Attivazione del sistema Wnt in linee cellulari di medulloblastoma umano: valutazione della risposta biologica e della risposta alle radiazioni ionizzanti

Medulloblastoma (MB) is a paediatric malignant brain tumour, sensitive to ionizing radiations (IR). However radiotherapy has detrimental effects on long-term survivors and the tumour is incurable in a third of patients, due to intrinsic radioresistance. Alterations of the Wnt pathway distinguish a molecular subgroup of MBs and nuclear beta-catenin, indicative of activated Wnt, is associated with good outcome in MB. Therefore there are increasing evidences about Wnt involvement in radio-response: IR induce activation of Wnt signalling with nuclear translocation of beta-catenin in MB cell lines. We studied effects of Wnt pathway activation in a MB cell line with p53 wild-type: UW228-1. Cells were stably transfected with a beta-catenin constitutively active and assessed for growth curves, mortality rate, invasiveness and differentiation. Firstly, activation of Wnt pathway by itself induced a slower cell growth and a higher mortality. After IR treatment, nuclear beta-catenin further inhibited cell growth, increasing mortality. Cell invasiveness was strongly inhibited by Wnt activation. Furthermore, Wnt cell population was characterized by club shaped cells with long cytoplasmic extensions containing neurofilaments, suggesting a neural differentiation of this cell line. These findings suggest that nuclear beta-catenin may leads to a less aggressive phenotype and increases radio-sensitivity in MB, accounting for its favourable prognostic value. In the future, Wnt/beta-catenin signalling will be considered as a molecular therapeutic target to develop new drugs for the treatment of MB.

Bioinformatic methods in applied genomic research

Here I will focus on three main topics that best address and include the projects I have been working in during my three year PhD period that I have spent in different research laboratories addressing both computationally and practically important problems all related to modern molecular genomics. The first topic is the use of livestock species (pigs) as a model of obesity, a complex human dysfunction. My efforts here concern the detection and annotation of Single Nucleotide Polymorphisms. I developed a pipeline for mining human and porcine sequences. Starting from a set of human genes related with obesity the platform returns a list of annotated porcine SNPs extracted from a new set of potential obesity-genes. 565 of these SNPs were analyzed on an Illumina chip to test the involvement in obesity on a population composed by more than 500 pigs. Results will be discussed. All the computational analysis and experiments were done in collaboration with the Biocomputing group and Dr.Luca Fontanesi, respectively, under the direction of prof. Rita Casadio at the Bologna University, Italy. The second topic concerns developing a methodology, based on Factor Analysis, to simultaneously mine information from different levels of biological organization. With specific test cases we develop models of the complexity of the mRNA-miRNA molecular interaction in brain tumors measured indirectly by microarray and quantitative PCR. This work was done under the supervision of Prof. Christine Nardini, at the “CAS-MPG Partner Institute for Computational Biology” of Shangai, China (co-founded by the Max Planck Society and the Chinese Academy of Sciences jointly) The third topic concerns the development of a new method to overcome the variety of PCR technologies routinely adopted to characterize unknown flanking DNA regions of a viral integration locus of the human genome after clinical gene therapy. This new method is entirely based on next generation sequencing and it reduces the time required to detect insertion sites, decreasing the complexity of the procedure. This work was done in collaboration with the group of Dr. Manfred Schmidt at the Nationales Centrum für Tumorerkrankungen (Heidelberg, Germany) supervised by Dr. Annette Deichmann and Dr. Ali Nowrouzi. Furthermore I add as an Appendix the description of a R package for gene network reconstruction that I helped to develop for scientific usage (http://www.bioconductor.org/help/bioc-views/release/bioc/html/BUS.html).

