Unique column combinations of a relational database table are sets of columns that contain only unique values. Discovering such combinations is a fundamental research problem and has many different data management and knowledge discovery applications. Existing discovery algorithms are either brute force or have a high memory load and can thus be applied only to small datasets or samples. In this paper, the wellknown GORDIAN algorithm and "Apriori-based" algorithms are compared and analyzed for further optimization. We greatly improve the Apriori algorithms through efficient candidate generation and statistics-based pruning methods. A hybrid solution HCAGORDIAN combines the advantages of GORDIAN and our new algorithm HCA, and it significantly outperforms all previous work in many situations. / Unique-Spaltenkombinationen sind Spaltenkombinationen einer Datenbanktabelle, die nur einzigartige Werte beinhalten. Das Finden von Unique-Spaltenkombinationen spielt sowohl eine wichtige Rolle im Bereich der Grundlagenforschung von Informationssystemen als auch in Anwendungsgebieten wie dem Datenmanagement und der Erkenntnisgewinnung aus Datenbeständen. Vorhandene Algorithmen, die dieses Problem angehen, sind entweder Brute-Force oder benötigen zu viel Hauptspeicher. Deshalb können diese Algorithmen nur auf kleine Datenmengen angewendet werden. In dieser Arbeit werden der bekannte GORDIAN-Algorithmus und Apriori-basierte Algorithmen zum Zwecke weiterer Optimierung analysiert. Wir verbessern die Apriori Algorithmen durch eine effiziente Kandidatengenerierung und Heuristikbasierten Kandidatenfilter. Eine Hybride Lösung, HCA-GORDIAN, kombiniert die Vorteile von GORDIAN und unserem neuen Algorithmus HCA, welche die bisherigen Algorithmen hinsichtlich der Effizienz in vielen Situationen übertrifft.

The general public is at risk of ionising-radiation exposure. The development of high-throughput methods to triage exposures is warranted. Current biodosimetry techniques are low-throughput and encumbered by time and technical expertise. Although there has been an emergence of gene-profiling tools for the purpose of photon biodosimetry, similar capacities do not exist for alpha-particle radiation. Herein is the first genomic study useful for alpha-particle radiation biodosimetric triage. This work has identified robust alpha-particle induced gene-based biomarkers in isolated, ex-vivo irradiated leukocytes from multiple donors. It was found that alpha-particle and photon radiation elicited similar transcriptional responses, which could potentially be distinguished by aggregate-signature analysis. Although no distinct genes were sole indicators of exposure type, clustering algorithms and principal component analysis were able to demarcate radiation type with some success. By comparing the biological effects elicited by photon and alpha-particle radiation, significant contributions have been made to the field of radiation biodosimetry.

Submitted by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:05:56Z No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Approved for entry into archive by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:06:29Z (GMT) No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Made available in DSpace on 2014-11-25T18:06:29Z (GMT). No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / As células-tronco (CTs) caracterizam-se por possuírem a capacidade de se autorrenovar e de dar origem a um ou mais tipos celulares diferenciados. Nos últimos anos, diversos trabalhos mostraram a existência de CTs em tecidos adultos, tornando-as uma alternativa interessante para uso em terapias celulares. Contudo, para melhor utilizar as CTs, é preciso primeiramente compreender como ocorre a diferenciação em um tipo celular específico e, principalmente, como é regulada a expressão gênica durante este processo. Em 2009, Ingolia e colaboradores apresentaram uma nova técnica conhecida como ribosome profiling, a qual consiste no isolamento e sequenciamento em larga escala dos fragmentos de RNA associados e protegidos pelos ribossomos, os quais têm um tamanho aproximado de 30 nucleotídeos (conhecido com footprint ribossomal). Ao mapear as sequências obtidas, é possível obter informações não apenas sobre quais sequências estão sendo traduzidas, mas também sobre a cinética da tradução e sua extensiva rede de regulação. Assim, o objetivo deste trabalho foi aplicar a técnica de ribosome profiling ao estudo do processo de diferenciação de CTs adultas. Como modelo de estudo, foram utilizadas CTs obtidas de tecido adiposo antes (t=0) e após a indução para diferenciação adipogênica por 3 dias (t=72h). O primeiro passo do trabalho foi a adaptação do