Identification of protein-coding and non-coding RNA expression profiles in CD34+ and in stromal cells in refractory anemia with ringed sideroblasts

Abstract Background Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods In this study, gene expression profiles of CD34+ cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34+ cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Differential expression of HIF-1α in CD44+CD24-/low breast ductal carcinomas

Abstract Background Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1α expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44+CD24-/low immunophenotype. Methods Immunohistochemical analysis of CD44+CD24-/low expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). Results In univariate analysis, CD44+CD24-/low expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44+CD24-/low immunophenotype showed relationship with HIF-1α (p = 0.039) and negativity for HER-2 (p = 0.013). Conclusion Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44+CD24-/low positive breast tumors.

Propagação vegetativa de progênies de meios-irmãos de angico-vermelho (Anadenanthera macrocarpa (Benth) Brenan) por miniestaquia

Objetivou-se com o presente estudo avaliar a eficiência da técnica de miniestaquia na propagação vegetativa de progênies de meios-irmãos de angico-vermelho (Anadenanthera macrocarpa (Benth) Brenan) quanto à produção de brotações e sobrevivência das minicepas, enraizamento das miniestacas apicais e intermediárias tratadas com diferentes doses do AIB (0; 2.000; 4.000 e 6.000 mg L-1), assim como determinar a velocidade de enraizamento em casa de vegetação. As minicepas foram obtidas a partir de mudas produzidas via sementes de seis progênies de meios-irmãos de angico-vermelho. Com base nos resultados obtidos, as minicepas apresentaram produtividade de 1,2 a 3,7 miniestacas/minicepa/coleta e sobrevivência de 84% a 98% ao longo das seis coletas realizadas. As miniestacas apicais foram superiores em relação às intermediárias, com maior predisposição ao enraizamento, no entanto o AIB não teve efeito significativo sobre o enraizamento das progênies estudadas. Quanto à velocidade de enraizamento, os resultados indicaram variação entre as progênies.

Produção de mudas clonais de eucalipto em espuma fenólica: crescimento inicial e mortalidade

A produção de mudas é um dos pontos cruciais nos empreendimentos florestais e diversos trabalhos procuraram identificar o método mais adequado de produção. Neste estudo, avaliou-se o uso da espuma fenólica na substituição de recipientes e de substrato na produção de mudas clonais de eucalipto. Foram implantados quatro experimentos, sendo dois de viveiro (experimentos 1 e 2) e dois de campo (experimentos 3 e 4) em locais e períodos distintos. Todos os experimentos foram compostos por quatro tratamentos: 1 - tubete de 55 cm³ + substrato comercial; 2 - espuma pequena (60 cm³); 3 - espuma média (75 cm³); e 4 - espuma grande (90 cm³). Nos experimentos, avaliouse a mortalidade das mudas e a biomassa do sistema radicular e da parte aérea. No experimento 1, as mudas produzidas nas espumas pequenas obtiveram menor índice de mortalidade (1%), enquanto no sistema convencional foi de 15% e observou-se superioridade na produção de biomassa aérea das mudas produzidas na espuma grande. No experimento 2, as mudas produzidas na espuma apresentaram maior sensibilidade ao excesso de água durante a fase de enraizamento, sendo a mortalidade de 40 % para os tratamentos com a utilização da espuma que foi superior ao tratamento convencional (16%). No experimento 3, conduzido no campo, não foi observada mortalidade das mudas ou diferenças na produção de biomassa entre os tratamentos. No experimento 4, observou-se maior resistência ao déficit hídrico nos tratamentos com as espumas média e grande e maior produção de biomassa aérea no tratamento com a espuma grande.

Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n = 87) and P. falciparum (n = 164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P = 0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P = 0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.

