1-42 oligomer induced cytotoxicity. In contrast, Aβ1-42 oligomers strongly inhibit PDGF-BB induced mitogenesis in both SH-SY5Y cells and primary neurons. Further investigation using Western blotting to measure PDGF receptor expression and phosphorylation in SH-SY5Y cells showed that Aβ1-42 oligomer can inhibit PDGF-BB induced phosphorylation of PDGF β-receptor on Tyr1021, a site that is crucial for PLCγ mediated mitogenesis. These findings not only explained the poor neuroprotective effect elicited by PDGF-BB against Aβ1-42 oligomers, but also led to a novel hypothesis that Aβ1-42 oligomer may interfere with neurotrophic factor induced neuronal survival, either selectively or perhaps globally. Further exploration on this hypothesis will be able to shed light on this potentially novel mechanism of pathogenesis in Alzheimer’s disease.

Heparan sulfate (HS) belongs to the glycosaminoglycan family of polysaccharides and is found attached to protein cores on cell surfaces and in the extracellular matrix. The HS backbone consists of alternating hexuronic acid and glucosamine units and undergoes a number of modification reactions creating HS chains with alternating highly and low modified domains, where high degree of modification correlates with high negative charge. Fibroblast growth factors (FGFs) and their receptors (FRs) both bind to HS, which affect formation of the FGF–FR complexes on the cell surfaces. Activated FRs can trigger several intracellular signaling pathways leading thereby to diverse cellular responses. Work presented in this thesis focuses on the effect of HS and its structures on FGF–FR complex formation and FGF-induced signaling. Studies with short, highly modified oligosaccharides and FGF1 and 2 combined with FR1c, 2c, 3c or 4 showed a correlation between the overall degree of modification and amount/stability of FGF–FR complexes. Our findings imply that several HS structures, differently modified but with the same negative charge density are equal in their ability to support complex formation. Co-application of oligosaccharides with FGF2 to HS-deficient cells and investigation of the thereby induced cell signaling confirmed our findings with a cell-free system. The oligosaccharide with the highest modification degree displayed the biggest impact on cell signaling, which was FGF2 concentration dependent. Studies with long HS polysaccharides with preserved high and low modified domains suggest that the proportion between these two types of domains and also the structure of the low modified domains are of importance for the FGF–HS–FR complex formation and cell activation capacity. This work illuminates several aspects in how HS structure influences the interplay between FGFs and FRs and contributes to the understanding of what factors affect a cell’s response following FGF stimulation.

Platelet-derived microparticles, released as a result of platelet activation, promote coagulation through the surface exposure of phosphatidylserine, acting as the catalytic site for the conversion of prothrombin to thrombin by the activated coagulation factors X and V. Although elevated numbers of circulating platelet-derived microparticles can be detected in a number of clinical disorders, the methods for the detection of these microparticles are far from standardised. In addition, recent reports have also speculated that not all microparticles may expose phosphatidylserine, demonstrating that the binding of Annexin V, a phosphatidylserine-specific binding protein, is not detectable on a population of microparticles. The initial stage of this thesis was to establish a flow cytometric method for the detection and enumeration of microparticles based on their capacity to bind Annexin V and to utilise this assay to investigate a number of the issues that have limited assay standardisation. The assay could be performed on either stimulated or unstimulated plasma or whole blood samples. Interestingly, plasma microparticle counts were significantly higher than whole blood microparticle counts. The effects of centrifugation alone could not be attributed as the sole source of this discrepancy. The antigenic characteristics of platelet-derived microparticles were also investigated, with platelet-derived microparticles demonstrated to express the platelet glycoproteins CD31, CD41a, CD42a and CD61. Platelet-derived microparticles also expressed CD42b, and this expression was significantly decreased when compared to their progenitor platelets. The expression of the platelet activation markers CD62p, CD63, CD40L and PAC-1 was dependent upon the sample milieu, suggesting that the centrifugation conditions required to generate platelet-poor plasma may lead to artefactual increases in the expression of platelet activation markers. An investigation of the role of the GpIIb/IIIa complex on the formation of platelet-derived microparticles was also performed. A monoclonal antibody to the GpIIb/IIIa complex (Abciximab) significantly inhibited in vitro collagen-stimulated platelet-derived microparticle formation. Interestingly, platelets obtained from two subjects with impaired GpIIb/IIIa activation, demonstrated normal microparticle formation following collagen stimulation, suggesting that the presence of GpIIb/IIIa complex, but not its activation, is required for collagen-induced microparticle formation. A novel mechanism for microparticle formation