MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

Abstract Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

Abstract Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.

Genotypic variation in Actinidia deliciosa fruit size and carbohydrate content

In a global and increasingly competitive fresh produce market, more attention is being given to fruit quality traits and consumer satisfaction. Kiwifruit occupies a niche position in the worldwide market, when compared to apples, oranges or bananas. It is a fruit with extraordinarily good nutritional traits, and its benefits to human health have been widely described. Until recently, international trade in kiwifruit was restricted to a single cultivar, but different types of kiwifruit are now becoming available in the market. Effective programmes of kiwifruit improvement start by considering the requirements of consumers, and recent surveys indicate that sweeter fruit with better flavour are generally preferred. There is a strong correlation between at-harvest dry matter and starch content, and soluble solid concentration and flavour when fruit are eating ripe. This suggests that carbon accumulation strongly influences the development of kiwifruit taste. The overall aim of the present study was to determine what factors affect carbon accumulation during Actinidia deliciosa berry development. One way of doing this is by comparing kiwifruit genotypes that differ greatly in their ability to accumulate dry matter in their fruit. Starch is the major component of dry matter content. It was hypothesized that genotypes were different in sink strength. Sink strength, by definition, is the effect of sink size and sink activity. Chapter 1 reviews fruit growth, kiwifruit growth and development and carbon metabolism. Chapter 2 describes the materials and methods used. Chapter 3, 4, 5 and 6 describes different types of experimental work. Chapter 7 contains the final discussions and the conclusions Three Actinidia deliciosa breeding populations were analysed in detail to confirm that observed differences in dry matter content were genetically determined. Fruit of the different genotypes differed in dry matter content mainly because of differences in starch concentrations and dry weight accumulation rates, irrespective of fruit size. More detailed experiments were therefore carried out on genotypes which varied most in fruit starch concentrations to determine why sink strengths were so different. The kiwifruit berry comprises three tissues which differ in dry matter content. It was initially hypothesised that observed differences in starch content could be due to a larger proportion of one or other of these tissues, for example, of the central core which is highest in dry matter content. The study results showed that this was not the case. Sink size, intended as cell number or cell size, was then investigated. The outer pericarp makes up about 60% of berry weight in ‘Hayward’ kiwifruit. The outer pericarp contains two types of parenchyma cells: large cells with low starch concentration, and small cells with high starch concentration. Large cell, small cell and total cell densities in the outer pericarp were shown to be not correlated with either dry matter content or fruit size but further investigation of volume proportion among cell types seemed justified. It was then shown that genotypes with fruit having higher dry matter contents also had a higher proportion of small cells. However, the higher proportion of small cell volume could only explain half of the observed differences in starch content. So, sink activity, intended as sucrose to starch metabolism, was investigated. In transiently starch storing sinks, such as tomato fruit and potato tubers, a pivotal role in carbon metabolism has been attributed to sucrose cleaving enzymes (mainly sucrose synthase and cell wall invertase) and to ADP-glucose pyrophosphorylase (the committed step in starch synthesis). Studies on tomato and potato genotypes differing in starch content or in final fruit soluble solid concentrations have demonstrated a strong link with either sucrose synthase or ADP-glucose pyrophosphorylase, at both enzyme activity and gene expression levels, depending on the case. Little is known about sucrose cleaving enzyme and ADP-glucose pyrophosphorylase isoforms. The HortResearch Actinidia EST database was then screened to identify sequences putatively encoding for sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoforms and specific primers were designed. Sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoform transcript levels were anlayzed throughout fruit development of a selection of four genotypes (two high dry matter and two low dry matter). High dry matter genotypes showed higher amounts of sucrose synthase transcripts (SUS1, SUS2 or both) and higher ADP-glucose pyrophosphorylase (AGPL4, large subunit 4) gene expression, mainly early in fruit development. SUS1- like gene expression has been linked with starch biosynthesis in several crop (tomato, potato and maize). An enhancement of its transcript level early in fruit development of high dry matter genotypes means that more activated glucose (UDP-glucose) is available for starch synthesis. This can be then correlated to the higher starch observed since soon after the onset of net starch accumulation. The higher expression level of AGPL4 observed in high dry matter genotypes suggests an involvement of this subunit in drive carbon flux into starch. Changes in both enzymes (SUSY and AGPse) are then responsible of higher starch concentrations. Low dry matter genotypes showed generally higher vacuolar invertase gene expression (and also enzyme activity), early in fruit development. This alternative cleavage strategy can possibly contribute to energy loss, in that invertases’ products are not adenylated, and further reactions and transport are needed to convert carbon into starch. Although these elements match well with observed differences in starch contents, other factors could be involved in carbon metabolism control. From the microarray experiment, in fact, several kinases and transcription factors have been found to be differentially expressed. Sink strength is known to be modified by application of regulators. In ‘Hayward’ kiwifruit, the synthetic cytokinin CPPU (N-(2-Chloro-4-Pyridyl)-N-Phenylurea) promotes a dramatic increase in fruit size, whereas dry matter content decreases. The behaviour of CPPU-treated ‘Hayward’ kiwifruit was similar to that of fruit from low dry matter genotypes: dry matter and starch concentrations were lower. However, the CPPU effect was strongly source limited, whereas in genotype variation it was not. Moreover, CPPU-treated fruit gene expression (at sucrose cleavage and AGPase levels) was similar to that in high dry matter genotypes. It was therefore concluded that CPPU promotes both sink size and sink activity, but at different “speeds” and this ends in the observed decrease in dry matter content and starch concentration. The lower “speed” in sink activity is probably due to a differential partitioning of activated glucose between starch storage and cell wall synthesis to sustain cell expansion. Starch is the main carbohydrate accumulated in growing Actinidia deliciosa fruit. Results obtained in the present study suggest that sucrose synthase and AGPase enzymes contribute to sucrose to starch conversion, and differences in their gene expression levels, mainly early in fruit development, strongly affect the rate at which starch is therefore accumulated. This results are interesting in that starch and Actinidia deliciosa fruit quality are tightly connected.