Analysis of genetic lineages and their correlation with virulence genes in enterococcus faecalis clinical isolates from root canal and systemic infections

Introduction: Enterococcus faecalis is a member of the mammalian gastrointestinal microbiota but has been considered a leading cause of hospital-acquired infections. In the oral cavity, it is commonly detected from root canals of teeth with failed endodontic treatment. However, little is known about the virulence and genetic relatedness among E. faecalis isolates from different clinical sources. This study compared the presence of enterococcal virulence factors among root canal strains and clinical isolates from hospitalized patients to identify virulent clusters of E. faecalis. Methods: Multilocus sequence typing analysis was used to determine genetic lineages of 40 E. faecalis clinical isolates from different sources. Virulence clusters were determined by evaluating capsule (cps) locus polymorphisms, pathogenicity island gene content, and antibiotic resistance genes by polymerase chain reaction. Results: The clinical isolates from hospitalized patients formed a phylogenetically separate group and were mostly grouped in the clonal complex 2, which is a known virulent cluster of E. faecalis that has caused infection outbreaks globally. The clonal complex 2 group comprised capsule-producing strains harboring multiple antibiotic resistance and pathogenicity island genes. On the other hand, the endodontic isolates were more diverse and harbored few virulence and antibiotic resistance genes. In particular, although more closely related to isolates from hospitalized patients, capsuleproducing E. faecalis strains from root canals did not carry more virulence/antibiotic genes than other endodontic isolates. Conclusions: E. faecalis isolates from endodontic infections have a genetic and virulence profile different from pathogenic clusters of hospitalized patients’ isolates, which is most likely due to niche specialization conferred mainly by variable regions in the genome.

Maturation of humoral immune responses : Studies on the effects of antigen type, apoptosis and age

The humoral immune response is dependent on the formation of antibodies. Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the germinal center (GC) reaction. The GC is the main site for class switch, somatic hypermutation and generation of memory cells. Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent antigens (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the T cell compartment is more affected by age than the B cell compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a plasma cell biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs. B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, lpr mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

[EN] Background. Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Indagine sulla presenza di Helicobacter pullorum in allevamenti avicoli italiani

From September 2005 to December 2006, in order to define the prevalence of Helicobacter pullorum in broiler chickens, laying hens and turkey, a total of 365 caecum contents of animals reared in 76 different farms were collected at the slaughterhouse. A caecum content of a ostrich was also sampled. In addition, with the aim of investigating the occurrence of H. pullorum in humans, 151 faeces were collected at the Sant’Orsola-Malpighi University Hospital of Bologna from patients suffering of gastroenteritis. A modified Steele–McDermott membrane filter method was used. Gram-negative curved rod bacteria were preliminary identified as H. pullorum by a PCR assay based on 16S rRNA, then subjected to a RFLP-PCR assay to distinguish between H. pullorum and H. canadensis. One isolate from each farm was randomly selected for phenotypic characterization by biochemical methods and 1D SDSPAGE analysis of whole cell proteins profiles. Minimum Inhibitory Concentration (MIC) for seven different antibiotics were also determined by agar dilution method. Moreover, to examine the intraspecific genomic variability, two strains isolated from 17 different farms were submitted to genotyping by Pulse-Field Gel Electrophoresis (PFGE). In order to assess the molecular basis of fluorquinolone resistance in H. pullorum, gyrA of H. pullorum CIP 104787T was sequenced and nucleotide sequences of the Quinolone Resistance Determining Region (QRDR) of a total of 18 poultry isolates, with different MIC values for ciprofloxacin and nalidixic acid, were compared. According to the PCR and PCR-RFLP results, 306 out of 366 animals examined were positive for H. pullorum (83,6%) and 96,1% of farms resulted infected. All positive samples showed a high number of colonies (>50) phenotipically consistent with H. pullorum on the first isolation media, which suggests that this microrganism, when present, colonizes the poultry caecum at an elevate load. No human sample resulted positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 74 isolates tested and with the type strain H. pullorum CIP 104787T. Regarding the MIC values, a monomodal distribution was found for ampicillin, chloramphenicol, gentamicin and nalidixic acid, whereas a bimodal trend was noticed for erythromycin, ciprofloxacin and tetracycline (indicating an acquired resistance for these antibiotics). Applying the breakpoints indicated by the CSLI, we may assume that all the H. pullorum tested are sensitive only to gentamicin. The intraspecific genomic variability observed in this study confirm that this species don’t have a clonal population structure, as motioned by other autors. The 2490 bp gyrA gene of H. pullorum CIP104787T with an Open Reading Frame (ORF) encoding a polypeptide of 829 amino acids was for the first time sequenced and characterized. All ciprofloxacin resistant poultry isolates showed ACA®ATA (Thr®Ile) substitution at codon 84 of gyrA corresponding to codons of gyrA 86, 87 and 83 of the Campylobacter jejuni, H. pylori and Escherichia coli, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin resistant phenotype of poultry isolates. This is the first report of isolation of H. pullorum in turkey and in ostrich, indicating that poultry species are the reservoir of this potential zoonotic microorganisms. In order to understand the potential role as food-borne human pathogen of H. pullorum, further studies must be carried on.

