Дисертації з теми "FAT10"

L'accumulation d'hépatocytes sénescents a été identifiée comme un facteur clé dans la progression des maladies du foie gras non alcooliques (NAFLDs), qui correspondent à un spectre de pathologies hépatiques chroniques, allant de la simple stéatose jusqu'au développement d'une stéato-hépatite non alcoolique (NASH), d'une cirrhose voire d'un carcinome hépatocellulaire (HCC). Cependant, les mécanismes et acteurs participant à la régulation de la sénescence au cours de la NASH sont encore peu décrits. L'objectif de cette thèse a donc été d'étudier les mécanismes contrôlant la sénescence des hépatocytes au cours du développement de la NASH. Grâce à des analyses transcriptomiques et protéiques, nous avons montré dans les foies de patients et de souris que la protéine FAT10 (human leukocyte antigen-F Adjacent Transcript 10), aussi appelée UBD (Ubiquitine D), est induite au cours de la NASH. Or, FAT10 est une protéine ubiquitin-like qui interagit avec différents partenaires jouant un rôle dans le métabolisme et la sénescence, nous avons donc émis l'hypothèse que FAT10 pourrait être impliquée dans le développement de la NASH, ainsi que dans l'induction et la propagation de la sénescence des hépatocytes. Dans un premier temps, nous avons montré dans les foies de patients NASH une corrélation positive entre l'expression de FAT10 et la gravité de la maladie. A l'inverse, l'expression de FAT10 diminue lorsque la maladie régresse. Nous avons montré spécifiquement dans les hépatocytes de souris NASH que l'expression de Fat10 corrèle négativement avec les voies du métabolisme des lipides, et que de manière intéressante, la diminution de l'expression de Fat10 dans les hépatocytes des souris NASH diminue la stéatose hépatique, en réduisant la taille et le nombre des gouttelettes lipidiques. Dans un second temps, nous avons montré une corrélation positive entre l'expression de FAT10 et celle des gènes de la sénescence au sein des foies de patients NASH. Cette corrélation est retrouvée spécifiquement dans les hépatocytes chez la souris atteinte de NASH. De plus, dans ce modèle murin, l'expression de Fat10 corrèle positivement avec l'activité SA-β-Gal (Senescence Associated-β-Galactosidase) du foie. In vitro, l'induction de la sénescence dans les hépatocytes humains par une irradiation ou un traitement à l'H2O2 permet d'induire la protéine FAT10 en tant qu'acteur du SASP (Senescence Associated Secretory Phenotype). De manière intéressante, l'inhibition de FAT10 dans ce modèle favorise l'induction et la propagation de la sénescence, par une augmentation de l'activité SA-β-Gal, une induction des gènes du SASP, un arrêt de la prolifération cellulaire accélérée, une induction des acteurs de la réponse aux dommages à l'ADN et une accumulation plus importante de gouttelettes lipidiques. A l'inverse, la surexpression stable de FAT10 dans les hépatocytes sénescents accélère la perte du statut sénescent (diminution de l'activité SA-β-Gal), et promeut l'échappement et l'acquisition d'un phénotype pro-cancéreux par les hépatocytes. Au final, ces données suggèrent que l'induction de FAT10 au sein des hépatocytes lors du développement de la NASH favorise la progression de la maladie, d'une part en altérant le métabolisme des lipides au sein des hépatocytes stéatosés, et d'autre part en favorisant progressivement l'échappement des hépatocytes sénescents à leur phénotype, ce qui pourrait conduire au développement de l'HCC
The accumulation of senescent hepatocytes has been identified as a key factor in the progression of non-alcoholic fatty liver diseases (NAFLDs), which correspond to a spectrum of chronic liver pathologies, ranging from simple steatosis to the development of non-alcoholic steatohepatitis (NASH), cirrhosis or even hepatocellular carcinoma (HCC). However, the mechanisms and actors involved in the regulation of senescence during NASH are still poorly described. The objective of this thesis was therefore to study the mechanisms controlling hepatocyte senescence during the development of NASH. Using transcriptomic and protein analyses, we have shown in the livers of patients and mice that the protein FAT10 (human leukocyte antigen-F Adjacent Transcript 10), also called UBD (Ubiquitin D), is induced during NASH. However, FAT10 is an ubiquitin-like protein that interacts with different partners playing a role in metabolism and senescence, we therefore hypothesized that FAT10 