Strategies to identify and further characterize novel and known genes involved in regulation of skeletogenesis

372 osteochondrodysplasias and genetically determined dysostoses were reported in 2007 [Superti-Furga and Unger, 2007]. For 215 of these conditions, an association with one or more genes can be stated, while the molecular changes for the remaining syndromes remain illusive to date. Thus, the present dissertation aims at the identification of novel genes involved in processes regarding cartilage/ bone formation, growth, differentiation and homeostasis, which may serve as candidate genes for the above mentioned conditions. Two different approaches were undertaken. Firstly, a high throughput EST sequencing project from a human fetal cartilage library was performed to identify novel genes in early skeletal development (20th week of gestation until 2nd year of life) that could be investigated as potential candidate genes. 5000 EST sequences were generated and analyzed representing 1573 individual transcripts, corresponding to known (1400) and to novel, yet uncharacterized genes (173). About 7% of the proteins were already described in cartilage/ bone development or homeostasis, showing that the generated library is tissue specific. The remaining profile of this library was compared to previously published libraries from different time points (8th–12th, 18th–20th week and adult human cartilage) that also showed a similar distribution, reflecting the quality of the presented library analyzed. Furthermore, three potential candidate genes (LRRC59, CRELD2, ZNF577) were further investigated and their potential involvement in skeletogenesis was discussed. Secondly, a disease-orientated approach was undertaken to identify downstream targets of LMX1B, the gene causing Nail-Patella syndrome (NPS), and to investigate similar conditions. Like NPS, Genitopatellar syndrome (GPS) is characterized by aplasia or hypoplasia of the patella and renal anomalies. Therefore, six GPS patients were enrolled in a study to investigate the molecular changes responsible for this relatively rare disease. A 3.07 Mb deletion including LMX1B and NR5A1 (SF1) was found in one female patient that showed features of both NPS and GPS and investigations revealed a 46,XY karyotype and ovotestes indicating true hermaphroditism. The microdeletion was not seen in any of the five other patients with GPS features only, but a potential regulatory element between the two genes cannot be ruled out yet. Since Lmx1b is expressed in the dorsal limb bud and in podocytes, proteomic approaches and expression profiling were performed with murine material of the limbs and the kidneys to identify its downstream targets. After 2D-gel electrophoresis with protein extracts from E13.5 fore limb buds and newborn kidneys of Lmx1b wild type and knock-out mice and mass spectrometry analysis, only two proteins, agrin and carbonic anhydrase 2, remained of interest, but further analysis of the two genes did not show a transcriptional down regulation by Lmx1b. The focus was switched to expression profiles and RNA from newborn Lmx1b wild type and knock-out kidneys was compared by microarray analysis. Potential Lmx1b targets were almost impossible to study, because of the early death of Lmx1b deficient mice, when the glomeruli, containing podocytes, are still immature. Because Lmx1b is also expressed during limb development, RNA from wild type and knock-out Lmx1b E11.5 fore limb buds was investigated by microarray, revealing four potential Lmx1b downstream targets: neuropilin 2, single-stranded DNA binding protein 2, peroxisome proliferative activated receptor, gamma, co-activator 1 alpha, and short stature homeobox 2. Whole mount in situ hybridization strengthened a potential down regulation of neuropilin 2 by Lmx1b, but further investigations including in situ hybridization and protein-protein interaction studies will be needed.

SANS (USH1G) in USH-Proteinnetzwerken von Photorezeptorzellen der Vertebratenretina

Das Usher Syndrom (USH) führt beim Menschen zur häufigsten Form erblicher Taub-Blindheit und wird aufgrund klinischer Merkmale in drei Typen unterteilt (USH1-3). Das Ziel dieser Arbeit war die Analyse der Expression und subzellulären Lokalisation des USH1G-Proteins SANS („Scaffold protein containing Ankyrin repeats and SAM domain“) in der Retina. Ein weiterer Fokus lag auf der Identifikation neuer Interaktionspartner zur funktionellen Charakterisierung von SANS. Im Rahmen der vorliegenden Arbeit konnte ein USH-Proteinnetzwerk identifiziert werden, das im Verbindungscilium und benachbarter Struktur, dem apikalen Innensegment von Photorezeptorzellen lokalisiert ist. Als Netzwerkkomponenten konnten die USH-Proteine SANS, USH2A Isoform b (USH2A), VLGR1b („Very Large G-protein coupled Receptor 1b“, USH2C) sowie Whirlin (USH2D) ermittelt werden. Innerhalb dieses Netzwerkes interagieren die Gerüstproteine SANS und Whirlin direkt miteinander. Die Transmembranproteine USH2A Isoform b und VLGR1b sind durch die direkte Interaktion mit Whirlin in ciliären-periciliären Membranen verankert und projizieren mit ihren langen Ektodomänen in den extrazellulären Spalt zwischen Verbindungscilium und apikalem Innensegment. Darüber hinaus konnte die Partizipation von SANS an Mikrotubuli-assoziiertem Vesikeltransport durch Identifikation neuer Interaktionspartner, wie dem MAGUK-Protein MAGI-2 („Membrane-Associated Guanylate Kinase Inverted-2“) sowie Dynaktin-1 (p150Glued) eruiert werden. Die Funktion des ciliären-periciliären USH-Proteinnetzwerkes könnte demnach in der Aufrechterhaltung benachbarter Membranstrukturen sowie der Beteiligung der Positionierung und Fusion von Transportvesikeln liegen.