protocolo de ribosome profiling para o estudo de CTs adultas, o qual consiste na lise celular, digestão do lisado com uma RNA nuclease (a qual irá degradar o RNA exposto, preservando os fragmentos protegidos pelo ribossomo), ultracentifrugação do homogenato sobre colchão de sacarose 1 M para sedimentação dos ribossomos, extração de RNA e isolamento dos fragmentos de 30 nucleotídeos. Também foi feita extração do RNA poliA. As amostras foram sequenciadas (SOLiD™) e os dados obtidos foram triados e mapeados contra um banco de dados de RNAm, utilizando-se a ferramenta CLC Genomics Workbench. Foram identificados mais de 8.000 transcritos para as amostras de ribosome profiling e mais de 17.000 para as de poliA. Ao calcular o fold change entre as condições t=0 e t=72h, foi possível verificar que mais de 50% dos genes foram detectados como diferencialmente expressos apenas por ribosome profiling. Observou-se que genes relacionados com vias de diferenciação adipogênica e de metabolismo de lipídeos encontravam-se regulados positivamente em ambas as amostras de RNA. Por outro lado, observou-se que vias de regulação do citoesqueleto de actina e de adesão focal estavam reguladas negativamente apenas nas amostras de ribosome profiling. Isso é interessante, uma vez que a inibição destas vias já foi descrita como importante para o processo de adipogênese. Além disso, foi observada uma forte redução na eficiência de tradução de genes relacionados com a tradução após 72 horas de indução para diferenciação. Os resultados obtidos no presente trabalho reforçam as evidências de que os mecanismos de regulação pós-transcricionais e traducionais têm um papel muito importante na regulação da diferenciação celular de CTs, sendo que a técnica de ribosome profiling permitiu obter informações mais detalhadas de como este processo pode estar acontecendo. / Stem cells (SC) are characterized by their capacity of both self-renewing and giving rise to new differentiated cells. SC are found in adult tissues, which are considered a putative source for cell therapy. However, little is known about the mechanisms involved in the trigger of SCs differentiation into a specific cell type. Understanding adult SCs differentiation process is a fundamental step to better use and to take advantage of their potential. In 2009, Ingolia and collaborators presented a new methodology of transcriptome analysis named ribosome profiling, which consists on the isolation and deep-sequencing of the mRNA fragments enclosed by ribosomes. When lysed cells are submitted to nuclease digestion, unprotected mRNA is degraded, while fragments within ribosomes are preserved and have a known footprint of 30 nucleotides. Sequencing these ribosome-protected fragments results in a high-precision measurement of in vivo translation, providing precise information about translation kinetics and its extensive regulation. The objective of this work was to apply the ribosome profiling methodology to the study of adipogenic differentiation in adult SCs. SCs were isolated from human adipose tissue from three donors and were cultured in a control medium (t=0) and induced to adipogenic differentiation for 72 hours (t=72h). The first step was to adapt and optimize the ribosome profiling protocol to the SC model, which consists in cell lysis, cell lysate digestion by nuclease (to degrade unprotected RNA, preserving ribosome-protected fragments), ultracentrifugation over a 1M sucrose cushion to pellet ribosomes, RNA extraction and 30 nucleotides fragments isolation. poliA RNA was also isolated. Samples were submitted to deep-sequencing (SOLiD™) and the reads obtained were trimmed and mapped onto the reference mRNA database using the CLC Genomics Workbench. Over 8000 transcripts were identified in ribosome profiling samples and over 17000 in poliA samples. Fold change analysis between t=0 and t=72h of both RNA samples showed that differential expression of more than 50% of the genes was identified only by ribosome profiling. Pathways related to adipogenesis and lipid metabolism were upregulated in both RNA samples. However, regulation of the actin cytoskeleton and focal adhesion proteins were downregulated only in ribosome profiling samples. Interestingly, downregulation of these pathways was already described as an important phenomenon to cell adipogenesis. Besides, we observed a strong reduction of translational efficiency of genes involved in translation at t=72h. Our results reinforce previous data, suggesting that posttranscriptional and translational regulation play a fundamental role in the regulation of SC differentiation process and that ribosome profiling is an important tool to better understand this process.

