Developmental self-construction and -configuration of functional neocortical neuronal networks.

The prenatal development of neural circuits must provide sufficient configuration to support at least a set of core postnatal behaviors. Although knowledge of various genetic and cellular aspects of development is accumulating rapidly, there is less systematic understanding of how these various processes play together in order to construct such functional networks. Here we make some steps toward such understanding by demonstrating through detailed simulations how a competitive co-operative ('winner-take-all', WTA) network architecture can arise by development from a single precursor cell. This precursor is granted a simplified gene regulatory network that directs cell mitosis, differentiation, migration, neurite outgrowth and synaptogenesis. Once initial axonal connection patterns are established, their synaptic weights undergo homeostatic unsupervised learning that is shaped by wave-like input patterns. We demonstrate how this autonomous genetically directed developmental sequence can give rise to self-calibrated WTA networks, and compare our simulation results with biological data.

Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation

Spinal cord neuronal restricted progenitor (NRP) cells, when transplanted into the neonatal anterior forebrain subventricular zone, migrate to distinct regions throughout the forebrain including the olfactory bulb, frontal cortex, and occipital cortex but not to the hippocampus. Their migration pattern and differentiation potential is distinct from anterior forebrain subventricular zone NRPs. Irrespective of their final destination, NRP cells do not differentiate into glia. Rather they synthesize neurotransmitters, acquire region-specific phenotypes, and receive synapses from host neurons after transplantation. Spinal cord NRPs express choline acetyl transferase even in regions where host neurons do not express this marker. The restricted distribution of transplanted spinal cord NRP cells and their acquisition of varied region-specific phenotypes suggest that their ultimate fate and phenotype is dictated by a combination of intrinsic properties and extrinsic cues from the host.

Molecular heterogeneity of progenitors and radial migration in the developing cerebral cortex revealed by transgene expression.

We have analyzed the developmental pattern of beta-galactosidase (beta-gal) expression in the cerebral cortex of the beta 2nZ3'1 transgenic mouse line, which was generated using regulatory elements of the beta 2-microglobulin gene and shows ectopic expression in nervous tissue. From embryonic day 10 onward, beta-gal was expressed in the medial and dorsal cortices, including the hippocampal region, whereas lateral cortical areas were devoid of labeling. During the period of cortical neurogenesis (embryonic days 11-17), beta-gal was expressed by selective precursors in the proliferative ventricular zone of the neocortex and hippocampus, as well as by a number of migrating and postmigratory neurons arranged into narrow radial stripes above the labeled progenitors. Thus, the transgene labels a subset of cortical progenitors and their progeny. Postnatally, radial clusters of beta-gal-positive neurons were discernible until postpartum day 10. At this age, the clusters were 250 to 500 microns wide, composed of neurons spanning all the cortical layers and exhibiting several neuronal phenotypes. These data suggest molecular heterogeneity of cortical progenitors and of the cohorts of postmitotic neurons originating from them, which implies intrinsic molecular mosaicism in both cortical progenitors and developing neurons. Furthermore, the data show that neurons committed to the expression of the transgene migrate along very narrow, radial stripes.

Dissecting signaling pathways that control glia migration in the developing Drosophila retina

The function of a complex nervous system relies on an intricate interaction between neurons and glial cells. However, as glial cells are generally born distant from the place where they settle, molecular cues are important to direct their migration. Glial cell migration is important in both normal development and disease, thus current research in the laboratory has been focused on dissecting regulatory events underlying that crucial process. With this purpose, the Drosophila eye imaginal disc has been used as a model. In response to neuronal photoreceptor differentiation, glial cells migrate from the CNS into the eye disc where they act to correctly wrap axons. To ensure proper development, attractive and repulsive signals must coordinate glial cell migration. Importantly, one of these signals is Bnl, a Fibroblast Growth Factor (FGF) ligand expressed by retinal progenitor cells that was suggested to act as a non-autonomous negative regulator of excessive glial cell migration (overmigration) by binding and activating the Btl receptor expressed by glial cells. Through the experimental results described in chapter 3 we gained a detailed insight into the function of bnl in eye disc growth, photoreceptor development, and glia migration. Interestingly, we did not find a direct correlation between the defects on the ongoing photoreceptors and the glia overmigration phenotype; however, bnl knockdown caused apoptosis of eye progenitor cells what was strongly correlated with glia migration defects. Glia overmigration due to Bnl down-regulation in eye progenitor cells was rescued by inhibiting the pro-apoptotic genes or caspases activity, as well as, by depleting JNK or Dp53 function in retinal progenitor cells. Thus, we suggest a cross-talk between those developmental signals in the control of glia migration at a distance. Importantly, these results suggest that Bnl does not control glial migration in the eye disc exclusively through its ability to bind and activate its receptor Btl in glial cells. We also discuss possible biological roles for the glia overmigration in the bnl knockdown background. Previous results in the lab showed an interaction between dMyc, a master regulator of tissue growth, and Dpp, a Transforming Growth Factor-β important for retinal patterning and for accurate glia migration into the eye disc. Thus, we became interested in understanding putative relationships between Bnl and dMyc. In chapter 4, we show that they positively cooperate in order to ensure proper development of the eye disc. This work highlights the importance of the FGF signaling in eye disc development and reveals a signaling network where a range of extra- and intra-cellular signals cooperate to non-autonomously control glial cell migration. Therefore, such inter-relations could be important in other Drosophila cellular contexts, as well as in vertebrate tissue development.

