A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions

The organization of chromosomes in sperm nuclei has been proposed to possess a unique “hairpin-loop” arrangement, which is hypothesized to aid in the ordered exodus of the paternal genome following fertilization. This study simultaneously assessed the 3D and 2D radial and longitudinal organization of telomeres, centromeres, and investigated whether chromosomes formed the same centromere clusters in sperm cells. Reproducible radial and longitudinal non-random organization was observed for all investigated loci using both 3D and 2D approaches in multiple subjects. We report novel findings, with telomeres and centromeres being localized throughout the nucleus but demonstrating roughly a 1:1 distribution in the nuclear periphery and the intermediate regions with \u3c15% occupying the nuclear interior. Telomeres and centromeres were observed to aggregate in sperm nuclei, forming an average of 20 and 7 clusters, respectively. Reproducible longitudinal organization demonstrated preferential localization of telomeres and centromeres in the mid region of the sperm cell. Preliminary evidence is also provided to support the hypothesis that specific chromosomes preferentially form the same centromere clusters. The more segmental distribution of telomeres and centromeres as described in this study could more readily accommodate and facilitate the sequential exodus of paternal chromosomes following fertilization

SEGMENTATION AND INFORMATICS IN MULTIDIMENSIONAL FLUORESCENCE OPTICAL MICROSCOPY IMAGES

Recent advances in the field of optical microscopy have enabled scientists to observe and image complex biological processes across a wide range of spatial and temporal resolution, resulting in an exponential increase in optical microscopy data. Manual analysis of such large volumes of data is extremely time consuming and often impossible if the changes cannot be detected by the human eye. Naturally it is essential to design robust, accurate and high performance image processing and analysis tools to extract biologically significant results. Furthermore, the presentation of the results to the end-user, post analysis, is also an equally challenging issue, especially when the data (and/or the hypothesis) involves several spatial/hierarchical scales (e.g., tissues, cells, (sub)-nuclear components). This dissertation concentrates on a subset of such problems such as robust edge detection, automatic nuclear segmentation and selection in multi-dimensional tissue images, spatial analysis of gene localization within the cell nucleus, information visualization and the development of a computational framework for efficient and high-throughput processing of large datasets. Initially, we have developed 2D nuclear segmentation and selection algorithms which help in the development of an integrated approach for determining the preferential spatial localization of certain genes within the cell nuclei which is emerging as a promising technique for the diagnosis of breast cancer. Quantification requires accurate segmentation of 100 to 200 cell nuclei in each patient tissue sample in order to draw a statistically significant result. Thus, for large scale analysis involving hundreds of patients, manual processing is too time consuming and subjective. We have developed an integrated workflow that selects, following 2D automatic segmentation, a sub-population of accurately delineated nuclei for positioning of fluorescence in situ hybridization labeled genes of interest in tissue samples. Application of the method was demonstrated for discriminating normal and cancerous breast tissue sections based on the differential positioning of the HES5 gene. Automatic results agreed with manual analysis in 11 out of 14 cancers, all 4 normal cases and all 5 non-cancerous breast disease cases, thus showing the accuracy and robustness of the proposed approach. As a natural progression from the 2D analysis algorithms to 3D, we first developed a robust and accurate probabilistic edge detection method for 3D tissue samples since several down stream analysis procedures such as segmentation and tracking rely on the performance of edge detection. The method based on multiscale and multi-orientation steps surpasses several other conventional edge detectors in terms of its performance. Subsequently, given an appropriate edge measure, we developed an optimal graphcut-based 3D nuclear segmentation technique for samples where the cell nuclei are volume or surface labeled. It poses the problem as one of finding minimal closure in a directed graph and solves it efficiently using the maxflow-mincut algorithm. Both interactive and automatic versions of the algorithm are developed. The algorithm outperforms, in terms of three metrics that are commonly used to evaluate segmentation algorithms, a recently reported geodesic distance transform-based 3D nuclear segmentation method which in turns was reported to outperform several other popular tools that segment 3D nuclei in tissue samples. Finally, to apply some of the aforementioned methods to large microscopic datasets, we have developed a user friendly computing environment called MiPipeline which supports high throughput data analysis, data and process provenance, visual programming and seamlessly integrated information visualization of hierarchical biological data. The computational part of the environment is based on LONI Pipeline distributed computing server and the interactive information visualization makes use of several javascript based libraries to visualize an XML-based backbone file populated with essential meta-data and results