The general public is at risk of ionising-radiation exposure. The development of high-throughput methods to triage exposures is warranted. Current biodosimetry techniques are low-throughput and encumbered by time and technical expertise. Although there has been an emergence of gene-profiling tools for the purpose of photon biodosimetry, similar capacities do not exist for alpha-particle radiation. Herein is the first genomic study useful for alpha-particle radiation biodosimetric triage. This work has identified robust alpha-particle induced gene-based biomarkers in isolated, ex-vivo irradiated leukocytes from multiple donors. It was found that alpha-particle and photon radiation elicited similar transcriptional responses, which could potentially be distinguished by aggregate-signature analysis. Although no distinct genes were sole indicators of exposure type, clustering algorithms and principal component analysis were able to demarcate radiation type with some success. By comparing the biological effects elicited by photon and alpha-particle radiation, significant contributions have been made to the field of radiation biodosimetry.

Microsoft Research / Although runtime systems and the dynamic compilation model have revolutionized the process of application development and deployment, the associated performance overheads continue to be a cause for concern and much research. In the first part of this thesis, we describe the design and implementation of an adaptive recompilation framework for Rotor, a shared source implementation of the Common Language Infrastructure (CLI) that can increase program performance through intelligent recompilation decisions and optimizations based on the program's past behavior. Our extensions to Rotor include a low overhead runtime-stack based sampling profiler that identifies program hotspots. A recompilation controller oversees the recompilation process and generates recompilation requests. At the first-level of a multi-level optimizing compiler, code in the intermediate language is converted to an internal intermediate representation and optimized using a set of simple transformations. The compiler uses a fast yet effective linear scan algorithm for register allocation. Hot methods can be instrumented in order to collect basic-block, edge and call-graph profile information. Profile-guided optimizations driven by online profile information are used to further optimize heavily executed methods at the second level of recompilation. An evaluation of the framework using a set of test programs shows that performance can improve by a maximum of 42.3% and by 9% on average. Our results also show that the overheads of collecting accurate profile information through instrumentation to an extent outweigh the benefits of profile-guided optimizations in our implementation, suggesting the need for implementing techniques that can reduce such overheads. A flexible and extensible framework design implies that additional profiling and optimization techniques can be easily incorporated to further improve performance. As previously stated, fine-grained and accurate profile information must be available at low cost for advanced profile-guided optimizations to be effective in online environments. In this second part of this thesis, we propose a generic framework that makes it possible for instrumentation based profilers to collect profile data efficiently, a task that has traditionally been associated with high overheads. The essence of the scheme is to make the underlying hardware aware of instrumentation using a special set of profile instructions and tuned microarchitecture. This not only allows the hardware to provide the runtime with mechanisms to control the profiling activity, but also makes it possible for the hardware itself to optimize the process of profiling in a manner transparent to the runtime. We propose selective instruction dispatch as one possible controlling mechanism that can be used by the runtime to manage the execution of profile instructions and keep profiling overheads under check. We propose profile flag prediction, a hardware optimization that complements the selective dispatch mechanism by not fetching profile instructions when the runtime has turned profiling off. The framework is light-weight and flexible. It eliminates the need for expensive book-keeping, recompilation or code duplication. Our simulations with benchmarks from the SPEC CPU2000 suite show that overheads for call-graph and basic block profiling can be reduced by 72.7% and 52.4% respectively with a negligible loss in accuracy.

