Our murine model of mild traumatic brain injury (mTBI) has shown improved survival after Pseudomonas aeruginosa (Psd) challenge as compared to controls (tail trauma or sham injury). Previous work suggests an mTBI-specific involvement of the neuro-immune axis which augments the innate immune response, increasing survival. Additional factors for the enhanced mTBI survival were explored via microarray analysis of lungs harvested 48 hours post-trauma, the point prior to Psd challenge in our model. At 48 hours post-trauma, mTBI lungs have a number of upregulated ATP synthesis and mitochondrial gene sets. Increased available energy could prime the mTBI lungs, allowing an earlier and more robust response to Psd infection, possibly contributing to the increased mTBI survival. This is supported by increased neutrophil recruitment in the bronchoalveolar lavage of mTBI mice four hours after Psd instillation. Downregulated gene sets related to cellular connections suggest that neutrophils recruited to the lung have an easier extravasation pathway into the air space of mTBI lungs compared to control. Based on genetic and neutrophil recruitment data, it is possible that mTBI creates an energetically prepared and easily accessible lung better tailored for recruiting and allowing entry of neutrophils in response to an infection compared to control.

Mutations in the human LMNA gene encoding A-type lamins cause a collection of diseases termed laminopathies, including several types of muscular dystrophy. Lamins are intermediate filaments, which line the inner membrane of nuclear envelope. Lamins maintain the nuclear shape and regulate gene expression through interactions with chromatin. Heterozygous mutations LMNA, which result in single amino acid substitutions within the C-terminal Ig-fold domain, were identified in patients with muscular dystrophy. These substitutions were modeled in Drosophila and found to cause muscle defects. We have taken a multi-disciplinary approach to understanding the molecular basis of these muscle defects. Using Nuclear Magentic Resonance (NMR) and Circular Dischroism (CD) we determined that the amino acid substitutions cause perturbations of the tertiary, but not secondary, structure of the Ig-fold. Microarray analysis of RNA isolated from muscle revealed that mutant lamins cause cause mis-regulation of genes involved oxidative stress and neuromuscular junction function. Collectively, these data demonstrate that perturbations within the lamin Ig-fold cause changes in gene expression, providing insights on pathways involved in pathogenesis and identifying new potential therapeutic targets.

Microarrays have been widely used to study differential gene expression at the genomic level. They can also provide genome-wide co-expression information. Robust approaches are needed for integration and validation of independently-collected datasets which may contribute to a common hypothesis. Previously, attempts at meta-analysis have contributed to solutions to this problem. As an alternative, for microarray data from multiple highly similar biological experimental designs, a more direct combined approach is possible. In this thesis, a novel approach is described for microarray combined analysis, including gene-level unification into a virtual platform followed by normalization and a method for ranking candidate genes based on co-expression information - called Coex-Rank. We applied this approach to our Sppar (a PPARγ mutant) dataset, which illustrated an improvement in statistical power and a complementary advantage of the Coex-Rank method from a biological perspective. We also performed analysis to other PPARγ-related microarray datasets. From the perspective of gene sets, we observed that up-regulated genes from mice treated with the PPARγ ligand rosiglitazone were significantly down-regulated in mice with a global knock-in dominant-negative mutation of PPARγ. Integrated with publicly available PPRE (PPAR Response Element) datasets, we found that the genes which were most up-regulated by rosiglitazone treatment and which were also down-regulated by the global knock-in mutation of PPARγ were robustly enriched in PPREs near transcription start sites. In addition, we identified several potential PPARγ targets in the aorta and mesenteric artery for further experimental validation, such as Rhobtb1 and Rgs5.

