Sensitivity of edge detection methods for quantifying cell migration assays

Quantitative imaging methods to analyze cell migration assays are not standardized. Here we present a suite of two–dimensional barrier assays describing the collective spreading of an initially–confined population of 3T3 fibroblast cells. To quantify the motility rate we apply two different automatic image detection methods to locate the position of the leading edge of the spreading population after 24, 48 and 72 hours. These results are compared with a manual edge detection method where we systematically vary the detection threshold. Our results indicate that the observed spreading rates are very sensitive to the choice of image analysis tools and we show that a standard measure of cell migration can vary by as much as 25% for the same experimental images depending on the details of the image analysis tools. Our results imply that it is very difficult, if not impossible, to meaningfully compare previously published measures of cell migration since previous results have been obtained using different image analysis techniques and the details of these techniques are not always reported. Using a mathematical model, we provide a physical interpretation of our edge detection results. The physical interpretation is important since edge detection algorithms alone do not specify any physical measure, or physical definition, of the leading edge of the spreading population. Our modeling indicates that variations in the image threshold parameter correspond to a consistent variation in the local cell density. This means that varying the threshold parameter is equivalent to varying the location of the leading edge in the range of approximately 1–5% of the maximum cell density.

Cell migration and proliferation on homogeneous and non-homogeneous domains : modelling on the scale of individuals and populations

Cell migration is a behaviour critical to many key biological effects, including wound healing, cancerous cell invasion and morphogenesis, the development of an organism from an embryo. However, given that each of these situations is distinctly different and cells are extremely complicated biological objects, interest lies in more basic experiments which seek to remove conflating factors and present a less complex environment within which cell migration can be experimentally examined. These include in vitro studies like the scratch assay or circle migration assay, and ex vivo studies like the colonisation of the hindgut by neural crest cells. The reduced complexity of these experiments also makes them much more enticing as problems to mathematically model, like done here. The primary goal of the mathematical models used in this thesis is to shed light on which cellular behaviours work to generate the travelling waves of invasion observed in these experiments, and to explore how variations in these behaviours can potentially predict differences in this invasive pattern which are experimentally observed when cell types or chemical environment are changed. Relevant literature has already identified the difficulty of distinguishing between these behaviours when using traditional mathematical biology techniques operating on a macroscopic scale, and so here a sophisticated individual-cell-level model, an extension of the Cellular Potts Model (CPM), is been constructed and used to model a scratch assay experiment. This model includes a novel mechanism for dealing with cell proliferations that allowed for the differing properties of quiescent and proliferative cells to be implemented into their behaviour. This model is considered both for its predictive power and used to make comparisons with the travelling waves which result in more traditional macroscopic simulations. These comparisons demonstrate a surprising amount of agreement between the two modelling frameworks, and suggest further novel modifications to the CPM that would allow it to better model cell migration. Considerations of the model’s behaviour are used to argue that the dominant effect governing cell migration (random motility or signal-driven taxis) likely depends on the sort of invasion demonstrated by cells, as easily seen by microscopic photography. Additionally, a scratch assay simulated on a non-homogeneous domain consisting of a ’fast’ and ’slow’ region is also used to further differentiate between these different potential cell motility behaviours. A heterogeneous domain is a novel situation which has not been considered mathematically in this context, nor has it been constructed experimentally to the best of the candidate’s knowledge. Thus this problem serves as a thought experiment used to test the conclusions arising from the simulations on homogeneous domains, and to suggest what might be observed should this non-homogeneous assay situation be experimentally realised. Non-intuitive cell invasion patterns are predicted for diffusely-invading cells which respond to a cell-consumed signal or nutrient, contrasted with rather expected behaviour in the case of random-motility-driven invasion. The potential experimental observation of these behaviours is demonstrated by the individual-cell-level model used in this thesis, which does agree with the PDE model in predicting these unexpected invasion patterns. In the interest of examining such a case of a non-homogeneous domain experimentally, some brief suggestion is made as to how this could be achieved.

Fractional models for the migration of biological cells in complex spatial domains

Travelling wave phenomena are observed in many biological applications. Mathematical theory of standard reaction-diffusion problems shows that simple partial differential equations exhibit travelling wave solutions with constant wavespeed and such models are used to describe, for example, waves of chemical concentrations, electrical signals, cell migration, waves of epidemics and population dynamics. However, as in the study of cell motion in complex spatial geometries, experimental data are often not consistent with constant wavespeed. Non-local spatial models have successfully been used to model anomalous diffusion and spatial heterogeneity in different physical contexts. In this paper, we develop a fractional model based on the Fisher-Kolmogoroff equation and analyse it for its wavespeed properties, attempting to relate the numerical results obtained from our simulations to experimental data describing enteric neural crest-derived cells migrating along the intact gut of mouse embryos. The model proposed essentially combines fractional and standard diffusion in different regions of the spatial domain and qualitatively reproduces the behaviour of neural crest-derived cells observed in the caecum and the hindgut of mouse embryos during in vivo experiments.

Introduction : the anthropology of migration

This introduction to the volume places the study of migration within the wider processes of social change and the complex actions of class, gender and ethnicity that are integrated into the experience of displacement. It argues that the articles in this volume contribute important new ethnographic and theoretical analyses to our knowledge and understanding of the constraints and the opportunities provided by cultural diversity in several societies by elaborating upon recent advances made in conceptualisations of migrancy. It places particular emphasis on developing phenomenological approaches to migration studies which incorporate the lived experience of migrants into any analysis. It also suggests that redressing the neglect of this area of study in Australia has the possibility of generating informed interruptions into the current political shift towards divisive public debates on immigration and cultural pluralism.