Les mitochondries jouent un rôle majeur dans le métabolisme de l'ATP des cellules eucaryotes. Le maintien de l'ADN mitochondrial (ADNmt) est fondamental pour la production d'énergie chez les organismes aérobie stricte. De grandes délétions de ADNmt sont à l'origine d'anomalies mitochondriales entrainant des maladies chez l'homme. Plusieurs gènes nucléaires impliqués dans le métabolisme de l’ADNmt ont été identifiés et caractérisés chez l'homme. Cependant, l’ensemble des facteurs et leurs activités requis pour le maintien de l'ADNmt reste largement inconnu. L'identification de ces facteurs et la détermination de leurs activités dans des systèmes modèles simples peuvent contribuer à l’étude du maintien de l'ADNmt et à la compréhension des mécanismes induisant des délétions de l’ADNmt chez l'homme. Le champignon filamenteux Podospora anserina est un modèle d'étude du maintien de l’ADNmt. Chez P. anserina, l’accumulation de délétions région-spécifiques de l’ADNmt (Δmt) est corrélée à la présence de la mutation AS1-4 dans le gène nucléaire codant la protéine cytosolique ribosomale S15. L'altération de la protéine S15 pourrait modifier la traduction de transcrits codant des protéines impliquées dans le maintien de l'ADNmt et indirectement causer l'accumulation des Δmt. Par une approche globale (translatome), nous avons analysé l’ensemble des transcrits associés aux ribosomes AS1-4 en cours de traduction. A partir des données obtenues, deux gènes candidats, PaIML2 et PaYHM2 potentiellement impliqués dans le maintien de l'ADNmt, ont été identifiés et étudiés. L'analyse fonctionnelle a été principalement développée pour PaYHM2. La protéine PaYHM2 partage 68% d’identité avec la protéine mitochondriale bi-fonctionnelle Yhm2 de levure, impliquée dans le transport de métabolites dans la mitochondrie et possèdant un domaine de liaison à l'ADN. J'ai démontré que le gène PaYHM2 est essentiel pour P. anserina, un organisme aérobie stricte et que la protéine PaYHM2 est mitochondriale. Par mutagénèse, j'ai montré que c'est la fonction de transport qui est essentielle à la survie du champignon et non pas la putative capacité à se lier à l'ADN. Les résultats obtenus suggèrent également que PaYHM2 participe au métabolisme de l'acétyl-CoA chez P. anserina. / Mitochondria play main role as adenosine triphosphate (ATP)-energy factories of the eukaryotic cells. To ensure energy production, mitochondrial DNA (mtDNA) maintenance is essential for all obligate-aerobe eukaryotic organisms. Large-scale mtDNA deletions are major causes of mitochondrial dysfunction in human diseases. Several nuclear genes implicated in mtDNA metabolism were identified and characterized in human. Nuclear-encoded factors and their activities required for mtDNA maintenance are, however largely unknown. Identification of these factors and discovery of their activities in simple model systems can contribute to the comprehension of mtDNA maintenance and of the mechanisms leading to mtDNA deletions in human. The filamentous fungus Podospora anserina is a useful model system for studying mtDNA maintenance. An S15 cytosolic ribosomal protein mutant in P. anserina, named AS1-4 mutant, shows a positive correlation with the accumulation of specific large mtDNA deletion (Δmt) at the time of death. Alteration of S15 protein might modify translation of transcripts encoding proteins related to mtDNA maintenance and indirectly cause Δmt accumulation. Polysome profiling (called translatome), a global approach giving genome-wide informations about modified transcripts on translation, was performed on AS1-4 mutant. From the data of this translatome, two candidate genes potentially related to mitochondrial DNA maintenance, the PaIML2 gene and PaYHM2 gene has been identified and functionally analyzed. The function of the PaYHM2 gene has been especially characterized in this project. This gene encodes a protein sharing 68% of identity with yeast Yhm2, a bi-functional protein as a mitochondrial carrier and as a protein with DNA-binding activity. I demonstrated that the PaYHM2 gene is essential for P. anserina, an obligate-aerobe organism and that the PaYHM2 protein localizes to mitochondria. Through mutagenesis approach, I showed that the transport function decides the essentiality of mitochondrial carrier PaYHM2 while the putative DNA binding activity of PaYHM2 protein is important for P. anserina. Furthermore, I found that the function of PaYHM2 probably participates in the cytosolic acetyl-CoA metabolism.

Die von der caprotec bioanalytics GmbH entwickelte und vermarktete Capture Compounds Mass SpectrometryTM (CCMS-Technologie) ermöglicht die spezifische Isolierung und Identifizierung von Proteinen, basierend auf ihre gezielte Wechselwirkung mit kleinen trifunktionalen Moleküle (sog. Capture CompoundsTM). Dadurch wird sowohl eine Reduzierung der Komplexität zellulärer Proteingemische als auch eine Anreicherung niedrig abundanter Proteine erreicht. In der vorliegenden Arbeit erfolgte die Synthese von neuartigen Capture Compounds, die dazu dienen sollen, unterschiedliche biochemische Fragestellungen zu beantworten und das Anwendungsspektrum der CCMS-Technologie stark erweitern. / The Capture Compound Mass SpectrometryTM (CCMS-Technology) is a novel technology developed and marketed by caprotec bioanalytics GmbH. CCMS allows the isolation of sub-proteomes based on specific interactions of target proteins with synthetic small molecules, called Capture Compounds (CCs). In this way, CCMS affords the functional reduction of complex protein mixtures that derived from e.g. cell lysates, and the enrichment and identification of low abundant proteins. In the present work, the synthesis of different novel Capture Compounds that helps studying different biological problems and overcoming present technological limitations of CCMS is described.