As human epithelial cells age in culture, protein levels of the tumor suppressor protein p16INK4a continue to increase resulting in growth arrest independent of telomere length. Telomere-independent senescence induced by the p16INK4a/Rb tumor suppressor pathway prevents many epithelial cells from becoming immortalized by telomerase alone. Differences in culture conditions have been hypothesized to modulate both p16INK4a expression and replicative capacity of human epithelial cells; however, the mechanism(s) of p16INK4a regulation under these conditions is unknown. We have demonstrated that p16INK4a expression is delayed and replicative capacity increased in human keratinocytes grown in co-culture with post-mitotic, fibroblast feeder cells as compared to keratinocytes grown on tissue culture plastic alone. We have found that p16INK4a induction in human keratinocytes cultured on plastic alone is associated with a migratory phenotype and that p16INK4a expression is selectively induced in cells possessing markers of keratinocyte migration. Furthermore, we have identified that tyrosine kinase activity and proper functioning of the urokinase plasminogen activation system are required for p16INK4a induction during keratinocyte migration whereas specific signaling through either Src-PTKs or FAK does not appear to regulate this phenomenon. We have shown that human keratinocytes possessing telomerase activity and co-cultured with feeder cells do become immortal without any apparent cellular crisis. In contrast to previous reports, however, we demonstrate that telomerase immortalized keratinocytes co-cultured with feeders do not consistently growth arrest upon transfer to the plastic culture condition and display an increased frequency of p16INK4a promoter methylation. In summary, p16INK4a-induced, telomere-independent senescence is associated with an epithelial migration response and provides a significant proliferation barrier to epithelial cell immortalization regardless of culture conditions. These results provide new insights into p16INK4a regulation and have significant implications for the study of epithelial tumor cell invasion and telomerase reactivation therapies.

Technische Neuerungen und steigende Ansprüche an die Gesundheit stellen die moderne Medizin immer wieder vor neue Herausforderungen und führen zur Entwicklung von neuen Therapiekonzepten wie dem Tissue Engineering. Vielfach kommen dabei adulte pluripotente Stammzellen zum Einsatz. Bei der Regeneration mesenchymalen Gewebes wie Knochen, Knorpel und Muskulatur leisten Mesenchymale Stammzellen (MSCs) einen entscheidenden Beitrag. Diese lassen sich aus allen mesenchymalen Geweben des Körpers gewinnen und stellen daher zwar keine homogene Zellpopulation dar, doch sie lassen sich aufgrund phänotypischer und molekularbiologischer Gemeinsamkeiten charakterisieren. In großer Zahl lassen sich MSCs aus dem Knochenmark gewinnen und werden als stromale MSCs bzw. mhMSCs (marrow-derived human MSCs) bezeichnet. Auf der Suche nach homogenen Subpopulationen von MSCs wurde in dieser Arbeit eine Zellpopulation aus Knochentrabekeln gewonnen, sogenannte bhMSCs (trabecular bone-derived MSCs), und anhand ihrer Genexpression mit mhMSCs verglichen. Dafür wurde RNA aus beiden Populationen in einem Microarray mit anschließender SAM (significance analysis of microarrays) analysiert um unterschiedliche Expressionsmuster zwischen mhMSCs und bhMSCs aufzuzeigen. Diese Ergebnisse wurden durch konventionelle Reverse Transkriptase Polymerase Kettenreaktion (RT-PCR) bestätigt, wobei das Augenmerk vor allem auf solche Gene gerichtet wurde, die differentiell exprimiert waren und zudem als Markergene ein Differenzierungspotential in bestimmte Gewebe wie Muskel und Knochen vorhersagen. Dabei konnte sowohl eine gute Übereinstimmung zwischen Microarray und RT-PCR demonstriert als auch die Hoffnung auf eine homogene (trabekuläre) MSC-Population mit anderen Differenzierungseigenschaften geweckt werden. Im Verlauf weitergehender Untersuchungen der SAM fiel eine unerklärlich hohe Expression von Immunglobulinketten in der mhMSC-Kultur (Passage 0) auf, die letztlich auf eine Kontamination der Zellkultur mit Plasmazellen schließen ließ. Da die Ergebnisse des Microarrays (Passage 0 Kultur) somit zu hinterfragen waren, wurde die Kontamination der Plasmazellen durch Passagieren der mhMSC-Zellkultur (Passage 1) beseitigt und erneut ein Microarray mit SAM durchgeführt. Dabei relativierten sich fast alle Expressionsunterschiede, die somit auf die Kontamination der Plasmazellen zurückgeführt werden mussten. Einzig drei Gene (CD24, TRIB2, AHNAK) wurden in diesem zweiten Array differentiell exprimiert, was sich bei CD24 und TRIB2 auch durch RT-PCR untermauern ließ. Es lässt sich also schlussfolgern, dass bhMSCs wahrscheinlich in der Zukunft des Tissue Engineering keinen Stellenwert haben werden, zumal ihre Gewinnung im Vergleich zu mhMSC deutlich aufwendiger ist. / Technical innovations and increasing demands on health confront modern medicine constantly with new challenges and lead to the development of new therapeutic concepts such as tissue engineering. Often adult pluripotent stem cells are used thereby. In the regeneration of mesenchymal tissues such as bone, cartilage and muscle Mesenchymal stem cells (MSCs) make a significant contribution. These can be harvested from all mesenchymal tissues of the body and do not represent a homogeneous cell population, but they can be characterized due to phenotypic and molecular