could be involved in the development of NASH, as well as in the induction and spread of hepatocyte senescence. First, we showed in the livers of NASH patients a positive correlation between the expression of FAT10 and the severity of the disease. Conversely, FAT10 expression decreases when the disease regresses. We showed specifically in hepatocytes of NASH mice that the expression of Fat10 negatively correlates with lipid metabolism pathways, and that interestingly, the decrease of Fat10 expression in NASH mice hepatocytes decreases hepatic steatosis, by reducing the size and number of lipid droplets. Secondly, we showed a positive correlation between the expression of FAT10 and of senescence genes in the livers of NASH patients. This correlation is found specifically in hepatocytes in mice. Furthermore, in this mouse model of NASH, Fat10 expression positively correlates with liver SA-β-Gal (Senescence Associated-β-Galactosidase) activity. In vitro, the induction of senescence in human hepatocytes by an irradiation or a treatment with H2O2 induces FAT10 protein as a SASP (Senescence Associated Secretory Phenotype) actor. Interestingly, FAT10 inhibition in this model promotes the induction and propagation of senescence, through an increase of SA-β-Gal activity, an induction of SASP genes, an accelerated cell proliferation arrest, an induction of the DNA damage response system and a greater accumulation of lipid droplets. Conversely, stable overexpression of FAT10 in senescent hepatocytes accelerates the loss of senescent status (decreased SA-β-Gal activity), and promotes the senescence escape and the acquisition of a pro-cancerous phenotype. In the end, all of these data suggest that the induction of FAT10 within hepatocytes during the development of NASH promotes the progression of the disease, on one hand by altering lipid metabolism within steatotic hepatocytes, and on the other hand by gradually promoting the escape of senescent hepatocytes, which could lead to the development of HCC

Chemical sediments in a variety of geomorphic environments (pedogenic, fluvial, palustrine, lacustrine and pan) were investigated from seven regions (SW Kalahari, Kgalagadi, Kalkweissrand, Etosha, Linyanti, Okavango and Ngami) in the Kalahari of southern Africa. These primary and multi-phase sediments were characterised by a range of pure and intergrade silcrete, calcrete and dolocrete fabrics which contained an array of structures indicative of the crystalline and biogenic processes responsible for their precipitation, epigenesis and paragenesis. Petrography, mineralogy and isotope geochemistry provided significant insight regarding the environmental and geochemical conditions at the time of precipitation. Petrography indicated that the majority of chemical sediments were undergoing epigenetic modification as a consequence of the desiccation induced transition from phreatic to vadose diagenetic and geochemical conditions. The role of rapidly infiltrating meteoric water, associated with unstable wetting fronts, is believed to be instrumental in vadose diagenesis and the precipitation of crystalline/alpha fabric carbonate. Salinisation within the capillary fringe and deeper vadose zone is believed to be responsible for the sequence dolomitisation of crystalline calcite within mature sequences. Highly saline pan conditions instigate the precipitation of authigenic dolomite, calcite and K-feldspar within the surface sediments and authigenic silica at depth. Phreatic water beneath the islands, floodplains and fluvial systems of the Okavango, which undergoes evapotranspirational and transmission salinisation and ultimately terminal desiccation, are similarly precipitating silcrete. Pedogenic processes principally associated with C₄ vegetation are responsible for the gratification of carbonate mud within desiccating lacustrine, palustrine and pan sediments. Within the thin sandy Kalahari soils, pedogenesis is limited, but biogenic/beta fabric precipitation linked to mycorrihizae and tree/shrub savanna vegetation is instrumental in the formation of hardpans and the modification of upper calcrete horizons. The dominance of a distinct assemblage (smectite/kaolinite or sepiolite/palygorskite) of authigenic clay minerals present within all the environments provides evidence of semi-arid precipitation within Mg and Ca enriched saline/rapidly evaporating water or brackish/reduced permeability environments.