New approaches to open problems in gene expression microarray data

In the past decade, the advent of efficient genome sequencing tools and high-throughput experimental biotechnology has lead to enormous progress in the life science. Among the most important innovations is the microarray tecnology. It allows to quantify the expression for thousands of genes simultaneously by measurin the hybridization from a tissue of interest to probes on a small glass or plastic slide. The characteristics of these data include a fair amount of random noise, a predictor dimension in the thousand, and a sample noise in the dozens. One of the most exciting areas to which microarray technology has been applied is the challenge of deciphering complex disease such as cancer. In these studies, samples are taken from two or more groups of individuals with heterogeneous phenotypes, pathologies, or clinical outcomes. these samples are hybridized to microarrays in an effort to find a small number of genes which are strongly correlated with the group of individuals. Eventhough today methods to analyse the data are welle developed and close to reach a standard organization (through the effort of preposed International project like Microarray Gene Expression Data -MGED- Society [1]) it is not unfrequant to stumble in a clinician's question that do not have a compelling statistical method that could permit to answer it.The contribution of this dissertation in deciphering disease regards the development of new approaches aiming at handle open problems posed by clinicians in handle specific experimental designs. In Chapter 1 starting from a biological necessary introduction, we revise the microarray tecnologies and all the important steps that involve an experiment from the production of the array, to the quality controls ending with preprocessing steps that will be used into the data analysis in the rest of the dissertation. While in Chapter 2 a critical review of standard analysis methods are provided stressing most of problems that In Chapter 3 is introduced a method to adress the issue of unbalanced design of miacroarray experiments. In microarray experiments, experimental design is a crucial starting-point for obtaining reasonable results. In a two-class problem, an equal or similar number of samples it should be collected between the two classes. However in some cases, e.g. rare pathologies, the approach to be taken is less evident. We propose to address this issue by applying a modified version of SAM [2]. MultiSAM consists in a reiterated application of a SAM analysis, comparing the less populated class (LPC) with 1,000 random samplings of the same size from the more populated class (MPC) A list of the differentially expressed genes is generated for each SAM application. After 1,000 reiterations, each single probe given a "score" ranging from 0 to 1,000 based on its recurrence in the 1,000 lists as differentially expressed. The performance of MultiSAM was compared to the performance of SAM and LIMMA [3] over two simulated data sets via beta and exponential distribution. The results of all three algorithms over low- noise data sets seems acceptable However, on a real unbalanced two-channel data set reagardin Chronic Lymphocitic Leukemia, LIMMA finds no significant probe, SAM finds 23 significantly changed probes but cannot separate the two classes, while MultiSAM finds 122 probes with score >300 and separates the data into two clusters by hierarchical clustering. We also report extra-assay validation in terms of differentially expressed genes Although standard algorithms perform well over low-noise simulated data sets, multi-SAM seems to be the only one able to reveal subtle differences in gene expression profiles on real unbalanced data. In Chapter 4 a method to adress similarities evaluation in a three-class prblem by means of Relevance Vector Machine [4] is described. In fact, looking at microarray data in a prognostic and diagnostic clinical framework, not only differences could have a crucial role. In some cases similarities can give useful and, sometimes even more, important information. The goal, given three classes, could be to establish, with a certain level of confidence, if the third one is similar to the first or the second one. In this work we show that Relevance Vector Machine (RVM) [2] could be a possible solutions to the limitation of standard supervised classification. In fact, RVM offers many advantages compared, for example, with his well-known precursor (Support Vector Machine - SVM [3]). Among these advantages, the estimate of posterior probability of class membership represents a key feature to address the similarity issue. This is a highly important, but often overlooked, option of any practical pattern recognition system. We focused on Tumor-Grade-three-class problem, so we have 67 samples of grade I (G1), 54 samples of grade 3 (G3) and 100 samples of grade 2 (G2). The goal is to find a model able to separate G1 from G3, then evaluate the third class G2 as test-set to obtain the probability for samples of G2 to be member of class G1 or class G3. The analysis showed that breast cancer samples of grade II have a molecular profile more similar to breast cancer samples of grade I. Looking at the literature this result have been guessed, but no measure of significance was gived before.

