Coronary Artery Segmentation and Motion Modelling

Conventional coronary artery bypass surgery requires invasive sternotomy and the use of a cardiopulmonary bypass, which leads to long recovery period and has high infectious potential. Totally endoscopic coronary artery bypass (TECAB) surgery based on image guided robotic surgical approaches have been developed to allow the clinicians to conduct the bypass surgery off-pump with only three pin holes incisions in the chest cavity, through which two robotic arms and one stereo endoscopic camera are inserted. However, the restricted field of view of the stereo endoscopic images leads to possible vessel misidentification and coronary artery mis-localization. This results in 20-30% conversion rates from TECAB surgery to the conventional approach. We have constructed patient-specific 3D + time coronary artery and left ventricle motion models from preoperative 4D Computed Tomography Angiography (CTA) scans. Through temporally and spatially aligning this model with the intraoperative endoscopic views of the patient's beating heart, this work assists the surgeon to identify and locate the correct coronaries during the TECAB precedures. Thus this work has the prospect of reducing the conversion rate from TECAB to conventional coronary bypass procedures. This thesis mainly focus on designing segmentation and motion tracking methods of the coronary arteries in order to build pre-operative patient-specific motion models. Various vessel centreline extraction and lumen segmentation algorithms are presented, including intensity based approaches, geometric model matching method and morphology-based method. A probabilistic atlas of the coronary arteries is formed from a group of subjects to facilitate the vascular segmentation and registration procedures. Non-rigid registration framework based on a free-form deformation model and multi-level multi-channel large deformation diffeomorphic metric mapping are proposed to track the coronary motion. The methods are applied to 4D CTA images acquired from various groups of patients and quantitatively evaluated

A New Mathematical Theory for the Dynamics of Large Tumor Populations, a Potential Mechanism for Cancer Dormancy & Recurrence and Experimental Observation of Melanoma Progression in Zebrafish

Cancer, a family of over a hundred disease varieties, results in 600,000 deaths in the U.S. alone. Yet, improvements in imaging technology to detect disease earlier, pharmaceutical developments to shrink or eliminate tumors, and modeling of biological interactions to guide treatment have prevented millions of deaths. Cancer patients with initially similar disease can experience vastly different outcomes, including sustained recovery, refractory disease or, remarkably, recurrence years after apparently successful treatment. The current understanding of such recurrences is that they depend on the random occurrence of critical mutations. Clearly, these biological changes appear to be sufficient for recurrence, but are they necessary? In contrast, we propose a new mathematical model that predicts long-term apparent dormancy followed by recurrence can occur because of tumor population dynamics without the need for a single random event such as the occurrence of a key mutation. Understanding the mechanisms and factors that influence the likelihood and timing of recurrence can strongly impact preferred treatments. Though cancer progression is a complex interplay of many complicated biological processes such as genetic changes, chemical signals and metabolic considerations, our model examines how the dynamic interplay of just three processes can dictate disease progress. We propose a population balance model to describe how populations of a large ensemble of tumors of different sizes evolve in time due to growth (mitosis), reduction (apoptosis or immunity) and metastasis, each with a size-dependent parameter. Mathematical analysis of the model’s parameter interactions leads to insights regarding the progression of metastatic cancer, including prediction of recurrence after long-term dormancy. We successfully tested the model against literature data on human hepatocellular carcinoma and then carried out extensive experiments on a zebrafish model of melanoma to validate the model. The experimental system consists of gender-segregated immune-competent and immune-suppressed translucent, stripeless zebra­fish (casper variant) inoculated with a fluorescent GFP-expressing transgenic melanoma cell line (ZMEL). We numerically solved the model’s partial differential equations for any given initial population size distribution and used it and the data to find best-fit parameters for growth, reduction and metastasis. This novel parameter optimization detected the differences between the immune status for each gender. Because the measured fish melanoma parameters are not in a range for which we predict dormancy and recurrence within fish lifetimes, this system cannot yet verify predictions on tumor recurrence. We complemented the above experiments with mathematical analysis, including both analytical and approximate solutions to the model for select parameters. We applied the theory of birth and death processes to estimate the probability and timing of recurrence. We have also developed a Markov chain simulation model to track the progression of discrete tumors. This formulation facilitates the study of tumor merging events by allowing individual tumors to be positioned in a virtual fish body. We are exploring alternate methods of obtaining tumor growth data from different species, including CT scans of mouse lungs to infer lung metastases sizes. Ultimately, the model may offer a flexible way to predict the progression of the cancer by using noninvasive imaging data to find model parameters

