Parasitic wasps attack a number of insect species on which they feed, either externally or internally. This requires very effective strategies for suppressing the immune response and a finely tuned interference with the host physiology that is co-opted for the developing parasitoid progeny. The wealth of physiological host alterations is mediated by virulence factors encoded by the wasp or, in some cases, by polydnaviruses (PDVs), unique viral symbionts injected into the host at oviposition along with the egg, venom and ovarian secretions. PDVs are among the most powerful immunosuppressors in nature, targeting insect defense barriers at different levels. During my PhD research program I have used Drosophila melanogaster as a model to expand the functional analysis of virulence factors encoded by PDV focusing on the molecular processes underlying the disruption of the host endocrine system. I focused my research on a member of the ankyrin (ank) gene family, an immunosuppressant found in bracovirus, which associates with the parasitic wasp Toxoneuron nigriceps. I found that ankyrin disrupts ecdysone biosynthesis by impairing the vesicular traffic of ecdysteroid precursors in the cells of the prothoracic gland and results in developmental arrest.

Obiettivo principale della ricerca è quello di aggiungere un tassello mancante, attraverso una nuova chiave di lettura, alla complessa attività artistico-teorica dell’artista futurista Enrico Prampolini (1894-1956). Questa tesi, oltre a riordinare e raccogliere tutto ciò che riguarda l’architettura e il rapporto di quest’ultima con le arti nell'opera di Prampolini, coglie l’occasione per puntualizzarne aspetti ancora poco esplorati, grazie anche all'analisi inedita dei documenti originali dell'artista conservati presso il CRDAV del Museo d’arte contemporanea di Roma. Partendo dall'analisi dei rapporti dell’artista modenese con le avanguardie straniere, la ricerca prosegue con la presa in esame dei manifesti, degli scritti e degli articoli noti e inediti legati alla teoria architettonica nella produzione dell’artista modenese. Il nucleo centrale della ricerca è costituito dagli elementi inediti emersi presso l’Archivio Prampolini consistenti in relazioni di progetti architettonici per un Piano urbanistico del Centro Alberghiero di Castel Fusano (1938) e per due alberghi nel centro di Roma (1938-1939). La seconda parte della ricerca si concentra sull'analisi del rapporto tra arte e architettura nel Futurismo e sul fondamentale contributo dato in tal senso da Prampolini, in particolare attraverso la “plastica murale”, come completamento dell’architettura futurista e fascista e la concezione dell’Arte Polimaterica. Nell'ultima parte della ricerca si affronta infine l'analisi del rapporto tra arte e architettura gestito in ambito istituzionale in Italia tra le due guerre, con l’emanazione, nel 1942, della legge detta “del 2%”. Aspetto finora inedito delle vicende legate alla “legge del 2%” è l'emergere del ruolo centrale di Prampolini nel contribuire al dibattito che portò alla sua approvazione, e non secondariamente nella ricerca di migliori condizioni economiche e maggiori occasioni di lavoro per gli artisti. La figura di Enrico Prampolini emerge dunque, da questa ricerca, come un nodo fondamentale per comprendere alcuni degli aspetti ancora inesplorati della cultura artistico-architettonica italiana degli anni Venti-Quaranta. / The main objective of this research is to add a missing piece, through a new interpretation, to the artistic and theoretical activity of the futurist Enrico Prampolini (1894-1956). This thesis , wants collect everything related to architecture and the relationship of the latter with the arts in the work of Prampolini and takes the opportunity to point out this aspects still poorly explored, thanks also to the analysis of unpublished documents’ artist kept at the CRDAV of the MACRO. The core of the research consists in the new elements emerged at the Prampolini Archives consisting in a descriptive reports of architectural plans for a development of the Centre of Hospitality of Castel Fusano (1938) and two hotels in the center of Rome (1938-1939). The second part of the research focuses on the analysis of the relationship between art and architecture in Futurism and the fundamental contribution in this direction by Prampolini, particularly through the "plastica murale" as completion of Futurist and Fascist architecture and the conception of the Polymaterials Art. In the last part of the research we deal with the analysis of the relationship between art and architecture managed in Italy in the institutional context, between the two Wars, with the enactment, in 1942, of the law known as the "2%". Appearance of until now unpublished events related to the "law of 2%" is the emergence of the central role of Prampolini in contributing to the debate that led to its approval, and not secondarily for search of better economic conditions and more job opportunities for artists. The figure of Enrico Prampolini therefore emerges from this research as a crucial question to understand some of the unexplored aspects of the Italian architectural and artistic culture of the Twenties-Forties.

