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Tübingen, Univ., Diss., 2003.

Malignant mesothelioma (MM) is an aggressive tumour, which is highly associated with previous asbestos exposure and is resistant to most conventional anticancer therapies. Previous studies have used a mouse model of to 01 p effective approaches to induction of anti-tumour immunity using modification of tumour cells by the introduction of genetic constructs expressing genes such as that for B7-1 so that tumour growth can be inhibited in vivo. Transfectant clones, AC29 B7-7 and AC29 B7-6, which showed equal levels of expression of B7-1 but were markedly different in tumorigenicity were assessed using suppression subtractive hybridization (SSH) in order to isolate transcripts which may have been differentially expressed in the two clones. SSH allowed isolation of a number of cDNAs which were apparently differentially expressed in the cell lines. These required characterisation in order to determine their possible relevance to tumorigenicity. Two cDNAs designated as 7-7-76 and 7-7-43 had been isolated previously and the aim of this project was to characterise these cDNAs by sequencing, searching for their homology relationships and investigating gene expression profiles. Preliminary searches revealed that clone 7-7-43 had homology to cyclin-dependent kinase regulatory subunit 1 which plays a role in the cell cycle. On the other hand, clone 77-76 showed only homology to an EST of hypertension related protein and therefore, further investigation was required to obtain the identity of clone 7-7-76. The first part of this project was to in investigate and evaluate gene expression on clone 7-7-43, using both relative RT-PCR and Northern blotting.' In the second part of this project, a more intense study of clone 7-7-76 was conducted. Clone 7-7-76 was investigated for its homology relationships and its gene expression profile. / Results obtained from relative RT-PCR suggested no difference in the expression of the either eDNA clone (7-7-43 and 7-7-76) between the MM clones AC29 B7-6 and AC29 B7-7, the cells used to derive these clones by SSH. Therefore, it was concluded that neither clone 7-7-43 nor 7-7-76 was differentially expressed in MM cells of differing immuno enicit RACE was employed in order to derive a longer sequence of clone 7-7-76 and the newly derived sequence of 7-7-76 was again used to search for homologies using a wider range of sequences for human and other species. These investigations on clone 7-7-76 showed it to correspond to the sequence of human mitofusin 2 which is involved in determining mitochondrial morphology The results determined in this project suggest that clones 7-7-43 and 7-7-76 are not differentially expressed in the range of MM cell lines tested. The data have however highlighted the potential of the SSH technique to easily derive cDNA clones worthy of investigation, but underline the possibility of false positive clones being isolated. The need for an efficient, accurate screening procedure such as real-time PCR is acknowledged.

The analysis of gene changes associated with formation of the mycorrhizal symbiosis between orchid and fungi could have broad implications for plant pathogen interactions. Fungi associated with North American terrestrial orchids were once included in the pathogenic genus Rhizoctonia. This suggests that orchids are able to overcome or utilize normally pathogenic pathways to establish symbioses. A differential display technique was employed to analyze gene changes in orchid in response to a fungus. Samples of RNA from roots of Cypripedium parviflorum var. pubescens (CyPP) grown in the presence or absence of a mycorrhizal fungus; Thanatephorus pennatus, were analyzed using AFLP differential display. Forty-four fragments were selected out of 5000 as being differentially expressed, but only 15 sequences were obtained. Most showed homology to ribosomal genes. Two represented genes believed to be regulated by the mycorrhizal interaction: trehalose-6-phosphate synthase/phosphatase (Tps), which showed down-regulation and nucleotide binding protein (NuBP), which showed up-regulation. The Tps partial clone identifies 2100 bp at the 3' end of the gene and encodes a protein of 667 amino acids. The NuBP gene is approximately 1200bp in length and encodes a protein of 352 amino acids. The Tps gene exists in multiple copies with high expression in roots and low expression in rhizomes and leaves. The NuBP gene exists as a single copy and has a low level of expression in rhizomes and leaves. Expression of Tps is induced by sucrose, but reduced by trehalose. Cultivation of CyPP with non-mycorrhizal fungi did not affect expression of Tps or NuBP. Trehalose induced NuBP expression whereas sucrose did not. A second species of mycorrhizal fungi induced expression of NuBP but reduced expression of Tps. Analysis of Tps expression in Arabidopsis was done using promoter:GUS fusions. The Tps promoter:GUS plants revealed that Tps expression is constitutive in roots. Regulation of Tps driven GUS is expressed throughout seedlings. GUS was not detected in leaves of older plants but was detected in anthers and stigmatic surfaces of flowers. Expression of GUS driven by Tps showed a strong wound response and was present in the junction between siliques and pedicels. / Ph. D.