Variation in peach fruit susceptibility to Monilinia laxa and gene expression

Brown rot caused by Monilinia laxa and Monilinia fructigena is considered one of the most important diseases affecting Prunus species. Although some losses can result from the rotten fruits in the orchard, most of the damage is caused to fruits during the post-harvest phase. Several studies reported that brown rot incidence during fruit development highly varies; it was found that at a period corresponding to the the pit hardening stage, fruit susceptibility drastically decreases, to be quickly restored afterwards. However the molecular basis of this phenomenon is still not well understood. Furthermore, no difference in the rot incidence was found between wound and un-wound fruits, suggesting that resistance associated more to a specifc biochemical response of the fruit, rather than to a higher mechanical resistance. So far, the interaction Monilinia-peach was analyzed through chemical approaches. In this study, a bio-molecular approach was undertaken in order to reveal alteration in gene expression associated to the variation of susceptibility. In this thesis three different methods for gene expression analysis were used to analyze the alterations in gene expression occurring in peach fruits during the pit hardening stage, in a period encompassing the temporary change in Monilinia susceptibility: real time PCR, microarray and cDNA AFLP techniques. In 2005, peach fruits (cv.K2) were weekly harvested during a 19-week long-period, starting from the fourth week after full bloom, until full maturity. At each sampling time, three replicates of 5 fruits each were dipped in the M.laxa conidial suspension or in distilled water, as negative control. The fruits were maintained at room temperature for 3 hours; afterwards, they were peeled with a scalpel; the peel was immediately frozen in liquid nitrogen and transferred to -80 °C until use. The degree of susceptibility of peach fruit to the pathogen was determined on 3 replicates of 20 fruits each, as percentage of infected fruits, after one week at 20 °C. Real time PCR analysis was performed to study the variation in expression of those genes encoding for the enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), cinnamate 4-hydroxylase (C4H), leucoanthocyanidine reductase (LAR), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and of the jasmonate pathway, such as lipoxygenase (LOX), both involved in the production of important defense compounds. Alteration in gene expression was monitored on fruit samples of a period encompassing the pit hardening stage and the corresponding temporary resistance to M.laxa infections, weekly, from the 6thto the 12th week after full bloom (AFB) inoculated with M. laxa or mock-inoculated. The data suggest a critical change in the expression level of the phenylpropanoid pathway from the 7th to the 8th week AFB; such change could be directly physiologically associated to the peach growth and it could indirectly determine the decrease of susceptibility of peach fruit to Monilinia rot during the subsequent weeks. To investigate on the transcriptome variation underneath the temporary loss of susceptibility of peach fruits to Monilinia rot, the microarray and the cDNA AFLP techniques were used. The samples harvested on the 8th week AFB (named S, for susceptible ones) and on the 12th week AFB (named R, for resistant ones) were compared, both inoculated or mock-inoculated. The microarray experiments were carried out at the University of Padua (Dept. of Environmental Agronomy and Crop Science), using the μPEACH1.0 microarray together with the suited protocols. The analysis showed that 30 genes (corresponding to the 0.6% of the total sequences (4806) contained in the μPeach1.0 microarray) were found up-regulated and 31 ( 0.6%) down regulated in RH vs. SH fruits. On the other hand, 20 genes (0.4%) were shown to be up-regulated and 13 (0.3%) down-regulated in the RI vs. SI fruit. No genes were found differentially expressed in the mock-inoculated resistant fruits (RH) vs. the inoculated resistant ones (RI). Among the up-regulated genes an ATP sulfurylase, an heat shock protein 70, the major allergen Pru P1, an harpin inducing protein and S-adenosylmethionine decarboxylase were found, conversely among the down-regulated ones, cinnamyl alcohol dehydrogenase, an histidine- containing phosphotransfer protein and the ferritin were found. The microarray experimental results and the data indirectly derived, were tested by Real Time PCR analysis. cDNA AFLP analysis was also performed on the same samples. 339 transcript derived fragments considered significant for Monilinia resistance, were selected, sequenced and classified. Genes potentially involved in cell rescue and defence were well represented (8%); several genes (12.1%) involved in the protein folding, post-transductional modification and genes (9.2%) involved in cellular transport were also found. A further 10.3% of genes were classified as involved in the metabolism of aminoacid, carbohydrate and fatty acid. On the other hand, genes involved in the protein synthesis (5.7%) and in signal transduction and communication (5.7%) were found. Among the most interesting genes found differentially expressed between susceptible and resistant fruits, genes encoding for pathogenesis related (PR) proteins were found. To investigate on the association of Monilinia resistance and PR biological function, the major allergen Pru P1 (GenBank accession AM493970) and its isoform (here named Pru P2), were expressed in heterologous system and in vitro assayed for their anti-microbial activity. The ribonuclease activity of the recombinant Pru P1 and Pru P2 proteins was assayed against peach total RNA. As the other PR10 proteins, they showed a ribonucleolytic activity, that could be important to contrast pathogen penetration. Moreover Pru P1 and Pru P2 recombinant proteins were checked for direct antimicrobial activity. No inhibitory effect of Pru P1 or Pru P2 was detected against the selected fungi.

Studio del pathway della Displasia Ectodermica Anidrotica (EDA)