The human DMD locus encodes dystrophin protein. Absence or reduced levels of dystrophin (DMD or BMD phenotype, respectively) lead to progressive muscle wasting. Little is known about the complex coordination of dystrophin expression and its transcriptional regulation is a field of intense interest. In this work we found that DMD locus harbours multiple long non coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms. These lncRNAs are tissue-specific and highly expressed during myogenesis, suggesting a possible role in tissue-specific expression of DMD gene isoforms. Their forced ectopic expression in human muscle and neuronal cells leads to a specific and negative regulation of endogenous dystrophin full lenght isoforms. An intriguing aspect regarding the transcription of the DMD locus is the gene size (2.4Mb). The mechanism that ensures the complete synthesis of the primary transcript and the coordinated splicing of 79 exons is still completely unknown. By ChIP-on-chip analyses, we discovered novel regions never been involved before in the transcription regulation of the DMD locus. Specifically, we observed enrichments for Pol II, P-Ser2, P-Ser5, Ac-H3 and 2Me-H3K4 in an intronic region of 3Kb (approximately 21Kb) downstream of the end of DMD exon 52 and in a region of 4Kb spanning the DMD exon 62. Interestingly, this latter region and the TSS of Dp71 are strongly marked by 3Me-H3K36, an histone modification associated with the regulation of splicing process. Furthermore, we also observed strong presence of open chromatin marks (Ac-H3 and 2Me-H3K4) around intron 34 and the exon 45 without presence of RNA pol II. We speculate that these two regions may exert an enhancer-like function on Dp427m promoter, although further investigations are necessary. Finally, we investigated the nuclear-cytoplasmic compartmentalization of the muscular dystrophin mRNA and, specifically, we verified whether the exon skipping therapy could influence its cellular distribution.

Studio prospettico su 75 pazienti con malattia paranale di Crohn che ha come obiettivo quello di confrontare i risultati tra le nuove terapie medico-chirurgiche emergenti. La prima procedura è comune a tutti i pazienti e consiste in un intervento di incisione degli ascessi, fistulectomia e posizionamento di setoni di drenaggio nei tramiti fistolosi per il controllo della sepsi.Successivamente i pazienti vengono divisi in cinque gruppi e sottoposti ai trattamenti per la chiusura dei tramiti fistolosi: terapia sistemica con Infliximab,terapia sistemica con Adalimumab,confezionamento di Flap endoanale, instillazione di colla di fibrina o posizionamento di protesi biologiche. Abbiamo osservato una chiusura completa dei tramiti fistolosi nel 60% dei pazienti trattati con Infliximab, 53% di quelli trattati con Adalimumab, 40% di quelli in terapia con colla di fibrina, 80% di quelli sottoposti a Flap endoanale e 60% di quelli trattati con protesi biologiche. Gli ottimi risultati raggiunti in con le diverse metodiche di trattamento chirurgico locale rappresentano una valida alternativa alla terapia con farmaci biologici. Tali nuove metodiche risultano anzi fondamentali per il trattamento di quei pazienti che dopo una terapia con farmaci biologici non hanno raggiunto una completa risoluzione del quadro (rescue therapy). Terapia biologica e nuove tecniche chirurgiche risultano pertanto complementari, la prima contribuendo al miglioramento della qualità della mucosa del canale anale e del retto basso sulla quale risulta quindi più agevole agire con le seconde con una percentuale di successo sempre maggiore. / Prospective study on 75 patients with perianal Crohn's disease that aims to compare the results between the new emerging medical and surgical therapies. The first procedure is common to all patients and consists of incision of abscesses, fistulectomy and setone placement in fistulas.Subsequently patients are divided into five groups and submitted to treatments to reach fistula healing: systemic therapy with Infliximab , systemic therapy with Adalimumab, endoanal advancement Flap, instillation of fibrin glue or placement of biological prostheses. We observed a complete closure of fistulas in 60% of patients treated with Infliximab , 53% of those treated with Adalimumab, 40% of those treated with fibrin glue , 80% of those who underwent endoanal flap and 60% of those treated with biological prostheses . The excellent results achieved with the different methods of local surgical treatment represent a valid alternative to treatment with biologics. These new methods are useful for the treatment of those patients that after treatment with biologic agents have not achieved a complete healing(rescue therapy). Biological therapy and new surgical techniques are therefore complementary, the first contributing to the healing of rectal and anal canal mucosa, on which is therefore easier to work with the second with an higher success rate.

