Manipulation of haemopoietic stem cells for clinical use

Normal haemopoiesis derives from a bone marrow pool of pluripotent stem cells capable of self renewal and multilineage differentiation. Further developments in autografting and gene therapy will rely on a clearer definition and understanding of these primitive cells and an ability to manipulate them effectively. Phenotypic and functional differences between bone marrow and peripheral blood stem cells have been identified and exploited. Techniques for the mobilisation and harvesting of stem cells are established in clinical practice but consensus is lacking on the optimum approach for different diseases. In malignancy, there is great interest in removing contaminating tumour cells from harvests in the hope of reducing relapse rates. Stem cell selection using the CD34 antigen has been commonly used for such purging strategies. The concentration of stem cells produced by CD34 selection provides an ideal product for further manipulation by cell expansion and /or genetic modification. These techniques should facilitate the future engineering of more specific therapeutic cell products.I have examined three specific aspects of the manipulation of haemopoietic stem cells with the aim of generating clinically useful therapeutic products. These were: a disease -specific mobilisation regimen in CML, clinical scale CD34 selection and transplantation in myeloma and genetic modification of stem cells to alter their function using PNH as a model disease.A disease -specific mobilisation procedure was studied in patients with CML in an attempt to collect mainly normal progenitors for subsequent transplantation. This in vivo purging strategy relies on the differential mobilisation of Ph +ve/Ph -ve stem cells following myelosuppression. The Hydroxyurea /G-CSF regimen used was markedly less toxic than existing methods and the results compared favourably, with 28% of the harvests entirely Ph -ve and 56% showing a major response. Cytogenetic responses have been demonstrated post transplant in some patients.Clinical scale ex vivo tumour purging was performed in myeloma patients by CD34 selection using an immunoaffinity column (CeprateTM). This procedure can effect a 3 -4 log tumour reduction and also yields cells suitable for further laboratory manipulation. I wished to confirm that the method was clinically acceptable. All but one patient achieved an adequate CD34 +ve cell dose for transplant post selection and engraftment was normal, confirming the safety and efficacy of the approach. Genetic modification of peripheral blood stem cells was achieved using a retroviral construct (CD59 -TM) capable of ameliorating the complement sensitivity in FNn. This was used successfully to transduce CFU -GM and BFU-E in peripheral blood and stem cell harvests from patients with haematological malignancy. Transduction efficiency was maximised by multiple cycles of infection in the presence of 113, 116 and SCF. A technique was developed for the simultaneous assessment of cell complement lysis and surface phenotype by flow cytometry to detect the presence and function of the novel construct. In FMI, the most primitive peripheral blood stem cells are of normal phenotype which has prompted suggestions from some authors that they might be collected and used for autologous transplantation. I have shown however, that G-CSF mobilises mainly cells of PM phenotype in these patients. This, combined with the observation that the overall progenitor numbers in these hypoplastic patients are low suggests that the prospects for autografting or gene therapy are poor in this disease

A Collection and Re-creation of Bahamian Traditional Dances

This research project provides a collection and re-creation of eleven traditional Bahamian dances. Since the early eighteenth century,Bahamian folk dancing has been an integral part of the native's social life and culture and has been heavily influenced by three cultural groups: African, American and British. People from these three cultures were the first immigrants to the island and their folk dances form the underlying structural base of the Bahamian folk dance tradition. Eventually these cultural elements were synthesised into a distinctive Bahamian folk style. In comparison to the amount of traditional dance information available from other Caribbean countries, the documentation on Bahamian folk dance tradition is sketchy at best. Adding to the lack of documentation is a continued decline in the spontaneous practice and cultural performances of these dances, plus little or no regular instruction to allow for the dances' survival in the culture. A sad consequence of cultural change is the loss or distortion of folk art. This is the present condition of the majority of traditional Bahamian folk dances. New trends in music as well as increased urbanization are causing many Bahamians to ignore or abandon their culture and heritage and as a result the dances which were once an important part of Bahamian society are now dying out. The awareness of the present condition of the dances has given me the necessary incentive to study ,examine and document the Bahamian Folk dance tradition. Central to this research project are two documentary video tape recordings of the Bahamian traditional dances. These tapes provide: necessary historical background,a step by step introduction to Bahamian dance, and important clues to the origins of them. The first tape explores the social,historical and cultural framework from which the Bahamian traditional dances have evolved. The second tape continues with a careful examination and documentation of each of the dances that will enable the viewer to actually recreate them and thus gain an even deeper understanding of the Bahamian folk style. I chose to use the medium of video through which to present my research because of its ability to capture the dances in a way that writing could not. Recording the dances on video allowed for the documentation of each dance as a whole entity in that the mannerisms, gestures, behaviour and steps were simultaneously recorded and preserved. As each dance has experienced a continued erosion of tradition at the hands of progress and increased modernization of the Bahamas,the video format has allowed me to record and thus preserve each dance before it is lost entirely