Bioimaging of animal embryogenesis: mathematical methods and computational algorithms

Some fundamental biological processes such as embryonic development have been preserved during evolution and are common to species belonging to different phylogenetic positions, but are nowadays largely unknown. The understanding of cell morphodynamics leading to the formation of organized spatial distribution of cells such as tissues and organs can be achieved through the reconstruction of cells shape and position during the development of a live animal embryo. We design in this work a chain of image processing methods to automatically segment and track cells nuclei and membranes during the development of a zebrafish embryo, which has been largely validates as model organism to understand vertebrate development, gene function and healingrepair mechanisms in vertebrates. The embryo is previously labeled through the ubiquitous expression of fluorescent proteins addressed to cells nuclei and membranes, and temporal sequences of volumetric images are acquired with laser scanning microscopy. Cells position is detected by processing nuclei images either through the generalized form of the Hough transform or identifying nuclei position with local maxima after a smoothing preprocessing step. Membranes and nuclei shapes are reconstructed by using PDEs based variational techniques such as the Subjective Surfaces and the Chan Vese method. Cells tracking is performed by combining informations previously detected on cells shape and position with biological regularization constraints. Our results are manually validated and reconstruct the formation of zebrafish brain at 7-8 somite stage with all the cells tracked starting from late sphere stage with less than 2% error for at least 6 hours. Our reconstruction opens the way to a systematic investigation of cellular behaviors, of clonal origin and clonal complexity of brain organs, as well as the contribution of cell proliferation modes and cell movements to the formation of local patterns and morphogenetic fields.

Study of two novel streptococcus pneumoniae operons: pilus islet 2 and the lantibiotic operon

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element resembling gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (containing the genes pitA, sipA, pitB, srtG1, and srtG2) codes for a novel functional pilus in pneumococcus. Therefore, there are two pilus islets identified so far in this pathogen (PI-1 and PI-2). Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. PI-2 is associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging in both industrialized and developing countries. Interestingly, strains belonging to clonal complex 271 (CC271) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that may play a role in the initial host cell contact to the respiratory tract. In addition, the pilus proteins are potential antigens for inclusion in a new generation of pneumococcal vaccines. Adherence by pili could represent important factor in bacterial community formation, since it has been demonstrated that bacterial community formation plays an important role in pneumococcal otitis media. In vitro quantification of bacterial community formation by S. pneumoniae was performed in order to investigate the possible role of pneumococcal pili to form communities. By using different growth media we were not able to see clear association between pili and community formation. But our findings revealed that strains belonging to MLST clonal complex CC15 efficiently form bacterial communities in vitro in a glucose dependent manner. We compared the genome of forty-four pneumococcal isolates discovering four open reading frames specifically associated with CC15. These four genes are annotated as members of an operon responsible for the biosynthesis of a putative lanctibiotic peptide, described to be involved in bacterial community formation. Our experiments show that the lanctibiotic operon deletion affects glucose mediated community formation in CC 15 strain INV200. Moreover, since glucose consumption during bacterial growth produce an acidic environment, we tested bacterial community formation at different pH and we showed that the lanctibiotic operon deletion affected pH mediated community formation in CC 15 strain INV200. In conclusion, these data demonstrate that the putative lanctibiotic operon is associated with pneumococcal CC 15 strains in vitro bacterial community formation.

