Brain metastases in gastro-oesophageal adenocarcinoma: insights into the role of the human epidermal growth factor receptor 2 (HER2).

BACKGROUND: Gastro-oesophageal adenocarcinomas rarely metastasize to the central nervous system (CNS). The role of the human epidermal growth factor receptor 2 (HER2) in patients with these cancers and CNS involvement is presently unknown. PATIENTS AND METHODS: A multicentre registry was established to collect data from patients with gastro-oesophageal adenocarcinomas and CNS involvement both retrospectively and prospectively. Inclusion in the study required a predefined clinical data set, a central neuro-radiological or histopathological confirmation of metastatic CNS involvement and central assessment of HER2 by immunohistochemistry (IHC) and in situ hybridisation (ISH). In addition, expression of E-cadherin and DNA mismatch repair (MMR) proteins were assessed by IHC. RESULTS: One hundred patients fulfilled the inclusion criteria. The population's median age was 59 years (interquartile range: 54-68), of which 85 (85%) were male. Twenty-five patients were of Asian and 75 of Caucasian origin. HER2 status was positive in 36% (95% CI: 26.6-46.2) of cases. Median time from initial diagnosis to the development of brain metastases (BMets) or leptomeningeal carcinomatosis (LC) was 9.9 months (95% CI: 8.5-15.0). Median overall survival from diagnosis was 16.9 months (95% CI: 14.0-20.7) and was not related to the HER2 status. E-cadherin loss was observed in 9% of cases and loss of expression in at least one DNA MMR proteins in 6%. CONCLUSIONS: The proportion of a positive HER2 status in patients with gastro-oesophageal adenocarcinoma and CNS involvement was higher than expected. The impact of anti-HER2 therapies should be studied prospectively.

Phylogeography of competing sexual and parthenogenetic forms of a freshwater flatworm: patterns and explanations

Background: Models of the maintenance of sex predict that one reproductive strategy, sexual or parthenogenetic, should outcompete the other. Distribution patterns may reflect the outcome of this competition as well as the effect of chance and historical events. We review the distribution data of sexual and parthenogenetic biotypes of the planarian Schmidtea polychroa. Results: S. polychroa lives in allopatry or sympatry across Europe except for Central and North-Western Europe, where sexual individuals have never been reported. A phylogenetic relationship between 36 populations based on a 385 bp fragment of the mitochondrial cytochrome oxidase I gene revealed that haplotypes were often similar over large geographic distances. In North Italian lakes, however, diversity was extreme, with sequence differences of up to 5% within the same lake in both sexuals and parthenogens. Mixed populations showed "endemic" parthenogenetic lineages that presumably originated from coexisting sexuals, and distantly related ones that probably result from colonization by parthenogens independent from sexuals. Conclusions: Parthenogens originated repeatedly from sexuals, mainly in Italy, but the same may apply to other Mediterranean regions (Spain, Greece). The degree of divergence between populations suggests that S. polychroa survived the ice ages in separate ice-free areas in Central, Eastern and Southern Europe and re-colonised Europe after the retreat of the major glaciers. Combining these results with those based on nuclear markers, the data suggest that repeated hybridisation between sexuals and parthenogenetic lineages in mixed populations maintains high levels of genetic diversity in parthenogens. This can explain why parthenogens persist in populations that were originally sexual. Exclusive parthenogenesis in central and western populations suggests better colonisation capacity, possibly because of inbreeding costs as well

Copy number variations of colorectal cancer by whole exome sequencing data

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death worldwide. About 85% of the cases of CRC are known to have chromosomal instability, an allelic imbalance at several chromosomal loci, and chromosome amplification and translocation. The aim of this study is to determine the recurrent copy number variant (CNV) regions present in stage II of CRC through whole exome sequencing, a rapidly developing targeted next-generation sequencing (NGS) technology that provides an accurate alternative approach for accessing genomic variations. 42 normal-tumor paired samples were sequenced by Illumina Genome Analyzer. Data was analyzed with Varscan2 and segmentation was performed with R package R-GADA. Summary of the segments across all samples was performed and the result was overlapped with DEG data of the same samples from a previous study in the group1. Major and more recurrent segments of CNV were: gain of chromosome 7pq(13%), 13q(31%) and 20q(75%) and loss of 8p(25%), 17p(23%), and 18pq(27%). This results are coincident with the known literature of CNV in CRC or other cancers, but our methodology should be validated by array comparative genomic hybridisation (aCGH) profiling, which is currently the gold standard for genetic diagnosis of CNV.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Evolution of Bordetella pertussis post vaccination

