Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

“What’s going on here?” The pedagogy of a data analysis session

Data analysis sessions are a common feature of discourse analytic communities, often involving participants with varying levels of expertise to those with significant expertise. Learning how to do data analysis and working with transcripts, however, are often new experiences for doctoral candidates within the social sciences. While many guides to doctoral education focus on procedures associated with data analysis (Heath, Hindmarsh, & Luff, 2010; McHoul & Rapley, 2001; Silverman, 2011; Wetherall, Taylor, & Yates, 2001), the in situ practices of doing data analysis are relatively undocumented. This chapter has been collaboratively written by members of a special interest research group, the Transcript Analysis Group (TAG), who meet regularly to examine transcripts representing audio- and video-recorded interactional data. Here, we investigate our own actual interactional practices and participation in this group where each member is both analyst and participant. We particularly focus on the pedagogic practices enacted in the group through investigating how members engage in the scholarly practice of data analysis. A key feature of talk within the data sessions is that members work collaboratively to identify and discuss ‘noticings’ from the audio-recorded and transcribed talk being examined, produce candidate analytic observations based on these discussions, and evaluate these observations. Our investigation of how talk constructs social practices in these sessions shows that participants move fluidly between actions that demonstrate pedagogic practices and expertise. Within any one session, members can display their expertise as analysts and, at the same time, display that they have gained an understanding that they did not have before. We take an ethnomethodological position that asks, ‘what’s going on here?’ in the data analysis session. By observing the in situ practices in fine-grained detail, we show how members participate in the data analysis sessions and make sense of a transcript.

The 2008 Tibet riots : perspectives, divided group protests and divergent media narratives

Awareness of the power of the mass media to communicate images of protest to global audiences and, in so doing, to capture space in global media discourses is a central feature of the transnational protest movement. A number of protest movements have formed around opposition to concepts and practices that operate beyond national borders, such as neoliberal globalization or threats to the environment. However, transnational protests also involve more geographically discreet issues such as claims to national independence or greater religious or political freedom by groups within specific national contexts. Appealing to the international community for support is a familiar strategy for communities who feel that they are being discriminated against or ignored by a national government.

Facial expression recognition using facial movement features

Facial expression is an important channel for human communication and can be applied in many real applications. One critical step for facial expression recognition (FER) is to accurately extract emotional features. Current approaches on FER in static images have not fully considered and utilized the features of facial element and muscle movements, which represent static and dynamic, as well as geometric and appearance characteristics of facial expressions. This paper proposes an approach to solve this limitation using ‘salient’ distance features, which are obtained by extracting patch-based 3D Gabor features, selecting the ‘salient’ patches, and performing patch matching operations. The experimental results demonstrate high correct recognition rate (CRR), significant performance improvements due to the consideration of facial element and muscle movements, promising results under face registration errors, and fast processing time. The comparison with the state-of-the-art performance confirms that the proposed approach achieves the highest CRR on the JAFFE database and is among the top performers on the Cohn-Kanade (CK) database.

Understanding the role of knowledge in the practice of expert nephrology nurses in Australia

This paper, which is abstracted from a larger study into the acquisition and exercise of nephrology nursing expertise, aims to explore the role of knowledge in expert practice. Using grounded theory methodology, the study involved 17 registered nurses who were practicing in a metropolitan renal unit in New South Wales, Australia. Concurrent data collection and analysis was undertaken, incorporating participants' observations and interviews. Having extensive nephrology nursing knowledge was a striking characteristic of a nursing expert. Expert nurses clearly relied on and utilized extensive nephrology nursing knowledge to practice. Of importance for nursing, the results of this study indicate that domain-specific knowledge is a crucial feature of expert practice.

Wide baseline correspondence extraction beyond local features

Robust, affine covariant, feature extractors provide a means to extract correspondences between images captured by widely separated cameras. Advances in wide baseline correspondence extraction require looking beyond the robust feature extraction and matching approach. This study examines new techniques of extracting correspondences that take advantage of information contained in affine feature matches. Methods of improving the accuracy of a set of putative matches, eliminating incorrect matches and extracting large numbers of additional correspondences are explored. It is assumed that knowledge of the camera geometry is not available and not immediately recoverable. The new techniques are evaluated by means of an epipolar geometry estimation task. It is shown that these methods enable the computation of camera geometry in many cases where existing feature extractors cannot produce sufficient numbers of accurate correspondences.