Molecular phylogeography of the European coastal plants Crithmum maritimum L., Halimione portulacoides (L.) Aellen, Salsola kali L. and Calystegia soldanella (L.) R. Br.

The intraspecific phylogeography of four European coastal plants, Crithmum maritimum, Halimione portulacoides, Salsola kali and Calystegia soldanella, was inferred from AFLP and ITS data. Only in C. maritimum, H. portulacoides and S. kali, a spatial genetic structure was revealed. The phylogeographic similarities and dissimilarities of these species include: (1) All three have distinct Black/Aegean and Adriatic Sea clusters. (2) Salsola kali and H. portulacoides show a distinct Atlantic/North Sea/Baltic Sea cluster, while Atlantic and eastern Spanish material of C. maritimum clustered together. (3) In the west Mediterranean, only S. kali forms a single cluster, while both H. portulacoides and C. maritimum display a phylogeographic break in the vicinity of the southern French coast. For S. kali, AFLP and ITS data concur in identifying separate Atlantic, east and west Mediterranean clades. All these patterns are postulated to result from both temperature changes during the last glacial and contemporary sea currents. No geographic AFLP structure was revealed in C. soldanella, both at the range-wide and population level. This was attributed to the remarkable seed dispersal ability of this species and possibly its longevity and clonal growth, preserving a random pattern of genetic variation generated by long-distance seed dispersal over long time periods.

Modulation of apoptosis by endogenously produced nitric oxide

Untersuchungen zur Expression der induzierbaren NO-Synthetase (NOS2) belegen eine häufige Expression dieses Enzyms in Tumoren unterschiedlicher Gewebe. Bislang ist jedoch ungeklärt, ob die Expression der NOS2 in Tumorzellen die apoptotische Eliminierung durch zytotoxische T-Zellen beeinflussen kann. In der vorliegenden Arbeit wurden die Folgen einer endogenen NO-Synthese auf die Apoptosesensitivität von HEK293-Zellen untersucht. Um primäre NO-Wirkungen von NO-induzierten, sekundären (kompensatorischen) Veränderungen zu trennen, wurde mit einem induzierbaren Vektorsystem gearbeitet. Die NOS2 wurde zunächst unter der Kontrolle eines Ecdyson-sensitiven Promoters in HEK293-Zellen kloniert. Es konnten regulierbare NOS2-Klone selektiert werden, die nach Ponasteronbehandlung dosisabhängig die NOS2 exprimieren und NO synthetisieren. Die NOS2-Expression wurde durch Western Blot Analyse und Immunfluoreszenzfärbung dargestellt und die NO-Produktion mit Hilfe der Griess-Reaktion gemessen. An den NOS2-induzierten Zellen wurde dann der Einfluss von NO auf die CD95-vermittelte Apoptose analysiert. Es zeigte sich nach Stimulation des CD95-Rezeptors eine deutliche Korrelation der Apoptoserate mit der NOS2-Expression. In Kokulturexperimenten mit Peptid-spezifischen zytotoxischen T-Zellen zeigte sich, dass NO-produzierende Zielzellen effektiver eliminiert werden konnten. Auch nach Behandlung der Zellen mit TRAIL ergab sich eine höhere Apoptoserate in NO-produzierenden Zellen. Die weitere Analyse der durch NO beeinflussten Signalwege ergab eine Beteiligung von ER-Stress-vermittelten Apoptosewegen. Dies zeigte sich an der Hochregulation des ER-Stress-Proteins Grp78 (BiP) nach NOS2-Expression und der Spaltung der am ER-lokalisierten Caspase-4. Darüber hinaus konnte der schnellere Verlust des mitochondrialen Membranpotentials in Abhängigkeit von der NOS2-Expression nachgewiesen werden. Weiterhin wurde die Wirkung einer dauerhaften NO-Exposition auf die Apoptosesensitivität der Zellen untersucht. Auch ohne zusätzliche CD95-Stimulation induzierte eine kontinuierliche NOS2-Expression nach wenigen Tagen in den EcR293-NOS2-Zellen Apoptose. Diese Dauerbehandlung führte zum nahezu vollständigen Absterben der Kulturen. Einige Zellen überlebten jedoch diese Behandlung und wuchsen zu Zellklonen. Diese NO-resistenten Klone konnten isoliert werden. Sie zeigten eine zusätzliche Resistenz für CD95-vermittelte Apoptosesignale und waren besser vor dem Angriff Peptid-spezifischer CTLs geschützt. Die Apoptoseresistenz blieb auch nach längerer Kultur erhalten und scheint auf NO-induzierter Genotoxizität zu beruhen. Anhand dieser Arbeit konnte gezeigt werden, dass allein durch chronische NO-Behandlung eine Selektion apoptoseresistenter Zellen stattfinden kann.