was also investigated, with platelet-derived microparticles demonstrated to form in response to the sclerosing agents sodium-tetradecyl sulphate and polidocanol. Interestingly, the removal of plasma proteins by the washing of platelets left platelets more susceptible to sclerosant-induced microparticle formation, suggesting that plasma proteins may protect platelets from microparticle formation. The procoagulant activity of platelet-derived microparticles was also investigated using a novel coagulation assay (XACT) specific for the procoagulant phospholipid. An evaluation of this assay demonstrated a significant correlation between Annexin V binding microparticle counts and procoagulant activity in both whole blood and plasma samples. There was more procoagulant activity in whole blood samples than in plasma samples, suggesting that the procoagulant phospholipid activity was also associated with erythrocytes or leukocytes. To further investigate this phenomenon, a whole blood flow cytometric assay was developed to assess Annexin V binding to erythrocytes, leukocytes, platelets and microparticles. This assay demonstrated that a large proportion of Annexin V binding (51.0%) was associated with erythrocytes. Interestingly, a proportion of the Annexin V binding erythrocytes (24.5%) and leukocytes (78.8%) were also associated with platelet CD61 antigen, suggesting that they also bound a platelet or platelet-derived microparticle. The effect of sample anticoagulant on microparticle procoagulant activity was investigated. Microparticle counts were most stable in EDTA anticoagulated samples, but were stable in sodium citrate for up to 15 minutes following sample collection. The procoagulant activity of microparticles was significantly inhibited by EDTA in collagen-stimulated platelet-rich plasma samples, when compared to sodium citrate anticoagulated samples. Although the initial method used to investigate microparticles was based upon their ability to bind Annexin V, it was consistently observed that a large proportion of events in the size region of a microparticle were Annexin V negative. An investigation was therefore commenced into the procoagulant activity of microparticles based on their capacity to bind Annexin V. The presence of Annexin V negative microparticles was confirmed by flow cytometry and the proportion of microparticles that bound Annexin V was dependent upon type of agonist used to stimulate microparticle formation. Varying the assay constituents (calcium concentration / Annexin V concentration / buffer type) did not alter the proportion of Annexin V binding microparticles. When compared to Annexin V positive microparticles, Annexin V negative microparticles expressed significantly higher levels of CD42b on their surface, but possessed significantly decreased expressions of CD62p, and CD63. A significant correlation between the percentage of Annexin V binding and XACT procoagulant activity was found (p=0.03). Furthermore, Annexin V binding inhibited greater than 98% of procoagulant phospholipid activity, suggesting that Annexin V binding was a true reflection of procoagulant activity. Microparticles could be sorted using either a flow cytometric or magnetic sorting strategy. By electron microscopy, Annexin V negative events isolated following magnetic sorting were vesicular structures and not small platelets or the remnants of activated platelets. In summary, this thesis has demonstrated the ability of the flow cytometer and XACT assays to detect microparticles and their procoagulant activity. It has also shown that the use of Annexin V to detect microparticles may warrant further investigation.

All animals have evolved mechanisms to recognise and eliminate nonself in order to defend against invading pathogens and to prevent chimerism, the fusion between genetically distinct conspecifics. Like other metazoans, sponges are known to rely on sophisticated systems to maintain their self-integrity. As poriferans are also considered one of the most ancient extant metazoan phyla, they represent a critical comparative model for understanding the early evolution of immunity and self/nonself recognition in animals. The Toll-like receptor (TLR) signalling cascade plays a crucial role in immunity, and recent findings in the sponge Suberites domuncula suggest that its origin could predate eumetazoan cladogenesis. My genome and expressed sequence tag (EST) screens of the demosponge Amphimedon queenslandica detected homologues to most components of this pathway, supporting the notion that a primordial TLR signalling cascade emerged at the dawn of the Metazoa. The sponge also encodes a couple of putative TLR-related proteins (AmqIgTIRs) that consist of at least one extracellular immunoglobulin (Ig) and an intracellular Toll/Interleukin-1 receptor/resistance (TIR) domain. The presence of other unconventional TLRs in S. domuncula and in cnidarian representatives, implies that an ancestral TLR probably existed in the last common ancestor of all living metazoans, and independent duplication and divergence events led to the variety of forms observed in animals. Among the putative transcription factors present in Amphimedon, which are known to be activated by the TLR signalling cascade in other eumetazoans, I detected a single member of the Rel/nuclear factor-kappaB (NF-κB) family, AmqNF-κB, which is also the only Rel homology