similarities. In large numbers MSCs can be harvested from the bone marrow and are called stromal MSCs or mhMSCs (marrow-derived human MSCs). Looking for homogeneous subpopulations of MSCs in this thesis was harvested a cell population derived from bone trabeculae, called bhMSCs (trabecular bone-derived MSCs), and was compared with mhMSCs based on their gene expression. RNA was isolated from both populations and analyzed in a microarray followed by SAM (significance analysis of microarrays) to point out different expression patterns between mhMSCs and bhMSCs. These results were confirmed by conventional reverse transcriptase polymerase chain reaction (RT-PCR). The attention was directed primarily to those genes that were differentially expressed and also predicted the differentiation potential to certain tissues such as muscle and bone as so-called marker genes. Both equivalence between microarray and RT-PCR was demonstrated and the hope of a homogeneous (trabecular) MSC population with other differentiating features was awakened. In the course of further investigations of the SAM an inexplicably high expression of immunoglobulin chains in the mhMSC culture (passage 0) was noticed, which indicated a contamination of the cell culture with plasma cells. Since the results of the microarray (passage 0 culture) were thus to question the contamination of the plasma cells was removed by passaging the mhMSC cell culture (passage 1) and a second microarray with SAM was performed. In this case, we could not find these expression differences between both populations anymore. Due to the contamination with plasma cells in the MSC culture all previous results were not valid any more. Only three genes (CD24, Trib2, AHNAK) were differentially expressed in this second array. It can be concluded, therefore, that bhMSCs likely in the future tissue engineering have no value, especially since their harvesting compared to mhMSC is much more complex.

Ascochyta blight, caused by Ascochyta rabiei (Pass.) Labrousse, is one of the most destructive diseases of chickpea (Cicer arietinum L.) worldwide. Despite the existence of highly resistant uncultivated genotypes, attempts to develop cultivars with a high level of durable resistance have been unsuccessful. This study investigated the chickpea defence response to A. rabiei using a functional genomics approach, which has the capacity to improve the overall understanding of the coordinated defence response at a molecular level. An existing cDNA library was used to generate a resource of Expressed Sequence Tags (ESTs) that, after clustering, comprised 516 unigenes. The unigenes were functionally annotated resulting in the identification of 20 specific defence-related unigenes, as well as numerous transcripts with possible involvement in the coordination of defence responses. To explore the expression patterns of the defence-related unigenes in an A. rabiei resistant and susceptible genotype, the unigenes were employed as probes in microarrays. Resulting expression data was analysed to identify differentially expressed unigenes over a time-course after infection. Comparison of the expression profiles from the resistant and susceptible genotype identified three putative genes that were exclusively up-regulated in the resistant genotype, thus may be involved in an effective defence response. Considering that a defence response can involve hundreds of genes, the entire set of chickpea unigenes were used to construct large-scale microarrays. To supplement the chickpea probes, 156 putative defence-related grasspea (Lathyrus sativus L.) ESTs and 41 lentil (Lens culinaris Med.) Resistance Gene Analogs (RGAs) were also included. Expression profiles for three chickpeas and one wild relative were generated over a time course. 97 differentially expressed ESTs were identified using a robust experimental system that included confirmation by quantitative RT-PCR. The results indicated that genes involved in the active defence response were similar to those governed by R-gene mediated resistance, including the production of reactive oxygen species and the hypersensitive response, down-regulation of 'housekeeping' gene expression, and expression of pathogenesis-related proteins. The comparison between resistant and susceptible genotypes identified certain gene expression 'signatures' that may be predictiv e of resistance. To further characterise the regulation of potential defence-related genes, the microarray was used to study expression profiles of the three chickpea genotypes (excluding the wild relative) after treatment with the defence signalling compounds, ethylene (E), salicylic acid (SA), and jasmonate (JA). 425 ESTs were differentially expressed, and comparison between genotypes revealed the presence of a wider range of inducible defence responses in resistant genotypes. Linking the results with the previous microarray results indicated the presence of other pathogen-specific signalling mechanisms in addition to E, SA and JA. The lower arsenal of defence-related gene expression observed in the susceptible genotype may be a result of 'breaks' in the pathways of defence-related gene activation. To draw together the findings of all experiments, a model was constructed for a hypothetical mechanism of chickpea resistance to A. rabiei. The model was synthesised based on the evidence gathered in this study and previously documented defence mechanisms in chickpea, and identified signal transduction as a key to resistance.