Methods development for the investigation of the mammalian genome radial architecture : the quantitative side

The nucleus of mammalian cells cradles the genome, an ensemble of nucleic acid macromolecular polymers that store information in a physical form. For a cell to perform life-sustaining processes, reading and utilizing the information encoded in the genome monomers’ sequence is necessary. Considerable attention has been paid to these processes since their discovery, leading to remarkable breakthroughs in our understanding of basic cell biology and the Genetics field’s birth. In the past two decades, the focus has shifted from this one-dimensional approach to a more spatio-temporal perspective. It is now clear that the genome has a complex architecture, with a multitude of organizational levels at different scales. Additionally, genome architecture interplays with gene expression, and alterations to its spatial organization associate with various pathologies like cancer, premature-aging diseases, and male infertility. In this thesis, we present the development of two methods enabling the investigation of genome architecture. In Paper I, we established iFISH, a full-stack workflow for easy DNA fluorescence in situ hybridization (FISH) setup and application. Specifically, iFISH includes a novel and accurately crafted database of 40 nt long oligonucleotide sequences for labeling specific human genomic loci. iFISH 40-mers provide a strikingly higher genomic coverage and shorter interoligo distance than other state-of-the-art databases. Moreover, the iFISH database of homologous sequences allows for the design of a 96-oligo probe in more than half of the ten kb-wide genomic regions and more than 85% of 15 kb-wide genomic regions (against a 15-30% for other databases). iFISH also includes a computational tool, easily accessible and usable via a web-based graphical user interface, for the automatic selection of optimal sets of oligos (i.e., probe design), for single-probe or homogeneous multi-probe (i.e., spotting) labeling. We applied our computational pipeline to design a total of 330 DNA FISH probes, covering all human chromosomes homogeneously, with an inter-probe distance of 10 Mb for chromosomes 1 to 16 and X and of 5 Mb for chromosomes 17 to 22. Additionally, we systematically and individually tested most probes, whose sequences are readily available for the community to download and utilize. Furthermore, we built upon cutting-edge sequence amplification methods to provide an inexpensive and straightforward protocol for the large-scale amplification of DNA FISH probes starting from relatively low concentrated oligopools. To this end, we designed a set of novel 20-mer sequences orthogonal to the human genome and compatible with the probe-specific PCR steps of the amplification protocol. Finally, we showcased the extensive applicability and flexibility of the iFISH workflow in human IMR90 fibroblast cells, revealing the importance of a dense label sampling for correct chromatin volume estimation, and in human embryonic stem cells, uncovering overall less distinct chromosome territories, and a remarkable lack of chromosome territoriality in a subset of cells. Altogether, these results support iFISH as an empowering set of tools and resources for the research community, freely accessible online at https://www.ifish4u.org. In Paper II, we presented Genomic loci Positioning by sequencing (a.k.a., GPSeq), a method for the genome-wide measurement of genomic loci position along the nuclear radius. GPSeq follows a straightforward protocol based on a simple and elegant concept: nuclear diffusion proceeds from the nucleus periphery towards its interior. We proved this concept by applying it to restriction enzyme diffusion, where we exploited a FISH-based method (YFISH) to visualize concentric genomic restriction signal waves generated by different digestion times. Specifically, GPSeq combines the sequencing of genomic loci restricted at different digestion time lengths into a so-called "GPSeq score," a reliable and accurate estimate of genomic loci centrality. We validated the GPSeq score against a collection of 68 DNA FISH probes, spanning 11 different chromosomes, data obtained from DamID-seq of Lamin B1, and also Hi-C chromatin contacts. Then, we utilized the radial maps drawn by GPSeq to reveal novel radial arrangements of different chromatin states and identify centrality predictors at different resolutions. Subsequently, we applied a novel 3D genome reconstruction algorithm to demonstrate how an additional centrality constraint can improve reconstructed structures’ quality. Specifically, 3D genome structures generated by a GPSeq-informed algorithm showed a higher correlation with FISH-based radial measurements and an arrangement of chromosome territories and genomic compartments that better reflects the underlying biology. Additionally, structures generated by the combination of GPSeq and Hi-C intrachromosomal contacts allowed the recovery of the inter-chromosomal contacts, further underscoring the necessity of additional constraints provided by orthogonal methods to Hi-C for a more reliable 3D genome reconstruction. Finally, we applied GPSeq to provide insight into the so-called "bodyguard hypothesis, " speculating that heterochromatin might act as a shield from exogenous mutagens for the more internally located active chromatin. In this regard, we showed that cancer-related single-nucleotide variants (SNVs) have a strikingly different radial arrangement than germline single-nucleotide polymorphisms (SNPs), with the former more peripherally located than the latter. We then showed that genomic regions involved with gene fusions in cancer tend to locate more internally and contact other chromosomes more frequently than other regions. We combined these observations and the fact that double-strand breaks (DSBs) tend to locate more internally, further confirmed from immunofluorescence experiments, to speculate that cancer-related SNVs and germline SNPs might come to be by different underlying mechanisms. Altogether, these results highlight the importance of genomewide high-resolution radial maps in the study of genome architecture, both as a standalone resource and as a complementary feature to chromatin contacts

