Archivio Aperto di Ateneo
Not a member yet
5899 research outputs found
Sort by
Regolazione della fisiologia cellulare attraverso la modulazione delle attività dei recettori degli estrogeni indotta da composti naturali e sintetici
No full text17-β-estradiol (E2) is the most active estrogen in humans and exerts profound effects on the
growth, differentiation, and functioning of many reproductive and non-reproductive tissues.
A number of synthetic substances known as xenoestrogens show estrogenic effects; among
them, bisphenol A (BPA) is one of the best characterized. Also naturally plant-produced molecules
[e.g., naringenin (Nar)] are known to display a mild estrogenic activity. Because of their ability to
interfere with many aspects of natural hormones-dependent control of body homeostasis they are also
known as endocrine disruptors (EDs).
Two estrogens receptors (ER) are known in humans to interact with endogenous E2 and EDs:
these two isoforms, the ERα and the ERβ, modify the expression of specific genes acting as ligandactivated
transcription factors. In addition, rapid effects of E2 depend on the presence of the ERs to the
plasma membrane, which is determined by Palmitoylation of the ERs and lead to the activation of
ERK/MAPK, PI3K/AKT and p38/MAPK pathways. Also EDs can bind and modulate genomic and
rapid ERs activities acting as agonist or antagonist depending on the isoform they bind.
Different layers of regulation modulate the complexity of this signaling network, through the
numerous ER post-translational modifications. Indeed, beside palmitoylation, in response to E2 binding
ERs are also phosphorylated on serine (S) residues. Another layer of complexity is introduced by the
control of ERs intracellular levels; the binding of E2 produces ERα ubiquitination, an event that leads
to the 26S-proteasome-mediated receptor degradation, drastically lowering the protein half life. This
mechanism is, however, not fully understood and many steps of the process are still to be cleared.
Because all the effects of E2 occurs through the above-mentioned ligand-dependent modulation
of ERs intracellular content, the goal of the present project was to understand the mechanisms
underlying the ligand-dependent modulation of ER intracellular levels to better clarify their modulation
abilities in ERα- and ERβ-driven physiological processes.
Results obtained with a wide spectrum of approaches demonstrate that Nar and BPA affect ERα
and ERβ protein intracellular content in this way influencing the resulting ERs-dependent effects. In
particular, while BPA mimics E2 effects in inducing the 26S proteasome-dependent ERα degradation,
Nar induces the receptor accumulation; importantly, this modulation seems to be connected with
degradation, as both E2 as well as EDs induce the ERα mRNA levels down-regulation. Studying ERα
palmitoylation, we found that this modification is the upstream structural determinant that guarantees
the physiological balance of the ERα protein levels. Our finding demonstrates that E2 maintains both a
constant level of S118 phosphorylation, whereas it triggers a significant reduction in total ERα content
and a parallel increase in ERα gene transcription and that the activation of the PI3K/AKT pathway but
not of the ERK/MAPK pathway regulates ERα phosphorylation. Reduction in S118 phosphorylation
also correlates with a faster E2-induced receptor elimination and is paralleled with ERα transcription
impairment. Indeed both palmitoylation and phosphorylation control ERα activity and stability and are
linked each other in a consequent process
Our experiments also show that the EDs produce as E2 an increase in ERE-mediated
transcription and that the ERα partial antagonist Nar determines ERα phosphorylation as well as the E2
mimetic BPA, that partially stabilizes the receptor. Analysis of the modality by which Nar and BPA
affect ERα protein intracellular content reveals that BPA mimics the E2 effects in inducing the 26S
proteasome-dependent ERα degradation while Nar induces the receptor accumulation by blocking ERα
proteolytic degradation.
More importantly, we also found that the Nar-dependent accumulation of ERα results in an
increased receptor transcriptional activity and that, upon Nar stimulation, E2 looses its capacity to
regulate ERα turnover and to physiologically control ERα gene transcription and that both EDs raise
ERβ cellular content alone and in co-administration with E2.
These discoveries indicate that in a cellular context exposed to Nar the absolute physiological
receptor response or the one in response to E2 is changed because of dysregulated receptor expression.