An indication that non-informative vision eliminates the Kinaesthetic Fusion Effect

This study investigated the Kinaesthetic Fusion Effect (KFE) first described by Craske and Kenny in 1981. The current study did not replicate these findings. Participants did not perceive any reduction in the sagittal separation of a button pressed by the index finger of one arm and a probe touching the other, following repeated exposure to the tactile stimuli present on both unseen arms. This study’s failure to replicate the widely-cited KFE as described by Craske et al. (1984) suggests that it may be contingent on several aspects of visual information, especially the availability of a specific visual reference, the role of instructions regarding gaze direction, and the potential use of a line of sight strategy when referring felt positions to an interposed surface. In addition, a foreshortening effect was found; this may result from a line-of-sight judgment and represent a feature of the reporting method used. The transformed line of sight data were regressed against the participant reported values, resulting in a slope of 1.14 (right arm) and 1.11 (left arm), and r > 0.997 for each. The study also provides additional evidence that mis-perceptions of the mediolateral position of the limbs specifically their separation and consistent with notions of Gestalt grouping, is somewhat labile and can be influenced by active motions causing touch of one limb by the other. Finally, this research will benefit future studies that require participants to report the perceived locations of the unseen limbs.

Asymptotic and numerical results for a model of solvent-dependent drug diffusion through polymeric spheres

A model for drug diffusion from a spherical polymeric drug delivery device is considered. The model contains two key features. The first is that solvent diffuses into the polymer, which then transitions from a glassy to a rubbery state. The interface between the two states of polymer is modelled as a moving boundary, whose speed is governed by a kinetic law; the same moving boundary problem arises in the one-phase limit of a Stefan problem with kinetic undercooling. The second feature is that drug diffuses only through the rubbery region, with a nonlinear diffusion coefficient that depends on the concentration of solvent. We analyse the model using both formal asymptotics and numerical computation, the latter by applying a front-fixing scheme with a finite volume method. Previous results are extended and comparisons are made with linear models that work well under certain parameter regimes. Finally, a model for a multi-layered drug delivery device is suggested, which allows for more flexible control of drug release.

Deceit and Bruno Taut's Glashaus

The historical context surrounding Bruno Taut's Glashaus has been established through work of authors like Reyner Banham, Dennis Sharp and Ian Boyd-Whyte. However, these English language translations, are mostly derived from secondary sources such as Adolf Behne and Paul Scheerbart. Surprisingly, Taut's own writings largely do not feature in this prevailing account of his work. Since 1990, strong doubts have arisen about this conventional picture of Taut's Glashaus. Manfred spiedel, for instance, minimizes Paul Scheerbart's contribution to the design by arguing that Scheerbart met Taut only a few months before the construction of the Glashaus, that is, after Taut had finished his preliminary sketches. Kurt Junghanns goes further and asserts that the Glashaus design was complete beefore Taut and Scheerbart ever met. In 2005, Kai Gutschow published The Culture of Criticism: Adolf Behne and the Development of Modern Architecture in Germany, 1910 - 1914. Most startling, Gutschow asserts that Behne acts as the propagandist for the Glashaus by fabricating its link with Expressionism. This is particularly troubling because nobody contributed more to establishing the link between the Glashaus, Bruno Taut and Expressionism than Behne. As a result of this new evidence, this paper concurs that the established historical understanding of the Glashaus is flawed. By returning to Taut's own writings, a reinterpretation can be offered that strongly links the Glashaus to the Victoria regia lily and Strasbourg Cathedral. The significance of this approach is that it re-establishes Taut's own rational behind the design of the Glashaus, and thus contributes to the re-evaluation of the generally accepted histories of the Modern movement.

Online discovery of similarity mappings

We consider the problem of choosing, sequentially, a map which assigns elements of a set A to a few elements of a set B. On each round, the algorithm suffers some cost associated with the chosen assignment, and the goal is to minimize the cumulative loss of these choices relative to the best map on the entire sequence. Even though the offline problem of finding the best map is provably hard, we show that there is an equivalent online approximation algorithm, Randomized Map Prediction (RMP), that is efficient and performs nearly as well. While drawing upon results from the "Online Prediction with Expert Advice" setting, we show how RMP can be utilized as an online approach to several standard batch problems. We apply RMP to online clustering as well as online feature selection and, surprisingly, RMP often outperforms the standard batch algorithms on these problems.