Discovery and restoration of Aberrant Nuclear structure and Genome behaviour in breast cancer cells

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The eukaryotic interphase nucleus is well organised and the genome positioned non-randomly. Nuclear structure is an important regulator of genome behaviour and function. Genome organisation and nuclear structure are compromised in diseases such as cancer and laminopathies. This study was to find out and to determine if there is any functional relationship between nuclear structure and genome mis-organisation in cancer cells. I have assessed the presence and distribution of specific nuclear structural proteins (A-type, B-type lamins and its receptor LBR, many of their binding proteins such as MAN1, LAP2α, LAP2, and Emerin and other nuclear proteins (PML, Nucleolin, and Ki67) using indirect immunofluorescence. From this study, it is found that the nuclear structure of breast cancer cells is often altered. The most severely affected proteins are the nuclear lamins B1 and B2 and they found as large foci within the nucleoplasm with little LBR expression to localise the lamin B. I also assessed the chromosome positioning (HSA 7, 10, 11, 14 and 17) and gene positioning (AKT1, CCND1, HSP90AA1, EGFR, ERRBB2/HER2 and PTEN) in breast cancer cell lines (T-47D, GI-101, Sk-Br-3 and BT-474) and in normal breast cell lines (MCF-10A) using 2D-FISH technique. I also assessed the position of the genes in nuclei and correlated with gene expression using qRT-PCR. Breast cell lines have treated with a drug named lovastatin and it was found that the cells have restored LBR expression and localisation of lamin B, leading to altered gene positioning and changed expression of breast cancer genes. Since the drug (lovastatin, 12 μM/48 hours) affects the prenylation as a post-translation modification process and lamins B biosythensis, it is found that B-type lamins and its receptor expression and distribution were improved and increased in expression by 2-fold in expression levels in the most affected cells (T-47D, and BT-474) compared to the normal cells (MCF-10A) and these cells also showed abnormal nuclei and dead cells. When analysing the nuclear positioning of the genes (AKT1, HSP90AA1 and ERRBB2/HER2), it is found that AKT1 was positioned periphery in BT-474 and T-47D cells and interiorly in the normal cells (MCF-10A) before treatment whereas the same gene was positioned periphery in T-47D and MCF-10A cells and interiorly in BT-474 after treatment with lovastatin. It is also found that HSP90AA1 was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 cells before and after treatment (no change). Moreover, ERRBB2/HER2 gene was positioned periphery in T-47D and BT-474 cells and interiorly in MCF-10A cells before treatment whereas the same gene was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 after treatment with the same drug. Regarding LMNB1, LMNB2, and LBR genes, the study focussed only on their expression levels and no work has done on their chromosome positioning as well as gene position before and after treatment. These three genes were over expressed when assessed by measuring the relative and fold changes in expression. Therefore, it is suggestive that 2D-FISH experiment to assess their localisation and their specific chromosome territories is required. The results shown in this thesis demonstrate the importance and roles of nuclear architecture specifically nuclear lamins and the integral nuclear membrane proteins (B-type lamins and LBR) in mediating correct genome organisation and function. The breast normal (immortalised cells) and cancerous cell lines showed different nuclear structures as lamin B affect the position of specific target chromosomes and genes. These results will strength the finding that the nuclear lamina is a significant nuclear structure which associates, organises, and regulates numerous vital nuclear processes and the stability of the genome