Thus, Nar modulation of ERα cellular content could further affect the E2-dependent regulation of
specific cellular processes leading to scenarios that strongly diverge from the physiological ones and
that the fine hormone-dependent modulation of ERs intracellular levels is intrinsically connected with
all the aspects of the molecular mechanisms (i.e., genomic and extra-nuclear) that ERs uses to
transduce the physiological E2 intracellular message
L’ingegneria proteica nello studio della struttura e funzione di (macro)molecole
No full textThe present Ph.D. project deals with the use of protein engineering
techniques to express in recombinant form and characterize disulphide rich
bioactive peptides, such as conotoxins. Moreover, taking advantage of their
high target specificity and stability, the potential application of this class of
macromolecules in “green” (environmental) and “white” (industrial)
biotechnology fields have been explored.
Protein engineering is a powerful tool to improve some proteins parameters,
acting on their sequence and structure, or to insert a desired function. There
are three basic approaches in protein engineering: the rational design is an
"intelligent" design that aims to change the structure or function of a
protein, starting from the analysis of its three-dimensional structure. Its
nucleotide sequence can be modified and the protein finally “built” and
characterized (Hellinga, 1997). The second approach is the directed
evolution or DNA breeding, a technique that simulates biological evolution
mechanisms (Stemmer, 1994). In fact it involves the generation of random
mutations in the gene coding for a protein, or shuffling the genes encoding
different domains, to generate a high number of new proteins that will be
then selected for a desired function. The third one, named de novo design,
allows to design and build artificial proteins which don’t exist in nature,
with novel functions and properties (Dahiyat and Mayo, 1997). This
strategy takes advantage of computational biology to draw novel proteins
both using natural scaffolds as templates and starting completely from
scratch (Dahiyat and Mayo, 1997).
The idea of using stable protein structural motifs as scaffold for reproducing
functional epitopes or stabilize bioactive conformations is currently one of
the most successful approaches of protein engineering. The knottin family
of proteins is particularly interesting from this point of view, since they
share a common structural fold while having very different sequences and
functions in animals and plants (Norton and Pallaghy, 1998). The multiple
disulphide bridges that characterize this protein family give the greatest
contribution to the structure stability, allowing a high variability of the
remaining amino acid sequence regions. Among knottins, the highest
tolerance to sequence variability is observed in conotoxins, small
disulphide-rich neurotoxic peptides extracted from cone snails venom.
Nature, during the evolution, has engineered this stable scaffold to express a
great variety of neurotoxins with a remarkable high target specificity for
different ion channels, both voltage-gated and ligand-gated (Olivera and
Teichert, 2007). As a result, their scaffold represents an excellent starting
point for design strategies aimed at developing new miniproteins with new properties as well as more stable/active conotoxins for therapeutics (Clark et al., 2005).
From the natural source, the purification of conotoxins proved to be very difficult because of the large number of specimens needed to obtain a sufficient amount of venom to process. Thus, during this thesis work, a recombinant expression system has been developed to express conotoxins as GST-fusion proteins, overcoming both the problem of the small amount of a single conotoxin that can be extracted from the Conus venom, and their propensity to form insoluble aggregates because of the formation of intermolecular disulfide bridges. As a test case, the new protocol has been used to express conotoxin Vn2 from the Mediterranean Sea Conus ventricosus, a 33 amino acids long highly hydrophobic neurotoxic peptide, and its Asp2His mutant (Spiezia et al., 2012). This first part of the work involved cloning of both the conotoxins in pET-CM plasmid and their purification by affinity chromatography, exploiting the GST tag. Since the worm-hunting C. ventricosus has an array of toxins acting on invertebrate ion channels, GST-conotoxins have been tested for their neurotoxic activity on the larvae of the moth Galleria mellonella, taken as an insect model system (Spiezia et al., 2012). Further, using ω-conotoxin GVIA as template, novel metal-binding peptides were designed to generate novel biocatalysts. Through computational modeling, two disulphide bridges in the original conotoxin have been replaced with His residues and the redesigned peptides, named Cupricyclin-1 and Cupricyclin-2, have been synthesized and characterized for their copper binding ability and superoxide dismutase activity (Barba et al., 2012). The last part of the work concerned the generation of Cupricyclin-1 mutants with the aim to improve its superoxide dismutase activity and to change metal-binding specificity from copper to iron and manganese. The new expression system developed in the first part of the present project was successfully used to express these Cupricyclin mutants as GST-fusion proteins. Following GST tag removal using thrombin cleavage, the mutants have been purified and their metal binding properties analyzed by optical and fluorescence spectroscopy and mass spectrometry.
