Application of stellar photometry to the analysis of microarray images

Improvements in the analysis of microarray images are critical for accurately quantifying gene expression levels. The acquisition of accurate spot intensities directly influences the results and interpretation of statistical analyses. This dissertation discusses the implementation of a novel approach to the analysis of cDNA microarray images. We use a stellar photometric model, the Moffat function, to quantify microarray spots from nylon microarray images. The inherent flexibility of the Moffat shape model makes it ideal for quantifying microarray spots. We apply our novel approach to a Wilms' tumor microarray study and compare our results with a fixed-circle segmentation approach for spot quantification. Our results suggest that different spot feature extraction methods can have an impact on the ability of statistical methods to identify differentially expressed genes. We also used the Moffat function to simulate a series of microarray images under various experimental conditions. These simulations were used to validate the performance of various statistical methods for identifying differentially expressed genes. Our simulation results indicate that tests taking into account the dependency between mean spot intensity and variance estimation, such as the smoothened t-test, can better identify differentially expressed genes, especially when the number of replicates and mean fold change are low. The analysis of the simulations also showed that overall, a rank sum test (Mann-Whitney) performed well at identifying differentially expressed genes. Previous work has suggested the strengths of nonparametric approaches for identifying differentially expressed genes. We also show that multivariate approaches, such as hierarchical and k-means cluster analysis along with principal components analysis, are only effective at classifying samples when replicate numbers and mean fold change are high. Finally, we show how our stellar shape model approach can be extended to the analysis of 2D-gel images by adapting the Moffat function to take into account the elliptical nature of spots in such images. Our results indicate that stellar shape models offer a previously unexplored approach for the quantification of 2D-gel spots. ^

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones

This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62.2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1% of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9% of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel pre-screening, colony isolation and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted. When this is not possible, pre-screening non-sequence verified clones with agarose gel electrophoresis provides an inexpensive and efficient method to eliminate contaminated clones from the probe set.

An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression

The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12–14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.

Host microarray analysis reveals a role for the Salmonella response regulator phoP in human macrophage cell death

Bacterial pathogens manipulate host cells to promote pathogen survival and dissemination. We used a 22,571 human cDNA microarray to identify host pathways that are affected by the Salmonella enterica subspecies typhimurium phoP gene, a transcription factor required for virulence, by comparing the expression profiles of human monocytic tissue culture cells infected with either the wild-type bacteria or a phoP∷Tn10 mutant strain. Both wild-type and phoP∷Tn10 bacteria induced a common set of genes, many of which are proinflammatory. Differentially expressed genes included those that affect host cell death, suggesting that the phoP regulatory system controls bacterial genes that alter macrophage survival. Subsequent experiments showed that the phoP∷Tn10 mutant strain is defective for killing both cultured and primary human macrophages but is able to replicate intracellularly. These experiments indicate that phoP plays a role in Salmonella-induced human macrophage cell death.

Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

Quantitative and qualitative changes in gene expression patterns characterize the activity of plaques in multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disorder of the CNS with both genetic and environmental contributing factors. Clinical symptoms are broadly characterized by initial onset, and progressive debilitating neurological impairment. In this study, RNA from MS chronic active and MS acute lesions was extracted, and compared with patient matched normal white matter by fluorescent cDNA microarray hybridization analysis. This resulted in the identification of 139 genes that were differentially regulated in MS plaque tissue compared to normal tissue. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue<0.0001, ρ=0.73, by Spearman's ρ analysis); while 70 transcripts were uniquely differentially expressed (≥1.5-fold) in either acute or chronic active tissues. These results included known markers of MS such as the myelin basic protein (MBP) and glutathione S-transferase (GST) M1, nerve growth factors, such as nerve injury-induced protein 1 (NINJ1), X-ray and excision DNA repair factors (XRCC9 and ERCC5) and X-linked genes such as the ribosomal protein, RPS4X. Primers were then designed for seven array-selected genes, including transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), GSTP1, crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1) and tubulin β-5 (TBB5), and real time quantitative (Q)-PCR analysis was performed. The results of comparative Q-PCR analysis correlated significantly with those obtained by array analysis (r=0.75, Pvalue<0.01, by Pearson's bivariate correlation). Both chronic active and acute plaques shared the majority of factors identified suggesting that quantitative, rather than gross qualitative differences in gene expression pattern may define the progression from acute to chronic active plaques in MS.

Transcriptome analyses of amoebic gill disease-affected atlantic salmon (Salmo salar) tissues reveal localized host gene suppression

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (± SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response. © 2008 Springer Science+Business Media, LLC.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways. © 2008 Elsevier Ltd. All rights reserved.