Live-cell visualisation of chromatin for the study of radiation-induced chromosome aberrations

This thesis was submitted for the degree of Master of Philosophy and awarded by Brunel University.Chromosomes can be visualised during the metaphase stage of the cell cycle however, there are various limitations associated with the visualisation of chromatin during interphase. This is due to the fact that during that stage of the cell cycle, chromatin is decondensed and difficult to resolve by using standard techniques. The employment of fluorescence microscopy has provided great insight into the examination of the structure and dynamics of fluorescently tagged chromatin regions in living cells during interphase. Thus the in vivo stable expression of fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) has the potential to address questions that lead to understanding dynamic responses of chromatin in response to DNA damage. In this study, chromatin sites within the nuclei of Chinese Hamster Ovary (CHO) cells were fluorescently labelled using the lac operon system, which works on the principle that EGFP-lac repressor protein will bind to sites of integrated lac operator sequence. To achieve this, CHO cells were transfected with pSV2-DHFR8.32 (lac operator) and p3‟SSdimerClonEGFP (lac repressor-EGFP fusion protein) plasmids by calcium phosphate transfection. 68 stable transfectants were stored and clone CHO-R-O-25 cells were used for further experimental analysis based on these cells containing discrete and intense fluorescent spots. It was shown that the physiological and functional characteristics of CHO-R-O-25 cells were comparable to those of control CHO DG44 cells. Time-lapse fluorescence microscopy was used to examine for any alterations in the physical structure of chromatin in response to DNA damage that was induced by radiation and a range of different chemicals. It was demonstrated that in cells exposed to DNA damaging agents, chromatin diffusion and expansions revealed by alterations in the intensity and size of the fluorescent spots within the nuclei of cells are owed to relaxation of chromatin structures in response to genome insults. The potential for this tool to study the organisational responses of chromatin in response to damage and the ulitimate relevance of any physical alterations to mechanisms for chromosome exchange formation, are described

Interaction of the host immune system with tumor cells in human papillomavirus associated diseases

