8,284 research outputs found
INVESTIGATION OF THE K2 ALGORITHM IN LEARNING BAYESIAN NETWORK CLASSIFIERS
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WNT-DEPENDENT REGENERATIVE FUNCTION IS INDUCED IN LEUKEMIA-INITIATING AC133BRIGHT CELLS
The Cancer Stem Cell model supported the notion that leukemia was initiated and maintained in vivo by a small fraction of leukemia-initiating cells (LICs). Previous studies have suggested the involvement of Wnt signaling pathway in Acute Myeloid Leukemia (AML) by the ability to sustain the development of LICs. A novel hematopoietic stem and progenitor cell marker, monoclonal antibody AC133, recognizes the CD34bright CD38- subset of human acute myeloid leukemia cells, suggesting that it may be an early marker for the LICs. During the first part of my phD program we previously evaluated the ability of leukemic AC133+ fraction, to perform engraftment following to xenotransplantation in immunodeficient mouse model Rag2-/-\u3b3c-/-. The results showed that the surface marker AC133 is able to enrich for the cell fraction that contains the LICs. In consideration of our previously reported data, derived from the expression profiling analysis performed in normal (n=10) and leukemic (n=33) human long-term reconstituting AC133+ cells, we revealed that the ligand-dependent Wnt signaling is induced in AML through a diffuse expression and release of WNT10B, a hematopoietic stem cells regenerative-associated molecule. In situ detection performed on bone marrow biopsies of AML patients, showed the activation of the Wnt pathway, through the concomitant presence of the ligand WNT10B and of the active dephosphorylated \u3b2-catenin form, suggesting an autocrine / paracrine-type ligand-dependent activation mechanism. In consideration of the link between hematopoietic regeneration and developmental signaling, we transplanted primary AC133+ AML A46 cells into developing zebrafish. This biosensor model revealed the formation of ectopic structures by activation of dorsal organizer markers that act downstream of the Wnt pathway. These results suggested that the misappropriating Wnt associated functions can promote pathological stem cell-like regeneration responsiveness. The analyses performed in situ retained information on the cellular localization, enabling determination of the activity status of individual cells and allowing the tumor environment view. Taking this issue into consideration, during the second part of my phD program, I set up the application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The mRNA in situ detection technique is based on padlock probes ligation and target priming rolling circle amplification allowing the single nucleotide resolution in heterogenous tissues. The mRNA in situ detection performed on bone marrow biopsies derived from AML patients, showed a diffuse localization pattern of WNT10B molecule in the tissue. Conversely, only the AC133bright cell population shows the Wnt signaling activation signature represented by the cytoplasmatic accumulation and nuclear translocation of the active form of \u3b2-catenin. In spite of this, we previously evidenced that the regenerative function of WNT signaling pathway is defined by the up-regulation of WNT10B, WNT10A, WNT2B and WNT6 loci, we identified the WNT10B as a major locus associated with the regenerative function and over-expressed by all AML patients. By the molecular evaluation of the WNT10B transcript, we isolated an aberrant splicing variant (WNT10BIVS1), that identify Non Core-Binding Factor Leukemia (NCBFL) class and whose potential role is discussed. Moreover, we demonstrate that the function of "leukemia stem cell", present in the cell population enriched for the marker AC133bright, is strictly related to regenerative function associated with WNT signaling, defining the key role of WNT10B ligand as a specific molecular marker for leuchemogenesis. This thesis defines the new suitable approaches to characterize the leukemia-initiating cells (LICs) and suggest the role of WNT10B as a new suitable target for AML
Extended depth of field imaging for high speed object analysis
A high speed, high-resolution flow imaging system is modified to achieve extended depth of field imaging. An optical distortion element is introduced into the flow imaging system. Light from an object, such as a cell, is distorted by the distortion element, such that a point spread function (PSF) of the imaging system is invariant across an extended depth of field. The distorted light is spectrally dispersed, and the dispersed light is used to simultaneously generate a plurality of images. The images are detected, and image processing is used to enhance the detected images by compensating for the distortion, to achieve extended depth of field images of the object. The post image processing preferably involves de-convolution, and requires knowledge of the PSF of the imaging system, as modified by the optical distortion element
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The role of homeobox gene in leukaemia
This thesis was submitted for the degree of Master of Philosophy and was awarded by Brunel UniversityHomeobox genes are known to be active during development and they are turned off after the early stages of developmental life. The HLXB9/MNX1 gene is a homeobox gene localized on human chromosome 7 and is involved in the development of pancreas and the nervous system. However, some leukaemia research groups have reported an over-expression of HLXB9 in leukaemia patients who carry the t(7;12) and in the GDM-1 cell line that carries the t(6;7). The mechanisms of leukaemogenesis in t(7;12) patients are still unclear. The t(7;12) is one of the recurrent cytogenetic abnormalities that is associated with infant acute myeloid leukaemia (AML) patients and has been linked to poor prognosis. The aim of this study was (i) to determine the involvement of HLXB9 in cell lines known to express this gene at the transcript level and (ii) to investigate the position on HLXB9 in AML patients with abnormalities of chromosome 7. This aim was achieved through a series of experiments involving the use of both conventional and molecular cytogenetics.
In the first place, the chromosomal abnormalities in leukaemia and lymphoma cell lines (GDM-1, K562 and Pfeiffer) have been analysed using G-banding and Multiplex FISH (M-FISH) techniques.
Furthermore, FISH using whole chromosome painting technique was performed on 7 AML patients to investigate chromosome 7 rearrangements.