Generate : Youth Culture and Migration Heritage in Western Sydney

Executive Summary This report is the first in-depth exploration of identity and popular culture among Middle Eastern and Asian youth. It documents preliminary research findings on the contribution of Middle Eastern and Asian youth to Sydney’s cultural life and migration heritage. While young people from these communities, the largest migrant communities in NSW, are often negatively portrayed, this research has focused on their social practices of cultural invention, opening up new and creative means of mobilising cultural difference. These young people’s cultural negotiations between migrant family background and the wider society require real engagement with difference and provide rich resources for invigorating the multicultural fabric of the nation. Their repertoire of cultural skills and their involvement in different cultural worlds are often viewed as evidence of not ‘belonging’ to the mainstream or dominant culture. However, the results of our research reveal that the ‘in-betweenness’ of these young people often enables them to move easily between different social and cultural groupings, embracing cultural diversity as inherent and integral to their everyday experience, that is, ‘normal’ to urban life. In this report, we document the changing nature of friendship networks and family relations, the particular meanings and uses of different languages and expressions, and the patterns of consumption of Middle Eastern and Asian youth. In these everyday activities these young people contribute to a changing migration heritage and are redefining what it means to be Australian.

Quantifying alterations in cell migration : tracking fluorescently-tagged migrating cells by FACs and live-imaging

Cell migration is fundamental to many different physiological processes including embryonic development, inflammation and wound healing. Given the range and importance cell migration plays a number of assays have been developed to measure different aspects of cell migration. Here we describe two different methods to analyze cell migration. The first method analyzes the migration of fluorescently tagged cells using Boyden chambers and FACs and the second looks at migration properties using time-lapse microscopy.

Help-seeking strategies of victim/survivors of human trafficking involving partner migration

Victim/survivors of human trafficking involving partner migration employ diverse help-seeking strategies, both formal and informal, to exit their exploitative situations. Drawing on primary research conducted by Lyneham and Richards (forthcoming), the authors highlight the importance of educating the community and professionals from a wide range of sectors—including health, mental health, child protection, social welfare, social work, domestic violence, migration, legal and law enforcement services—about human trafficking and the help-seeking strategies of victims/survivors in order to support them to leave exploitative situations. Enhancing Australia’s knowledge of victim/survivors’ help-seeking strategies will better inform government and community responses to this crime, improve detection and identification of human trafficking matters and subsequent referral to appropriate victim services.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

Performance-driven live migration of multiple virtual machines in datacenters

Live migration of multiple Virtual Machines (VMs) has become an indispensible management activity in datacenters for application performance, load balancing, server consolidation. While state-of-the-art live VM migration strategies focus on the improvement of the migration performance of a single VM, little attention has been given to the case of multiple VMs migration. Moreover, existing works on live VM migration ignore the inter-VM dependencies, and underlying network topology and its bandwidth. Different sequences of migration and different allocations of bandwidth result in different total migration times and total migration downtimes. This paper concentrates on developing a multiple VMs migration scheduling algorithm such that the performance of migration is maximized. We evaluate our proposed algorithm through simulation. The simulation results show that our proposed algorithm can migrate multiple VMs on any datacenter with minimum total migration time and total migration downtime.

Identification of a long non-coding RNA gene, GHSROS (growth hormone secretagogue receptor opposite strand), which stimulates cell migration in non-small cell lung cancer cell lines

The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

Formation of the heterocumulene anion SCCCN- by a cyano migration from the radical anion of 1,2-dicyanoethylenedithiolate

The anionic heterocumulene SCCCN- was generated in the gas phase by collisional activation of the radical anion of 1,2-dicyanoethylenedithiolate. The mechanism of this reaction, as well as the structures of neutral and anionic products, was investigated by hybrid density functional theory (DFT) calculations. Dissociation to form SCCCN- and SCN is proposed to occur by a radical directed cyano migration reaction, with calculations suggesting this is the lowest energy fragmentation pathway available to the precursor anion. In contrast, the even-electron protonated 1,2-dicyanoethylenedithiolate anion fragmented by loss of HCN.

Ion-molecule reactions reveal facile radical migration in peptides

Ion-molecule reactions between molecular oxygen and peptide radicals in the gas phase demonstrate that radical migration occurs easily within large biomolecules without addition of collisional activation energy.

Anionic migration reactions in aromatic systems effected by collisional activation : Deprotonated anilides containing methoxycarbonyl substituents

Long-range cross-ring reactions are of minor importance in the collision-induced mass spectra (MS/MS) of [M - H]- ions of CH2OCO-C6H4-NHCOR systems: e.g. the loss of 'CD3CO2CH3' from CH3OCO-C6H4-(N) over bar COCD3. Major processes involve (i) losses of radicals to form stable radical anions, e.g. loss of a ring hydrogen atom and losses from the ester (CH3 ., CH3O . and . CO2CH3), (ii) losses of neutral molecules from the amide moiety [e.g. CO (R = H) and CH2CO (R = CH3), and proximity effects when the two substituents are ortho [e.g. loss of (CH3OD+CO2) from o-CH3OCO-C6H4 (N) over bar COCD3].

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.