The interactions of biomolecules and cells at solid-liquid interfaces play a pivotal role in a range of biomedical applications and have hence been studied in detail. An improved understanding of these interactions results in the ability to manipulate biomolecules and concurrently cells spatially and temporally at surfaces with high precision. Spatial control can be achieved using patterned surface chemistries whilst temporal control is achieved by switchable surfaces. The combination of these two surface properties offers unprecedented control over the behaviour of biomolecules and cells at the solid-liquid interface. This is particularly relevant for cell microarray applications, where a range of biological processes must be duly controlled in order to maximise the efficiency and throughput of these devices. Of particular interest are transfected cell microarrays (TCMs), which significantly widen the scope of microarray genomic analysis by enabling the high-throughput analysis of gene function within living cells Initially, this thesis focuses on the spatially controlled, electro-stimulated adsorption and desorption of DNA. Surface modification of a silicon chip with an allylamine plasma polymer (ALAPP) layer resulted in a surface that supported DNA adsorption and sustained cell attachment. Subsequent high density grafting of poly(ethylene glycol) (PEG) formed a layer resistant to biomolecule adsorption and cell attachment. PEG grafted surfaces also showed significantly reduced attachment of DNA with an equilibrium binding constant of 23 ml/mg as compared with 1600 ml/mg for ALAPP modified surfaces. Moreover, both hydrophobic and electrostatic interactions were shown to contribute to the binding of DNA to ALAPP. Spatial control over the surface chemistry was achieved using excimer laser ablation of the PEG coating which enabled the production of patterns of re-exposed ALAPP with high resolution. Preferential electro-stimulated adsorption of DNA to the ALAPP regions and subsequent desorption by the application of a negative bias was observed. Furthermore, this approach was investigated for TCM applications. Cell culture experiments demonstrated efficient and controlled transfection of cells. Electro-stimulated desorption of DNA was shown to yield enhanced solid phase transfection efficiencies with values of up to 30%. The ability to spatially control DNA adsorption combined with the ability to control the binding and release of DNA by application of a controlled voltage enables an advanced level of control over DNA bioactivity on solid substrates and lends itself to biochip applications. As an alternative approach to surface patterning, the fabrication and characterisation of chemical patterns using a technique that can be readily integrated with methods currently used for the formation of microarrays is also presented. Here, phenylazide modified polymers were printed onto low fouling ALAPP-PEG modified surfaces. UV irradiation of these polymer arrays resulted in the crosslinking of the polymer spots and their covalent attachment to the surface. Cell attachment was shown to follow the patterned surface chemistry. Due to the use of a microarray contact printer it was easily possible to deposit DNA on top of the polymer microarray spots. A transfected cell microarray was generated in this way, demonstrating the ability to limit cell attachment to specific regions and the suitability of this approach for high density cell assays. In order to allow for the high-throughput characterisation of the resultant polymer microarrays, surface plasmon resonance imaging was utilised to study the adsorption and desorption of bovine serum albumin, collagen and fibronectin. This analysis enabled insights into the underlying mechanisms of cell attachment to the polymers studied. For the system analysed here, electrostatic interactions were shown to dominate cellular behaviour.