Anhidrotic Ectodermal Dysplasia (EDA), is the most frequent form among Ectodermal Dysplasias, hereditary genetic disorders causing ectodermal appendages defective development. Indeed, EDA is characterized by defective formation of hair follicles, sweat glands and teeth both in human patients and animals. EDA, the gene mutated in Anhidrotic Ectodermal Dysplasia, encodes Ectodysplasin, a TNF family member that activates NF-kB mediated transcription. This disease can occur with mutations in other EDA-NF-kB pathway members, as EDA receptor, EDAR and its adapter, EDARADD. Moreover, mutations in TRAF6, NEMO, IKB and NF-kBs genes are responsible for Immunodeficiency associated EDA (EDA-ID). Several molecules, as SHH, WNT/DKK, BMP and LTβ, have already been reported to be EDA pathway regulators or effectors although the knowledge of the full spectrum of EDA targets remains incomplete. During the first part of the research project a gene expression analysis was performed in primary keratinocytes from Wild-type and Tabby (EDA model mouse) mice to identify novel EDA target genes. Earlier expression profiling at various developmental time points in Tabby and Wild-type mouse skin reported genes differentially expressed in the two samples and, to increase the resolution to find genes whose expression may be restricted to epidermal cells, the study was extended to primary keratinocyte cultures established from E19 Wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 “preliminary candidate” genes whose expression was significantly affected by Eda defect. By comparing expression profiles to those from Eda-A1 (where Eda-A1 is highly expressed) transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. This work confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in primary keratinocytes and in Wild-type and Tabby whole skin, by Q-PCR and Western blotting analyses. Thus, this study detected novel candidate pathways downstream of EDA. In the second part of the research project, plasmid constructs were produced and analyzed to create a transgenic mouse model for Immunodeficiency associated EDA disease (XL-EDA-ID). In particular, plasmids containing mouse Wild-type and mutated Nemo cDNA under K-17 epidermis-specific promoter control and a Flag tag, were prepared, on the way to confine transgene expression to mice epidermis and to determine EDA phenotype without immunodeficiency for a comparison to Tabby model phenotype. EDA-ID mutations reported in patients and selected for this study are: C417R (C409R in mouse), causing Zinc Finger protein domain destabilization and A288G (A282G in mouse) affecting oligomerization of the protein. Moreover, the ex-novo mutation, ZnF, C-terminal Zinc Finger domain deletion, was tested. Thus, the constructs were analyzed by transient transfection, Western blotting and luciferase assays techniques, detecting Nemo Wild-type and mutant protein products and residue NF-kB activity in presence of mutants, after TNF stimulation. In particular, MEF_Nemo-/- cell line was used to monitor NF-kB activity without endogenous Nemo gene. Results show reduced NF-kB activity in presence of mutated Nemo forms compared to Wild-type: 81% for A282G (A288G in human); 24% for C409R (C417R in human); 15% for ZnF. C409R mutation (C417R in human), reported in 6 EDA-ID human patients, was selected to prepare transgenic model mouse. Mice (white, FVP) born following K17-promoter-Flag-Nemo_C409R plasmid region pronuclear injection, were analyzed for the transgene presence in the genotype and a preliminar examination of their phenotype was performed. In particular, one mouse showed considerable coat defects if compared to Wild-type mice. This preliminar analysis suggests a possible influence of Nemo mutant over-expression in epidermis without immunodeficiency. Still, more microscopic studies to analyze hair subtypes, Guard, Awl and Zigzag (usually alterated inTabby mouse model), Immunohistochemistry experiments to detect epidermis restricted Nemo expression and sweat glands analysis, will follow. This and other transgene positive mice will be crossed with black mice C57BL6 to obtain at least two indipendent agouti lines to analyze. Theses mice will be used in EDA target genes detection through microarrays. Following, plasmid constructs containing other Nemo mutant forms (A282G and ZnF) might be studied by the same experimental approaches to prepare more transgenic model mice to compare to Nemo_C409R and Tabby mouse models.

Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

Controllo molecolare del differenziamento osteoblestico per applicazioni nel campo della rigenerazione del tessuto osseo

The aim of the study was to identify expression signatures unique for specific stages of osteoblast differentiation in order to improve our knowledge of the molecular mechanisms underlying bone repair and regeneration. We performed a microarray analysis on the whole transcriptome of human mesenchymal stem cells (hMSCs) obtained from the femoral canal of patients undergoing hip replacement. By defining different time-points within the differentiation and mineralization phases of hMSCs, temporal gene expression changes were visualised. Importantly, the gene expression of adherent bone marrow mononuclear cells, being the undifferentiated progenitors of bone cells, was used as reference. In addition, only the cultures able to form mineral nodules at the final time-point were considered for the gene expression analyses. To obtain the genes of our interest, we only focused on genes: i) whose expression was significantly upregulated; ii) which are involved in pathways or biological processes relevant to proliferation, differentiation and functions of bone cells; iii) which changed considerably during the different steps of differentiation and/or mineralization. Among the 213 genes identified as differentially expressed by microarray analysis, we selected 65 molecular markers related to specific steps of osteogenic differentiation. These markers are grouped into various gene clusters according to their involvement in processes which play a key role in bone cell biology such as angiogenesis, ossification, cell communication, development and in pathways like TGF beta and Wnt signaling pathways. Taken together, these results allow us to monitor hMSC cultures and to distinguish between different stages of differentiation and mineralization. The signatures represent a useful tool to analyse a broad spectrum of functions of hMSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration. Morover, this work will enhance our understanding of bone development and will enable us to recognize molecular defects that compromise normal bone function as occurs in pathological conditions.