Further studies will be necessary to elucidate the redox properties of these novel Cupricyclins and their potential as biosensors for the detection of heavy metals or as therapeutics in oxidative stress injuries, given the ability of Cupricyclin-1 to dismutate O2- radicals
Stars, seasons and weather in Somali pastoral traditions
No full textThe text deals with astrology, astronomy, the conception of time (calendar), botany and political, economic and social life through examples taken from Somali folk literature.Buugu wuxuu si qoto dheer uga hadlayaa mowduucyo la xiriira xiddiska, cilmufalagga, aragtida laga qabo waqtiga (kalanderka) iyo nolosha xagga siyaasadda, dhaqaalaha iyo bulshada, waxaana loo adeegsaday tusaalooyin ka mid ah suugaanta dadweynaha soomaaliyeed.Il testo tratta tematiche relative ad astrologia, astronomia, alla concezione del tempo (calendario) e alla vita politica, economica e sociale attraverso esempi di letteratura popolare somala.Mimeographed text - Mogadishu.Qoraal lagu daabacay jikolostiil - Muqdisho.Testo ciclostilato - Mogadiscio
Hiindey Aaminay
No full text"Hiindey Aaminay". F. Giannattasio ayaa duubay 08/03/85, isagoo jooga guri ku yaalla Shangaani (Moqdisho), waqti la xusayay qof geeriyooday: digri nabiammaan. Waxaa duubistaas ka qayb qaatay Aweys Maxamed Waasuge, oo kala shaqaynaayay Giannattasio xagga baarista muusikada soomaaliyeed, isagoo markaas ka ahaa Masraxa Qaranka Soomaaliyeed soosaare 1984-89-_"Hiindey Aaminay" (O madre Amina [la madre del Profeta]). Registrazione effettuata da Francesco Giannattasio l'8 marzo 1985 a Mogadiscio, in un'abitazione privata del quartiere Shingaani, in occasione di una commemorazione funebre (xus): seduta (leyli = dall'arabo "serale", serata) di Nebi-ammaan (canti in lode del Profeta), sorta di digri domiciliare. Partecipa alla registrazione Aweys Maxamed Waasuge, allora regista del Teatro Nazionale Somalo e, dal 1984 al 1989, collaboratore di Giannattasio alla ricerca sulla musica somala._-_"Hiindey Aaminay" (Oh mother Amina [Prophet's mother]). Recorded by Francesco Giannattasio on March 8th, 1985 in Mogadishu in a private home in the Shingaani district, on the occasion of a memorial service (xus): Nebi-ammaan session (leyli = from Arabic "evening"), songs in praise of the Prophet, a sort of domestic dhikr. In the presence of Aweys Maxamed Waasuge, director of the Somali National Theatre and Giannattasio's collaborator in his research on Somali music from 1984 to 1989
Cashiiradayay ninkii Cabdi Nuur
No full text"Cashiiradayay ninkii Cabdi Nuur", wiglo. F. Giannattasio ayaa 30/04/85, isagoo jooga gurigiisa ku yaalla Liido ee Moqdisho, ka duubay Diiriye Faarax Cali oo loo yaqaan "Xaaji Baalbaal". Waxaa duubistaas ka qayb qaatay Aweys Maxamed Waasuge, oo kala shaqaynaayay Giannattasio xagga baarista muusikada soomaaliyeed, isagoo markaas ka ahaa Masraxa Qaranka Soomaaliyeed soosaare 1984-89._-_"Cashiiradayay ninkii Cabdi Nuur" (O miei parenti, quell’uomo Cabdi Nuur), incipit; genere poetico: 'wiglo'. Registrazione effettuata da Francesco Giannattasio il 30 aprile 1985 a Mogadiscio (nella propria abitazione al Liido), con Diiriye Faarax Cabdi, detto Xaaji "Baalbaal". Partecipa alla registrazione Aweys Maxamed Waasuge, allora regista del Teatro Nazionale Somalo e, dal 1984 al 1989, collaboratore di Giannattasio alla ricerca sulla musica somala._-_"Cashiiradayay ninkii Cabdi Nuur" (Oh my relatives, that man Cabdi Nuur), opening words; poetic genre: 'wiglo'. Recorded by Francesco Giannattasio on April, 30th 1985 in Mogadishu (at Liido home), with Diiriye Faarax Cabdi, called Xaaji "Baalbaal". In the presence of Aweys Maxamed Waasuge, director of the Somali National Theatre and Giannattasio's collaborator in his research on Somali music from 1984 to 1989