The mammary explant culture model has been a major experimental tool for studying hormonal requirements for milk protein gene expression as markers of secretory differentiation. Experiments with mammary explants from pregnant animals from many species have established that insulin, prolactin, and glucocorticoid are the minimal set of hormones required for the induction of maximal milk protein gene expression. However, the extent to which mammary explants mimic the response of the mammary gland in vivo is not clear. Recent studies have used microarray technology to study the transcriptome of mouse lactation cycle. It was demonstrated that the each phase of mouse lactation has a distinct transcriptional profile but making sense of microarray results requires analysis of large amounts of biological information which is increasingly difficult to access as the amount of literature increases. / The first objective is to examine the possibility of combining literature and genomic analysis to elucidate potentially novel hypotheses for further research into lactation biology. The second objective is to evaluate the strengths and limitations of the murine mammary explant culture for the study and understanding of murine lactogenesis. The underlying question to this objective is whether the mouse mammary explant culture is a good model or representation to study mouse lactogenesis. / The exponential increase in publication rate of new articles is limiting access of researchers to relevant literature. This has prompted the use of text mining tools to extract key biological information. Previous studies have reported extensive modification of existing generic text processors to process biological text. However, this requirement for modification had not been examined. We have constructed Muscorian, using MontyLingua, a generic text processor. It uses a two-layered generalizationspecialization paradigm previously proposed where text was generically processed to a suitable intermediate format before domain-specific data extraction techniques are applied at the specialization layer. Evaluation using a corpus and experts indicated 86-90% precision and approximately 30% recall in extracting protein-protein interactions, which was comparable to previous studies using either specialized biological text processing tools or modified existing tools. This study also demonstrated the flexibility of the two-layered generalization-specialization paradigm by using the same generalization layer for two specialized information extraction tasks. / The performance of Muscorian was unexpected since potential errors from a series of text analysis processes is likely to adversely affect the outcome of the entire process. Most biomedical entity relationship extraction tools have used biomedical-specific parts-of-speech (POS) tagger as errors in POS tagging and are likely to affect subsequent semantic analysis of the text, such as shallow parsing. A comparative study between MontyTagger, a generic POS tagger, and MedPost, a tagger trained in biomedical text, was carried out. Our results demonstrated that MontyTagger, Muscorian's POS tagger, has a POS tagging accuracy of 83.1% when tested on biomedical text. Replacing MontyTagger with MedPost did not result in a significant improvement in entity relationship extraction from text; precision of 55.6% from MontyTagger versus 56.8% from MedPost on directional relationships and 86.1% from MontyTagger compared to 81.8% from MedPost on un-directional relationships. This is unexpected as the potential for poor POS tagging by MontyTagger is likely to affect the outcome of the information extraction. An analysis of POS tagging errors demonstrated that 78.5% of tagging errors are being compensated by shallow parsing. Thus, despite 83.1% tagging accuracy, MontyTagger has a functional tagging accuracy of 94.6%. This suggests that POS tagging error does not adversely affect the information extraction task if the errors were resolved in shallow parsing through alternative POS tag use. / Microarrays had been used to examine the transcriptome of mouse lactation and a simple method for microarray analysis is correlation studies where functionally related genes exhibit similar expression profiles. However, there has been no study to date using text mining to sieve microarray analysis to generate new hypotheses for further research in the field of lactational biology. Our results demonstrated that a previously reported protein name co-occurrence method (5-mention PubGene) which was not based on a hypothesis testing framework, is generally more stringent than the 99th percentile of Poisson distribution-based method of calculating co-occurrence. It agrees with previous methods using natural language processing to extract protein-protein interaction from text as more than 96% of the interactions found by natural language processing methods coincide with the results from 5-mention PubGene method. However, less than 2% of the gene co-expressions analyzed by microarray were found from direct co-occurrence or interaction information extraction from the literature. At the same time, combining microarray and literature analyses, we derive a novel set of 7 potential functional protein-protein interactions that had not been previously described in the literature. We conclude that the 5-mention PubGene method is more stringent than the 99th percentile of Poisson distribution method for extracting protein-protein interactions by co-occurrence of entity names and literature analysis may be a potential filter for microarray analysis to isolate potentially novel hypotheses for further research. / The availability of transcriptomics data from time-course experiments on mouse mammary glands examined during the lactation cycle and hormone-induced lactogenesis in mammary explants has permitted an assessment of similarity of gene expression at the transcriptional level. Global transcriptome analysis using exact Wilconox signed-rank test with continuity correction and hierarchical clustering of Spearman coefficient demonstrated that hormone-induced mammary explants behave differently to mammary glands at secretory differentiation. Our results demonstrated that the mammary explant culture model mimics in vivo glands in immediate responses, such as hormone-responsive gene transcription, but generally did not mimic responses to prolonged hormonal stimulus, such as the extensive development of secretory pathways and immune responses normally associated with lactating mammary tissue. Hence, although the explant model is useful to study the immediate effects of stimulating secretory differentiation in mammary glands, it is unlikely to be suitable for the study of secretory activation.

