The role of MYCN-mediated transcriptional repression in neuronal physiopathology

MYC is a transcription factor that can activate transcription of several targets by direct binding to their promoters at specific DNA sequences (E-box). Recent findings have also shown that it can exert its biological role by repressing transcription of other set of genes. C-MYC can mediate repression on its target genes through interaction with factors bound to promoter regions but not through direct recognition of typical E-Boxes. In this thesis, we investigated whether MYCN can also repress gene transcription and how this is mechanistically achieved. Moreover, expression of TRKA, P75NTR and ABCC3 is attenuated in aggressive MYCN-amplified tumors, suggesting a causal link between elevated MYCN activity and transcriptional repression of these three genes. We found that MYCN is physically associated with gene promoters in vivo in proximity of the transcriptional start sites and this association requires interactions with SP1 and/or MIZ-1. Furthermore, we show that this interaction could interfere with SP1 and MIZ-1 activation functions by recruiting co-repressors such as DNMT3a or HDACs. Studies in vitro suggest that MYCN interacts through distinct domains with SP1, MIZ-1 and HDAC1 supporting the idea that MYCN may form different complexes by interacting with different proteins. Re-expression of endogenous TRKA and P75NTR with exposure to the TSA sensitizes neuroblastoma to NGF-mediated apoptosis, whereas ectopic expression of ABCC3 decreases cell motility without interfering with growth. Finally, using shRNA whole genome library, we dissected the P75NTR repression trying to identify novel factors inside and/or outside MYCN complex for future therapeutic approaches. Overall, our results support a model in which MYCN can repress gene transcription by direct interaction with SP1 and/or MIZ-1, and provide further lines of evidence on the importance of transcriptional repression induced by Myc in tumor biology.

Epidemiology and population genetics of Podosphaera fusca and Golovinomyces orontii, causal agents of cucurbit powdery mildew

In 2010, 2011 and 2012 growing seasons, the occurrence of the ascomycetes Podosphaera fusca and Golovinomyces orontii, causal agents of powdery mildew disease, was monitored on cultivated cucurbits located in Bologna and Mantua provinces to determine the epidemiology of the species. To identify the pathogens, both morphological and molecular identifications were performed on infected leaf samples and a Multiplex-PCR was performed to identify the mating type genes of P. fusca isolates. The investigations indicated a temporal succession of the two species with the earlier infections caused by G. orontii, that seems to be the predominant species till the middle of July when it progressively disappears and P. fusca becomes the main species infecting cucurbits till the end of October. The temporal variation is likely due to the different overwintering strategies of the two species instead of climatic conditions. Only chasmothecia of P. fusca were recorded and mating type alleles ratio tended to be 1:1. Considering that only chasmothecia of P. fusca were found, molecular-genetic analysis were carried out to find some evidence of recombination within this species by MLST and AFLP methods. Surprisingly, no variations were observed within isolates for the 8 MLST markers used. According to this result, AFLP analysis showed a high similarity within isolates, with SM similarity coefficient ranging between 0.91-1.00 and also, sequencing of 12 polymorphic bands revealed identity to some gene involved in mutation and selection. The results suggest that populations of P. fusca are likely to be a clonal population with some differences among isolates probably due to agricultural practices such as fungicides treatments and cultivated hosts. Therefore, asexual reproduction, producing a lot of fungal biomass that can be easily transported by wind, is the most common and useful way to the spread and colonization of the pathogen.

Obsessed With Work: A multi-causal and multi-rater approach to workaholism

Workaholism is defined as the combination of two underlying dimensions: working excessively and working compulsively. The present thesis aims at achieving the following purposes: 1) to test whether the interaction between environmental and personal antecedents may enhance workaholism; 2) to develop a questionnaire aimed to assess overwork climate in the workplace; 3) to contrast focal employees’ and coworkers’ perceptions of employees’ workaholism and engagement. Concerning the first purpose, the interaction between overwork climate and person characteristics (achievement motivation, perfectionism, conscientiousness, self-efficacy) was explored on a sample of 333 Dutch employees. The results of moderated regression analyses showed that the interaction between overwork climate and person characteristics is related to workaholism. The second purpose was pursued with two interrelated studies. In Study 1 the Overwork Climate Scale (OWCS) was developed and tested using a principal component analysis (N = 395) and a confirmatory factor analysis (N = 396). Two overwork climate dimensions were distinguished, overwork endorsement and lacking overwork rewards. In Study 2 the total sample (N = 791) was used to explore the association of overwork climate with two types of working hard: work engagement and workaholism. Lacking overwork rewards was negatively associated with engagement, whereas overwork endorsement showed a positive association with workaholism. Concerning the third purpose, using a sample of 73 dyads composed by focal employees and their coworkers, a multitrait-multimethod matrix and a correlated trait-correlated method model, i.e. the CT-C(M–1) model, were examined. Our results showed a considerable agreement between raters on focal employees' engagement and workaholism. In contrast, we observed a significant difference concerning the cognitive dimension of workaholism, working compulsively. Moreover, we provided further evidence for the discriminant validity between engagement and workaholism. Overall, workaholism appears as a negative work-related state that could be better explained by assuming a multi-causal and multi-rater approach.