A question which has arisen clinically is the inability to generate adequate vertical force during the second half of the stance phase in cerebral palsy gait. Neither the mechanism nor the consequences of this inability are understood. It implies that the centre of mass (CoM) is inadequately supported with the potential for collapse of the limb in single support. Normal walking is achieved by the sophisticated control of the neuromuscular and skeletal systems with the purpose of advancing the body with minimum energy expenditure. These control systems are affected by damage to the brain in children with cerebral palsy and result in a multitude of problems which affect their gait making it difficult to determine cause and effect. This study aimed to improve the understanding of the ground reaction forces and motion of the CoM. Objectives The principle objectives were to analyse the supporting forces of the CoM during gait in normal adults, children, and children with diplegic cerebral palsy, by examining the inter-relationships of the vertical and horizontal components of the ground reaction force with the vertical component of motion and temporal parameters of the CoM. Design The study of adults and children’s data was both prospective and retrospective. The study of cerebral palsy data was retrospective. A new software programme was developed to extract specific gait parameters from both normal and cerebral palsy gait patterns. A novel approach to analyse the interaction of motion and force data in normal walking was subsequently developed. Background In normal gait, the vertical component of the ground reaction force forms a double hump where both humps are equal in magnitude and greater than body weight. It is widely accepted that the first hump (FZ1) is associated with deceleration of the downward motion of the CoM as the foot “impacts” the ground and the second hump (FZ2) is the action of “push off” as the body is propelled upwards and forwards. Walking requires force contributions from both legs but traditionally computed three-dimensional (3D) gait analysis packages presents the force data with both legs superimposed, rather than sequentially in time. This gives the impression that there is no period of overlap of the forces and thus ignores the critically important period where the CoM is transferred from one leg to the other during double support. By changing the presentation to view it sequentially in time, plus superimposing the CoM kinematics onto the same scale allowed their interactions to be observed and objectively measured. Methods The kinematic and kinetic data of 53 normal adults, 33 normal children, and 57 diplegic cerebral palsy children were acquired for the study. A new gait cycle, based on CoM motion, was defined to allow analysis of the forces in relation to CoM motion. Software was developed to extract specified parameters from the gait data. Impulse calculations over specific periods (based on the vertical motion of the CoM) allowed analysis of the vertical force contribution of each leg to the vertical support of the CoM. Results The results showed that inadequate generation of vertical force in children with diplegic cerebral palsy was widespread. In approximately 40% of such children there was a reduced ability to generate an adequate FZ2, which resulted in a reduced ability to control of the descent velocity of the CoM. The mean reduction in descent velocity in cerebral palsy was only 54% compared to 86% in adults. Integration of the vertical force and motion of the CoM showed that FZ2 was associated with controlling the descent velocity of the CoM. This reverses the traditionally accepted concept of “impact” and is contrary to the belief that FZ2 is associated with ‘push off’. Conclusions and Relevance This study emphasises the importance of the role of the supporting leg in the second half of stance. Failure to generate an adequate FZ2, which is currently ignored in clinical practice, should be given priority in patient management. This new knowledge has significant implications from a clinical perspective, not only in diplegic cerebral palsy, but in pathologies such as amputees, spina-bifida, and other neuro-muscular conditions.

This thesis investigates selected seventeenth century writings from England and New England to explore the varying significances afforded to the Biblical figure of Adam in theories of the polity. It notes the strong impulse in this period of political upheaval to find foundations and patterns for the polity by returning to the original of Adam’s existence in the Garden of Eden as recorded in Genesis and reinterpreted in Christian tradition. An overview of the times and their political and religious order identifies contemporary preoccupations – legitimacy of sovereignty, security of person and property, freedom of religious conscience, direction of history, expansion to new parts of the world and encounters with new people – that coloured interpretations and political applications of Adam. Against this background, the thesis presents contemporary conversations in which interpretations of Adam played a significant part, proposing a categorisation of Adam as state (the pattern and condition of man and polity) and Adam as story (Creation, Fall, Redemption, Restoration). Under the general heading of Adam as State the conversations include discussions about patriarchalism, natural law and rights, covenant and conscience; under Adam as Story they are concerned with millennial expectation, mystical discourses of the inner man, and the incorporation of the American Indian into a shared narrative that begins in Eden. These themes are developed in detailed studies of selected works by individual authors; Roger Williams, John Eliot, Gerrard Winstanley, John Milton and John Locke. In these works varied approaches are observed as the authors interrogate, expand or contract the story and traditions of Adam in their interpretations of its significance to their times and nation; in particular distinctions are made between theories that use man’s natural state or fallen condition as the foundation of the polity, and those that give eschatological significance to the actions of men, to events and political change. The diversity of methods and theories that result demonstrates the creativity and wide ranging possibility of Adamic political thought at this time.