Role of Notch signalling in human hepatocellular carcinoma

The Notch signalling is a cellular pathway that results conserved from Drosophila to Homo sapiens controlling a wide range of cellular processes in development and in differentiated organs. It induces cell proliferation or differentiation, increased survival or apoptosis, and it is involved in stemness maintainance. These functions are conserved, but exerted with a high tissue and cellular context specificity. Signalling activation determs nuclear translocation of the receptor’s cytoplasmic domain and activation of target genes transcription. As many developmental pathway, Notch deregulation is involved in cancer, leading to oncogenic or tumour suppressive role depending on the functions exerted in normal tissue. Notch1 and Notch3 resulted aberrantly expressed in human hepatocellular carcinoma (HCC) that is the more frequent tumour of the liver and the sixth most common tumour worldwide. This thesis has the aim to investigate the role of the signalling in HCC, with particular attention to dissect common and uncommon regulatory pathways between Notch1 and Notch3 and to define the role of the signalling in HCC. Nocth1 and Notch3 were analysed on their regulation on Hes1 target and involvement in cell cycle control. They showed to regulate CDKN1C/p57kip2 expression through Hes1 target. CDKN1C/p57kip2 induces not only cell cycle arrest, but also senescence in HCC cell lines. Moreover, the involvement of Notch1 in cancer progression and epithelial to mesenchymal transition was investigated. Notch1 showed to induce invasion of HCC, regulating EMT and E- Cadherin expression. Moreover, Notch3 showed specific regulation on p53 at post translational levels. In vitro and ex vivo analysis on HCC samples suggests a complex role of both receptors in regulate HCC, with an oncogenic role but also showing tumour suppressive effects, suggesting a complex and deep involvement of this signalling in HCC.