Deeqay qoordheeray qoladiinnu waa Quraysh
No full text"Deeqay qoordheeray qoladiina waa Quraysh", buraanbur, la codsaday, waxyaabaha loo adeegsaday: codad tanag la garaacayo, sacab iyo alalaas. F. Giannattasio ayaa ka duubay 20/09/98, isagoo jooga Baabile, gobolka IV (Oromo) ee Itoobiya, dad badankooda soomaali ah (Daarood Ogaadeen iyo qabaa'il kale ee soomaali Galbeed). Waxaa duubista ka qayb qaatay, iyadoo lagu sugnaa guri gaar ah, Axmed Faarax C. "Idaajaa", Simone Tarsitani iyo Giorgio Banti._-_"Deeqay qoordheeray qoladiinnu waa Quraysh" (O Deeqa dal collo lungo, la vostra tribù è quella di Quraysh), incipit; buraambur: poesia femminile (su richiesta). Modo di esecuzione: voci, tamburo (tanica di plastica), mani, alalaas (trilli di glottide). Registrazione effettuata da Francesco Giannattasio il 20 settembre 1998 a Baabile, nella IV regione (Oromia) dell’Etiopia, gruppo etnico prevalente: somali (Daarood Ogaadeen e altre cabile della Soomaali Galbeed). Partecipavano alla registrazione, svoltasi presso un’abitazione privata, Axmed Faarax Cali “Idaajaa”, Simone Tarsitani e Giorgio Banti._-_"Deeqay qoordheeray qoladiinnu waa Quraysh" (Oh long-necked Deeqa, your tribe is the Quraysh tribe), opening words; buraambur, female poetry, on request. Performance: vocals, drum (plastic tank), hands, alalaas (glottis trills). Recorded by Francesco Giannattasio on September, 20th 1998 in Baabile, in the 4th region (Oromia) of Ethiopia, in a private house; main group/clan: Somalis (Daarood Ogaadeen and other sub-clans of the ‘Soomaali Galbeed’). In the presence of Axmed Faarax Cali “Idaajaa”, Simone Tarsitani and Giorgio Banti
Musica di danza (2)
No full textMusica di danza, culto del Nuumbi (in funzione). Registrazione effettuata da Francesco Giannattasio il 25 gennaio 1985 nel villaggio di Buulo Sheekh (Qoriooley - Shabeellada Hoose), relativa a una seduta rituale del culto di possessione del Nuumbi. Modo di esecuzione: shareero (lumiya), tre tamburi, battito di mani, voci. Partecipa alla registrazione Aweys Maxamed Waasuge, allora regista del Teatro Nazionale Somalo e, dal 1984 al 1989, collaboratore di Giannattasio alla ricerca sulla musica somala._-_Muusika qoobkaciyaar, ciyaarta Nuumbiga. F. Giannattasio ayaa 25/01/1985 cayaartan Nuumbiga ka duubay goob ay ka dhaceysay oo ahayd Buulo Sheekh (Qoryooley – Sh/Hoose), waxyaabaha loo adeegsadayna waxaa ka mid ah: shareero, saddex durbaan, sacab iyo codad. Waxaa duubistaas ka qayb qaatay Awees Maxamed Waasuge, oo markaas ka ahaa soosaare Masraxa Qaranka Soomaaliyeed, islamarkiina kaaliye u ahaa Giannattasio xagga baarista muusikada muddada u dhexeysay 1984-1989._-_Music of dance, Nuumbi possession ceremony. Ritual session of Nuumbi possession ceremony recorded by Francesco Giannattasio on January 25th 1985 in Buulo Sheekh village (Qoriooley - Lower Shabeel). Performance: shareero (lumiya), three drums, hands, voices. In the presence of Aweys Maxamed Waasuge, director of the Somali National Theatre and Giannattasio's collaborator in his research on Somali music from 1984 to 1989