Evolution of Bordetella pertussis post vaccination Whooping cough or pertussis is caused by the gram-negative bacterium Bordetella pertussis. It is a highly contiguous disease in the human respiratory tract. Characteristic of pertussis is a paroxysmal cough with whooping sound during gasps of breath after coughing episodes. It is potentially fatal to unvaccinated infants. The best approach to fight pertussis is to vaccinate. Vaccinations against pertussis have been available from the 1940s. Traditionally vaccines were whole-cell pertussis (wP) preparations as part of the combined diphtheria-tetanus-pertussis (DTP) vaccines. More recently acellular pertussis (aP) vaccines have replaced the wP vaccines in many countries. The aP vaccines are less reactogenic and can also be administered to school children and adults. There are several publications reporting variation in the i>B. pertussis virulence factors that are also aP vaccine antigens. This has occurred in the genes coding for pertussis toxin and pertactin about 15 to 30 years after the introduction of pertussis vaccines to immunisation programs. Resurgence of pertussis has also been reported in many countries with high vaccination coverage. In this study the evolution of B. pertussis was investigated in Finland, the United Kingdom, Poland, Serbia, China, Senegal and Kenya. These represent countries with a long history of high vaccination coverage with stable vaccines or changes in the vaccine formulation; countries which established high vaccination coverage late; and countries where vaccinations against pertussis were started late. With bacterial cytotoxicity and cytokine measurements, comparative genomic hybridisation, pulsed-field gel electrophoresis (PFGE), genotyping and serotyping it was found that changes in the vaccine composition can postpone the emergence of antigenic variants. It seems that the change in PFGE profiles and the loss of genetic material in the genome of B. pertussis are similar in most countries and the vaccine-induced immunity is selecting non-vaccine type strains. However, the differences in the formulation of the vaccines, the vaccination programs and in the coverage of pertussis vaccination have affected the speed and timing of these changes.