domain (RHD)-containing gene present in the sponge. This gene encodes a protein that is equipped with both a RHD and ankyrin (ANK) repeats, suggesting that the ancestral metazoan NF-κB was configured identically to contemporary vertebrate and sponge forms, and that the truncated NF-κB found in Nematostella vectensis resulted from the secondary loss of ANK. Aside from immunity, the Toll and TLR pathways contribute to a variety of biological processes in bilaterians, however their functions have only been investigated in detail in a limited number of metazoan model organisms. While studies have tested the immune role of various sponge genes, including components of the TLR cascade, no research has yet established whether they are also involved in development. Therefore, I investigated the expression of some of the immunity-related genes I isolated in Amphimedon in a developmental and immune context to shed light on the potential ancestral function(s) of the proteins they encode. Using in situ hybridisation, I demonstrate that AmqIgTIR2, AmqMyD88, AmqTollip, AmqPellino and AmqNF-ĸB are expressed during A. queenslandica early development. In contrast, the spatial and temporal expression of AmqIgTIR1 suggests it might encode a receptor that is specifically involved in the detection of metamorphic cues in larvae. A real-time quantitative PCR (qPCR) study performed on a pool of adult sponge cDNAs indicates that the expression levels of AmqIgTIR1, AmqIgTIR2, AmqMyD88 and AmqTollip are significantly affected by a nine-hour incubation in 50 µg/ml of lipopolysaccharide (LPS), and to a lesser extent by 105 colony forming units (cfu)/ml of live Vibrio harveyi. The gene expression of AmqIgTIR1 and AmqIgTIR2 suggests that they may encode proteins with antagonistic immunological functions. While AmqPellino and AmqNF-ĸB do not appear to be affected by LPS and Vibrio exposure, it is possible that these genes do not participate in the early immune response of poriferans. Together, my data indicate that the sponge genes surveyed might encode proteins that perform developmental, sensory and immunological functions, suggesting their roles could have also been multifaceted in the last common ancestor to all living metazoans. As is observed in other invertebrates, poriferans display an ontogenic shift in allorecognition; genetically different individuals can fuse during early development but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of poriferan allorecognition response. By following the fates of fluorescently tagged cells derived from genetically distinct Amphimedon larvae that are fused together at metamorphosis, I establish that there is a rapid ontogenic shift in the sponge allogeneic response about two weeks after the initiation of metamorphosis. Moreover, the molecular basis of the poriferan allorecognition system is possibly involved in creating differential cell affinities, which underlie the construction of the sponge body plan. Compatible with this scenario is the observation that cells from postlarvae that are allowed to develop for two weeks before contact do not fuse, and form a distinct boundary between genotypes. The molecules responsible for sponge cell reaggregation, the aggregation factors (AFs), have been proposed to drive the allorecognition response in poriferans. Notably, the Amphimedon genome encodes six putative AFs, of which five occur in a cluster. These findings indicate that the polymorphic variation observed in other poriferan AFs is probably the result of allelic variations of multiple genes belonging to the same family.

Impaired implantation and placental development have been implicated in several disorders of pregnancy such as unexplained miscarriage, preeclampsia, and intrauterine growth retardation. Insulin-Like Growth Factor (IGF)-II has previously been shown to promote blastocyst development and placental growth and function. We were interested in how IGF-II interacts with other factors throughout blastocyst development, implantation and placentation in the mouse to improve pregnancy outcome. In vitro embryo culture increases the risk of pregnancy complications associated with poor placentation. Recent research has focussed on optimising the culture conditions to more resemble that of the in vivo environment. IGF-II, Urokinase Plasminogen Activator (uPA) and Plasminogen individually have all been shown to be important for embryo development. However, it is likely that a combination of factors is required to counteract the negative effects of in vitro culture. Here we show that IGF-II, uPA and Plasminogen, in combination, significantly improve mouse blastocyst hatching rates and implantation rates on day 8 and doubles the number of mothers that are pregnant after embryo transfer. Following implantation, IGF-II is suggested to play a role in promoting placental development and function. We demonstrate that IGF-II is co-localised with both IGF receptors throughout early pregnancy in trophoblasts and in the developing blood vessels and adjacent stromal cells in the mesometrial decidua. This suggests that IGF-II may play a role in both decidual angiogenesis and placentation. We suggest that perhaps murine trophoblasts secrete molecules such as IGF-II to promote angiogenesis in the decidua early in pregnancy to compensate for their shallow invasion and allow for adequate trophoblast remodelling later in