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton. Results: We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence) associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion: The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path into the investigation of the gene networks associated with cuticle formation.

Quantitative and molecular genetics of juvenile wood traits in radiata and slash/Caribbean pines.

The Juvenile Wood Initiative (JWI) project has been running successfully since July 2003 under a Research Agreement with FWPA and Letters of Association with the consortium partners STBA (Southern Tree Breeding Association), ArborGen and FPQ (Forestry Plantations Queensland). Over the last five and half years, JWI scientists in CSIRO, FPQ, and STBA have completed all 12 major milestones and 28 component milestones according to the project schedule. We have made benchmark progress in understanding the genetic control of wood formation and interrelationships among wood traits. The project has made 15 primary scientific findings and several results have been adopted by industry as summarized below. This progress was detailed in 10 technical reports to funding organizations and industry clients. Team scientists produced 16 scientific manuscripts (8 published, 1 in press, 2 submitted, and several others in the process of submission) and 15 conference papers or presentations. Primary Scientific Findings. The 15 major scientific findings related to wood science, inheritance and the genetic basis of juvenile wood traits are: 1. An optimal method to predict stiffness of standing trees in slash/Caribbean pine is to combine gravimetric basic density from 12 mm increment cores with a standing tree prediction of MoE using a time of flight acoustic tool. This was the most accurate and cheapest way to rank trees for breeding selection for slash/Caribbean hybrid pine. This method was also recommended for radiata pine. 2. Wood density breeding values were predicted for the first time in the STBA breeding population using a large sample of 7,078 trees (increment cores) and it was estimated that selection of the best 250 trees for deployment will produce wood density gains of 12.4%. 3. Large genetic variation for a suite of wood quality traits including density, MFA, spiral grain, shrinkage, acoustic and non-acoustic stiffness (MoE) for clear wood and standing trees were observed. Genetic gains of between 8 and 49% were predicted for these wood quality traits with selection intensity between 1 to 10% for radiata pine. 4. Site had a major effect on juvenile-mature wood transition age and the effect of selective breeding for a shorter juvenile wood formation phase was only moderate (about 10% genetic gain with 10% selection intensity, equivalent to about 2 years reduction of juvenile wood). 5. The study found no usable site by genotype interactions for the wood quality traits of density, MFA and MoE for both radiata and slash/Caribbean pines, suggesting that assessment of wood properties on one or two sites will provide reliable estimates of the genetic worth of individuals for use in future breeding. 6. There were significant and sizable genotype by environment interactions between the mainland and Tasmanian regions and within Tasmania for DBH and branch size. 7. Strong genetic correlations between rings for density, MFA and MoE for both radiata and slash/Caribbean pines were observed. This suggests that selection for improved wood properties in the innermost rings would also result in improvement of wood properties in the subsequent rings, as well as improved average performance of the entire core. 8. Strong genetic correlations between pure species and hybrid performance for each of the wood quality traits were observed in the hybrid pines. Parental performance can be used to identify the hybrid families which are most likely to have superior juvenile wood properties of the slash/Caribbean F1 hybrid in southeast Queensland. 9. Large unfavourable genetic correlations between growth and wood quality traits were a prominent feature in radiata pine, indicating that overcoming this unfavourable genetic correlation will be a major technical issue in progressing radiata pine breeding. 10. The project created the first radiata pine 18 k cDNA microarray and generated 5,952 radiata pine xylogenesis expressed sequence tags (ESTs) which assembled into 3,304 unigenes. 11. A total of 348 genes were identified as preferentially expressed genes in earlywood or latewood while a total of 168 genes were identified as preferentially expressed genes in either juvenile or mature wood. 12. Juvenile earlywood has a distinct transcriptome relative to other stages of wood development. 13. Discovered rapid decay of linkage disequilibrium (LD) in radiata pine with LD decaying to approximately 50% within 1,700 base pairs (within a typical gene). A total of 913 SNPS from sequencing 177,380 base pairs were identified for association genetic studies. 14. 149 SNPs from 44 genes and 255 SNPs from a further 51 genes (total 95 genes) were selected for association analysis with 62 wood traits, and 30 SNPs were shortlisted for their significant association with variation of wood quality traits (density, MFA and MoE) with individual significant SNPs accounting for between 1.9 and 9.7% of the total genetic variation in traits. 15. Index selection using breeding objectives was the most profitable selection method for radiata pine, but in the long term it may not be the most effective in dealing with negative genetic correlations between wood volume and quality traits. A combination of economic and biological approaches may be needed to deal with the strong adverse correlation.

Microarrays in molecular profiling of cancer - focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.