Relaxin stimulates leukocyte adhesion and migration through a relaxin receptor LGR7-dependent mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration

Settlement geography of African refugee communities in Southeast Queensland : an analysis of residential distribution and secondary migration

Before 2001, most Africans immigrating to Australia were white South Africans and Zimbabweans who arrived as economic and family-reunion migrants (Cox, Cooper & Adepoju, 1999). Black African communities are a more recent addition to the Australian landscape, with most entering Australia as refugees after 2001. African refugees are a particularly disadvantaged immigrant group, which the Department of Immigration and Multicultural Affairs (in the Community Relations Commission of New South Wales, 2006) suggests require high levels of settlement support (p.23). Decision makers and settlement service providers need to have settlement data on the communities so that they can be effective in planning, budgeting and delivering support where it is most needed. Settlement data are also useful for determining the challenges that these communities face in trying to establish themselves in resettlement. There has been no verification of existing secondary data sources, however, or previous formal study of African refugee settlement geography in Southeast Queensland. This research addresses the knowledge gap by using a mixed-method approach to identify and describe the distribution and population size of eight African communities in Southeast Queensland, examine secondary migration patterns in these communities and assess the relationship between these geographic features and housing, a critical factor in successful settlement. Significant discrepancies exist between the primary data gathered in the study and existing secondary data relating to population size and distribution of the communities. Results also reveal a tension between the socio-cultural forces and the housing and economic imperatives driving secondary migration in the communities, and a general lack of engagement by African refugees with structured support networks. These findings have a wide range of implications for policy and for groups that provide settlement support to these communities.

Migration of breast cancer cells : understanding the roles of volume exclusion and cell-to-cell adhesion

We study MCF-7 breast cancer cell movement in a transwell apparatus. Various experimental conditions lead to a variety of monotone and nonmonotone responses which are difficult to interpret. We anticipate that the experimental results could be caused by cell-to-cell adhesion or volume exclusion. Without any modeling, it is impossible to understand the relative roles played by these two mechanisms. A lattice-based exclusion process random-walk model incorporating agent-to-agent adhesion is applied to the experimental system. Our combined experimental and modeling approach shows that a low value of cell-to-cell adhesion strength provides the best explanation of the experimental data suggesting that volume exclusion plays a more important role than cell-to-cell adhesion. This combined experimental and modeling study gives insight into the cell-level details and design of transwell assays.

Mechanistic investigations into interactions between IGF-I and IGFBPs and their impact on facilitating cell migration on vitronectin

Numerous studies have reported links between insulin-like growth factors (IGFs) and the extra-cellular matrix protein vitronectin (VN). We ourselves have reported that IGF-I binds to VN via IGF-binding proteins (IGFBPs) to stimulate HaCaT and MCF-7 cell migration. Here, we detail the functional evaluation of IGFBP-1, -2, -3, -4 and -6 in the presence and absence of IGF-I and VN. The data presented here, combined with our prior data on IGFBP-5, suggest that IGFBP-3, -4 and -5 are the most effective at stimulating cell migration in combination with IGF-I and VN. In addition, we demonstrate that different regions within IGFBP-3 and -4 are critical for complex formation. Furthermore, we examine whether multi-protein complexes of IGF-I and IGFBPs associated with fibronectin and collagen IV are also able to enhance functional biological responses.

