Dissertations / Theses: 'Modelli murino'

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CAVIOLA, GIADA. "Impatto dell’ambiente materno precoce e del sesso sul comportamento e metabolismo in due modelli murini KO." Doctoral thesis, Università degli studi di Modena e Reggio Emilia, 2020. http://hdl.handle.net/11380/1200998.

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Le interazioni gene-ambiente svolgono un ruolo critico nella modulazione dello sviluppo dell’organismo: in particolare l’ambiente materno precoce sembra essere un fattore chiave dello sviluppo neuro-comportamentale. Il legame tra la madre e il figlio infatti è considerato la relazione sociale più comune e duratura nei mammiferi, filogeneticamente conservato tra le diverse specie. Evidenze sperimentali indicano che l’ambiente materno precoce può modulare la regolazione dell’espressione genica; diversi studi hanno dimostrato che animali che avevano ricevuto maggiori cure materne durante il primo periodo post nascita, presentavano minori livelli di ansia, minore produzione di ACTH e corticosterone, minore invecchiamento nell’ippocampo e maggiore attività immunitaria in risposta allo stress. Negli esprimenti condotti abbiamo approfondito come l’interazione tra ambiente materno precoce e lo specifico background genetico, in relazione al sesso, agisca sul metabolismo e il comportamento. Abbiamo esaminato quindi due diversi modelli murini con modificazioni geniche: il primo è un modello caratterizzato da un knockout condizionale per il recettore Y1r nei neuroni eccitatori del proencefalo; il secondo è un knockout selettivo per TLQP21 (ΔTLQP-21), peptide derivato di VGF. Per testare l’ipotesi che gli effetti della delezione genica possono variare in relazione all’ambiente materno precoce, nel primo esperimento Npy KO e controlli di entrambi i sessi sono stati dati in adozione alla nascita a madri ad alto o basso grado di cure materne (rispettivamente CD1 swiss, FVB – C57/Bl6, Balb/c). In età adulta i topi sono stati sottoposti a test comportamentali per valutare l’aspetto emozionale, sociale ed aggressivo, e a test metabolici in risposta a dieta standard o ad alto contenuto di grassi. Nel secondo esperimento abbiamo eseguito un’analisi dettagliata dell’effetto dell’ambiente materno precoce sugli animali controllo del modello Npy Y1r KO. Nel terzo esperimento infine abbiamo osservato il comportamento materno e il successo riproduttivo in topi ΔTLQP-21 eterozigoti o omozigoti, ed eseguito una caratterizzazione della prole analizzando comportamento emozionale e simil-depressivo negli individui adulti di entrambi i sessi. In generale i risultati del primo studio hanno mostrato che i maschi, ma non le femmine, avevano un aumento dei comportamenti simil-ansiosi e pesavano meno dei loro rispettivi controlli solo quando allevati da madri adottive ad alto grado di cure materne. Nel secondo esperimento abbiamo dimostrato che l’ambiente materno agisce sullo sviluppo della prole ma l’impatto non è ascrivibile soltanto alle variazioni delle cure materne; infatti anche la quantità e la qualità del latte materno e fattori nutrizionali potrebbero essere variabili importanti che influenzano il comportamento e il metabolismo in età adulta. Infine, i risultati del terzo studio hanno indicato che la mutazione nel gene TLQP21 influenza il comportamento materno: genitori che avevano fenotipo mutante ed eterozigote mostravano un livello minore di cure materne (definito dal tempo trascorso nella posizione di arched back) se confrontati con topi wild-type. Tale differenza ha effetto anche sul fenotipo comportamentale degli animali adulti, in particolare riguardo al comportamento emozionale e alle relative differenze sessuali. Saranno dunque necessari ulteriori studi per comprendere il ruolo della nutrizione e del latte materno nello sviluppo comportamentale della prole negli animali Ko e Wt del modello Npy, e per capire in maniera dettagliata quale sia il ruolo dell’ambiente materno nei topi ΔTLQP-21.I risultati ottenuti sottolineano quanto sia importante prendere in considerazione la complessa interazione tra gene, ambiente materno precoce ed effetti legati al sesso quando si lavora con modelli murini geneticamente modificati per identificare funzioni specifiche dei geni.