Ein genereller Ansatz zur Charakterisierung von biologischen Systemen bietet die Untersuchung des Metaboloms, dessen Analyse als „Metabolomics“ bezeichnet wird. “Omics”- Technologien haben das Ziel, ohne Selektionskriterien möglichst alle Bestandteile einer biologischen Probe zu detektieren (identifizieren und quantifizieren), um daraus Rückschlüsse auf nicht vorhersehbare und somit neuartige Korrelationen in biologischen Systemen zu ziehen. Ein zentrales Dogma in der Biologie besteht in der Kausalität zwischen Gen – Enzym – Metabolite. Perturbationen auf einer Ebene rufen systemische Antworten hervor, die in einem veränderten Phänotyp münden können. Metabolite sind die Endprodukte von zellulären regulatorischen Prozessen, deren Abundanz durch die Resonanz auf genetische Modifikationen oder Umwelteinflüsse zurückzuführen ist. Zudem repräsentieren Metabolite ultimativ den Phänotyp eines Organismus und haben die Fähigkeit als Biomarker zu fungieren. Die integrale Analyse verschiedenster Stoffwechselwegen wie Krebszyklus, Pentosephosphatzyklus oder Calvinzyklus offeriert die Identifikation von metabolischen Mustern. In dieser Arbeit wurden sowohl das targeted Profiling via GC-TOF-MS als auch das untargeted Profiling via GC-TOF-MS und LC-FT-MS als analytische Strategien genutzt, um biologische Systeme anhand ihrer Metabolite zu charakterisieren und um physiologische Muster als Resonanz auf endogene oder exogene Stimuli zu erkennen. Dabei standen die metabolische, phänotypische und genotypische Plastizität von Pflanzen im Fokus der Untersuchungen. Metabolische Varianzen eines Phänotyps reflektieren die genotyp-abhängige Resonanz des Organismus auf umweltbedingte Parameter (abiotischer und biotischer Stress, Entwicklung) und können mit sensitiven Metabolite Profiling Methoden determiniert werden. Diese Anwendungen haben unter anderem auch zum Begriff des „Environmental Metabolomics“ geführt. In Kapitel 2 wurde der Einfluss biotischer Interaktionen von endophytischen Bakterien auf den Metabolismus von Pappelklonen untersucht; Kapitel 3 betrachtet die metabolische Plastizität von Pflanzen im Freiland auf veränderte biotische Interaktionsmuster (Konkurrenz/Diversität/Artenzusammensetzung); Abschließend wurde in Kapitel 4 der Einfluss von spezifischen genetischen Modifikationen an Peroxisomen und den daraus resultierenden veränderten metabolischen Fluss der Photorespiration dargestellt. Aufgrund der sensitiven Analyse- Technik konnten metabolische Phänotypen, die nicht zwingend in einen morphologischen Phänotyp mündeten, in drei biologischen Systemen identifiziert und in einen stoffwechselphysiologischen Kontext gestellt werden. Die drei untersuchten biologischen Systeme – in vitro- Pappeln, Grünland- Arten (Arrhenatherion-Gesellschaft) und der Modellorganismus (Arabidopsis) – belegten anschaulich die Plastizität des Metabolismus der Arten, welche durch endogene oder exogene Faktoren erzeugt wurden. / A general approach to characterise biological systems offers the analysis of the metabolome, named “metabolomics”. “Omics”- technologies are untargeted approaches without any selection criteria which aim to detect every potential analyte in a sample in order to draw conclusions about new correlations in biological systems. A central dogma in biology is the causality between gene – enzyme – metabolite. Perturbations on one level are reflected in systemic response, which possibly result in a changed phenotype. Metabolites are end products of its gene expression and metabolism, whose abundance is determined as a resonance of genetic modifications or environmental disturbance. Furthermore metabolites represent the ultimate phenotype of an organism and are able to act as a biomarker. The integral analysis of distinct metabolic pathways like TCA, Pentose phosphate and Calvin cycle consequently leads to the identification of metabolic patterns. In this work targeted profiling via GC-TOF-MS as well as untargeted profiling via GC-TOF-MS and LC-FT-MS were used as analytical strategies to characterise biological systems on the basis of their metabolites and to identify physiological patterns as resonance of endogenic or exogenic stimuli. The focus of the investigations concentrates on the metabolic, phenotypic and genotypic plasticity of plants. Metabolic variance of a phenotype is reflected in the genotypic dependence response of an organism on environmental parameters which may be detected via sensitive metabolic profiling methods. In chapter 2 the influence of biotic interaction of endophytic bacteria on the metabolism of their poplar host was analyzed; chapter 3 explores the metabolic plasticity of field-grown grassland species as a consequence of biotic interaction pattern (competition / diversity / species composition); In conclusion, chapter 4 illustrates the influence of specific genetic modifications on peroxisomes and the consequent changed metabolic flux in the photorespiration pathway. Due to the sensitive analytic methods, metabolic phenotypes in all three biological systems could be identified and classified in a physiological context. The three biological systems – in vitro poplar plants, field-grown grassland species and the model organism Arabidopsis – demonstrate the plasticity of the metabolism of species in response to stimuli.