Insulin-like growth factor-i : vitronectin complex-induced changes in gene expression effect breast cell survival and migration

Recent studies have demonstrated that IGF-I associates with VN through IGF-binding proteins (IGFBP) which in turn modulate IGF-stimulated biological functions such as cell proliferation, attachment and migration. Since IGFs play important roles in transformation and progression of breast tumours, we aimed to describe the effects of IGF-I:IGFBP:VN complexes on breast cell function and to dissect mechanisms underlying these responses. In this study we demonstrate that substrate-bound IGF-I:IGFBP:VN complexes are potent stimulators of MCF-7 breast cell survival, which is mediated by a transient activation of ERK/MAPK and sustained activation of PI3-K/AKT pathways. Furthermore, use of pharmacological inhibitors of the MAPK and PI3-K pathways confirms that both pathways are involved in IGF-I:IGFBP:VN complex-mediated increased cell survival. Microarray analysis of cells stimulated to migrate in response to IGF-I:IGFBP:VN complexes identified differential expression of genes with previously reported roles in migration, invasion and survival (Ephrin-B2, Sharp-2, Tissue-factor, Stratifin, PAI-1, IRS-1). These changes were not detected when the IGF-I analogue (\[L24]\[A31]-IGF-I), which fails to bind to the IGF-I receptor, was substituted; confirming the IGF-I-dependent differential expression of genes associated with enhanced cell migration. Taken together, these studies have established that IGF-I:IGFBP:VN complexes enhance breast cell migration and survival, processes central to facilitating metastasis. This study highlights the interdependence of ECM and growth factor interactions in biological functions critical for metastasis and identifies potential novel therapeutic targets directed at preventing breast cancer progression.

Mental health of newly arrived Burmese refugees in Australia : contributions of pre-migration and post-migration experience

Objective: This study documents the mental health status of people from Burmese refugee backgrounds, recently arrived in Australia; then examines the contributions of gender, premigration and postmigration factors in predicting mental health. Method: Structured interviews, including a demographic questionnaire, the Harvard Trauma Questionnaire, Postmigration Living Difficulties Checklist and Hopkins Symptom Checklist assessed premigration trauma, postmigration living difficulties, depression, anxiety, somatisation and traumatisation symptoms in a sample of 70 adults across five Burmese ethnic groups. Results: Substantial proportions of participants reported psychological distress in symptomatic ranges including: posttraumatic stress disorder (9%); anxiety (20%), and; depression (36%), as well as significant symptoms of somatisation (37%). Participants reported multiple and severe premigration traumas. Postmigration living difficulties of greatest concern included communication problems and worry about family not in Australia. Gender did not predict mental health. Level of exposure to traumatic events and postmigration living difficulties each made unique and relatively equal contributions to traumatisation symptoms. Postmigration living difficulties made unique contributions to depression, anxiety and somatisation symptoms. Conclusions: While exposure to traumatic events impacted on participants’ mental wellbeing, postmigration living difficulties had greater salience in predicting mental health outcomes of people from Burmese refugee backgrounds. Reported rates of posttraumatic stress disorder symptoms were consistent with a large review of adults across seven western countries. High levels of somatisation pointed to a nuanced expression of distress. Findings have implications for service provision in terms of implementing appropriate interventions to effectively meet the needs of this newly arrived group in Australia.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

A Raman spectroscopic study of bukovskýite Fe2(AsO4)(SO4)(OH)•7H2O-a mineral phase with a significant role in arsenic migration

The Raman spectrum of bukovskýite, Fe3+2(OH)(SO4)(AsO4)•7H2O has been studied and compared with the Raman spectrum of an amorphous gel containing specifically Fe, As and S elements and is understood as an intermediate product in the formation of bukovskýite. Observed bands are assigned to the stretching and bending vibrations of (SO4)2- and (AsO4)3- units, stretching and bending vibrations and librational modes of hydrogen bonded water molecules, stretching and bending vibrations of hydrogen bonded (OH)- ions and Fe3+-(O,OH) units. Approximate range of O-H...O hydrogen bond lengths is inferred from the Raman spectra. Raman spectra of crystalline bukovskýite and of the amorphous gel differ in that the bukovskýite spectrum is more complex, observed bands are sharp, the degenerate bands of (SO4)2- and (AsO4)3- are split and more intense. Lower wavenumbers of  H2O bending vibration in the spectrum of the amorphous gel may indicate the presence of weaker hydrogen bonds compared with those in bukovskýite.