Interactions between genes and environment are recognized to play a critical role in modulating the development of the organism. More in details early maternal environment seems to be a key factor in shaping neuro-behavioral development; the mother-infant bond is the strongest and more common social relationship in mammal species and phylogenetically highly conserved. Experimental evidence demonstrated that maternal behavior may act on the regulation of genetic expression; in adult life, the offspring that had received high levels of maternal cares showed lower anxiety, less ACTH and Corticosterone production, less aging effect on hyppocampus and more immune activity in response to stress. In the present experiments we investigated the interaction between early maternal environment and specific genetic background, in relation to sex on metabolism and behaviour. We examined two different genetically modified mice models; the first one is a mouse model characterized by a conditional knock-out of the Y1 receptor in the excitatory neurons of the forebrain; the second one is a knockin mouse model of selective loss of the VGF peptide TLQP-21. To test the hypothesis that the effects of gene deletion may vary in relation to early maternal environment, in the first experiment male and female, Npy Ko and control mice were fostered at birth to dams that displayed high or low levels of maternal care (CD1 swiss, FVB – C57/Bl6, Balb/c respectively). As adults mice were tested for emotional, aggressive and social behavior and metabolic changes in response to standard or high fat diets. In the second experiment we performed a detailed analysis of the effect of early maternal environment on control animals of the Npy Y1r KO model. In the third experiment we observed maternal behavior and reproductive success in ΔTLQP-21 heterozygotes or homozygotes breeders and characterized their offspring by an analysis of emotional and depression-like behaviors in adult males and females. Overall the results of the first study showed that males, but not females, had an increase in axiety-like behavior and weighted less than their controls, but only when reared by foster mothers displaying high levels of maternal care. In the second experiment, we demonstrated that early maternal environment affected offspring development but its impact can not be ascribed entirely to the variation in maternal cares; in fact also maternal milk quality/quantity and nutritional factors could be important variables influencing behavior and metabolism in adult’s life. Lastly, results in the third study indicated that the mutation in TLQP21 gene influence maternal behavior, with mutant or heterozygotes breeders displaying lower amount of maternal care (defined by the time spent in the arched back position) when compared with wild type mice. Such a difference affected the offspring subsequent behavioral phenotype as adults, particularly emotional behaviour and sex related differences . Further study will be necessary to investigate the role of nutrition and maternal milk in metabolic and behavioural development of offspring in both NPY KO and WT animals, and to better understand the role of early maternal environment in ΔTLQP-21 mouse. The present findings, however, highlight the importance to take into consideration and control for the complex interaction between genes, early maternal environment and sex-related effects when working with GM mouse models to uncover specific gene functions.

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Festa, Anna. "Controlli di qualità e rappresentazione 3D delle microtomografie a raggi X applicate allo studio dei modelli murini per la progeria." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2018. http://amslaurea.unibo.it/15565/.

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La progeria è una malattia genetica rarissima che genera un invecchiamento precoce, con una incidenza di 1 caso su 4 milioni di nascite e circa 100 casi noti attualmente in tutto il mondo. L’età media di sopravvivenza è di circa 13.5 anni, con una aspettativa di vita tra gli 8 e i 21 anni. La massa ossea ridotta e le anomalie scheletriche sono tra i principali disordini dell’invecchiamento precoce. Al fine di sviluppare una migliore comprensione della patogenesi e della progressione della sindrome, oltre che per progredire nelle possibili terapie, sono stati sviluppati modelli animali che replicano in-vivo ed in condizioni controllate la genesi e l’evoluzione della malattia. Uno dei modelli animali più diffusi è quello murino, il cui sistema scheletrico può essere studiato mediante microtomografia a raggi X. Attualmente considerata come il gold standard, grazie alla sua elevata risoluzione spaziale, la micro-CT fornisce importanti indicazioni sulla struttura trabecolare e corticale del tessuto osseo. Le misure microtomografiche dei segmenti ossei murini sono affette da variabilità intrinseca legata ai parametri di scansione, al posizionamento del campione da parte dell'operatore, alla selezione manuale o automatizzata della ROI, alla scelta del filtro e dei parametri di segmentazione. Questo lavoro di tesi, svolto presso il Laboratorio di Tecnologia Medica (LTM) dell’Istituto Ortopedico Rizzoli (IOR) di Bologna, parte da una approfondita ricerca bibliografica in merito ad accuratezza e ripetibilità delle misure ossee morfometriche e densitometriche del modello murino analizzato mediante micro-CT. Sono stati quindi definiti e applicati opportuni protocolli di controllo qualità, di scansione e di rendering 3D al fine di avere la possibilità di confronto riguardo i modelli murini analizzati, in particolare per il femore e il cranio, e opportunità di indagine più approfondita tramite la visualizzazione tridimensionale dei campioni in analisi.

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de, la Fuente Hernández Noa. "Generación de modelos murinos de adenocarcinoma pancreático con mayor eficiencia metastásica para el estudio de agentes antitumorales." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/673338.