The economic analysis of state aid: Some open questions

The last few years have seen both a significant shift in EU state aid policy towards a more sophisticated economic approach and a great enrichment of the economic literature on state aid. This paper examines the control of state aid in the EU in the light of the new literature. It begins with a discussion of the objectives of state aid control, taking account of the principle of subsidiarity. We discuss whether state aid control should concentrate on limiting the effects of aid on trade and competition (i.e. harm to rivals) or whether the purpose is broader and includes also considerations such as avoiding government failures and encouraging Member States to use state aid more sparingly and target it more efficiently. This leads to a brief analysis of how the appropriate welfare standard to be applied in state aid control would vary according to the weight given to different objectives. The paper then considers the objectives pursued by governments in granting aid with particular emphasis on the concept of market failure. The nature and magnitude of the market failure addressed by a state aid, together with the design of the aid, will strongly influence the extent of any anti-competitive effects. The last two parts (4 and 5) of the paper are devoted to specific problems of assessing anti-competitive effects using the types of information normally available to the aid-granting authorities and the European Commission. Part 4 discusses the problem of assessing the effects on competition of aid schemes and broad classes of aid, when the beneficiaries and even the affected markets are not known. Because Member States grant a vast number of individual aids every year, the Commission has to apply simple criteria to screen out those aids that are unlikely to have significant anti-competitive effects. We survey a range of indicators that can be used for this purpose and conclude that they all have drawbacks. Part 5 deals with the problems of assessing individual awards of aid which have failed the screening test, discussing how the main characteristics of firms (e.g. market share, vertical integration) and markets (such as product differentiation and market growth) may influence a state aid's impact on competition. Keywords: European Union, state aid, subsidies, competition policy.European Union, state aid, subsidies, competition policy, Buelens, Garnier, Meiklejohn, Johnson

Quantitative magnetic resonance image analysis via the EM algorithm with stochastic variation

Quantitative Magnetic Resonance Imaging (qMRI) provides researchers insight into pathological and physiological alterations of living tissue, with the help of which researchers hope to predict (local) therapeutic efficacy early and determine optimal treatment schedule. However, the analysis of qMRI has been limited to ad-hoc heuristic methods. Our research provides a powerful statistical framework for image analysis and sheds light on future localized adaptive treatment regimes tailored to the individual's response. We assume in an imperfect world we only observe a blurred and noisy version of the underlying pathological/physiological changes via qMRI, due to measurement errors or unpredictable influences. We use a hidden Markov random field to model the spatial dependence in the data and develop a maximum likelihood approach via the Expectation--Maximization algorithm with stochastic variation. An important improvement over previous work is the assessment of variability in parameter estimation, which is the valid basis for statistical inference. More importantly, we focus on the expected changes rather than image segmentation. Our research has shown that the approach is powerful in both simulation studies and on a real dataset, while quite robust in the presence of some model assumption violations.Comment: Published in at http://dx.doi.org/10.1214/07-AOAS157 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

A Bayesian Image Analysis of the Change in Tumor/Brain Contrast Uptake Induced by Radiation via Reversible Jump Markov Chain Monte Carlo

This work is motivated by a pilot study on the change in tumor/brain contrast uptake induced by radiation via quantitative Magnetic Resonance Imaging. The results inform the optimal timing of administering chemotherapy in the context of radiotherapy. A noticeable feature of the data is spatial heterogeneity. The tumor is physiologically and pathologically distinct from surrounding healthy tissue. Also, the tumor itself is usually highly heterogeneous. We employ a Gaussian Hidden Markov Random Field model that respects the above features. The model introduces a latent layer of discrete labels from an Markov Random Field (MRF) governed by a spatial regularization parameter. We further assume that conditional on the hidden labels, the observed data are independent and normally distributed, We treat the regularization parameter of the MRF, as well as the number of states of the MRF as parameters, and estimate them via the Reversible Jump Markov chain Monte Carlo algorithm. We propose a novel and nontrivial implementation of the Reversible Jump moves. The performance of the method is examined in both simulation studies and real data analysis. We report the pixel-wise posterior mean and standard deviation of the change in contrast uptake marginalized over the number of states and hidden labels. We also compare the performance with a Markov chain with fixed number of states and a parallel Expectation-Maximization approach from a frequentist perspective