Der Einfluss der T-Zellavidität und der TLR-vermittelten Aktivierung Dendritischer Zellen der Haut auf die Funktion zytotoxischer T-Zellen

Zytotoxische T-Zellen (CTL) vermitteln die effektive und gezielte Lyse Virus-infizierter und maligner Zellen. Um den Einfluss der T-Zellavidität auf die Immunodominanz von CTL-Antworten zu untersuchen, wurde ein neues Mausmodell mit zwei T-Zellrezeptor (TCR)-transgenen Stämmen etabliert. Die T-Zellen der beiden Stämme exprimieren denselben TCR in unterschiedlicher Dichte. Studien mit diesem Modell zeigten, dass bei identischen Vorläuferfrequenzen die hochaviden T-Zellen nach Immunisierung auf Kosten der niederaviden T-Zellen proliferieren. Sind die niederaviden T-Zellen jedoch im Überschuss vorhanden, so können sie die Antwort dominieren. Hieraus lässt sich schließen, dass die CTL Immunodominanz durch die Avidität und die Vorläuferfrequenz der T-Zellklone bestimmt wird. Darüber hinaus wurde ein neues transkutanes Immunisierungsverfahren (TCI) entwickelt. Die Applikation eines CTL Epitops zusammen mit dem synthetischen Toll-like Rezeptor 7 Liganden Imiquimod führt zur effizienten Aktivierung TCR-transgener CTLs. Eine in Wild-typ Mäusen generierte CTL-Antwort gegen das SIINFEKL Epitop aus dem Ovalbumin vermittelt die effiziente Lyse inokulierter Ovalbumin-transgener Thymomzellen. Schließlich konnte gezeigt werden, dass regulatorische T-Zellen die durch TCI vermittelte CTL Aktivierung inhibieren.

Genetic analysis of tumour initiation and progression in a Drosophila model of epithelial cancer

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. The molecular crosstalk occurring between precancerous and normal cells strongly influences the early steps of the tumourigenic process as well as later stages of the disease. Precancerous cells are often removed by cell death from normal tissues but the mechanisms responsible for such fundamental safeguard processes remain in part elusive. To gain insight into these phenomena I took advantage of the clonal analysis methods available in Drosophila for studying the phenotypes due to loss of function of the neoplastic tumour suppressor lethal giant larvae (lgl). I found that lgl mutant cells growing in wild-type imaginal wing discs are subject to the phenomenon of cell competition and are eliminated by JNK-dependent cell death because they express very low levels of dMyc oncoprotein compared to those in the surrounding tissue. Indeed, in non-competitive backgrounds lgl mutant clones are able to overgrow and upregulate dMyc, overwhelming the neighbouring tissue and forming tumourous masses that display several cancer hallmarks. These phenotypes are completely abolished by reducing dMyc abundance within mutant cells while increasing it in lgl clones growing in a competitive context re-establishes their tumourigenic potential. Similarly, the neoplastic growth observed upon the oncogenic cooperation between lgl mutation and activated Ras/Raf/MAPK signalling was found to be characterised by and dependent on the ability of cancerous cells to upregulate dMyc with respect to the adjacent normal tissue, through both transcriptional and post-transcriptional mechanisms, thereby confirming its key role in lgl-induced tumourigenesis. These results provide first evidence that the dMyc oncoprotein is required in lgl mutant tissue to promote invasive overgrowth in developing and adult epithelial tissues and that dMyc abundance inside versus outside lgl mutant clones plays a key role in driving neoplastic overgrowth.