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L’ adenocarcinoma ductal de pàncrees (ACDP) és un tumor agressiu, de biologia tumoral i genètica complexa, amb mal pronòstic per el seu diagnòstic tardà i quimioresistència. El receptor de quimiocines, CXCR4 és un marcador de cèl·lules mare tumorals metastàtiques, associat amb un augment de la invasió, la disseminació tumoral i la quimioresistència en tumors pancreàtics. Aquest receptor seria, per tant, una diana excel·lent per al adreçament d’una teràpia antitumoral i antimetastática. La motivació per a la realització d’aquest projecte de tesi ha estat la necessitat de desenvolupar treballs de recerca per millorar el diagnòstic, el pronòstic i el tractament de l’ACDP. En aquest treball s’han generat models en ratolí amb una major eficiència metastàtica que permeten ampliar l’estudi d’aquesta patologia i que s’han utilitzat, a més, per validar dos tractaments de l’àmbit de la nanomedicina: una membrana constituïda per nanofibres que permet un alliberament local i controlada de fàrmac cap al tumor primari i una nanopartícula per al lliurament dirigida d’un citotòxic cap a les cèl·lules mare tumorals CXCR4+, implicades en el procés de metàstasi. Els objectius específics d’aquest projecte han estat: l’obtenció de mostres de ACDP humà i l’estudi de l’expressió de CXCR4; la creació de models en ratolí de ACDP d’alta eficiència metastàtica a partir de línies cel·lulars de ACDP i mostres tumorals humanes amb alta expressió de CXCR4; l’avaluació, en xenógrafs ortotòpics pancreàtics, de l’eficàcia terapèutica d’una membrana de nanofibres produïda per la polimerització d’àcid poli (làctic-co-glicòlic, PLGA) i carregada en la seva matriu amb el citotòxic SN38 (CEB-01-SN38 ) en tractament únic o en combinació amb quimioteràpia, i finalment, l’estudi de la citotoxicitat i biodistribució in vivo de la nanopartícula T22-O-Gemcitabina (T22-O-GEM), adreçada a l’eliminació selectiva de les cèl·lules mare metastàtiques.Per a això, la metodologia ha inclòs experiments in vitro (cultiu cel·lular i l’obtenció de línies tumorals PANC-1 i MIA PaCa-2 amb expressió de CXCR4 i l’obtenció d’una línia tumoral a partir d’un tumor de ACDP de pacient), i experiments in vivo (la generació de models subcutanis de ADCP i models ortotòpics metastàtics, la implantació de membranes CEB-01, l’administració de quimioteràpia, el seguiment de l’ creixement tumoral i avaluació de les metàstasis per bioluminescència), i finalment, l’avaluació de la citotoxicitat i biodistribució in vivo de T22-GFP-GEM. Les nostres conclusions són que la implantació de cèl·lules tumorals amb sobreexpressió de CXCR4 augmenta significativament la disseminació tumoral i és útil per a l’avaluació preclínica d’ antitumorals, que la implantació ortotòpica de biòpsies humanes amb alta expressió de CXCR4 és un model més predictiu per a la clínica, que la implantació de la membrana CEB-01 amb SN-38 permet el control de l’ creixement tumoral local i la seva combinació amb quimioteràpia pot controlar la progressió tumoral i disminuir la càrrega tumoral metastàtica, i que la nanopartícula T22-O-GEM presenta adreçament cap a cèl·lules mare tumorals metastàtiques i indueix mort cel·lular. Aquest tractament podria aconseguir un major efecte antitumoral comparat amb el tractament estàndard amb Gemcitabina, disminuint, a més, la toxicitat depenent de dosi.
El adenocarcinoma ductal de páncreas (ACDP) es un tumor agresivo, de biología tumoral y genética compleja. Presenta un mal pronóstico por su diagnóstico tardío y quimiorresistencia. El receptor de quimiocinas, CXCR4 es un marcador de células madre tumorales metastásicas. Varios estudios lo han asociado con un aumento de la invasión, la diseminación tumoral y la quimiorresistencia en tumores pancreáticos. Este receptor sería, por tanto, una diana excelente para el direccionamiento de una terapia antitumoral y antimetastásica. La motivación para la realización de este proyecto de tesis ha sido la necesidad de desarrollar trabajos de investigación que permitan encontrar nuevas herramientas para mejorar el diagnóstico, el pronóstico y el tratamiento del ACDP. En este trabajo se han generado modelos en ratón con una mayor eficiencia metastásica que permiten ampliar el estudio de esta patología. Estos modelos se han utilizado, además, para validar dos tratamientos del ámbito de la nanomedicina: una membrana constituída por nanofibras que permite una liberación local y controlada de fármaco en el tumor primario tras la resección quirúrgica, y una nanopartícula para la entrega dirigida de un citotóxico hacia las células madre tumorales CXCR4+, implicadas en el proceso de metástasis, permitiendo, en ambas estrategias, un aumento de la concentración farmacológica del compuesto antitumoral, y disminuyendo la toxicidad sistémica asociada al tratamiento. Los objetivos específicos de este proyecto han sido: la obtención de muestras de ACDP humano y el estudio de la expresión de CXCR4; la creación de modelos en ratón de ACDP de alta eficiencia metastásica