The diploma thesis deals with the questions of battery powering, measuring of analog physical values using microcontroller and recording of data on a memory SD card Bibliographic reference: REZEK, P. Battery powered datalogger. Brno: Brno University of Technology, Faculty of Electrical Engineering and Communication, 2008. 65 p. Supervisor: Ing. Zdeněk Bradáč, Ph.D.

The aim of Master’s thesis Control of the communication on the USB is realization hardware and software designed for microcontroller C8051F120 what is enabling recording of data on memory medium Flash drive. It includes study of USB, transfer protocols, which are needed and file system. The thesis deals about host type USB controllers and about USB controller MAX3421E fy Maxim, which has been choosen for development of software. It is required ability to create directories and files including recording data to file and ability of reading data from files in the file system FAT16.

Research Doctorate - Doctor of Philosophy (PhD)
Fat cadherins comprise the largest of all known members of cadherin superfamily. They are present in all multicellular organisms and retain a high degree of structural conservation. In Drosophila there are two Fat genes: Fat and Fat-like, whilst in vertebrates there are four members called Fat1, Fat2, Fat3 and Fat4. In Drosophila, the archetype Fat (Ft) cadherin is upstream of a discrete branch Hippo signalling where it also functionally intersects with planar cell polarity (PCP), the process which organizes cells within the plane of an epithelial sheet. A second Drosophila Fat gene, Ft2, is only involved in PCP. Evolutionary divergence has seen expansion to four vertebrate members (Fat1, Fat2, Fat3 and Fat4) with most literature addressing their roles in PCP. In contrast to Drosophila, connections between the vertebrate Fat cadherins and Hippo signalling are not completely established nor fully explored. Neurogenesis describes the cellular processes required for the development and maintenance of the central nervous system. Here the Hippo pathway is emerging as a critical nexus that balances self-renewal of neural progenitors against differentiation. However, while neurogenesis in Drosophila involves Fat-Hippo signalling, the upstream elements in vertebrate Hippo signalling are poorly understood. Prominent expression of Fat1 cadherin is evident within the developing vertebrate neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggests Fat1 may play a critical role in differentiation. Based on these findings, my overarching hypothesis is that the primary function of Fat1 cadherin in the nervous system is to drive neuronal differentiation. This fundamentally occurs through inhibiting self-renewal of neuronal stem cells and/or promoting neuronal differentiation. This function may potentially be driven through one or more cell signalling pathways known to be involved in neurogenesis. The emerging role of the Hippo signalling pathway in stem cell compartments is of particular interest to this notion, along with overlapping effects of Hippo on other major signalling pathways, particularly Shh, BMP and TGF-β pathways. To explore this hypothesis, Chapter 3 investigated the possible roles of FAT1 during neuronal differentiation of SH-SY5Y cells in vitro. These results showed that FAT1 but not other FAT cadherins was induced by neuronal differentiation of SH-SY5Y cells. Using gene-silencing techniques employing shRNA it was then established that FAT1 depletion reduced both neurite initiation and elongation. Moreover, FAT1 knockdown cells displayed comparatively higher rates of proliferation and survival at higher densities. Changes in proliferation were confirmed by altered levels of cell cycle regulators. This indicated that FAT1 was involved in the neuronal differentiation process where it is required for neuritogenesis as well as inhibiting proliferation in a density-dependent manner. Chapter 4 then investigated the possible signalling pathways that are governed by FAT1 during neuronal differentiation. As cell density effects are the hallmark of the Hippo pathway, the involvement of FAT1 engaging Hippo signalling during SH-SY5Y differentiation was examined. Using shRNA-mediated depletion of FAT1 it was inferred that FAT1 served to activate core Hippo kinase components and affected the activities of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target genes ANKRD1 and CTGF. Further investigations indicated that FAT1-signalling did not involve Shh or BMP signalling pathways, however there was crosstalk shown between FAT1-Hippo and elements of the TGF-β pathway. The increase in nuclear TAZ was accompanied with increased nuclear levels of the TGF-β effector Smad3. While FAT1 expression did not influence the levels of TAZ itself, inhibiting FAT1 