FREITAS, Helano Carioca. Envolvimento da interleucina-18 (IL-18) na patogênese da mucosite gastrointestinal induzida pelo cloridrato de irinotecano (CPT-11). 2007. 105 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2007. / Submitted by denise santos (denise.santos@ufc.br) on 2012-04-02T15:54:30Z No. of bitstreams: 1 2007_dis_hcfreitas.pdf: 13077439 bytes, checksum: c3cb63e93c6e7c247752adbdd02afc64 (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2012-04-04T11:27:30Z (GMT) No. of bitstreams: 1 2007_dis_hcfreitas.pdf: 13077439 bytes, checksum: c3cb63e93c6e7c247752adbdd02afc64 (MD5) / Made available in DSpace on 2012-04-04T11:27:30Z (GMT). No. of bitstreams: 1 2007_dis_hcfreitas.pdf: 13077439 bytes, checksum: c3cb63e93c6e7c247752adbdd02afc64 (MD5) Previous issue date: 2007 / The term GIM refers to the adverse effects of cancer chemotherapy on mucosal surfaces, affecting different portions of the alimentary tract. 15% to 40% of patients in chemotherapy present some degree of mucositis. By now, much of GIM pathophysiology remains unknown. IL-18 is a pleiotropic cytokine that exerts regulatory functions over the immune system and is also involved in inflammation. Purpose: The aim of the present study was to elucidate the involvement of IL-18 in the pathogenesis of CPT-11-induced mucositis. Materials and methods: Male IL-18 wild type (Wt) or IL-18 knockout (KO) Balb/C mice received CPT-11 (60mg/kg/day, i.p.) or vehicle (0.5ml, i.p.) in a four day schedule. A group of IL-18Wt also received IL-18 binding protein (IL-18Bp, 200 µg, i.p., 1h before CPT-11). Animals were sacrificed on the fifth day. The following parameters were assessed: histologycal analysis, diarrhea, survival curve, leucogram, myeloperoxidase (MPO) activity assay, ileum levels of TNF-α and IL-1β by ELISA, immunohistochemistry for TNF-α and IL-1β and contractility assay. Results: IL-18Wt animals receiving CPT-11 presented diarrhea and intense histological alterations in the ileum (shortening of villi, flattening and vacuolization of enterocytes, cript necrosis and the presence of inflammatory infiltrate). There was also increased MPO activity, increased levels of TNF-α and IL-1β and strong immunostaining for TNF-α and IL-1β in the ileum. In addition, the CPT-11 treated mice presented increased intestinal contractility when stimulated with cholinergic drugs (bethanecol, BCh and acetylcholine, ACh). In contrast, IL-18KO animals treated with the same dose of CPT-11 presented less diarrhea and less intestinal histological alterations. There was no increase in MPO activity nor in TNF levels, but in IL-1 levels. In addition, TNFα and IL-1β immunostaining was weaker IL-18KO animals. Also, the intestinal contractility in IL-18KO animals was not exarcerbated after a cholinergic challenge. IL-18Wt animals receiving CPT-11 and IL-18Bp did not present significant diarrhea and there was no significant alterations on intestinal contractility after Ach administration. Although MPO activity