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Dex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer

Computer-aided image quality assessment in automated 3D breast ultrasound images

Automated 3D breast ultrasound (ABUS) is a valuable, non-ionising adjunct to X-ray mammography for breast cancer screening and diagnosis for women with dense breasts. High image quality is an important prerequisite for diagnosis and has to be guaranteed at the time of acquisition. The high throughput of images in a screening scenario demands for automated solutions. In this work, an automated image quality assessment system rating ABUS scans at the time of acquisition was designed and implemented. Quality assessment of present diagnostic ultrasound images has rarely been performed demanding thorough analysis of potential image quality aspects in ABUS. Therefore, a reader study was initiated, making two clinicians rate the quality of clinical ABUS images. The frequency of specific quality aspects was evaluated revealing that incorrect positioning and insufficiently applied contact fluid caused the most relevant image quality issues. The relative position of the nipple in the image, the acoustic shadow caused by the nipple as well as the shape of the breast contour reflect patient positioning and ultrasound transducer handling. Morphological and histogram-based features utilized for machine learning to reproduce the manual classification as provided by the clinicians. At 97 % specificity, the automatic classification achieved sensitivities of 59 %, 45 %, and 46 % for the three aforementioned aspects, respectively. The nipple is an important landmark in breast imaging, which is generally---but not always correctly---pinpointed by the technicians. An existing nipple detection algorithm was extended by probabilistic atlases and exploited for automatic detection of incorrectly annotated nipple marks. The nipple detection rate was increased from 82 % to 85 % and the classification achieved 90 % sensitivity at 89 % specificity. A lack of contact fluid between transducer and skin can induce reverberation patterns and acoustic shadows, which can possibly obscure lesions. Parameter maps were computed in order to localize these artefact regions and yielded a detection rate of 83 % at 2.6 false positives per image. Parts of the presented work were integrated to clinical workflow making up a novel image quality assessment system that supported technicians in their daily routine by detecting images of insufficient quality and indicating potential improvements for a repeated scan while the patient was still in the examination room. First evaluations showed that the proposed method sensitises technicians for the radiologists' demands on diagnostically valuable images

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer

PRELIMINARY FINDINGS OF A POTENZIATED PIEZOSURGERGICAL DEVICE AT THE RABBIT SKULL

The number of available ultrasonic osteotomes has remarkably increased. In vitro and in vivo studies have revealed differences between conventional osteotomes, such as rotating or sawing devices, and ultrasound-supported osteotomes (Piezosurgery®) regarding the micromorphology and roughness values of osteotomized bone surfaces. Objective: the present study compares the micro-morphologies and roughness values of osteotomized bone surfaces after the application of rotating and sawing devices, Piezosurgery Medical® and Piezosurgery Medical New Generation Powerful Handpiece. Methods: Fresh, standard-sized bony samples were taken from a rabbit skull using the following osteotomes: rotating and sawing devices, Piezosurgery Medical® and a Piezosurgery Medical New Generation Powerful Handpiece. The required duration of time for each osteotomy was recorded. Micromorphologies and roughness values to characterize the bone surfaces following the different osteotomy methods were described. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and atomic force microscopy. The selective cutting of mineralized tissues while preserving adjacent soft tissue (dura mater and nervous tissue) was studied. Bone necrosis of the osteotomy sites and the vitality of the osteocytes near the sectional plane were investigated, as well as the proportion of apoptosis or cell degeneration. Results and Conclusions: The potential positive effects on bone healing and reossification associated with different devices were evaluated and the comparative analysis among the different devices used was performed, in order to determine the best osteotomes to be employed during cranio-facial surgery