pregnancy. The first trimester human placenta experiences a low oxygen environment. The Hypoxia-Inducible Factors (HIFs) mediate the response to low oxygen, inducing genes such as IGF-II. Currently, the role of oxygen in mouse placentation, the mechanisms by which HIFs promote placentation or their interaction with IGF-II in the placenta is unknown. Here, we demonstrate that the early mouse implantation site is exposed to low oxygen levels similar to those seen in humans and expresses HIF-1 protein. We were interested then in the interaction between IGF-II, oxygen and HIFs in trophoblasts in vitro. Prolonged exposure to low oxygen reduced trophoblast outgrowth, and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. Interestingly, we found that antisense (as) Hif-1 may mediate the response to prolonged hypoxia in murine trophoblasts. Importantly, Hif-1 and Hif-2 were differentially regulated by oxygen and IGF-II in cultured trophoblast cells suggesting a novel interaction between IGF-II and oxygen. In conclusion, it appears that IGF-II is a central growth factor which interacts with other molecules to regulate a wide variety of process in early pregnancy to promote blastocyst development, implantation and placentation. The results outlined in this thesis demonstrate a novel interaction between IGF-II, uPA and Plasminogen in promoting blastocyst development and implantation which may be used to improve pregnancy outcome following ART. In addition, we have also identified a novel interaction between IGF-II, oxygen and the HIF system which may regulate trophoblast function. This has important implications not only for placental research, but also for cancer research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326731 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Impaired implantation and placental development have been implicated in several disorders of pregnancy such as unexplained miscarriage, preeclampsia, and intrauterine growth retardation. Insulin-Like Growth Factor (IGF)-II has previously been shown to promote blastocyst development and placental growth and function. We were interested in how IGF-II interacts with other factors throughout blastocyst development, implantation and placentation in the mouse to improve pregnancy outcome. In vitro embryo culture increases the risk of pregnancy complications associated with poor placentation. Recent research has focussed on optimising the culture conditions to more resemble that of the in vivo environment. IGF-II, Urokinase Plasminogen Activator (uPA) and Plasminogen individually have all been shown to be important for embryo development. However, it is likely that a combination of factors is required to counteract the negative effects of in vitro culture. Here we show that IGF-II, uPA and Plasminogen, in combination, significantly improve mouse blastocyst hatching rates and implantation rates on day 8 and doubles the number of mothers that are pregnant after embryo transfer. Following implantation, IGF-II is suggested to play a role in promoting placental development and function. We demonstrate that IGF-II is co-localised with both IGF receptors throughout early pregnancy in trophoblasts and in the developing blood vessels and adjacent stromal cells in the mesometrial decidua. This suggests that IGF-II may play a role in both decidual angiogenesis and placentation. We suggest that perhaps murine trophoblasts secrete molecules such as IGF-II to promote angiogenesis in the decidua early in pregnancy to compensate for their shallow invasion and allow for adequate trophoblast remodelling later in pregnancy. The first trimester human placenta experiences a low oxygen environment. The Hypoxia-Inducible Factors (HIFs) mediate the response to low oxygen, inducing genes such as IGF-II. Currently, the role of oxygen in mouse placentation, the mechanisms by which HIFs promote placentation or their interaction with IGF-II in the placenta is unknown. Here, we demonstrate that the early mouse implantation site is exposed to low oxygen levels similar to those seen in humans and expresses HIF-1 protein. We were interested then in the interaction between IGF-II, oxygen and HIFs in trophoblasts in vitro. Prolonged exposure to low oxygen reduced trophoblast outgrowth, and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. Interestingly, we found that antisense (as) Hif-1 may mediate the response to prolonged hypoxia in murine trophoblasts. Importantly, Hif-1 and Hif-2 were differentially regulated by oxygen and IGF-II in cultured trophoblast cells suggesting a novel interaction between IGF-II and oxygen. In conclusion, it appears that IGF-II is a central growth factor which interacts with other molecules to regulate a wide variety of process in early pregnancy to promote blastocyst development, implantation and placentation. The results outlined in this thesis demonstrate a novel interaction between IGF-II, uPA and Plasminogen in promoting blastocyst development and implantation which may be used to improve pregnancy outcome following ART. In addition, we have also identified a novel interaction between IGF-II, oxygen and the HIF system which may regulate trophoblast function. This has important implications not only for placental research, but also for cancer research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326731 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.