Robotics, Vision and Control : Fundamental Algorithms in MATLAB

The practice of robotics and computer vision each involve the application of computational algorithms to data. The research community has developed a very large body of algorithms but for a newcomer to the field this can be quite daunting. For more than 10 years the author has maintained two open-source MATLAB® Toolboxes, one for robotics and one for vision. They provide implementations of many important algorithms and allow users to work with real problems, not just trivial examples. This new book makes the fundamental algorithms of robotics, vision and control accessible to all. It weaves together theory, algorithms and examples in a narrative that covers robotics and computer vision separately and together. Using the latest versions of the Toolboxes the author shows how complex problems can be decomposed and solved using just a few simple lines of code. The topics covered are guided by real problems observed by the author over many years as a practitioner of both robotics and computer vision. It is written in a light but informative style, it is easy to read and absorb, and includes over 1000 MATLAB® and Simulink® examples and figures. The book is a real walk through the fundamentals of mobile robots, navigation, localization, arm-robot kinematics, dynamics and joint level control, then camera models, image processing, feature extraction and multi-view geometry, and finally bringing it all together with an extensive discussion of visual servo systems.

Evaluation of texture and geometry for dimensional facial expression recognition

Facial expression recognition (FER) algorithms mainly focus on classification into a small discrete set of emotions or representation of emotions using facial action units (AUs). Dimensional representation of emotions as continuous values in an arousal-valence space is relatively less investigated. It is not fully known whether fusion of geometric and texture features will result in better dimensional representation of spontaneous emotions. Moreover, the performance of many previously proposed approaches to dimensional representation has not been evaluated thoroughly on publicly available databases. To address these limitations, this paper presents an evaluation framework for dimensional representation of spontaneous facial expressions using texture and geometric features. SIFT, Gabor and LBP features are extracted around facial fiducial points and fused with FAP distance features. The CFS algorithm is adopted for discriminative texture feature selection. Experimental results evaluated on the publicly accessible NVIE database demonstrate that fusion of texture and geometry does not lead to a much better performance than using texture alone, but does result in a significant performance improvement over geometry alone. LBP features perform the best when fused with geometric features. Distributions of arousal and valence for different emotions obtained via the feature extraction process are compared with those obtained from subjective ground truth values assigned by viewers. Predicted valence is found to have a more similar distribution to ground truth than arousal in terms of covariance or Bhattacharya distance, but it shows a greater distance between the means.

On the data consumption benefits of accepting increased uncertainty

In the context of learning paradigms of identification in the limit, we address the question: why is uncertainty sometimes desirable? We use mind change bounds on the output hypotheses as a measure of uncertainty and interpret ‘desirable’ as reduction in data memorization, also defined in terms of mind change bounds. The resulting model is closely related to iterative learning with bounded mind change complexity, but the dual use of mind change bounds — for hypotheses and for data — is a key distinctive feature of our approach. We show that situations exist where the more mind changes the learner is willing to accept, the less the amount of data it needs to remember in order to converge to the correct hypothesis. We also investigate relationships between our model and learning from good examples, set-driven, monotonic and strong-monotonic learners, as well as class-comprising versus class-preserving learnability.

SwarmPS: Rapid, semi-automated single particle selection software

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that ∼104–105 particle projections are required to attain a 3 Å resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present SwarmPS, a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (∼102), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. SwarmPS is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.

Pattern recognition using invariants defined from higher order spectra : 2-D image inputs

A new algorithm for extracting features from images for object recognition is described. The algorithm uses higher order spectra to provide desirable invariance properties, to provide noise immunity, and to incorporate nonlinearity into the feature extraction procedure thereby allowing the use of simple classifiers. An image can be reduced to a set of 1D functions via the Radon transform, or alternatively, the Fourier transform of each 1D projection can be obtained from a radial slice of the 2D Fourier transform of the image according to the Fourier slice theorem. A triple product of Fourier coefficients, referred to as the deterministic bispectrum, is computed for each 1D function and is integrated along radial lines in bifrequency space. Phases of the integrated bispectra are shown to be translation- and scale-invariant. Rotation invariance is achieved by a regrouping of these invariants at a constant radius followed by a second stage of invariant extraction. Rotation invariance is thus converted to translation invariance in the second step. Results using synthetic and actual images show that isolated, compact clusters are formed in feature space. These clusters are linearly separable, indicating that the nonlinearity required in the mapping from the input space to the classification space is incorporated well into the feature extraction stage. The use of higher order spectra results in good noise immunity, as verified with synthetic and real images. Classification of images using the higher order spectra-based algorithm compares favorably to classification using the method of moment invariants