expression did increase cellular levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ/TGF-β signalling with Hippo signalling mediated through FAT1. Chapter 5 then sought to substantiate the results obtained with SH-SY5Y cells in an alternate in vitro model of neuronal differentiation involving NTera2 cells. The results obtained were entirely in concordance with findings of the SH-SY5Y model. Of all four FAT cadherins, FAT1 was selectively induced. Moreover, FAT1 depletion using siRNA inhibited the initiation of neurites and increased transcription of the Hippo target genes ANKRD1 and CTGF. Therefore these data support the conclusion that FAT1 is involved in neuritogenesis and affects Hippo signalling during neuronal differentiation. As confirmation of these experimental findings, Chapter 6 then investigated the expression and regulation of the Fat cadherin in a number of different physiological models of neuronal differentiation. In silico analyses were undertaken against a number of publically available microarray datasets. These analyses involved differentiation studies of human stem cells in vitro together with in vitro and in vivo and mouse models. In all cases it was shown that Fat1 expression was increased during differentiation thereby validating the findings in the SH-SY5Y and NTera2 differentiation models. Collectively the results from this thesis establish that FAT1 regulates the neuronal differentiation process through governing two key aspects of neurogenesis; these being neuritogenesis and the suppression of cell proliferation. Moreover, establishing that FAT1 acts new upstream Hippo element, it is shown that FAT1 mediates these functions through the Hippo effector TAZ. Furthermore, this serves to inhibit the crosstalk between TAZ and TGF-β signalling during the early stages of neuronal differentiation.

Research Doctorate - Doctor of Philoshphy (PhD)
First identified in Drosophila as a tumour-suppressor gene, Fat cadherin (Ft) and the closely related Fat2 (Ft2) have been identified as giant members of the cadherin superfamily. Ft engages the Hippo signalling pathway during development and both receptors have been shown to function in different aspects of cell polarity and migration. There are four vertebrate homologues, Fat1-Fat4, all closely-related in structure to Drosophila ft and ft2. Over the past decade knock-out mouse studies, genetic manipulation and large sequencing projects have aided our understanding of the function of vertebrate Fat cadherins in tissue development and disease. The majority of studies of this family have focused on Fat1, with evidence now showing it can bind to ENA/VASP, β-catenin and Atrophin proteins to influence cell polarity and motility; Homer1 and 3 proteins to regulate actin accumulation in neuronal synapses; and Scribble to influence the Hippo signalling pathway. Fat2 and Fat3 can regulate cell migration in a tissue specific manner and Fat4 appears to influence both planar cell polarity and Hippo signalling recapitulating the activity of Drosophila Ft. Knowledge about the exact downstream signalling pathways activated by each family member remains in its infancy, but it is becoming clearer that each may have tissue specific and redundant roles in development. Importantly there is also evidence building to suggest that Fat cadherins may be lost or gained in certain cancers. This thesis represents the first in-depth biochemical investigation of human FAT1 cadherin, particularly its comparative expression in normal versus cancer cells. The first chapter studied the expression profile of all FAT cadherins in a panel of 20 cultured melanoma cells where all melanoma cell lines variably, but universally express FAT1 at the mRNA level and less commonly Fat2, Fat3 and Fat4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA levels relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that human FAT1 was site-1 (S1) cleaved into a non-covalent heterodimer before achieving cell surface expression. A similar processing event had been reported in Drosophila Ft indicating that this was an evolutionary conserved mechanism. The use of inhibitors also established such cleavage is catalysed by a member of the proprotein convertase family, likely furin. However, in melanoma cells the non-cleaved pro-form of FAT1 was also expressed on the cell surface together with the S1-cleaved heterodimer. The appearance of both processed and non-processed forms of FAT1 on the cell surface demarked two possible biosynthetic pathways. Moreover FAT1 processing in melanoma cells generated a potentially functional proteolytic product in melanoma cells: a persistent 65kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. Localisation studies of FAT1 both in vitro and in vivo showed melanoma cells display