Heritage through Fiction: Dracula Tourism in Romania

Heritage and tourism have become inextricably linked. This link can be seen as producing inauthentic and falsified tradition, and it can therefore be seen as a threat to cultural heritage. On the other hand the link can be seen as a positive thing, as something which helps to preserve herit-age, culture and folklore in a changing and globalising world. This dissertation investigates heritage in the context of Dracula Tourism in Romania. Dracula tourism is tourism where tourists visit places connected with either the fictional vampire Dracula or the historical Dracula, a Romanian historical ruler Vlad the Impaler. The main research question of this study is how can Romanian heritage and culture be shown and promoted through a seemingly superficial Dracula tourism which is based on Western popular cul-ture? And is it possible to find Romanian heritage through popular fiction in Dracula tourism? The main sources for this work are based on the fieldwork done by the author in 2010 and 2011 and the web pages of ten Romanian travel agencies that offer Dracula tourism. The stories and images found on the web pages and used by the tour guides form the bulk of the research material. The emphasis and perspective of this research is folkloristic. Critical discourse analysis and multimodal discourse analysis form the main theoretical approach of this dissertation. In addition the research material is approached through intertextuality, folklore process, hybridisation, authenticity and social constructionism. This dissertation aims to offer new perspectives on the research literature concerning tourism and heritage and to offer a folkloristic view of tourism research. It also aims to offer new perspectives to folkloristics in terms of the research on the use of folklore and tradition and offer new perspectives on the use and definition of the concept of authenticity. Although the research subject of this thesis is Dracula tourism in Romania, the findings can be utilised and applied in a larger context and field of research. The key research findings show that heritage can be found within Dracula Tourism in three forms: as defined from above (UNESCO World Heritage Sites), as local heritage and as a form of opposition. The Romanian travel agencies researched in this dissertation use Dracula tourism as a gateway into Romanian history, culture, tradition and heritage. Kulttuuriperintö ja turismi yhdistyvät toisiinsa erottamattomasti. Toisaalta tämän yhteyden on nähty tuottavan epäautenttista ja väärennettyä perinnettä, ja tällöin sen on koettu muodostavan uhan kulttuuriperinnölle. Toisaalta yhteys on mielletty myös positiivisena asiana, sillä sen on nähty toimivan globalisoituvassa maailmassa kulttuuriperintöä, kulttuuria ja kansanperinnettä säilyttävänä tekijänä. Väitöskirjassa tutkitaan sitä, miten kulttuuriperintö ilmenee Dracula-turismissa Romaniassa. Dracula- turismi on turismia, joka liittyy joko fiktiiviseen vampyyrikreivi Draculaan tai historiallisena Draculana tunnettuun romanialaiseen hallitsijaan Vlad Seivästäjään. Väitöskirjan päätutkimuskysymyksenä on, miten romanialaista kulttuuriperintöä ja kulttuuria voidaan tuoda esiin näennäisesti pinnallisen ja länsimaiseen populaarikulttuuriin pohjautuvan Dracula-turismin kautta. Työn päälähteet pohjautuvat tutkijan vuosina 2010 ja 2011 tekemiin kenttätöihin sekä kymmenen Dracula-turismia tarjoavan romanialaisen matkatoimiston WWW-sivustoihin. Internet-sivuilta löytyvät tarinat ja kuvat sekä matkaoppaiden käyttämät tarinat muodostavat tutkimuksen tutkimusaineiston. Tutkimuksen painotus ja näkökulma ovat folkloristisia. Tutkimuksen teoreettinen viitekehys muodostuu kriittisestä diskurssianalyysistä sekä multimodaalisesta diskurssianalyysistä. Näiden lisäksi tutkimusaineistoa analysoidaan intertekstuaalisuuden, folkloreprosessin, hybridisaation, autenttisuuden ja sosiaalisen konstruktion käsitteiden avulla. Väitöskirjatutkimus tarjoaa uusia näkökulmia turismia ja kulttuuriperintöä käsittelevään tutkimukseen sekä tarjoaa matkailun tutkimukseen folkloristisen lisänäkökulman. Folkloristisen tutkimuksen näkökulmasta työn keskiössä ovat perinteen ja folkloren hyödyntäminen sekä autenttisuuden käsitteen määrittely ja käyttö, joihin työssä otetaan kantaa. Vaikka tutkimus käsittelee Dracula-turismia Romaniassa, ovat tutkimustulokset käytettävissä ja sovellettavissa myös laajemmin. Työn keskeiset tutkimustulokset osoittavat, että kulttuuriperintö ilmenee Dracula-turismissa kolmella eri tavalla: ylätasolla kuten esimerkiksi UNESCO:n kohteissa, alatasolla paikallisten ihmisten tai ihmisryhmien määrittelemänä sekä vastustuksen muotona. Väitöskirjassa tutkitut matkatoimistot käyttävät Dracula-turismia ikään kuin porttina romanialaiseen historiaan, kulttuuriin, perinteeseen ja kulttuuriperinteeseen.

Hunting for differentially expressed genes

Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Several methods have been used for this purpose, including differential hybridization, cDNA subtraction, differential display and, more recently, DNA chips. Subtractive hybridization has significantly improved after the polymerase chain reaction was incorporated into the original method and many new protocols have been established. Recently, the availability of the well-known coding sequences for some organisms has greatly facilitated gene expression analysis using high-density microarrays. Here, we describe some of these modifications and discuss the benefits and drawbacks of the various methods corresponding to the main advances in this field.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs=0.317, P=0.006), hemoglobin levels (rs=0.210, P=0.049), and percentage of peripheral blood blasts (rs=0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs=–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

The analysis of gene transcripts associated with conidiation in the insect pathogenic fungus, Metarhizium anisopliae /