Wilmstumor- und Metanephrogenese: differenzielle Genexpressionsanalysen und weitere Charakterisierung identifizierter Kandidatengene

In der vorliegenden Dissertation wurden verschiedene Kandidatengene für den Wilmstumor (WT), eine Tumorerkrankung der Niere, identifiziert und charakterisiert. Da dieses frühkindliche Malignom aus einer inkorrekt ablaufenden Metanephrogenese resultiert, wurden die Genexpressionsmuster verschiedener humaner Wilmstumor- und Normalnierengewebe (adulte sowie fetale Niere) mit Hilfe der Technik des differential display verglichen und die als differenziell exprimiert identifizierten Gene kloniert und charakterisiert. Bei TM7SF1 handelt es sich um ein neues Gen, dessen Transkription im Zuge der Metanephrogenese angeschaltet wird. Das von ihm codierte putative Protein kann aufgrund von Strukturvorhersagen vermutlich zur Familie G Protein-gekoppelter Rezeptoren gezählt werden. Die ableitbare Funktion als Signalmolekül der Nierenentwicklung, sowie seine Lokalisation in einem WT-Lokus (1q42-q43) machen TM7SF1 zu einem aussichtsreichen Kandidatengen für den WT. Darüber hinaus konnten die Voraussetzungen für funktionelle Tests, die eine weitere Charakterisierung von TM7SF1 erlauben, geschaffen werden (Identifikation und Klonierung des murinen Homologen, stabil überexprimierende WT-Zelllinien, Antikörper gegen den Aminoterminus des putativen Proteins). Mit TCF2 wurde ein weiteres Gen identifiziert, dessen Produkt in Prozessen der Metanephrogenese eine Rolle spielt. Die signifikante Herunterregulation der TCF2-Expression in der großen Mehrzahl der untersuchten WTs, die innerhalb der vorliegenden Arbeit gezeigte Regulation durch das WT1-Genprodukt, sowie seine genomische Lokalisation in einem Intervall für die familiäre Form des WT (FWT1 in 17q12-q21) zeigen das Potenzial von TCF2, als Kandidatengen für den FWT zu gelten. Darüber hinaus wurde mit GLI3 ein in verschiedenen WTs stark exprimiertes Gen identifiziert. Sein Produkt ist eine Komponente des entwicklungsbiologisch relevanten und in verschiedene Tumorerkrankungen involvierten sonic hedgehog-Signaltransduktionsweges. Mit FE7A3 und CDT151 konnten zwei differenziell exprimierte cDNAs identifiziert werden, die Teile neuer Gene darstellen und die in WT-Loci kartiert werden konnten. Aufgrund von Homologievergleichen im Bereich der identifizierten offenen Leserahmen konnte eine mögliche Bedeutung der putativen Genprodukte für die WT-Pathogenese als Zelladhäsionsmolekül (FE7A3) bzw. als mit der Proliferation assoziiertem Transkriptionsfaktor (CDT151) herausgearbeitet werden. Neben den komparativen Genexpressionsuntersuchungen wurde in einem zweiten Ansatz die transkriptionelle Regulation des einzigen bisher klonierten Wilmstumorgens (WT1) analysiert. Mit Hilfe vergleichender Reportergenanalysen in WT1-exprimierenden und nicht-exprimierenden Zelllinien konnten neue für die transkriptionelle Regulation von WT1 relevante Bereiche identifiziert werden. Darüber hinaus wurde der für die Transkriptionsfaktoren SP1 und SP3 an anderen Promotoren beschriebene funktionelle Antagonismus für die WT1-Expression untersucht und in Gelretardationsanalysen mit dem WT1-Expressionsstatus oben genannter Zelllinien korreliert.

DNA-Array-Technologie

Es wurde ein genomischer DNA-Array der Modellpflanze Arabidopsis thaliana mit einer 13.800 EST-Klone umfassenden cDNA-Bibliothek entwickelt und in der Genexpressionsanalyse der pflanzlichen Pathogenabwehr eingesetzt. Mittels PCR-Amplifikation sind 13.000 PCR-Produkte der cDNA-Fragmente hergestellt worden, mit denen 66 genomische Arabidopsis-Arrays auf Nylon und Polypropylen als Trägermaterial hergestellt werden konnten. Die Validierung mit Fluoreszenz- und Radiaktivhybridisierung sowie der Vergleich von drei Normalisierungsmethoden führte zu reproduzierbaren Ergebnissen bei hohem Korrelationskoeffizienten. Die etablierte DNA-Array-Technologie wurde zur Genexpressionsanalyse der pathogeninduzierten Abwehrmechanismen der Pflanze Arabidopsis thaliana in den ersten 24 Stunden nach Infektion mit dem avirulenten Bakterium Pseudomonas syringae pv. tomato eingesetzt. In einer Auswahl von 75 Genen der Stoffwechselwege Glycolyse, Citrat-Cyclus, Pentosephosphat-Cyclus und Glyoxylatmetabolismus konnte für 25 % der Gene, im Shikimat-, Tryptophan- und Phenylpropanoidsyntheseweg für 60 % der Gene eine erhöhte Transkriptionsrate nachgewiesen werden. Die Ergebnisse dieser Arbeit stimmen mit experimentellen Daten verschiedener unabhängiger Studien zur pflanzlichen Pathogenantwort überein. Darüberhinaus sind erstmals Transkriptionsprofile von bisher auf Transkriptionsebene nicht untersuchten Genen erstellt worden. Diese Ergebnisse bestätigen die transkriptionelle Aktivierung ganzer Stoffwechselwege und gewähren erstmals einen Einblick in die koordinierte differentielle Transkription ganzer Stoffwechselwege während der Pathogenabwehr.