high levels of cytosolic FAT1 protein whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. The mechanisms deriving the unprocessed FAT1 and the p65 product were then further investigated to uncover the potential biological activities of these cancer specific products. The second chapter investigated the mechanisms behind dual processing of FAT1 in cancer cells including the mechanism of FAT1 heterodimerisation. Generally the S1 processing step and accompanying receptor heterodimerisation is thought to occur constitutively but the functional significance of this process in transmembrane receptors has been unclear and controversial. Using siRNA against a number of different proprotein convertases it was established that the S1-cleavage of FAT1 is catalysed only by furin. Mass spectrographic analysis identified the precise location of the cleavage site occurring between the laminin G and the second EGF domain on the extracellular domain of FAT1, consistent with an evolutionarily conserved region found in Drosophila DE-cadherin known to be involved in heterodimerisation. Utilising furin overexpressing studies in melanoma together with the furin deficient LoVo cells, indicated the likely reason behind partial heterodimerisation of FAT1 was deficiency in furin activity. Moreover, it was also determined from these experiments that only the heterodimer form of FAT1 was subject to a second cleavage step (S2) and subsequent release of the extracellular domain. This indicated that S1-processing was a prerequisite for FAT1 ectodomain shedding and established a general biological precedent with implications for the shedding of other transmembrane receptors that undergo heterodimerisation. Part of this work also established an ELISA assay against the extracellular domain of FAT1 that may find utility to investigate shed FAT1 as a potential new cancer biomarker in blood. Previous studies in Drosophila had shown that the interaction between Ft and its ligand, the large cadherin Dachsous (Ds) is regulated through ectodomain phosphorylation mediated by the atypical kinase, Four-jointed (Fj). The third chapter investigated the process of ectodomain phosphorylation of FAT1 on the basis that this important regulatory mechanism may be conserved. Using the known Fj-phosphorylation motif, in silico analyses were undertaken to determine if phosphorylation sites were conserved in human FAT cadherins. This search identified nine potential sites in FAT1 as potential substrates for the sole homologue of Fj in humans, FJX1. Using general antibodies against phospho-serine and phospho-threonine it was revealed that the extracellular domain of FAT1 was multiply phosphorylated on these residues. However, silencing FJX1 using either siRNA or stable shRNA transduction did not indicate any role for FJX1 in FAT1 ectodomain phosphorylation. Nevertheless, given that many regulatory processes are conserved between Drosophila and vertebrate Fat cadherins, the establishment that ectodomain phosphorylation occurs in FAT1 provides the strong likelihood that this process will be important in regulating the interaction of FAT1 with its presently unknown ligand. This knowledge may therefore provide an essential starting point for identifying the ligand of FAT1 and in helping to understand how their interaction is regulated between cells.

碩士
國立陽明大學
口腔生物研究所
103
In the past years, the incidence and mortality rate of oral squamous cell carcinoma (OSCC) have ascended in Taiwan. Therefore, to understand the mechanistic of oral carcinogenesis is a crucial issue. Previously, our laboratory identified the frequent FAT1 mutation in OSCC. FAT1 can be oncogene or even tumor suppressor by regulating cell morphology, motility, and transcription activity in various types of cancers. In OSCC, FAT1 seems to be a tumor suppressor gene. However, the mechanism is still unclear. Pathogenetic specimens whole for NGS analysis of OSCC was profound. After exclusive of SNP and somatic mutation, samples’ point mutations on four sites were found in about 42 to 13 %. As consequence, the topic of my thesis is to identify the association between FAT1 mutation and oral cancer progression. Western Blot and Real-time quantitative PCR (Q-PCR) was used to assay the expression, and identified that the endogenous expression of FAT1 in OSCC comparing to normal oral keratinocyte is downregulated in both protein and mRNA level. When knocking down FAT1 expression with siRNA or shRNA, the cells have higher proliferation, migration, and invasion activity. Furthermore, overexpression of truncated FAT1, seems to reduced malignant level. In the future, we will identify the mechanisms of FAT1 in OSCC. We would like to unraveled that mutated FAT1 could be a potential marker and beneficial for diagnosis and treatment.