Secondary metabolites in Gerbera hybrida

Plants produce a diversity of secondary metabolites, i.e., low-molecular-weight compounds that have primarily ecological functions in plants. The flavonoid pathway is one of the most studied biosynthetic pathways in plants. In order to understand biosynthetic pathways fully, it is necessary to isolate and purify the enzymes of the pathways to study individual steps and to study the regulatory genes of the pathways. Chalcone synthases are key enzymes in the formation of several groups of flavonoids, including anthocyanins. In this study, a new chalcone synthase enzyme (GCHS4), which may be one of the main contributors to flower colour, was characterised from the ornamental plant Gerbera hybrida. In addition, four chalcone synthase-like genes and enzymes (GCHS17, GCHS17b, GCHS26 and GCHS26b) were studied. Spatial expression of the polyketide synthase gene family in gerbera was also analysed with quantitative RT-PCR from 12 tissues, including several developmental stages and flower types. A previously identified MYB transcription factor from gerbera, GMYB10, which regulates the anthocyanin pathway, was transferred to gerbera and the phenotypes were analysed. Total anthocyanin content and anthocyanidin profiles of control and transgenic samples were compared spectrophotometrically and with HPLC. The overexpression of GMYB10 alone was able to change anthocyanin pigmentation: cyanidin pigmentation was induced and pelargonidin pigmentation was increased. The gerbera 9K cDNA microarray was used to compare the gene expression profiles of transgenic tissues against the corresponding control tissues to reveal putative target genes for GMYB10. GMYB10 overexpression affected the expression of both early and late biosynthetic genes in anthocyanin-accumulating transgenic tissues, including the newly isolated gene GCHS4. Two new MYB domain factors, named as GMYB11 and GMYB12, were also upregulated. Gene transfer is not only a powerful tool for basic research, but also for plant breeding. However, crop improvement by genetic modification (GM) remains controversial, at least in Europe. Many of the concerns relating to both human health and to ecological impacts relate to changes in the secondary metabolites of GM crops. In the second part of this study, qualitative and quantitative differences in cytotoxicity and metabolic fingerprints between 225 genetically modified Gerbera hybrida lines and 42 non-GM Gerbera varieties were compared. There was no evidence for any major qualitative and quantitative changes between the GM lines and non-GM varieties. The developed cell viability assays offer also a model scheme for cell-based cytotoxicity screening of a large variety of GM plants in standardized conditions.

Prognostic importance of the tumour microenvironment in follicular lymphoma patients treated with immunochemotherapy

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma. It is an indolent and clinically heterogeneous disease, which is generally considered incurable. Currently, immunochemotherapy has significantly improved the outcome of FL patients. This is based on the combination of rituximab, a monoclonal anti-CD20 antibody, with chemotherapy, and is used at present as a standard first-line therapy in FL. Thus far, however, patients have been selected for treatment based on clinical risk factors and indices that were developed before the rituximab era. Therefore, there is a growing need to understand the molecular mechanisms underlying the disease, which would not only provide information to predict survival in the rituximab era, but also enable the design of more targeted therapeutic strategies. In this study, our aim was to identify genes predicting the outcome in FL patients treated with immunochemotherapy. Thus, we performed a cDNA microarray with 24 FL patients. When gene expression differences from diagnostic tumour samples were related to the clinical outcome, we identified novel genes with a prognostic impact on survival. The expression of selected genes was further characterized with quantitative PCR and immunohistochemistry (IHC). Interestingly, the prognostic influence of these genes was often associated with their expression in non-malignant cells instead of tumour cells. Based on the observed gene expression patterns, we analyzed the abundance and prognostic value of non-malignant immune cells in 95-98 FL patients treated with immunochemotherapy. We observed that a high content of tumour-associated macrophages was a marker of a favourable prognosis. In contrast, the accumulation of mast cells correlated with a poor outcome and was further associated with tumour vascularity. Increased microvessel density also correlated with an inferior outcome. In addition, we used the same microarray data with a systems biology approach to identify signalling pathways or groups of genes capable of separating patients with favourable or adverse outcomes. Among the transcripts, there were many genes associated with signal transducers and activators of the transcription (STAT5a) pathway. When IHC was used as validation, STAT5a expression was mostly observed in T-cells and follicular dendritic cells, and expression was found to predict a favourable outcome. In cell cultures, rituximab was observed to induce the expression of STAT5a-associated interleukins in human lymphoma cell lines, which might provide a possible link for the cross-talk between rituximab-induced FL cells and their microenvironment. In conclusion, we have demonstrated that the microenvironment has a prognostic role in FL patients treated with immunochemotherapy. The results also address the importance of re-evaluating the prognostic markers in the rituximab era of lymphoma therapies.