Comparative genomic sequencing analysis of a region in human chromosome 11p15.3, mouse chromosome 7 and analysis of the novel STK33, Stk33 gene

The comparative genomic sequence analysis of a region in human chromosome 11p15.3 and its homologous segment in mouse chromosome 7 between ST5 and LMO1 genes has been performed. 158,201 bases were sequenced in the mouse and compared with the syntenic region in human, partially available in the public databases. The analysed region exhibits the typical eukaryotic genomic structure and compared with the close neighbouring regions, strikingly reflexes the mosaic pattern distribution of (G+C) and repeats content despites its relative short size. Within this region the novel gene STK33 was discovered (Stk33 in the mouse), that codes for a serine/threonine kinase. The finding of this gene constitutes an excellent example of the strength of the comparative sequencing approach. Poor gene-predictions in the mouse genomic sequence were corrected and improved by the comparison with the unordered data from the human genomic sequence publicly available. Phylogenetical analysis suggests that STK33 belongs to the calcium/calmodulin-dependent protein kinases group and seems to be a novelty in the chordate lineage. The gene, as a whole, seems to evolve under purifying selection whereas some regions appear to be under strong positive selection. Both human and mouse versions of serine/threonine kinase 33, consists of seventeen exons highly conserved in the coding regions, particularly in those coding for the core protein kinase domain. Also the exon/intron structure in the coding regions of the gene is conserved between human and mouse. The existence and functionality of the gene is supported by the presence of entries in the EST databases and was in vivo fully confirmed by isolating specific transcripts from human uterus total RNA and from several mouse tissues. Strong evidence for alternative splicing was found, which may result in tissue-specific starting points of transcription and in some extent, different protein N-termini. RT-PCR and hybridisation experiments suggest that STK33/Stk33 is differentially expressed in a few tissues and in relative low levels. STK33 has been shown to be reproducibly down-regulated in tumor tissues, particularly in ovarian tumors. RNA in-situ hybridisation experiments using mouse Stk33-specific probes showed expression in dividing cells from lung and germinal epithelium and possibly also in macrophages from kidney and lungs. Preliminary experimentation with antibodies designed in this work, performed in parallel to the preparation of this manuscript, seems to confirm this expression pattern. The fact that the chromosomal region 11p15 in which STK33 is located may be associated with several human diseases including tumor development, suggest further investigation is necessary to establish the role of STK33 in human health.

Genomische Struktur und Promotorcharakterisierung des humanen ADAM10-Gens

Das ADAM10-Gen kodiert für eine membrangebundene Disintegrin-Metalloproteinase, die das Amyloidvorläuferprotein spaltet. Im Mausmodell konnte bewiesen werden, dass die Überexpression von ADAM10 die Plaquebildung vermindern und das Langzeitgedächtnis verbessert. Aus diesem Grund ist es für einen möglichen Therapieansatz für die Alzheimer’sche Erkrankung erforderlich, die Organisation des humanen ADAM10-Gens und seines Promotors aufzuklären. Beim Vergleich der genomischen Sequenzen von humanem und murinem ADAM10 zeigte sich eine hohe Übereinstimmung. Beide Gene umfassen 160 kbp und bestehen aus 16 Exons. Die ersten 500 bp stromaufwärts vom Translationsstartpunkt zwischen dem Menschen, der Maus und der Ratte sind hoch konserviert. Diese Region beinhaltet spezifische regulatorische Elemente, die die ADAM10-Transkription modulieren. In den ersten 2179 bp stromaufwärts vom humanen ADAM10-Translationsstartpunkt fanden sich einige potentiellen Transkriptionsfaktor-bindungsstellen (Brn-2, SREBP, Oct-1, Creb1/cJun, USF, Maz, MZF-1, NFkB und CDPCR3HD). Es wurde eine charakteristische GC-Box und eine CAAT-Box, aber keine TATA-Box identifiziert. Nach Klonierung dieser 2179 bp großen Region wurde eine starke Promotoraktivität, insbesondere in neuronalen Zelllinien, gefunden. Bei der Analyse von Deletionskonstrukten wurde die Region zwischen -508 und -300 als essentiell für die Transkriptionsaktivierung bestimmt. Die Promotoraktivität wird zudem streng herunterreguliert, wenn in die Region 317 bp stromaufwärts vom Startpunkt der Translation eine Punktmutation eingeführt wird. Diese per Computeranalyse als USF-Bindungsstelle deklarierte Region spielt eine zentrale Rolle bei der ADAM10-Transkription. Im EMSA wurde eine Protein-DNA-Interaktion für diese Region gezeigt. Durch transienten Transfektionen in Schneider Drosophila Insektenzellen konnte nachgewiesen werden, dass die Überexpression von Sp1 und USp3 für die ADAM10-Promotoraktivität entscheidend ist. In EMSA-Studien bestätigte sich eine Protein-DNA-Interaktion für die Region -366 bp stromaufwärts vom Translationsstartpunkt. Die Punktmutation in der CAAT-Box veränderte die die Promotoraktivität nicht. Da weiterhin für diese potentielle Bindungsstelle kein Bindungsfaktor vorausgesagt wurde, scheint die CAAT-Box keine Bedeutung bei der Promotorregulation zu spielen. Schließlich fand sich im EMSA eine Protein-DNA-Interaktion für die Bindungsstelle 203 bp stromaufwärts vom Translationsstartpunkt. Diese in Computeranalysen als RXR-Bindungsstelle identifizierte Region ist ebenfalls von Bedeutung in der Promotorregulation. Auf der Suche nach Substanzen, die die ADAM10-Promotoraktivität beeinflussen, wurde ein negativer Effekt durch die apoptoseauslösende Substanz Camptothecin und ein positiver Effekt durch die zelldifferenzierungsauslösende Substanz all-trans Retinsäure festgestellt. Mit dieser Arbeit wurde die genomische Organisation des ADAM10-Gens zusammen mit dem zugehörigen Promotor aufgeklärt und ein neuer Regulationsmechanismus für die Hochregulation der Expression der alpha-Sekretase ADAM10 gefunden. Im Weiteren sollen nun die genauen Mechanismen bei der Hochregulation der alpha-Sekretase ADAM10 durch Retinsäure untersucht und durch Mikroarray-Analysen an RNA-Proben transgener Mäuse, welche ADAM10 überexpremieren, neue therapeutische Ansätze zur Behandlung der Alzheimer´schen Erkrankung identifiziert werden.