Human papillomaviruses (HPV) are very common in the sexually active population and contribute to 610,000 cancers per year occurring at different locations. The initial step of HPV-related carcinogenesis is the induction of transforming processes in the host cells mediated by the viral oncoproteins E6 and E7 that interfere with critical host cell pathways. The transforming infection is highlighted by overexpression of the tumor suppressor protein p16INK4a. Only a small number of precancerous lesions progress while the majority can be controlled by the host’s immune system and undergo regression. Progressing lesions under the immunoselective pressure seem to acquire characteristics that enable them to circumvent the host’s immune attack and promote disease progression. Immune evasion might be mediated by the immune microenvironment of the tumor as well as by tumor cell intrinsic features. The here presented thesis addressed different questions and strategies with regard to the role of the immune system in HPV-associated diseases and can be subdivided in two main parts: In the first part immunologic characteristics of precancerous lesions and cancers are investigated to gain insight into possible immune evasion mechanisms developed during disease progression. In the second part treatment options to positively influence the balance between immune evasion and anti-tumoral immune responses are evaluated. In the first part a) the immunohistochemical characterization of cervical precancers and cancers for infiltration with different T cell phenotypes revealed that generally increasing T cell densities occur late in carcinogenesis – and not yet with the onset of early transforming infection - and are accompanied by immunosuppressive regulatory T cells (Tregs). Mean cell densities for Tregs in the stroma significantly increased from 121.6 cells/mm2 (range: 24-286.8 cells/mm2) in low-grade lesions to 308.8 cells/mm2 (24-724.8 cells/mm2) in high-grade lesions and 673.6 cells/mm2 (52.8-1564.8 cells/mm2) in cancer which points to their immunosuppressive role during carcinogenesis. The demonstrated large variances in T cell densities within one diagnostic category, however, point to a remarkable heterogeneity of the immune control with potential interesting prognostic implications. On keratinocytes themselves b) a selective loss for human leukocyte antigen (HLA) class I heavy chain A expression was observed in about 55% high-grade cervical intra-epithelial neoplasia (CIN) and 65% of cervical cancers. HLA class II de novo expression was found in 50% of low-grade CIN and in about 85% of high-grade CIN and cervical cancers. These alterations could represent another fundamental mechanism contributing to immune evasion. A c) longitudinal analysis of immune infiltrates in patients treated with imiquimod, an immuno-modulatory Toll-like receptor (TLR) agonist, revealed that the patient’s local immune constitution might be decisive for a possible response to immune-enhancing treatment strategies. Importantly, in patients responding to imiquimod immune cell densities increased during the treatment as epithelial CD3+ T cell counts (from 160.8 to 371.1 cells/mm2) and CD8+ T cell counts (from 113.8 to 174.1 cells/mm2) demonstrated. The d) development and establishment of an automated cell quantification tool for high-throughput analysis allows the search for immune evasion markers and strategies to be continued in an objective, standardized and faster way. In consideration of the clinical efficacy of imiquimod and the observed stimulatory effects on the immune infiltrate density in part one of this thesis e) a new second generation TLR-agonist (TMX-202) potentially having less side-effects than imiquimod was tested for the first time in an in vitro T cell stimulation model in part two of this thesis. Its potential to stimulate innate and adaptive immunity was demonstrated by an enhanced killing capacity of T cells that were stimulated with HPV-related antigens loaded on dendritic cells and then co-incubated with HPV16-positive CaSki cells. Based on the dense infiltration with Tregs observed in part one of the presented thesis the f) immune stimulating effects of Treg depletion was tested in an autologous in vitro model. In this regard, one major aim of the thesis was the generation of a new HPV-positive tumor cell line derived from an oropharyngeal squamous cell carcinoma that serves as model system for HPV-associated tumors. In combination with peripheral blood lymphocytes obtained from the same patient this autologous system allowed to address Treg depletion as an immunotherapeutic approach. The results demonstrated that this strategy might enhance the cell-mediated immune response against tumor cells and emphasize the role that this particular T cell phenotype is obviously playing in the carcinogenesis of HPV-associated tumors. Based on the results obtained in the first part of the thesis it is well conceivable that the combination of different immunologic markers contributes to the definition of a prognostic biomarker tool for progression and regression of precancerous lesions. Such a prognostic “immune score” has a high clinical relevance and allows risk-adapted treatment decisions minimizing the costs and long-term sequelae of surgical interventions. In particular the newly developed microscopy based method in this work allowing for the automated histological high-throughput quantification of infiltrating immune cells in cervical intraepithelial neoplasia provides an important methodical tool to realize this long term goal. The immuno-stimulating effects of the novel TLR7-agonist TMX-202 and Treg depletion demonstrated in the second part of this thesis by in vitro models indicate that immunomodulatory approaches could play an important role for the treatment of HPV-associated cancers in the future. In this regard, the established novel tumor cell line in combination with autologous immune cells provides a valuable in vitro model system for HPV-associated cancers that can be used to investigate further immunotherapeutic intervention and treatment strategies

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Faculty Impact Statements, 2009