Conidia of the insect pathogenic fungus, Metarhizium anisopliae play an important role in pathogenicity because they are the infective propagules that adhere to the surface of the insect, then germinate and give rise to hyphal penetration of the insect cuticle. Conidia are produced in the final stages of insect infection as the mycelia emerge from the insect cadaver. The genes associated with conidiation have not yet been studied in this fiingus. hi this study we used the PCR-based technique, suppression subtractive hybridization (SSH) to selectively amplify conidial-associated genes in M. anisopliae. We then identified the presence of these differentially expressed genes using the National Center for Biotechnology Information database. One of the transcripts encoded an extracellular subtilisin-like protease, Prl, which plays a fundamental role in cuticular protein degradation. Analysis of the patterns of gene expression of the transcripts using RT-PCR indicated that conidial-associated cDNAs are expressed during the development of the mature conidium. RT-PCR analysis was also performed to examine in vivo expression of Prl during infection of waxworm larvae {Galleria mellonelld). Results showed expression of Prl as mycelia emerge and produce conidia on the surface of the cadaver. It is well documented that Prl is produced during the initial stages of transcuticular penetration by M. anisopliae. We suggest that upregulation of Prl is part of the mechanism by which reverse (from inside to the outside of the host) transcuticular penetration of the insect cuticle allows subsequent conidiation on the cadaver.

Les voies de signalisation utérines à l'émergence de la diapause embryonnaire chez le vison américain

La diapause embryonnaire se manifeste par un arrêt réversible du développement embryonnaire durant la période de préimplantation et induit un retard de l’implantation. Chez le vison américain, une diapause embryonnaire obligatoire caractérise chaque gestation. Si les mécanismes de contrôle de la diapause embryonnaire obligatoire chez cette espèce sont bien connus, le rôle utérin impliqué dans la réactivation de l’embryon demeure, quant à lui, encore inconnu. Le sujet de ce doctorat a consisté dans un premier temps à explorer l’environnement utérin à la sortie de la diapause embryonnaire afin de caractériser, dans un deuxième temps, les principaux acteurs utérins qui provoquent la réactivation de l’embryon. Nous avons effectué une analyse du transcriptome utérin à l’émergence de la diapause embryonnaire ce qui a permis de construire une librairie de 123 séquences d’ADNc utérines différentiellement exprimées à la réactivation de l’embryon et homologues à des séquences de gènes connues chez d’autres espèces. Ces gènes sont impliqués dans la régulation du métabolisme (25 %), de l’expression génique (21 %), de la transduction de signal (15 %), du cycle cellulaire (15 %), du transport (10 %) et de la structure cellulaire (9 %), reflétant ainsi d’importantes modifications utérines à la réactivation embryonnaire. Nous avons validé l’expression différentielle de dix gènes ainsi identifiés : GDF3 (growth and differentiation 3), ALCAM (activated leukocyte cell adhesion molecule), ADIPOR1 (adiponectin receptor 1), HMGN1 (high mobility group N1), TXNL1 (thioredoxin like 1), TGM2 (tissue transglutaminase 2), SPARC (secreted protein acidic rich in cystein), et trois gènes codant pour AZIN1 (antizyme inhibitor 1), ODC1 (ornithine decarboxylase 1) et SAT1 (spermidine/spermine N1-acetyltransferase), des enzymes impliquées dans la biosynthèse des polyamines. Le patron de l’expression spatio-temporel de SPARC et d’HMGN1 illustrent spécifiquement un remodelage tissulaire et de la chromatine au niveau utérin à la sortie de la diapause embryonnaire. Ayant mesuré une augmentation des concentrations utérines en polyamines à la reprise du développement embryonnaire, nous avons émis l’hypothèse que les polyamines seraient impliquées dans les événements menant à la sortie de la diapause. L’inhibition de la biosynthèse des polyamines par un traitement à l’ α-difluoromethylornithine (DFMO) a provoqué une diminution significative de la proliferation cellulaire dans les embryons à la réactivation, un retard du moment de l’implantation, mais n’a pas affecté le succès de la reproduction. De manière similaire, nous avons induit un état de dormance dans les cellules de trophoblaste de vison en présence DFMO dans le milieu de culture, et constaté que cet état était réversible. En conclusion, cette étude a non seulement ouvert de nouveaux horizons quant à la compréhension du rôle utérin dans les événements menant à la sortie de la diapause embryonnaire, mais a démontré pour la première fois, l’existence de facteurs utérins indispensables à la réactivation de l’embryon: les polyamines.

