Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

In questa tesi descriviamo una procedura automatizzata per l’analisi della corteccia motoria dello scimpanzè, del Macaca fascicularis e del cavallo, basata su un nuovo metodo computerizzato di analisi delle sezioni colorate attraverso il metodo di Nissl, al fine di studiare la corteccia cerebrale in specie differenti. Le microfotografie delle sezioni sono state elaborate con una procedura standardizzata usando il software ImageJ. Questa procedura ha previsto la suddivisione degli strati corticali, dal primo al sesto, in diversi frames. Per misurare la complessità delle cellule nervose (cioè quanto una cellula fosse diversa dalle adiacenti) abbiamo utilizzato un modello di rappresentazione statistica non-parametrica che mostra come la complessità può essere espressa in termini di un adeguato indice di dispersione statistica quale il MAD (mean absolute deviation). Abbiamo quindi dimostrato che gli strati piramidali della corteccia motoria del cavallo sono più irregolari di quelli di scimpanzè e Macaca fascicularis. La combinazione dell’analisi automatica delle immagini e delle analisi statistiche consente pertanto di confrontare e classificare la complessità della corteccia motoria attraverso diverse specie. Il modello viene proposto come strumento al fine di contribuire a stabilire le somiglianze cerebrali tra umani e animali, rispettando il principio delle 3R. / In this thesis we describe an automated procedure based on a new computerized method of partitioning Nissl-stained sections of the motor cortex of the chimpanzee, crab-eating monkey, and horse, to study the neocortex in different species. Microphotographs of the sections were first processed using a standard procedure in ImageJ, then the stained neuronal profiles were analyzed within continuously adjoining frames from the first to the sixth layer of neocortex. To measure the neuronal complexity (how a given cell is different from its neighbors) we used a general non-parametric data representation model showing that the complexity can be expressed in terms of a suitable measure of statistical dispersion such as the mean absolute deviation. We demonstrated that the pyramidal layers of the motor cortex of the horse are more irregular than those of the monkeys studied. The combination of automated image analysis and statistical analysis made it possible to compare and rank the motor cortex complexity across different species. Therefore, we are confident that our work will help to establish brain similarities between humans and animals used for alimentary purpose, whose brain is often discarded. This, in turn, will allow to carry out the experimental brain research obeying the 3Rs principle.