Afin d'identifier de nouvelles cibles organo-spécifiques de l'endothélium, nous avons comparé l'expression de gènes entre deux lignées de hautes cellules endothéliales et mis en évidence la protéine ARM-1. Des expériences de RT-PCR ont montré une expression spécifique d'ARM-1 dans certaines lignées de cellules endothéliales et de lymphocytes. La surexpression d'ARM-1 dans des cellules endothéliales de peau, n'exprimant pas cette protéine, augmente de façon préférentielle l'adhésion de certaines lignées lymphocytaires sur ces cellules. Ceci renforce l'hypothèse d'un rôle particulier d'ARM-1 au sein des cellules endothéliales. Une étude de fractionnement cellulaire a montré qu'ARM-1 était une protéine sécrétée, pouvant être associée à la membrane. Des expériences d'immunolocalisation n'ont cependant démontré aucune expression d'ARM-1 à la surface cellulaire. Ces résultats suggèrent une action indirecte d'ARM-1 dans l'adhésion plutôt qu'un rôle direct en tant que molécule d'adhésion.

<p>Glioblastoma multiforme is a malignant brain tumor characterized by heterogeneity.Interactions between heterogeneous tumor cells are supposed to affect the behavior of awhole tumor cell population. In this thesis an <i>in vitro</i> model system of clonal glioma celllines originating from one glioblastoma tumor was used, and the behavior of cells incocultures was studied and compared the behavior of cells grown separately. The resultsindicate the presence of two types of interactions. In one, paracrine signals acted via extra-cellular media. This was associated with increased growth of the whole co-culture followedby a selective force driving one clone to dominance. In the other type, the cell clones grewside by side without signs of paracrine signalling, in a balance resulting in an increasedterminal cell density. Further investigations focused on mechanisms of interactions in thiscombination.</p><p>Two cell clones were chosen, a GFAP⁺ and a GFAP^-, for further experiments. Withdifferential display PCR it was possible to investigate their specific gene expressionpatterns. Seventeen cDNA fragments were differentially expressed, among them twocorresponded to known transcription factors, ATF3 and prox-1, one to a cytoskeletal protein,α-tropomyosin. The collection also contained eight ESTs (Expressed Sequence Tags) wherethe corresponding genes are unknown at present. Expression of the isolated sequences werealso analyzed in a panel of 12 different glioma cell lines and the results illustrate thecomplexity of gene expression and of tumor heterogeneity. Genes, the expression levels ofwhich were modulated in co-cultures and/or were cell density dependent, were alsoidentified.</p><p>PDGF B is suggested to play a role in sarcomas. The gene codes for an mRNA transcriptwith long UTRs, parts of which are deleted in the homologous oncogene <i>v-sis</i>. The UTRs ofPDGF B mRNAs in human sarcomas were investigated for deletions similar to <i>v-sis</i> thatmight result in increased protein levels. A new transcript variant was identified, lacking a149 base region in the 3'UTR, but its presence was not associated with increased levels ofprotein. Alterations in the 5'UTR were found more likely to be associated with increasedprotein levels.</p>

Glioblastoma multiforme is a malignant brain tumor characterized by heterogeneity.Interactions between heterogeneous tumor cells are supposed to affect the behavior of awhole tumor cell population. In this thesis an in vitro model system of clonal glioma celllines originating from one glioblastoma tumor was used, and the behavior of cells incocultures was studied and compared the behavior of cells grown separately. The resultsindicate the presence of two types of interactions. In one, paracrine signals acted via extra-cellular media. This was associated with increased growth of the whole co-culture followedby a selective force driving one clone to dominance. In the other type, the cell clones grewside by side without signs of paracrine signalling, in a balance resulting in an increasedterminal cell density. Further investigations focused on mechanisms of interactions in thiscombination. Two cell clones were chosen, a GFAP+ and a GFAP-, for further experiments. Withdifferential display PCR it was possible to investigate their specific gene expressionpatterns. Seventeen cDNA fragments were differentially expressed, among them twocorresponded to known transcription factors, ATF3 and prox-1, one to a cytoskeletal protein,α-tropomyosin. The collection also contained eight ESTs (Expressed Sequence Tags) wherethe corresponding genes are unknown at present. Expression of the isolated sequences werealso analyzed in a panel of 12 different glioma cell lines and the results illustrate thecomplexity of gene expression and of tumor heterogeneity. Genes, the expression levels ofwhich were modulated in co-cultures and/or were cell density dependent, were alsoidentified. PDGF B is suggested to play a role in sarcomas. The gene codes for an mRNA transcriptwith long UTRs, parts of which are deleted in the homologous oncogene v-sis. The UTRs ofPDGF B mRNAs in human sarcomas were investigated for deletions similar to v-sis thatmight result in increased protein levels. A new transcript variant was identified, lacking a149 base region in the 3'UTR, but its presence was not associated with increased levels ofprotein. Alterations in the 5'UTR were found more likely to be associated with increasedprotein levels.