In vivo- und in vitro-Untersuchungen zur Immunantwort und Parasitenentwicklung bei Infektionen mit dem Darmparasiten Cryptosporidium parvum

Cryptosporidium parvum ist ein intrazellulärer protozoischer Darmparasit (Apikomplexa), der weltweit zu den bedeutendsten Erregern von Diarrhöen beim Menschen und einer Reihe von Nutztieren zählt. Vor allem immunkompromittierte Personen wie zum Beispiel AIDS-Patienten erleiden schwere, chronische bis lebensbedrohende Erkrankungen. Da nach wie vor keine effektive Therapie gegen eine Kryptosporidiose in Form eines spezifisch wirkenden Chemotherapeutikums oder einer Vakzine existiert, ist es notwendig, die Immunantwort des Wirtes gegen den Parasiten und dessen Bindung, Invasion und die intrazelluläre Entwicklung in den Epithelzellen eingehend zu studieren, um neue Ansatzpunkte zu entwickeln. Wohingegen Menschen zeitlebens suszeptibel für eine Infektion mit C. parvum sind, entwickeln Mäuse eine natürliche Resistenz und können als adulte Tiere nicht mehr infiziert werden. Daher sind Mausmodelle der Kryptosporidiose auf neonatale oder immunsupprimierte und immundefiziente adulte Mäuse beschränkt. Bei der Überwindung einer C. parvum-Infektion sind Effektoren der natürlichen und adaptiven Immunität beteiligt. Die zentrale Rolle spielen CD4+-T-Zellen, sowie Interferon-gamma und Interleukin-12. Im Rahmen dieser Arbeit wurden Infektionen in IFN-gamma (GKO)- und IL-12 p40 (IL12KO)-Knockout-Mäusen (C57BL/6) etabliert, für die bereits gezeigt wurde, dass sie eine Suszeptibilität gegenüber einer Erstinfektion besitzen. Erstmals wurden die beiden Infektionsmodelle parallel unter denselben Bedingungen analysiert, um Rückschlüsse auf die Funktion und die Bedeutung der beiden Th1-Zytokine IFN-gamma und IL-12 bei der Auseinandersetzung mit dem Parasiten und der Überwindung einer Infektion ziehen zu können. Es wurden deutliche Unterschiede im Infektionsverlauf, bei der Höhe und Dauer der Parasitenausscheidung und der induzierten systemischen und mukosalen Antikörperantwort beobachtet. Zum ersten Mal konnte gezeigt werden, dass neben IL12KO auch GKO in der Lage sind, eine erste Infektion zu überwinden und eine Resistenz gegenüber einer erneuten Konfrontation mit dem Parasiten zu entwickeln. Alle Ergebnisse deuten darauf hin, dass die Etablierung einer protektiven Immunität gegen eine Kryptosporidiose generell unabhängig von der Anwesenheit der Zytokine IFN-gamma und IL-12 ist, der Verlust von IFN-gamma jedoch schwerer wiegt. Bei GKO-Mäusen persistierte der Parasit in Form einer niedriggradigen chronischen Infektion. Die beiden Infektionsmodelle stellten sich als ideales System für die Etablierung einer effektiven Immunisierungsstrategie heraus. Intranasale Immunisierungen, welche neben einer systemischen auch eine mukosale Immunantwort induzieren können, schienen einen richtigen Ansatz darzustellen. Intraperitoneale und subkutane Immunisierungen führten zwar zur Ausbildung einer starken spezifischen IgG-Antwort im Serum, diese war jedoch nicht in der Lage, einen Schutz vor einer Infektion zu vermitteln. Neben den in vivo Untersuchungen wurde des Weiteren auch die intrazelluläre Entwicklung von C. parvum in einem in vitro Kultursystem verfolgt. Zum ersten Mal wurde die Genexpression von vier Oberflächenproteinen der invasiven Zoitenstadien und eines Oozystenwandproteins parallel durch RT-PCR analysiert. Es konnte gezeigt werden, dass alle untersuchten Gene während der intrazellulären Entwicklung differentiell exprimiert werden, was eine unterschiedliche Funktion der Proteine während des Entwicklungszyklus nahe legt. Das Expressionsmuster der verschiedenen Gene charakterisiert bestimmte Abschnitte innerhalb des Entwicklungszyklus. Dabei wurden Marker für die Invasion (CP17) sowie für die asexuelle (GP900) und sexuelle Replikation (COWP) identifiziert.