was not increased in IL-18Bp treated animals, the ileal mucosa presented moderate to severe histological alterations. Conclusion: IL-18 participates in the CPT-11-induced GIM. IL-18Bp attenuates some inflammatory (cell infiltrate) and functional (diarrhea and contractility) events of CPT-11-induced GIM. The contractility alterations in CPT-11 treated animals seem to have an inflammatory related component. / MGI é o termo que descreve os efeitos da quimioterapia antineoplásica nas mucosas, podendo acometer o trato alimentar de maneira global ou localizada. 15 a 40% dos pacientes em quimioterapia apresentam algum grau de mucosite. Muito da fisiopatologia da MGI permanece desconhecido. A IL-18 é uma citocina pleiotrópica, com função regulatória sobre o sistema imune e envolvimento nas fases iniciais da inflamação. Objetivo: Avaliar o envolvimento de IL-18 na patogênese da MGI induzida por CPT-11. Materiais e Métodos: camundongos Balb/C IL-18Wt ou IL-18KO, machos, foram tratados durante quatro dias consecutivos com CPT-11 (60mg/Kg, i.p.) ou veículo (0,5 mL, i.p.). Um grupo de animais IL-18Wt recebeu, além do CPT-11, a proteína ligante de IL-18 (IL-18Bp; 200 µg, i.p.). Os seguintes parâmetros foram avaliados: diarréia, leucograma, sobrevida, análise histopatológica, atividade de mieloperoxidade (MPO), dosagem de citocinas (TNF-α, IL-1β ) por ELISA e imunoistoquímica para TNF-α e IL-1β nas mucosas ileais. Resultados: CPT-11 induziu diarréia significante e promoveu alterações intestinais exuberantes (encurtamento de vilos, achatamento e vacuolização de enterócitos, necrose em criptas e infiltrado inflamatório de leucócitos polimorfonucleares e células mononucleares) Observou-se aumento da atividade de MPO e dos níveis tissulares de TNF-α e IL-1β dosados por ELISA e intensa imunomarcação com anticorpos anti-TNFα e anti-IL-1β nesses animais. Além disso, tais animais apresentaram resposta contrátil intestinal exacerbada ao estímulo com colinérgicos (acetilcolina e betanecol). Os animais IL-18KO tratados com CPT-11 apresentaram diarréia estatisticamente menos severa e menor intensidade de alterações morfométricas e histológicas. Não houve aumento de TNF-α, mas houve de IL-1β , a atividade de MPO não diferiu da observada nos animais que receberam veículo e não houve aumento de resposta contrátil ao estímulo colinérgico. Os animais que receberam CPT-11 e IL-18Bp apresentaram diarréia estatisticamente menos intensa que aqueles que receberam apenas CPT-11, resposta contrátil estatisticamente menos acentuada e menor atividade de MPO. Entretanto, as alterações morfométricas e histológicas não diferiram das encontradas nos animais tratados só com CPT-11. Conclusão: IL-18 está envolvida na patogênese da MGI induzida por CPT-11. IL-18Bp atenua os eventos envolvidos na MGI induzida por CPT-11 e é um possível candidato a modulador farmacológico desse processo. As alterações contráteis no intestino promovidas pelo CPT-11 parecem ter um componente inflamatório.