Each issue [in the Research Series] has a distinctive titl

Advanced analytical diagnostics applied to human osteological remains

Le ossa antiche, recuperate dai contesti archeologici e preservati all’interno dei Musei, rappresentano una preziosa fonte di informazioni sull'alimentazione, lo stato di salute, la mobilità delle popolazioni antiche nonché sulla demografia e condizioni ambientali del passato, utili a ricercatori e accademici. A seguito dello sviluppo di moderne tecnologie delle scienze omiche, i reperti osteologici sono sempre più richiesti e questo ha comportato un aumento dell'analisi del DNA antico (aDNA). I metodi di campionamento per l'estrazione del DNA antico sono prevalentemente distruttivi e spesso possono compromettere i reperti osteologici per ulteriori future analisi o per studi in altri campi di ricerca. Oltre al campionamento invasivo e distruttivo, in condizioni di scarsa conservazione dell’osso archeologico causata da alterazioni tafonomiche e diagenetiche, il sequenziamento del DNA antico può essere un'operazione estremamente costosa. Dati gli elevati costi della procedura di sequenziamento dell'aDNA, in questo lavoro di ricerca è stato condotto uno studio analitico mediante spettroscopia a raggi infrarossi (FTIR) per sviluppare un metodo di pre-screening affidabile, veloce ed economico per determinare la presenza/assenza di molecole genetiche in un campione osseo archeologico. La spettroscopia IR è uno strumento utile in quanto è rapida, minimamente distruttiva, economica e sensibile alle variazioni delle proprietà strutturali delle componenti organiche (collagene) e inorganiche (nano cristalli di bioapatite) che costituiscono l’osso. A livello ultrastrutturale, le componenti organiche e inorganiche possono stabilire forti legami con il DNA , stabilizzandolo e determinando la sua sopravvivenza nel tempo. Da campioni archeologici (di epoche e provenienze diverse) estremamente alterati a moderne ossa fresche, abbiamo valutato la sensibilità e l'efficacia di nuovi parametri IR per caratterizzare la diagenesi subita dalle ossa tenendo in considerazioni i cambiamenti delle condizioni climatico–ambientali e di seppellimento. Il lavoro è stato esteso per esaminare le modificazioni indotte dalla diagenesi sulla struttura secondaria del collagene conservato, valutandone gli effetti sui cristalli di bioapatite. I risultati ottenuti dimostrano che il parametro IR che descrive l’ordine/disordine atomico, utilizzato in questa ricerca, è vantaggioso per il monitoraggio di variazioni minime nella struttura e nelle proprietà chimiche della bioapatite nonché indirettamente nel collagene. Questo metodo potrebbe migliorare il processo di selezione dei campioni ossei nonché la loro idoneità per analisi specifiche, ad es. analisi genetiche, paleoproteomiche e degli isotopi stabili sulla base delle analisi spettrali. Viene qui proposto inoltre un modello predittivo funzionale con i parametri infrarossi utilizzati, al fine di determinare il parametro più predittivo per la prensenza/assenza di DNA, utile per ridurre i costi delle analisi genetiche. Dai dati ottenuti, la qualità/quantità di aDNA risulterebbe non essere determinabile a causa dell'influenza di fattori ambientali locali.Ancient bone tissues, recovered from archaeological contexts and preserved within the Museums, represent a valuable source of information on health, diet, mobility of ancient populations as well as on demographics and environmental conditions of the past, useful for researchers and academics. Following the development of modern technologies of omic sciences, osteological finds are increasingly requested and this has led to an increase in the analysis of ancient DNA (aDNA). Sampling methods for ancient DNA extraction are predominantly destructive and may often compromise osteological findings for further future analysis or for studies in other research fields. In addition to invasive and destructive sampling, in poor conservation conditions of the archaeological bone caused by taphonomic and diagenetic alterations, the sequencing of ancient DNA can be an extremely expensive operation. Given the high costs of the aDNA sequencing procedure, an analytical study by means of infrared spectroscopy (FTIR) was conducted in this research work to develop a reliable, fast and inexpensive pre-screening method to determine presence/absence of genetic molecules in an archaeological bone sample. Infrared spectroscopy is a useful tool fast, minimally destructive, inexpensive and sensitive to changes in the structural properties of the organic (collagen) and inorganic (bioapatite nanocrystals) components that make up bone. At the ultrastructural level, the organic and inorganic components of bone may stabilize strong bounds with DNA, stabilizing it and determining its survival over time. The sensitivity and efficiency of new IR parameters was tested on fresh bones and extremely altered archaeological samples, characterized by different chronology and origin. The diagenesis undergone by the bones was characterized taking into account changes in climatic-environmental and burial conditions. The research was expanded by examining changes induced by diagenesis on the secondary structure of collagen preserved, evaluating their effects on bioapatite crystals. The results obtained demonstrate that the IR parameter used in this research, that describes the atomic order/disorder, is advantageous for monitoring minimal changes in the structure and chemical properties of bioapatite as well as indirectly in collagen. This method may improve the selection process of bone samples as well as their suitability for specific analyzes, e.g. genetic, paleo-proteomic and stable isotope analysis on the basis of infrared spectra. A functional predictive model with the infrared parameters used, in order to determine the most predictive parameter for the presence/absence of DNA, allowing to reduce the costs of genetic analyzes, was proposed here. The results obtained, shows that the quality/quantity of aDNA cannot be determined due to the influence of local environmental factors