Le cancer est une cause principale de décès et d'importants eorts doivent être réalisés pour vaincre la maladie. La technologie des microarrays est un puissant outil de recherche en oncologie pour comprendre les mécanismes de la progression tumorale qui est due à une perturbation de la régulation des gènes. Par conséquent, l'étude de leur niveau d'expression dans les tumeurs offre une perspective pour comprendre les mécanismes biologiques de la maladie et identier de nouveaux facteurs pronostiques et prédictifs qui aideront le clinicien à choisir la thérapie de chaque patients. Par ailleurs, les tumeurs présentent un changement du nombre de copies d'ADN dont la quantication est aussi possible par microarray. L'utilisation des données de microarray nécessite un traitement statistique approprié permettant de transformer les données brutes en données interprétables biologiquement et cliniquement. Ainsi, nous avons développé des méthodes statistiques qui visent à normaliser et extraire l'information biologique issue des microarrays dédiés à l'étude du nombre de copies d'ADN des tumeurs. Nos méthodes ont permis la caractérisation des tumeurs de haut-risque métastatique dans le mélanome uvéal. Par ailleurs, un des enjeux de l'analyse biostatistique des données de microarrays consiste en l'analyse intégrée de différents types de prols moléculaires. Ainsi, une méthode statistique qui combine les données d'expression de gènes et du nombre de copie d'ADN obtenues par microarrays a été développée dans un contexte de classication supervisée. Les propriétés statistiques de la méthode ont été étudiées et ses performances estimées sur des données simulées et réelles.

<p>In this thesis we attempted to elicit patterns of gene expression that influence phenotype, and that may also have been altered by thousands of years of domestication and selection, between red junglefowl and White Leghorn chickens. Red junglefowl are the wild ancestor to all domesticated chickens, and poultry in general are highly valued as a research animal and food resource. The project was also begun in order to complement an earlier study of an intercross between White Leghorn and red junglefowl, which identified several regions that were linked with phenotypic differences between the two birds.</p><p>We began by creating our own cDNA microarray via generating four cDNA libraries from red junglefowl/White Leghorn brain and testis. We generated 12,549 unique transcripts. This included 400 new putative transcripts specific to chickens, and 180 transcripts that were not found in any other database. When investigating polymorphisms between White Leghorn and red junglefowl we found a SNP rate of 1.9/kb coding region, and a synonymous and non-synonymous percentage for these SNPs of 80 and 20% respectively.</p><p>In the last two studies we used the cDNA microarray to measure gene expression differences between White Leghorn and red junglefowl in both hypothalamus/thalamus and liver. We found that there appears to be a significant number of genes down-regulated in White Leghorn hypothalamus/thalamus, plus an over-representation of up-regulated genes from well-known pathways, as compared with red junglefowl. We hypothesize that domestication/selection may be connected with this characteristic. We also found that the p-arm of chicken chromosome 4, which is an ancestral microchromosome, was over represented with differentially expressed genes in hypothalamus/thalamus. A number of differentially expressed genes are shared between the two tissues, and these genes are expressed in same manner between red junglefowl and White Leghorn.</p>