1. Despite extensive research on oral squamous cell carcinoma (OSCC), its five-year survival rate has not improved for the last two decades. Effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, DDRT-PCR analysis was used to identify molecular markers, which could be used as therapeutic targets. 2. DDRT-PCR in combination with reverse Northern analysis identified 25 differentially expressed genes in oral tumors. Fourteen genes did not show homology to any known gene in the database and therefore may represent non-specific genomic DNA sequences or novel genes that have not yet been identified. The remaining 11 genes showed homology to known genes such as DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, MTATP6, ZKSCAN1, TNKS2, PAM, TUBB2C and C14orf154. TNKS2, PAM, TUBB2C and C14orf154 showed downregulation and the remaining seven genes were upregulated in oral tumor samples. 3. To reconfirm the results of DDRT-PCR and reverse Northern blot analyses, Northern blot analysis was carried out on matched normal and tumor samples for a few genes. As expected, PCNA, NJMU-R1 and ZKSCAN1 showed upregulation, whereas TUBB2C showed downregulation in the tumor sample. PCNA was also found to be upregulated in tumor samples at the protein level. 4. The expression of eight differentially expressed genes (viz., DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, TNKS2, PAM and TUBB2C) was also validated in a panel of 16 matched normal and tumor samples. The mean mRNA expression levels of GLTP, PCNA, RBM28, NJMU-R1 and DIAPH1 were significantly greater in tumor samples than in normal samples. The mean expression levels of TNKS2, PAM and TUBB2C were significantly lower in tumor samples than in normal samples. 5. As some of the genes like NJMU-R1, RBM28, GLTP and PAM are found to differentially regulated in a majority of the tumors, they could be used as potential markers in oral cancer. 6. Tuberin and hamartin have been placed as a complex in the insulin signaling pathway and are known to negatively regulate this pathway. Since overexpression of TSC2 has been previously shown to exert antitumor effect on two oral cancer cell lines, and some components of the insulin signaling pathway have already been implicated in head and neck cancers, we reasoned that both TSC genes and other key players of this pathway might be differentially regulated in oral tumors. Northern blot analysis showed downregulation of the TSC2 gene in an oral tumor sample. In order to further validate the expression pattern of the TSC2 gene, a semiquantative RT-PCR analysis was carried out in a panel of 16 matched normal and tumor samples. The mean expression level of TSC2 was significantly lower in tumor samples than in normal tissue samples. The mean expression level of its interacting partner TSC1 was also significantly lower in tumor samples than in normal tissue samples, suggesting the involvement of these genes in the etiology of oral cancer. TSC1 and TSC2 were also downregulated in eight matched normal and tumor samples at the protein level. We wanted further to determine the expression of both TSC genes in cell lines. Interestingly, TSC2 did not show a detectable level of expression in an oral cancer cell line SCC 131, whereas it was expressed in two other oral cancer cell lines KB and SCC 104 as well as in four non-oral cell lines: A549, HEK-293T, HeLa and HepG2 at the protein level. The TSC2 expression in KB was, however, lower than in other cell lines. TSC1 was expressed in all the cell lines, albeit at different levels. The TSC1 expression was lower in SCC 131 as compared to two other cell lines KB and SCC 104. 7. Given the fact that both are tumor suppressors, it was hypothesized that LOH, inactivating somatic mutations and/or promoter methylation might be playing a role for their downregulation in oral tumors. Mutation analysis of all the coding regions of both the TSC genes failed to detect any mutation in a panel of 25 tumor samples. However, seven normal population variants were identified in different patients. Our analysis of the matched peripheral blood and tumor DNA samples from 52 patients showed LOH at both the TSC loci. At the TSC1 locus, 17/48 (35.42%) tumors showed an allelic loss for one or more markers. At the TSC2 locus, LOH was found in 18/48 (37.5%) informative cases. Nine patients (9/48, 18.75%) had LOH at both the TSC loci. Since PTEN is another tumor suppressor in the insulin signaling pathway, we then sought to determine if LOH is also present in the PTEN candidate region in a panel of 50 matched samples. Microsatellite analysis using three markers showed a low LOH rate of 13% in tumor samples. 