Einfluss von oxidativem Stress auf die DNA-Reparatur in Säugerzellen

Gegenstand dieser Arbeit war es, das Zusammenspiel zwischen DNA-Reparatur und zellulärem anitoxidativen Abwehrsystem in Melanomzellen und gesunden Hautfibroblasten näher zu untersuchen. Dabei konnte gezeigt werden, dass die dominierenden DNA-Läsionen im Falle einer Bestrahlung mit sichtbarem Licht (400 – 800 nm) Fpg-sensitive Läsionen, zu denen die Basenmodifikation 7,8-Dihydro-8-oxoguanin (8-oxoG) gehört, und im Falle der UVA-Bestrahlung Cyclobutan-Pyrimidindimere (CPDs) sind. Sowohl Melanomzellen als auch Hautfibroblasten waren problemlos in der Lage, die durch sichtbares Licht und UVA-Strahlung induzierten oxidativen DNA-Modifikationen zu reparieren. Jedoch reagierten Melanomzellen in einer adaptiven Antwort mit einer Erhöhung ihres Glutathion-Gehalts auf ein Maximum (nach circa 10 - 14 h) nach Bestrahlung mit sichtbarem Licht, wohingegen die Hautfibroblasten einen massiven Einbruch direkt nach Bestrahlung und eine extrem lange Erholungsphase über 48 h aufzuweisen hatten. Die darauffolgende Untersuchung der DNA-Reparaturkapazität der Zellen unter Bedingungen von oxidativem Stress mit vorangegangener Depletion intrazellulären Glutathions zeigten eine dramatische, nahezu vollständige Hemmung der Reparatur durch UVA- bzw. Sonnenlicht-induzierter Fpg-sensitiver DNA-Modifikationen (8-oxoG) - sowohl in Melanomzellen als auch in Hautfibroblasten. Dieser Effekt ließ sich durch den Zusatz von Dithiothreitol (DTT), nach erfolgter Bestrahlung der Glutathion-depletierten Zellen, wieder komplett revertieren. Diese Ergebnisse weisen darauf hin, dass an der Reparatur ein redoxempfindliches Protein oder zellulärer Cofaktor beteiligt sein muß. Zudem konnte durch Untersuchungen der Nukleotidexzisionsreparatur (NER) und der Einzelstrangbruchreparatur nach dem gleichen Versuchsdesign gezeigt werden, dass es sich hierbei sehr wahrscheinlich um einen für die Basenexzisionsreparatur (BER) von 7,8-dihydro-8-oxo-guanine (8-oxoG) exklusiven Effekt handelte. Zwei der wichtigsten Reparaturproteine der BER, nämlich hOGG1 und APE1, wurden anschließend auf ihre Funktionsfähigkeit hin untersucht, da es naheliegend war, dass der Reparaturhemmung ein Funktionsverlust eines dieser beiden Enzyme zugrunde liegen könnte. Im Falle des APE1-Proteins konnte dies ausgeschlossen werden, da mit Hilfe der Alkalischen Elution die volle Funktionsfähigkeit für die Reparatur von AP-Läsionen nachgewiesen werden konnte. Interessanterweise zeigte aber das hOGG1-Protein eine zwischen der dritten und vierten Stunde nach Bestrahlung Glutathion-depletierter Zellen stark abfallende Aktivität der 8-oxoG-Glykosylasefunktion. Die Western-Blot-Analyse ergab allerdings keinen Hinweis auf eine Proteinoxidation von hOGG1. Möglicherweise wird nicht hOGG1 selbst, wohl aber ein anderes, für eine konzertierte Abfolge der einzelnen Reparaturschritte entscheidend notwendiges Protein innerhalb der Zelle durch ROS leicht oxidiert. In jedem Fall bleibt festzustellen, dass Glutathion eine wichtige Aufgabe hinsichtlich einer voll funktionsfähigen Basenexzisionreparatur zuzukommen scheint. Die Ergebnisse unterstreichen die mögliche Bedeutung von oxidativem Stress für die Entstehung von Krebs durch Sonnenlicht, insbesondere durch UVA, da die durch die Strahlung (und eventuell auftretende Entzündung) gebildeten ROS nicht nur DNA-Schäden induzieren, sondern auch ihre Reparatur verhindern können.

Characterization of circulating steroids and endocannabinoids levels in obese subjects by liquid chromatography tandem mass spectrometry

Nowadays obesity can be defined as a global epidemic. The precise identification of circulating biomarkers involved in this pathology could be essential to early diagnose potential co-morbidities and to better address the development of future therapeutic strategies. Published evidences show that circulating steroid hormones and endocannabinoids might have a role in the physiopathology of obesity; however, a precise and reliable quantification of these molecules is still lacking. In the first part of the present thesis, we developed a sensitive, specific and accurate quantification method for nine steroid hormones using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system. This method has been used first for a comparative study with immunoassays, currently used in the clinical practice to quantify these molecules and then to redefine circulating reference intervals in healthy subjects. Furthermore, we measured circulating steroid hormones in three groups of subjects: normo-weight, over-weight and obese, defining different steroid hormones profiles depending on the obesity state. The role of circulating endocannabinoids in humans is still unclear, however there are several evidences concerning their involvement in obesity. In the second part of the thesis, we determined changes of circulating endocannabinoids in obese patients after a weight loss induced by bariatric surgery, currently the most effective long-term treatment for obesity, using LC/MS-MS. We measured basal and dynamic endocannabinoids plasma levels in 12 patients with severe obesity before, one month after and six months after the Roux-en-Y gastric bypass intervention, currently one of the most performed types of bariatric surgery. All together the findings illustrated in this thesis project will help better define the role of steroid hormones and endocannabinoids in the framework of obesity in humans and the role that each type of molecule might have in its pathophysiology.