L’importance du conflit identitaire majeur et de la perte d’identité sur le changement de trajectoire de vie

Un nombre important d’individus subit des conséquences négatives en lien avec une appartenance à un groupe peu adapté socialement (p. ex., membre d’un gang de rue). Certains parviennent à mettre fin à cette identification, alors que d'autres n’y arrivent pas. Nous proposons que les individus qui réussissent le peuvent grâce à l’intégration d’une nouvelle identité, davantage adaptée, et conflictuelle avec leur identité d’origine. Dans ce mémoire, nous mettons de l’avant l’argument que lors de conflit identitaire majeur entre deux identités, le processus d’intégration identitaire est soustractif. Cinq sous hypothèses ont été testées lors de deux études effectuées avec des participants vivant un conflit identitaire majeur. Un niveau élevé de conflit identitaire prédit un faible niveau d’identification envers l’identité au statut le moins élevé (hypothèse 1). Un lien prédictif est postulé entre le statut perçu d’une identité et le niveau d’identification à cette identité (hypothèse 2). Un niveau d’intégration identitaire élevé de la nouvelle identité prédit un faible niveau d’identification envers l’identité au statut le moins élevé (hypothèse 3). Un niveau d’intégration identitaire élevé de la nouvelle identité prédit un faible niveau de déviance (étude 1) et d’alcoolisme (étude 2) (hypothèse 4). Finalement, un niveau d’intégration identitaire élevé de la nouvelle identité prédit un niveau de bien-être élevé (hypothèse 5). Les résultats de la première étude (N=42), effectuée sur un échantillon de jeunes filles placées en Centre Jeunesse, vont dans le sens des hypothèses 2 et 3. Les résultats de la deuxième étude (N=28), effectuée sur un échantillon d’individus membres des Alcooliques Anonymes, vont dans le sens des hypothèses 2 et 5.

Hybridations fin-de-siècle : les Contes et nouvelles de Rachilde entre décadentisme et symbolisme

Notre mémoire prend pour objet d’étude les dix-sept textes brefs rassemblés dans le recueil Contes et nouvelles suivis du Théâtre, écrits durant la période symboliste et dans lesquels on constate un phénomène d’hybridation entre les genres narratif et dramatique ainsi qu’entre thèmes symbolistes et décadents. Le premier chapitre s’attache à l’étude de Contes et nouvelles suivis du Théâtre dans la perspective des poétiques du recueil afin de constituer les éléments qui en font une « anthologie de l’entre-deux » du corpus rachildien de textes brefs. En adoptant la perspective de la poétique des genres, il sera question dans le deuxième chapitre d’étudier l’hybridation des genres littéraires, plus précisément entre la prose narrative et le texte dramatique, donnant lieu à des textes en « prose dramatique » qui se situent entre texte à lire et à mettre en scène. Enfin, dans le troisième chapitre nous examinerons les thèmes et les motifs récurrents de l’imaginaire rachildien où s’entremêlent symbolisme et décadentisme, après nous être intéressée à « l’écriture artiste » sous la plume de Rachilde, qui permet de rendre « visibles » les différentes formes de spectres, d’illusions et d’ombres qui parsèment les textes de Contes et nouvelles suivis du Théâtre.

Le personnage-figurant : absence de monde et corps spectral dans l’œuvre de Béla Tarr

Ce mémoire examine le rapport du visible à l’invisible dans l’œuvre du cinéaste hongrois Béla Tarr afin de montrer comment cette œuvre fait sa plus grande place, quoique le plus souvent sous la forme d’une absence-présence, à la question du néant et de la mort. À travers une étude tripartite, tour à tour concernant le traitement cinématographique et thématique du monde, du temps et du personnage et de leurs fonctions, l’objectif de cette recherche est celui de montrer comment cette œuvre soustractive interroge le rapport de l’existant à un monde qui fait défaut. C’est la promesse, en partie telle que théorisée par Peter Sloterdijk, qui permettra ultimement d’éclairer la démarche tragique des personnages tarriens : lutte contre la mort au moyen d’un constant effort de soi et ce, jusqu’à l’épuisement radical. La notion de personnage-figurant développée dans ce mémoire se propose d’articuler ensemble les valeurs figuratives et thématiques communes aux différents personnages qui traversent l’œuvre afin d’en mieux comprendre, malgré les ruptures stylistiques et narratives, la continuité. Enfin, le dialogue engagé ici entre l’œuvre du cinéaste et certains concepts-clé de la tradition philosophique (le monde, le soi, le temps) entend montrer comment la première ouvre des pistes pertinentes pour une réflexion sur le sujet contemporain et son époque.