Research Doctorate - Doctor of Philosophy (PhD)
Childhood leukaemia is one of the success stories of modern oncology with cure rates of around 80% although a significant number of cases still relapse and die. The therapeutic approaches for adult leukaemia’s are far less successful and collectively the unmet needs of these patients’ drives the need for improved clinical management approaches (diagnostics, prognostics and treatments). Here one of the main obstacles involves the specific identification of leukaemic progenitors but because they originate from their normal counterparts, the discovery of biomarkers that exclusively recognise leukaemic cells has proven to be a demanding task. Continuing the search for new and improved leukaemia biomarkers, this thesis addressed the general hypothesis that Fat1 represents a novel marker for both diagnostic and therapeutic applications in leukaemia. Fat1 is the largest known member of the cadherins, a large superfamily known for their ability to mediate cell-cell and cell substrate adhesion. Seminal studies have shown that the Drosophila homologue of Fat1 functions as a tumour suppressor and engages in fundamental signalling pathways controlling growth and differentiation during development. In Chapter 2, the expression of Fat1 was examined using quantitative PCR and Western blotting in a panel of leukaemic cell lines together with normal blood cells. Fat1 expression was identified in a number of acute lymphoid (ALL) and myeloid (AML) cell lines whereas no expression could be detected in PBMC’s or HSCs from peripheral blood and bone marrow of normal individuals. In silico mining of microarray expression data confirmed the expression of Fat1 in clinical ALL specimens, and further analyses using quantitative PCR in clinical samples revealed the presence of Fat1 mRNA transcripts in 11% of AML, 29% of B-ALL and 63% of T-ALL, respectively. An intensive search throughout normal haematopoiesis found mostly negligible levels of Fat1 mRNA in all of the major haematopoietic lineages, indicating that Fat1 represents a truly differentially expressed leukaemia-antigen. Further investigation across two independent matched diagnosis-relapse cohorts of pre-B ALL found that high Fat1 expression at the time of diagnosis was an independent prognostic marker of poor outcome (relapse-free and overall survival) therefore also suggesting some role for Fat1 in the biology of relapse. In the clinical setting, the measurement of MRD fulfils an important role in the clinical management of ALL, both as a prognostic feature and to direct the proper timing of therapy. To exploit the overexpression of Fat1 in pre-B- and T-ALL, Chapter 3 examined the potential of Fat1 to be used as a marker of MRD. Analysing microarray data from 125 matched diagnosis/relapse samples across three independent data sets, Fat1 mRNA was detectable in an average of 31.3% of diagnosed pre-B-ALL, of which 67.5% of cases remained positive at relapse. Furthermore, some 20% of cases with undetectable levels of Fat1 mRNA at diagnosis became positive upon relapse. Of note we also found that 15.6% of these cases lack any known chromosomal translocations and gene mutations. We also found 83.3% of T-ALL cases were positive for Fat1 expression at diagnosis which 77.7% of them remained positive at relapse. Towards proof of concept, a quantitative polymerase chain reaction assay was developed. Using standardised samples where Fat1-postive leukemic cells were mixed with normal peripheral blood cells, Fat1 mRNA could be detected at a sensitivity of 1 in 10,000 to 100,000 cells. Meeting the established clinical criteria, the results therefore suggest Fat1 could be employed as a marker of MRD for a major subset of patients with ALL including patients without known genetic aberrations. As illustrated by the example of Fat1, no one leukaemia marker is sufficient to identify all cases of the disease. Therefore in Chapter 4 it was hypothesised that a small number of overexpressed genes could provide complete MRD coverage for all patients. This idea was then tested using genome wide expression arrays in combination with a computational approach to identify compatible sets of genes. Using differentially expressed genes, i.e. those expressed in preB-ALL but not in normal B or hematopoietic stem cells, an integer-programming model was used to identify sets of genes that collectively identify the maximum number of preB-ALL cases. As a further refinement we validated the expression of candidate genes in distinct hematopoietic subpopulations for lack of expression in normal cells. Accordingly 13 overexpressed genes were identified in nine sets of three genes. Collectively each unique set provided potential MRD coverage for at least 99.3% of the entire disease cohort. None of the 13 genes have previously been reported in context of pediatric preB-ALL. However, the majority of the identified genes have been implicated directly or indirectly in tumorigenesis or have been recognised as tumor biomarkers in various cancer settings. We also characterised these genes for their continued overexpression in leukemic cells at relapse with a number of identified MRD sets of likely clinical utility.