Thirdly, the involvement of the homeobox gene HLXB9 has been investigated in the acute myeloid leukaemia (AML) derived cell line GDM-1 and in 4 AML patients. Fluorescence in situ hybridization (FISH) analysis was carried out using a specific probe for the HLXB9 gene on the AML patients in single and dual colour FISH in combination with an additional probe distal to HLXB9 on the GDM-1 cell line. FISH analysis showed no involvement of the HLXB9 gene in any rearrangement or breaks at chromosomal level on the AML cell line (GDM-1) and AML patients. Nevertheless, a breakpoint either proximal or distal to HLXB9 has been identified.
In particular, the breakpoint in the GDM-1 cell line has been confirmed on between the two probes used. This thesis poses the basis for further studies to investigate the mechanisms of oncogenesis in leukaemias with over-expression of HLXB9 in relation to possible breakage of chromosome 7 in the vicinity of the gen
Molecular cytogenetic evaluation of uveal melanoma cell lines and archival tissue
Uveal melanoma is the most common intraocular tumor in adults and often results in unilateral blindness and/or death. Previous cytogenetic characterizations of this tumor have consistently revealed chromosomal abnormalities involving chromosomes 3, 6, and 8; reports of other abnormalities vary in frequency. We further defined cytogenetic abnormalities of this tumor using molecular cytogenetic techniques on 10 uveal malignant melanoma cell lines and 100 formalin-fixed paraffin-embedded (FFPE) tumors.;The synthesis of comparative genomic hybridization (CGH) and spectral karyotyping (SKY) results revealed that chromosomal rearrangement plays a significant role in the generation of DNA sequence copy number abnormalities throughout the genome, but none of the cell lines demonstrated monosomy 3. Centromeric fluorescence in situ hybridization (FISH) for chromosome 3 revealed approximately one signal per cell, but further evaluation with telomeric probes demonstrated multiple signals per cell, suggesting chromosomal rearrangement without the loss of an entire chromosome 3. Based on combined CGH, SKY and FISH data, we propose that chromosome 3 is more frequently involved in chromosomal rearrangements rather than whole-chromosome loss in uveal melanoma.;CGH was similarly employed to elucidate characteristic DNA-sequence copy number abnormalities of FFPE archival cases of uveal melanoma, and correlate genomic imbalance abnormalities with a prognosis. We set out to study 100 archival uveal melanoma cases, each with comprehensive patient follow-up, by correlation of copy number imbalances with survival. Fifty-one patients survived more than 9 years without evident metastasis, and the remaining 49 patients died with metastatic disease. Viable probe was generated from 82 of the 100 cases, allowing correlation of CGH findings with patient histories for all but 18 of the cases. Significant copy number imbalances were tested for univariate prognostic significance. The most powerful predictor of prognosis was gain of 18q11.2, which was subsequently compared with other significant chromosomal regions, as well as histological and clinical factors in a multivariate analysis. Multivariate analyses revealed that the gain of 18q11.2 and concomitant loss of 1p33 was the strongest indicator of a poor prognosis.;Our large-scale molecular cytogenetic analysis of cell lines and archival material contributes significantly to the characterization of uveal melanoma. This research is intended to direct further gene-specific study of malignancy in uveal melanoma
Computational analysis of pathological images enables a better diagnosis of TFE3 Xp11.2 translocation renal cell carcinoma
TFE3 Xp11.2 translocation renal cell carcinoma (TFE3-RCC) generally progresses more aggressively compared with other RCC subtypes, but it is challenging to diagnose TFE3-RCC by traditional visual inspection of pathological images. In this study, we collect hematoxylin and eosin- stained histopathology whole-slide images of 74 TFE3-RCC cases (the largest cohort to date) and 74 clear cell RCC cases (ccRCC, the most common RCC subtype) with matched gender and tumor grade. An automatic computational pipeline is implemented to extract image features. Comparative study identifies 52 image features with significant differences between TFE3-RCC and ccRCC. Machine learning models are built to distinguish TFE3-RCC from ccRCC. Tests of the classification models on an external validation set reveal high accuracy with areas under ROC curve ranging from 0.842 to 0.894. Our results suggest that automatically derived image features can capture subtle morphological differences between TFE3-RCC and ccRCC and contribute to a potential guideline for TFE3-RCC diagnosis
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Molecular Cytogenetic Applications in Analysis of the Cancer Genome
Cancer cells exhibit nonrandom and complex chromosome abnormalities. The role of genomic changes in cancer is well established. However, the identification of complex and cryptic chromosomal changes is beyond the resolution of conventional banding methods. The fluorescence microscopy afforded by imaging technologies, developed recently, facilitates a precise identification of these chromosome alterations in cancer. The three most commonly utilized molecular cytogenetics methods comparative genomic hybridization, spectral karyotype, and fluorescence in situ hybridization, that have already become benchmark tools in cancer cytogenetics, are described in this chapter. Comparative genomic hybridization is a powerful tool for screening copy-number changes in tumor genomes without the need for preparation of metaphases from tumor cells. Multicolor spectral karyotype permits visualization of all chromosomes in one experiment permitting identification of precise chromosomal changes on metaphases derived from tumor cells. The uses of fluorescence in situ hybridization are diverse, including mapping of alteration in single copy genes, chromosomal regions, or entire chromosomes. The opportunities to detect genetic alterations in cancer cells continue to evolve with the use of these methodologies both in diagnosis and research
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