Nuclear organisation and epigenetic regulation of gene expression in Dictyostelium discoideum

A series of vectors for the over-expression of tagged proteins in Dictyostelium were designed, constructed and tested. These vectors allow the addition of an N- or C-terminal tag (GFP, RFP, 3xFLAG, 3xHA, 6xMYC and TAP) with an optimized polylinker sequence and no additional amino acid residues at the N or C terminus. Different selectable markers (Blasticidin and gentamicin) are available as well as an extra chromosomal version; these allow copy number and thus expression level to be controlled, as well as allowing for more options with regard to complementation, co- and super-transformation. Finally, the vectors share standardized cloning sites, allowing a gene of interest to be easily transfered between the different versions of the vectors as experimental requirements evolve. The organisation and dynamics of the Dictyostelium nucleus during the cell cycle was investigated. The centromeric histone H3 (CenH3) variant serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. A number of Dictyostelium histone H3-domain containing proteins as GFP-tagged fusions were expressed and it was found that one of them functions as CenH3 in this species. Like CenH3 from some other species, Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins. The targeting domain, comprising α-helix 2 and loop 1 of the histone fold is required for targeting CenH3 to centromeres. Compared to the targeting domain of other known and putative CenH3 species, Dictyostelium CenH3 has a shorter loop 1 region. The localisation of a variety of histone modifications and histone modifying enzymes was examined. Using fluorescence in situ hybridisation (FISH) and CenH3 chromatin-immunoprecipitation (ChIP) it was shown that the six telocentric centromeres contain all of the DIRS-1 and most of the DDT-A and skipper transposons. During interphase the centromeres remain attached to the centrosome resulting in a single CenH3 cluster which also contains the putative histone H3K9 methyltransferase SuvA, H3K9me3 and HP1 (heterochromatin protein 1). Except for the centromere cluster and a number of small foci at the nuclear periphery opposite the centromeres, the rest of the nucleus is largely devoid of transposons and heterochromatin associated histone modifications. At least some of the small foci correspond to the distal telomeres, suggesting that the chromosomes are organised in a Rabl-like manner. It was found that in contrast to metazoans, loading of CenH3 onto Dictyostelium centromeres occurs in late G2 phase. Transformation of Dictyostelium with vectors carrying the G418 resistance cassette typically results in the vector integrating into the genome in one or a few tandem arrays of approximately a hundred copies. In contrast, plasmids containing a Blasticidin resistance cassette integrate as single or a few copies. The behaviour of transgenes in the nucleus was examined by FISH, and it was found that low copy transgenes show apparently random distribution within the nucleus, while transgenes with more than approximately 10 copies cluster at or immediately adjacent to the centromeres in interphase cells regardless of the actual integration site along the chromosome. During mitosis the transgenes show centromere-like behaviour, and ChIP experiments show that transgenes contain the heterochromatin marker H3K9me2 and the centromeric histone variant H3v1. This clustering, and centromere-like behaviour was not observed on extrachromosomal transgenes, nor on a line where the transgene had integrated into the extrachromosomal rDNA palindrome. This suggests that it is the repetitive nature of the transgenes that causes the centromere-like behaviour. A Dictyostelium homolog of DET1, a protein largely restricted to multicellular eukaryotes where it has a role in developmental regulation was identified. As in other species Dictyostelium DET1 is nuclear localised. In ChIP experiments DET1 was found to bind the promoters of a number of developmentally regulated loci. In contrast to other species where it is an essential protein, loss of DET1 is not lethal in Dictyostelium, although viability is greatly reduced. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed apparent cell type patterning with a bias towards pre-stalk cell types.

Diez visiones sobre la vejez : del enfoque deficitario y de deterioro al enfoque positivo.

Resumen del autor. Resumen en castellano y en inglés