The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. Subplate plays a fundamental role in cortical development. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of corticofugal and thalamic axons. At later stages, these cells are involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. In my thesis, I aimed to further characterize the embryonic murine subplate by establishing a gene expression profile of this population at embryonic day 15.5 (E15.5) using laser capture microdissection combined with microarrays. I found over 250 transcripts with presumed higher expression in the subplate at E15.5. Using quantitative RT-PCR, in situ hybridization and immunohistochemistry, I have confirmed specific expression in the E15.5 subplate for 13 selected genes which have not been previously associated with this compartment. In the reeler mutant, the expression pattern of a majority of these genes was shifted in accordance with the altered position of subplate cells. These genes belong to several functional groups and likely contribute to the maturation and electrophysiological properties of subplate cells and to axonal growth and guidance. The roles of two selected genes - cadherin 10 (Cdh10) and Unc5 homologue c (Unc5c) - were explored in more detail. Preliminary results suggest an involvement of Cdh10 in subplate layer organization while Unc5c could mediate the waiting period of subplate corticothalamic axons in the internal capsule. Finally, I compared the expression of a selection of subplate-specific genes (subplate markers) between mouse and rat and found some surprising species differences. Confirmed subplate markers were used to monitor subplate injury in a rat model of preterm hypoxiaischemia and it appeared that deep cortical layers including subplate showed an increased vulnerability over upper layers. Further characterization of subplate-specific genes will allow us to broaden our understanding of molecular mechanisms underlying subplate properties and functions in normal and pathological development.

Una dieta scorretta incrementa il rischio di malattie come l’insulino resistenza e l’obesità. Questa tesi ha l’obiettivo di valutare l’effetto di diete sbilanciate sulla fisiologia ed espressione genica in topi e suini allo svezzamento. Topi C57BL/6 sono stati sacrificati dopo 2 settimane, dopo essere stati alimentati con dieta iper-lipidica e dieta controllo. L’espressione genica è stata stimata usando la tecnologia microarray. Quattro dei sette geni identificati differenzialmente espressi tra il controllo e l’iper-lipidico sono coinvolti nella regolazione della via metabolica del sistema circadiano, che recentemente è stato mostrato avere effetti sul metabolismo lipidico e processo infiammatorio. Il secondo studio ha avuto lo scopo di capire gli effetti dello svezzamento con o senza l’aggiunta di acidificante nella dieta. I suinetti allo svezzamento (T0) sono stati comparati con i suinetti dopo una settimana (T1). Il gruppo post-svezzamento è stato alimentato con una dieta convenzionale, e metà di questi hanno ricevuto un supplemento di acido sorbico. L’aggiunta di acido sorbico nella dieta non ha causato nessuna differenza a livello fisiologico e di espressione genica. 205 geni sono stati identificati come differenzialmente espressi in T1 comparato con T0, evidenziando una forte risposta all’adattamento metabolico e agli stress subiti durante lo svezzamento. / An incorrect diet increases the risk of diseases as insulin resistance and obesity. This thesis aims at assessing the effects of unbalanced diets on gut physiology and gene expression in pig and mouse during weaning. The first research explored the impact of a high fat diet in C57BL/6 mice. High-fat-fed mice and control-fed mice were sacrificed after two weeks of treatment. Gene expression level was assessed by 90K Combimatrix microarray technology. Four of seven genes found differentially expressed between control and high fat diet mice are involved in the regulatory pathway of the circadian clock system, which was recently shown to affect lipid metabolism and inflammatory processes. Those genes were successfully validated by real time PCR. The second study aimed at understanding the weaning effect with or without acidifier addition in the diet. Piglets at weaning (T0) were compared to piglets after one week (T1). The post-weaning group was fed a conventional diet, half of which received in addition sorbic acid. The sorbic acid supplementation evidenced no effects in terms of physiology and gene expression. 205 genes were significantly differentially expressed in T1 when compared with T0, evidencing a response to the metabolic adaptation and the stress suffered during weaning.

The intimate relationship between the higher-order chromatin organisation and the regulation of gene expression is increasingly attracting attention in the scientific community. Thanks to high-resolution microscopy, genome-wide molecular biology tools (3C, ChIP-on-chip), and bioinformatics, detailed structures of chromatin loops, territories, and nuclear domains are gradually emerging. However, to fully reveal a comprehensive map of nuclear organisation, some fundamental questions remain to be answered in order to fit all the pieces of the jigsaw together. The underlying mechanisms, precisely organising the interaction of the different parts of chromatin need to be understood. Previous work in our lab hypothesised and verified the “transcription factory” model for the organisation of mammalian genomes. It is widely assumed that active polymerases track along their templates as they make RNA. However, after allowing engaged polymerases to extend their transcripts in tagged precursors (e.g., Br-U or Br-UTP), and immunolabelling the now-tagged nascent RNA, active transcription units are found to be clustered in nuclei, in small and numerous sites we call “transcription factories”. Previous work suggested the transcription machinery acts both as an enzyme as well as a molecular tie that maintains chromatin loops, and the different classes of polymerases are concentrated in their own dedicated factories. This thesis aims to further characterise transcription factories. Different genes are transcribed by different classes of RNA polymerase (i.e., I, II, or III), and the resulting transcripts are processed differently (e.g., some are capped, others spliced). Do factories specialise in transcribing particular subsets of genes? This thesis developed a method using replicating minichromosomes as probes to examine whether transcription occurs in factories, and whether factories specialise in transcribing particular sets of genes. Plasmids encoding the SV40 origin of replication are transfected into COS-7 cells, where they are assembled into minichromosomes. Using RNA fluorescence in situ hybridisation (FISH), sites where minichromosomes are transcribed are visualised as discrete foci, which specialise in transcribing different groups of genes. Polymerases I, II, and III units have their own dedicated factories, and different polymerase II promoters and the presence of an intron determine the nuclear location of transcription. Using chromosome conformation capture (3C), minichromosomes with similar promoters are found in close proximity. They are also found close to similar endogenous promoters and so are likely to share factories with them. In the second part of this thesis, I used RNA FISH to confirm results obtained by tiling microarrays. Addition of tumour necrosis factor alpha (TNF alpha) to human umbilical vein endothelial cells induces an inflammatory response and the transcription of a selected sub-set of genes. My collaborators used tiling arrays to demonstrate a wave of transcription that swept along selected long genes on stimulation. RNA FISH confirmed these results, and that long introns are co-transcriptionally spliced. Results are consistent with one polymerase being engaged on an allele at any time, and with a major checkpoint that regulates polymerase escape from the first few thousand nucleotides into the long gene.