Farmacogenomica della Clofarabina nel trattamento delle Leucemie Acute pediatriche: identificazione di nuovi bersagli molecolari e del profilo genomico associato all’efficacia terapeutica del farmaco antitumorale

In quest’ultimi decenni si è assistito ad un notevole miglioramento nella terapia delle Leucemie Acute (LA) pediatriche, nonostante tutto si assiste oggi ad una fase di plateau della curva di sopravvivenza e le leucemie continuano a costituire la principale causa di morte pediatrica per malattia. Ulteriori progressi nel trattamento delle LA potrebbero essere ottenuti mediante studi di farmacogenomica che, identificando le componenti genetiche associate alla risposta individuale ai trattamenti farmacologici, consentono il disegno di terapie personalizzate e tumore-specifiche, ad alta efficacia e bassa tossicità per ciascun paziente. Il lavoro svolto è stato, dunque, finalizzato allo studio della farmacogenomica del farmaco antitumorale Clofarabina (CLO) nel trattamento delle LA pediatriche al fine di identificare marcatori genetici predittivi di risposta delle cellule leucemiche al farmaco, delucidare i meccanismi di resistenza cellulare ed individuare nuovi bersagli verso cui indirizzare terapie più mirate ed efficaci. L’analisi in vitro della sensibilità alla CLO di blasti provenienti da pazienti pediatrici affetti da Leucemia Acuta Linfoblastica (LAL) e Mieloide (LAM) ha consentito l’identificazione di due sottopopolazioni di cellule LAL ad immunofenotipo T a diversa sensibilità alla CLO. Mediante DNA-microarrays, si è identificata la “signature” genetica specificamente associata alla diversa risposta delle cellule LAL-T al farmaco. Successivamente, la caratterizzazione funzionale dei geni differenziali e l’analisi dei pathways hanno consentito l’identificazione specifica di potenziali biomarcatori di risposta terapeutica aprendo nuove prospettive per la comprensione dei meccanismi di resistenza cellulare alla CLO e suggerendo un nuovo bersaglio terapeutico per le forme LAL-T a bassa sensibilità al farmaco. In conclusione, nel lavoro svolto si sono identificati set di geni e pathways di rilievo biologico per la risposta delle cellule LAL-T alla CLO suggerendo marcatori genetici in grado di identificare i soggetti eleggibili per il trattamento o verso cui disegnare terapie innovative. Il lavoro è paradigma per l’applicazione della farmacogenomica in altre neoplasie.

Molekulargenetische Untersuchungen zur Reblausresistenz bei der Unterlagsrebe Börner

Dass Pflanzen gegen phytopathogene Infektionen resistent sind, ist das Ergebnis von multip-len Abwehrreaktionen. Eine solche ist auch die Hypersensitivitätsreaktion (HR). Sie ist die Folge eines Befalls von Börner mit Rebläusen und zeigt sich an Blättern und Wurzeln der resistenten Unterlagsrebe in Form von lokalen Nekrosen. Die Erzeugung von neuen, trans-genen reblausresistenten Unterlagsreben verlangt präzise Kenntnisse über die Mechanismen der Reblausresistenz. Um Resistenzgene zu identifizieren, wurden im Rahmen dieser Arbeit differenzielle Genexpressionsanalysen eingesetzt. Diese waren die Microarray Analyse mit der Geniom one Technik und die real time (RT) -PCR. Sie erlaubten eine Gegenüberstellung der Genexpression in behandeltem Wurzelgewebe mit der Expression im Normalgewebe der Unterlagsrebe Börner. Als experimenteller Induktor der HR in Börner diente die Indol-3-Essigsäure (IES), ein Bestandteil des Reblausspeichels. Frühere Untersuchungen zur Reb-lausresistenz zeigten, dass bei einer Behandlung mit IAA an Wurzeln von Börner Nekrosen entstehen, nicht jedoch an Wurzeln von der reblaustoleranten Unterlagssorte SO4 oder dem reblausanfälligem Edelreis. Das war der Grund, SO4 und Riesling als Vergleichsobjekte zu Börner für diese Studie auszuwählen. So sollte die Bedeutung der Rolle von IES als Auslö-ser der Resistenzmechanismen in Börner erklärt werden. Insgesamt konnten deutliche Unter-schiede in den Reaktionen der drei Rebsorten auf die IES Behandlung aufgedeckt werden. Während in Börner eine hohe Anzahl an Genen und diese intensiv auf den IES Reiz reagiert, fallen die Gene bei SO4 und Riesling zahlenmäßig kaum ins Gewicht und die Reaktionen der beiden Sorten auf IES zudem eher schwach aus. In der Summe waren es 27 Gene, die für die Reblausresistenz in Börner verantwortlich sein könnten. So konnte eine IES bedingte Aktivierung von Genen beobachtet werden, die bei der Produktion von Phytoalexinen be-deutsam sind, wie z.B. die phenylalanine ammonia-lyase, die lipoxygenase und die stilbene synthase. Weiter ließ sich eine Regulation von allgemein Stress assoziierten Genen und von Zellwandproteinen und eine Induktion von Signalkomponenten, etwa des Transkriptionsfak-tors ethylene response factor, nachweisen. Eine deutliche Hochregulation von Au-xintransportern in den IES behandelten Börnerwurzeln gab zudem Anhaltspunkte auf sorten-spezifische Unterschiede in der zellulären Aufnahme und Abgabe der IES. Durch die Ausar-beitung des Zusammenspiels der durch IES regulierten Gene konnten in dieser Arbeit wert-volle Hinweise auf die Mechanismen der Reblausresistenz in Börner gewonnen werden.