Quantitative Magnetic Resonance Image Analysis via the EM Algorithm with Stochastic Variation

Quantitative Magnetic Resonance Imaging (qMRI) provides researchers insight into pathological and physiological alterations of living tissue, with the help of which, researchers hope to predict (local) therapeutic efficacy early and determine optimal treatment schedule. However, the analysis of qMRI has been limited to ad-hoc heuristic methods. Our research provides a powerful statistical framework for image analysis and sheds light on future localized adaptive treatment regimes tailored to the individual’s response. We assume in an imperfect world we only observe a blurred and noisy version of the underlying “true” scene via qMRI, due to measurement errors or unpredictable influences. We use a hidden Markov Random Field to model the unobserved “true” scene and develop a maximum likelihood approach via the Expectation-Maximization algorithm with stochastic variation. An important improvement over previous work is the assessment of variability in parameter estimation, which is the valid basis for statistical inference. Moreover, we focus on recovering the “true” scene rather than segmenting the image. Our research has shown that the approach is powerful in both simulation studies and on a real dataset, while quite robust in the presence of some model assumption violations

Determination of the plasmid size and location of d-endotoxin genes of Bacillus thuringiensis by pulse field gel electrophoresis

The genes encoding the d-endotoxins of Bacillus thuringiensis are located on plasmids ranging in size from 45 to 1000 kb. Plasmid size and variety are diagnostic features for characterizing subspecies of this aerobic spore-forming crystalliferous entomopathogen. Two of 25 B. thuringiensis isolates obtained from Middle Tennessee were characterized into subspecies on the basis of size, number, and varieties of plasmids they host using pulse field gel electrophoresis (PFGE). By using specific pulse angle (120°), field strength (5 V/cm), pulse time (26 h), and agarose concentration (1.2%) these DNA molecules were separated from other genomic DNA. The purified DNA product the agarose gel was tested for homology to 49 d-endotoxin gene using PCR.. The PFGE data for 5 of the 25 isolates collected in Tennessee showed distinct banding patterns. Two of the isolates had only 1 band whereas the others had more than 4 ranging from 45 to 1000 kb in size. Isolate 6 and 10 both yielded one 49 kb fragment that contained the cry1A gene.African Journal of Biotechnology Vol. 4 (7), pp. 580-585, 200

Longitudinal image analysis of tumour–healthy brain change in contrast uptake induced by radiation

The work is motivated by a quantitative magnetic resonance imaging study of the differential tumour–healthy tissue change in contrast uptake induced by radiation. The goal is to determine the time in which there is maximal contrast uptake (a surrogate for permeability) in the tumour relative to healthy tissue. A notable feature of the data is its spatial heterogeneity. Zhang and co-workers have discussed two parallel approaches to ‘denoise’ a single image of change in contrast uptake from baseline to one follow-up visit of interest. In this work we extend the image model to explore the longitudinal profile of the tumour–healthy tissue contrast uptake in multiple images over time. We fit a two-stage model. First, we propose a longitudinal image model for each subject. This model simultaneously accounts for the spatial and temporal correlation and denoises the observed images by borrowing strength both across neighbouring pixels and over time. We propose to use the Mann–Whitney U -statistic to summarize the tumour contrast uptake relative to healthy tissue. In the second stage, we fit a population model to the U -statistic and estimate when it achieves its maximum. Our initial findings suggest that the maximal contrast uptake of the tumour core relative to healthy tissue peaks around 3 weeks after initiation of radiotherapy, though this warrants further investigation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79255/1/j.1467-9876.2010.00718.x.pd

Thermal Structures Technology Development for Reusable Launch Vehicle Cryogenic Propellant Tanks

Analytical and experimental studies conducted at the NASA, Langley Research Center (LaRC) for investigating integrated cryogenic propellant tank systems for a reusable launch vehicle (RLV) are described. The cryogenic tanks are investigated as an integrated tank system. An integrated tank system includes the tank wall, cryogenic insulation, thermal protection system (TPS) attachment sub-structure, and TPS. Analysis codes are used to size the thicknesses of cryogenic insulation and TPS insulation for thermal loads, and to predict tank buckling strengths at various ring frame spacings. The unique test facilities developed for the testing of cryogenic tank components are described. Testing at cryogenic and high-temperatures verifies the integrity of materials, design concepts, manufacturing processes, and thermal/structural analyses. Test specimens ranging from the element level to the subcomponent level are subjected to projected vehicle operational mechanical loads and temperatures. The analytical and experimental studies described in this paper provide a portion of the basic information required for the development of light-weight reusable cryogenic propellant tanks