Biomolecular studies in Alzheimer’s Disease models: investigations in vitro and in vivo

The Alzheimer’s disease (AD), the most prevalent form of age-related dementia, is a multifactorial and heterogeneous neurodegenerative disease. The molecular mechanisms underlying the pathogenesis of AD are yet largely unknown. However, the etiopathogenesis of AD likely resides in the interaction between genetic and environmental risk factors. Among the different factors that contribute to the pathogenesis of AD, amyloid-beta peptides and the genetic risk factor apoE4 are prominent on the basis of genetic evidence and experimental data. ApoE4 transgenic mice have deficits in spatial learning and memory associated with inflammation and brain atrophy. Evidences suggest that apoE4 is implicated in amyloid-beta accumulation, imbalance of cellular antioxidant system and in apoptotic phenomena. The mechanisms by which apoE4 interacts with other AD risk factors leading to an increased susceptibility to the dementia are still unknown. The aim of this research was to provide new insights into molecular mechanisms of AD neurodegeneration, investigating the effect of amyloid-beta peptides and apoE4 genotype on the modulation of genes and proteins differently involved in cellular processes related to aging and oxidative balance such as PIN1, SIRT1, PSEN1, BDNF, TRX1 and GRX1. In particular, we used human neuroblastoma cells exposed to amyloid-beta or apoE3 and apoE4 proteins at different time-points, and selected brain regions of human apoE3 and apoE4 targeted replacement mice, as in vitro and in vivo models, respectively. All genes and proteins studied in the present investigation are modulated by amyloid-beta and apoE4 in different ways, suggesting their involvement in the neurodegenerative mechanisms underlying the AD. Finally, these proteins might represent novel potential diagnostic and therapeutic targets in AD.

A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Investigating the role of Copy Number Variants in Specific Language Impairment and identification of new candidate genes

Specific language impairment (SLI) is a complex neurodevelopmental disorder defined as an unexpected failure to develop normal language abilities for no obvious reason. Copy number variants (CNVs) are an important source of variation in the susceptibility to neuropsychiatric disorders. Therefore, a CNV study within SLI families was performed to investigate the role of structural variants in SLI. Among the identified CNVs, we focused on CNVs on chromosome 15q11-q13, recurrently observed in neuropsychiatric conditions, and a homozygous exonic microdeletion in ZNF277. Since this microdeletion falls within the AUTS1 locus, a region linked to autism spectrum disorders (ASD), we investigated a potential role of ZNF277 in SLI and ASD. Frequency data and expression analysis of the ZNF277 microdeletion suggested that this variant may contribute to the risk of language impairments in a complex manner, that is independent of the autism risk previously described in this region. Moreover, we identified an affected individual with a dihydropyrimidine dehydrogenase (DPD) deficiency, caused by compound heterozygosity of two deleterious variants in the gene DPYD. Since DPYD represents a good candidate gene for both SLI and ASD, we investigated its involvement in the susceptibility to these two disorders, focusing on the splicing variant rs3918290, the most common mutation in the DPD deficiency. We observed a higher frequency of rs3918290 in SLI cases (1.2%), compared to controls (~0.6%), while no difference was observed in a large ASD cohort. DPYD mutation screening in 4 SLI and 7 ASD families carrying the splicing variant identified six known missense changes and a novel variant in the promoter region. These data suggest that the combined effect of the mutations identified in affected individuals may lead to an altered DPD activity and that rare variants in DPYD might contribute to a minority of cases, in conjunction with other genetic or non-genetic factors.

Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

Funktionsaufklärung von Atmungsproteinen in Insekten

Globine sind kleine globuläre Proteine mit nahezu ubiquitärem Vorkommen in allen Tiergruppen. Sie weisen eine typische Sandwichstruktur auf, die in der Regel aus acht α-Helices mit einer zentralen prosthetischen Häm-Gruppe besteht und die Proteine zur Bindung gasförmiger Liganden befähigt. Die Funktionen der Globine reichen von O2-Transport und – Speicherung, über eine Beteiligung bei der Entgiftung reaktiver Sauerstoff- und Stickstoffspezies bis hin zu sensorischen physiologischen Aufgaben. Innerhalb der Klasse der Insekten schien das Vorhandensein von Globinen zunächst auf Insekten mit offensichtlich hypoxischen Habitaten beschränkt zu sein. Die Entdeckung des Globins glob1 in Drosophila melanogaster deutete jedoch eine sehr viel weitere Verbreitung der Globine in Insekten an, die sich durch die Identifizierung von Globingenen in einer Vielzahl von normoxisch lebenden Insekten, wie z.B. Apis mellifera oder Aedes aegypti bestätigte. D. melanogaster besitzt drei Globine, glob1, glob2 und glob3. Glob1 ist eng mit anderen intrazellulären Insektenglobinen verwandt, was zu der Annahme führte, dass es sich bei glob1 um das ursprüngliche und bei glob2 und glob3 um abgeleitete D. melanogaster Globine handelt. Glob1 wird in allen Entwicklungsstadien exprimiert, wobei die Hauptexpressionsorte der Fettkörper und das Tracheensystem sind. Die Transkription des glob1 startet von zwei alternativen Promotoren (Promotor I und II), wodurch in Kombination mit alternativem Splicing vier Transkriptvarianten (Isoform A-D) entstehen, deren Translation jedoch in einer Proteinvariante (glob1) resultiert. Hypoxische Bedingungen führen zu einer vermutlich HIF (=‚hypoxia-inducible factor‘) -vermittelten Abnahme der glob1 Genexpression, wohingegen Hyperoxie eine leichte Zunahme der glob1 mRNA Menge bewirkt. Der mithilfe des UAS/Gal4- Systems erzeugte, RNAi-vermittelte glob1 Knockdown führt zu einer schlechteren Überlebensrate adulter Fliegen unter hypoxischen Bedingungen, einer verkürzten Erholungszeit nach hypoxischem Stupor in Weibchen sowie zu einer erhöhten Resistenz gegenüber dem ROS (=‘reactive oxygen species‘) -generierenden Herbizid Paraquat in Larven und adulten Weibchen. Diese Beobachtungen sprechen für eine Funktion des Drosophila glob1 innerhalb der O2-Versorgung. Unter hyperoxischen Bedingungen hingegen wurde kein Unterschied zwischen Fliegen mit wildtypischer und manipulierter glob1-Expression festgestellt, wodurch eine Beteiligung des glob1 bei der Entgiftung reaktiver Sauerstoffspezies als mögliche Funktion vorerst ausscheidet. Bei glob2 und glob3 handelt es sich um duplizierte Gene. Auf phylogenetischen Rekonstruktionen basierend konnte die Entstehung der Globin-Duplikate auf ein Duplikationsereignis vor der Radiation des Subgenus Sophophora vor mindestens 40 Millionen Jahren zurückgeführt werden. Die durchgeführten Analysen zur molekularen Sequenzevolution der Globin-Duplikate deuten darauf hin, dass glob2 und glob3 nach der Duplikation eine Kombination aus Sub- und Neo-Funktionalisierungsprozessen durchlaufen haben. Glob2 und glob3 zeigen eine deckungsgleiche mRNA Expression, die auf die männliche Keimbahn beschränkt ist. Aufgrund des hohen Konservierungsgrads der für die Häm- und O2-Bindung essentiellen Aminosäuren kann von der Funktionalität beider Proteine ausgegangen werden. Die streng auf die männliche Keimbahn begrenzte Expression von glob2 und glob3 deutet auf eine Rolle der Globin-Duplikate innerhalb der Spermatogenese hin, die möglicherweise in einem Schutz der Spermatogenese vor oxidativem Stress besteht. Auch eine Beteiligung beim korrekten Ablauf der Spermien-Individualisierung, beispielsweise durch Regulation von Apoptoseprozessen wäre denkbar.

Oncology nurses' perceptions about involving patients in the prevention of chemotherapy administration errors

To explore oncology nurses' perceptions and experiences with patient involvement in chemotherapy error prevention.

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues

The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.