Recurrent disruptive mutations in chromodomain helicase DNA-binding protein 2 and 8 (CHD2 and CHD8) are emerging as prominent risk factors for Epilepsy and ASD, respectively. While both CHD2 and CHD8 play important roles in chromatin regulation and transcription, not fully dissected are the molecular consequences of the inactivating mutations described in patients. Here, we first investigated how chromatin reacts to CHD8 suppression by analysing a panel of histone modifications in human induced pluripotent stem cell-derived neural progenitors (hiNPC). CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affected highly expressed, CHD8-bound genes. Strikingly, the levels of transcription in cells presenting reduced H3K36me3 in the gene body appeared unchanged, however the process of alternative splicing was instead significantly affected. In particular, we have found aberrant alternative splicing patterns – mainly affecting alternative first and exon skipping events - of ~ 2000 protein coding genes implicated in “RNA splicing”, “mitotic cell cycle phase transition” and “mRNA processing”. Mechanistically, mass-spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator Heterogeneous Nuclear Ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain CHD8-suppression splicing phenotype, partially implicating SETD2, H3K36me3 methyltransferase. Most of the mutations in CHD2 and CHD8 genes are disruptive, leading to protein haploinsufficiency. Thus, any molecular manipulation eliciting an increase in CHD2/CHD8 proteins, could prove beneficial for therapeutic development. Here, we intended to provide a Proof-of-Principle of how SINEUP - recently described class of non-coding RNAs able to augment, in a specific and controlled way, the expression of target proteins - can increase the translation of CHD2/CHD8 proteins and possibly rescue haploinsufficiency-associated phenotypes. With this purpose, we designed and cloned SINEUP targeting human CHD2/CHD8 isoforms and tested their efficacy in human induced pluripotent stem (iPS), induced neural progenitor cells (hiNPC) expressing normal and reduced levels of the target proteins and in patients’ fibroblasts bearing CHD8 heterozygous loss-of-function mutations. While stimulation by different CHD2/CHD8-SINEUP molecules didn’t elicit any effect in wild-type cells with physiologic levels of CHD2/CHD8, CHD2/CHD8-SINEUP were fully effective in haploinsufficient conditions, when reduced levels of the target proteins were expressed. Functionally, CHD8-SINEUP were able to revert molecular phenotypes associated to CHD8-suppression, i.e. the transcriptional dysregulation of the ASD-related genes, MBD3 and SHANK3, and restore the genome-wide reduction of H3K36me3 enrichment. Strikingly, chd8-SINEUP injection in vivo, in chd8-morpholino treated, developing zebrafish embryos, confirmed that stimulation of internal methionine could rescue chd8-suppression induced macrocephaly phenotype. In conclusion, CHD2/CHD8-SINEUP molecules represent a Proof-of-Concept towards the development of an RNA-based therapy for neurodevelopmental syndromes, with implications for and beyond ASD and epilepsy, surely relevant to a large repertory of presently incurable brain genetic diseases.

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Tesis para optar al grado de Magíster en Estudios Latinoamericanos / Proyecto FONDECYT 1120083

Intestinal health is essential for the health of the body since the gastro-intestinal mucosa is the main site of interaction with the external environment, as well as the major area colonized by the microbiota. Intestinal health relies on proper barrier function, epithelial integrity and related mechanisms of protection (mucous layer, tight junctions, immune and inflammatory system). In pigs, during the weaning transition, intestinal inflammation and barrier integrity play a crucial role in regulating intestinal health and, consequently, pig’s health, growth and productivity. The aim of the project was to assess the impact of different nutritional strategies on the intestinal health of weaning piglets with reference to the inflammatory status and epithelial integrity. Therefore, in vivo trials were conducted to test the in-feed supplementation with zinc, tributyrin, or organic acids and nature-identical compounds (NIC) to weaning piglets. All the dietary interventions positively impacted the intestinal inflammatory status and, as a consequence, improved epithelial integrity by modulating tight junctions proteins (zinc or tributyrin) or by enhancing barrier properties measured with Ussing chambers (organic acids and NIC). These findings highlight that intestinal inflammation and barrier function are strictly linked, and that the control of inflammation is essential for adequate barrier function. In addition, in zinc trial and organic acids and NIC trial, better intestinal health could successfully result in better growth performance, as aimed for pig production improvement. To conclude, this work shows that dietary supplementation with bio-active substances such as zinc, tributyrin or organic acids and NIC may improve intestinal health of weaning piglets modulating intestinal inflammatory stress and barrier integrity and allowing better piglet’s health, growth and productivity.