One of the major problems concerning the production of food crops is the controlling of plant diseases to maintain the high quality and yield. Wheat diseases are caused by parasitic bacteria, fungi and viruses that are a major hazard in wheat production. Therefore, understanding of any resistance mechanism is prerequisite for the successful utilization of wheat crop species in modern agriculture. The phenomenon of induced resistance by fungi, bacteria, microbial elicitors and chemicals has been investigated widely and resulted in many discoveries that conclude a general realization that the disease resistance signaling pathway in plants shares a number of common elements with those leading to innate immunity but a few of them have been characterized at the molecular level yet. Therefore our goal in this study is to identify genes activated or repressed after treatment of wheat plants with biological elicitor fungus, Trichoderma harzianum, and chemical inducers, benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) and &szlig / -aminobutyric acid (BABA). mRNA differential display technique, which is a powerful tool to identify those genes that are differentially expressed between the two cell types has been extensively used in this study. The variety &#039 / Avocet S&#039 / is used to identify putative genes activated or repressed after treatment of wheat plants with biological elicitor fungus, Trichoderma harzianum, and chemical inducers, BTH and BABA comparing to untreated &#039 / Avocet S&#039 / wheat plants. The differentially expressed cDNA bands were cloned and sequenced. Nucleotide sequences of differentially expressed cDNA bands were searched in the Genbank. Sequence alignments between the fragments that represent a certain gene were also searched in ClustalX-1.81 computer programs. The sequences of the differentially expressed fragments were also confirmed by real time PCR that verify the gene expression differences observed between the biologically or chemically treated and untreated plants as a result of defense induction. The confirmed genes were found to be involved directly or indirectly in the induced disease resistance. These genes are important in terms of understanding the mechanism of systemic acquired resistance (SAR) signalling defense and helpful in producing transgenic wheat.

In this study, it was aimed to identify chickpea (Cicer arietinum) genes or gene fragments expressed upon Ascochyta rabiei infection using a tolerant chickpea cultivar ILC195 and fungal isolates with varying level of pathogenicity. PCR amplification of resistance gene analogs (RGA) and disease related genes, and mRNA differential display reverse transcription (DDRT) were used to get these expressed gene fragments in chickpea. The constitutively or differentially expressed PCR product fragments were cloned and sequenced. Out of nearly 300 clones, 160 sequences (expressed sequence tags, ESTs) could be analyzed and these sequences were disclosed in this study. About 100 of these ESTs were classified according to predicted &ldquo / molecular function&rdquo / , &ldquo / biological process&rdquo / and &ldquo / cellular component&rdquo / . The most common ppredicted functions of the products coded by these ESTs were &ldquo / Protein Fate&rdquo / , &ldquo / Metabolism&rdquo / , &ldquo / Cell Rescue, Defense and Virulence&rdquo / , &ldquo / Transcription&rdquo / , &ldquo / Transport&rdquo / , &ldquo / Energy&rdquo / , and &ldquo / Cell Fate&rdquo / . Six ESTs were subjected to Real-Time quantitative RT-PCR analysis to compare the response of ILC195 infected by one A.rabiei isolate with another resistant chickpea genotype (FLIP84-92C)/A.rabiei pathotype system. Some of these genes were differentially expressed among different chickpea/A.rabiei isolate combinations. Highly upregulated ESTs in all these combinations were a formate dehydrogenase (metabolism and detoxification), a serine carboxypeptidase (protein fate and communication) and a hypothetical protein probably similar to acyl-CoA synthetases. A genetic mapping study was carried out with EST specific primers and two EST markers were assigned in the current chickpea genetic map. However, no genetic linkage of them was detected with known chickpea quantitative trait loci for A.rabiei resistance.

Zinc is a metal involved in structure of many enzymes, in the growth and differentiation of plants. Wheat is one of the most consumed cereals. Some wheat cultivars can&amp / #8217 / t deal with zinc deficiency and this situation not only reduces grain yield but also weakens the resistance of cereals to diseases and impairs the nutritional quality of the grain. Some wheat cultivars are not affected by zinc deficiency. In this study, &amp / #8216 / differential display&amp / #8217 / , used for determination differentially expressed genes between two samples, was performed. The most zinc efficient bread wheat cultivar Kira&ccedil / -66 was grown in hydroponics medium and samples were taken at different time periods. RNA isolations were done and differential display technique was performed. After examining the results, differentially expressed bands were selected and sequenced. DNA sequence analysis were done in available databases which showed that three of the bands were fragments of putative zinc transporters. In this study we have found threee putative gene fragments using differential display technique on zinc efficient plants grown under differeing zinc concentrations. These fragments showed homology with zinc transporter, ABC transporter and ADH (Alcohol Dehydrogenase). It is known that all of these three genes are involved in zinc efficiency mechanism. Further studies will be conducted on these gene fragments.