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Waaya-aragnimadeydii 15ka Sano iyo Siyaasadda Puntland 1998-2012
No full textQoraaga, oo ah Caasha Geelle, waxay buuggan kaga hadlaysaa waaya-aragnimadeeda 15 sano iyo siyaasadda Puntaland, iyo weliba la soo shaqayneedii ururrada rayidka ah.La scrittrice, Asha Gelle, descrive in questo suo libro i suoi 15 anni di esperienza politica nel governo locale del Puntland (Somalia), soprattutto la sua collaborazione con le organizzazioni di servizio sociale.The author, Asha Gelle, describes her 15 years of political experience in the local government of Puntland (Somalia), especiallu her cooperation with social service organizations
Sviluppo di strategie per la ricerca di inibitori dei partner cellulari di interazione della proteina Nef di HIV-1
No full textThis Ph.D. project has been focused on the study of HIV-1 Nef protein.
Nef is a regulatory phosphoprotein and studies on animal models and
seropositive patients have demonstrated its importance as virulence factor.
Nef, in fact, plays a pivotal role in maintaining high viral load and in
progression to AIDS, and is already object of interest of the HIV scientific
community in the search of new targets to prevent or block HIV disease.
Nef exerts three main functions in HIV-1 target cells (i.e.CD4+ T-helper
lymhpocytes, monocytes/macrophages, dendritic cells, microglial cells): 1)
the modulation of cellular signalling pathways; 2) the enhancement of viral
infectivity; 3) the regulation of expression of cell surface receptors,
including the well-studied down-regulation of CD4 and certain class I major
histocompatibility complex (MCH-I) antigens.
Altogether these effects increase viral infectivity and spreading contributing
to render the virus able to escape the immune system response.
Nef presents a two-domains structure: a structurally flexible N-terminal
membrane anchor domain and a well conserved and folded C-terminal core
domain. In addition it is post-translationally modified by phosphorylation
and by myristoylation which is important for the Nef signalling activity. Nef
exerts its functions working as a molecular adaptor, interacting and
influencing the activity of more than 30 intracellular partners and several
Nef interaction sites have been identified. As a consequence of Nef ability
to bind multiple targets certain Nef effects dominate over others in time- or
cell type- dependent manner.
Macrophages are the first cells to be infected by HIV and represent the main
reservoir of the virus. In these cells Nef induces the synthesis and the
release of a specific sub-set of chemokines and cytokines able to recruit Tcells
on the infectious site rendering them susceptible to HIV infection.
Moreover, recent studies, carried out by the research group where it has
been performed this Ph.D. project, have demonstrated that Nef is efficiently
internalized by human non infected monocytes derived macrophages
(MDMs) in in vitro cultures and mediates the NF-kB-mediated synthesis of
specific cytokines and chemokines that in turn lead to the activation by
phosphorylation of STAT (Signal Transducer and Activator of
Transcription)-1, -2 and -3. Importantly, it has been demonstrated that the
Nef interaction site designated as acidic cluster (A60QEEEE65) is required
for this process. In addiction, modelling analysis and silencing experiments
indicate that this region represents a putative binding motif for specific TNF
receptor associated factor (TRAF) adaptor family members. It has been
proposed, in fact, that in macrophages Nef intersects the CD40 signalling
pathway and TRAF are involved in the signalling events downstream this
receptor. Therefore, the study of the Nef interactome, of the molecular basis
underlying the Nef acidic cluster/TRAFs interactions, and the finding of
molecules able to block the main Nef interaction sites is certainly of great
interest both to clarify the so far enigmatic biology of Nef and to open new
perspectives in the field of HIV-AIDS therapy.
In detail, this project has been centred on the study of Nef anchor domain
and of its acidic cluster with the aim to further investigate on the role of
these regions in the Nef-mediated signalling.
To achieve these goals, the first part of the project has been focused on the
production of a Nef target region designated as N-Term76-Nef that has been
used first to carry out experiments on cellular systems, and secondly as
target for phage display experiments in order to identify Nef binding
peptides. Thus, an efficient protocol for the expression and the purification
of the N-Term76-Nef has been setup and as result the production of a high
level of purity of the target protein has been obtained.
During the second part of the work N-Term76-Nef has been used for the
treatment of THP-1 cells in order to evaluate the role of the Nef anchor
domain in the Nef signalling effects. It has been demonstrated that NTerm76-
Nef region alone is able to affect the macrophages cell signalling as
the Nef full-length protein does. This finding highlights on the importance
of the Nef anchor domain in the Nef functions and opens new intriguing
perspectives regarding the biology of Nef.
Finally, phage display of random peptide experiments have been performed
to identify peptide to be used both for the characterization of the Nef
interactome and for the search of potential Nef binding inhibitors.
To this purpose, a 50 amino acid long phage random peptide library has
been designed and produced and N-Term76-Nef has been used as target for
the affinity selection cycles that has been performed through bio-panning
methodology. The selection cycles have been carried out until the
obtainment of a final phage sub-library enriched for the interested phage.
Sequencing analysis of the selected phage genomes have been performed
while modelling analysis and pull-down experiments are currently under
planning and design with the final goal to isolate peptides displaying the
best binding affinity for Nef. Once found, the more suitable peptides will be
tested in cell culture systems to verify their potential Nef inhibitory activity