8. As the OSCC cell line SCC 131 did not show a detectable level of TSC2 expression, we treated this cell line with methylation inhibition drug 5-azacytidine. The treatment restored the expression of TSC2 and increased the expression of TSC1, suggesting that the promoter methylation and LOH are the important mechanisms for their downregulation. In order to see if the downregulation of the TSC genes is due to their promoters being methylated in tumors from the patients, we examined the methylation status of their promoters in 16 oral tumors, three normal oral tissues, two peripheral blood DNA samples from normal individuals and two cell lines HeLa and SCC 131 by COBRA. Our repeated efforts to amplify the TSC1 promoter using different DNA polymerases failed. However, we were able to successfully amplify the 571 bp long TSC2 promoter. Our analysis showed methylation of the TSC2 promoter in all tumors and two cell lines. As expected, the TSC2 promoter was not methylated in normal oral tissues and control blood DNA samples. Our bisulfite sequencing data suggested a low level and a considerable heterogeneity of methylation. 9. Using Fisher’s exact test, no correlation was found between LOH at the TSC loci and different clinical parameters such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis. 10. Using Fisher’s exact test, no correlation was found between the TSC2 promoter methylation and its downregulation in 16 tumor samples. We believe that this could be due to small sample size. 11. Since TSC1 and TSC2 are important regulators of the insulin pathway, it was hypothesized that other key players of this pathway might also be dysregulated in oral cancer. To this end, the expression pattern of some of the major regulators of the insulin pathway (viz., PI3K, AKT, PDK1, RHEB, mTOR, S6K1, S6, eIF4E, 4E-BP1, PTEN, 14-3-3ﾟ and IRS1) was investigated using semiquantative RT-PCR in a panel of 16 matched normal and tumor samples. The mean expression levels of the following genes showed significant upregulation in tumor samples: AKT, PI3K, PDK1, RHEB, mTOR, S6K1, S6 and eIF4E. On the other hand, 4E-BP1 and PTEN showed significant downregulation in tumor tissues. No significant difference in the expression was found for 14-3-3ﾟ and IRS1 between tumor and normal tissues. The expression pattern of some of these genes was also analyzed at the protein level using Western blot analysis and eight matched normal and tumor tissues. The level of total AKT was upregulated in 2/8 tumor samples only. However, phosphorylated-AKT (Thr308) showed upregulation in 6/8 samples. p70S6K1 and phosphorylated-p70S6K1 (Thr389) were upregulated in 8/8 and 6/8 tumor samples, respectively. Increase in the phosphorylated forms of both AKT and its downstream effector p70S6K1 suggested an increase in their kinase activity, indicating a constitutive activation of this pathway in oral cancer. 12. Based on our findings of mutation analysis, LOH study, 5-azacytidine treatment of an oral cancer cell line and COBRA analysis, we suggest that LOH at the TSC gene loci and promoter methylation are important mechanisms for the downregulation of the TSC genes. Loss of function of these genes may thus contribute to the constitutive activation of the insulin signaling pathway in oral cancer, leading to overall cell growth and proliferation. Our studies have shown that several key members of this pathway show aberrant expression in a subset of cancers of the oral cavity and can provide useful therapeutic targets. Several inhibitors of the insulin signaling pathway, such as rapamycin and its derivatives which inhibit mTOR and the PI3K inhibitor wortmannin, are now being actively evaluated for clinical trials for other cancers. We suggest that these inhibitors could also be evaluated for the treatment of oral cancer in future. Our differential display analysis has served to identify several genes that may be important for the onset and progression of oral cancer. Further analysis of these genes is warranted.

No description available.