a partir de líneas celulares de ACDP y muestras tumorales humanas con alta expresión de CXCR4; la evaluación, en xenógrafos ortotópicos pancreáticos, de la eficacia terapéutica de una membrana de nanofibras producida por la polimerización de ácido poli (láctico-co-glicólico, PLGA) y cargada en su matriz con el citotóxico SN-38 (CEB-01-SN38) en tratamiento único o en combinación con quimioterapia,y finalmente, el estudio de la citotoxicidad y biodistribución in vivo de la nanopartícula T22-O-Gemcitabina (T22-O-GEM), dirigida a la eliminación selectiva de las células madre metastásicas. Para ello, la metodología ha incluido experimentos in vitro (cultivo celular y la obtención de líneas tumorales PANC-1 y MIA PaCa-2 con expresión de CXCR4 y la obtención de una línea tumoral a partir de un tumor de ACDP de paciente), y experimentos in vivo: la generación de modelos subcutáneos de ADCP y modelos ortotópicos metastásicos, la implantación de membranas CEB-01, la administración de quimioterapia, el seguimiento del crecimiento tumoral y evaluación de las metástasis por bioluminiscencia), y finalmente, la evaluación de la citotoxicidad y biodistribución in vivo de T22-GFP-Gemcitabina y el análisis de la expresión de CXCR4 en muestras de pacientes con ACDP. Nuestras conclusiones son que la implantación de células tumorales con sobreexpresión de CXCR4 aumenta significativamente la diseminación tumoral y es útil para la evaluación preclínica de compuestos antitumorales, que la implantación ortotópica de biopsias humanas con alta expresión de CXCR4 es un modelo más predictivo para la clínica, que la implantación de la membrana CEB-01 cargada con el fármaco SN-38 permite el control del crecimiento tumoral local y su combinación con quimioterapia puede controlar la progresión tumoral y disminuir la carga tumoral metastásica, y que la nanopartícula T22-O-GEM presenta direccionamiento hacia células madre tumorales metastásicas e induce muerte celular. Este tratamiento podría conseguir un mayor efecto antitumoral comparado con el tratamiento estándar con Gemcitabina, disminuyendo, además, la toxicidad dependiente de dosis observada en los quimioterápicos utilizados habitualmente, no sólo en ACDP sino en otras neoplasias.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplasia with a complex tumor behaviour and genetics. It has a poor prognosis due to its late diagnosis and chemo- resistance. CXCR4, a marker of metastatic tumor stem cells, has been associated with an increase in invasion, tumor spread and chemoresistance in pancreatic tumors. This receptor could be a good target for the addressing of an anti-tumor and antimetastatic therapy. The motivation to perform this thesis has been the need to develop research work that will allow to find new tools that improve the diagnosis, prognosis and treatment of PDAC. In this work, mouse models have been generated with greater metastatic efficiency to expand the study of this pathology. They have also been used to validate two treatments in the field of nanomedicine, which allow a local controlled release of the drug in the primary tumor after surgical resection, and targeted administration of cytotoxic to CXCR4+ tumor stem cells, associated with metastasis, allowing an increase in pharmacological concentration and decreasing systemic toxicity. The specific objectives of this project have been to obtain samples from human PDAC and to study the expression of CXCR4, the development of high-efficiency metastatic mouse models of PDAC from PDAC cell lines and human tumor samples CXCR4+. Those models were used to evaluate the therapeutic efficacy of a nanofiber membrane produced by poly acid polymerization (lactic-co-glycolic) loaded with cytotoxic SN-38 (CEB-01-SN38) and treated alone or in combination with chemotherapy, and the study of in vivo cytotoxicity and biodistribution of the nanoparticle T22-O-Gemcitabine (T22-O-GEM), aimed at selective elimination of metastatic stem cells. To this end, the methodology has included conducting in vitro experiments: cell culture and obtaining PANC-1 and MIA PaCa-2 tumor lines with high CXCR4 expression and a tumor line from a patient's PDAC tumor, as well as in vivo experiments developing subcutaneous PDAC models and metastatic orthotopic models, implantation of CEB-01 membranes, administration of chemotherapy, monitoring of tumor growth and determination of metastases by bioluminescence, and finally, the evaluation of cytotoxicity and in vivo biodistribution of T22-GFP—GEM. Our conclusions are that implantation of tumor cells with CXCR4 overexpression significantly increases tumor spread being usefulness for preclinical evaluation of antitumor compounds. The orthotopic implantation of human biopsies with high expression of CXCR4 is a more predictive and translational model for preclinical assays. The implantation of the CEB-01 membrane loaded with the drug SN-38, can control the local tumor growth alone or combined with chemotherapy, decreasing metastatic tumor spread, and finally, the T22-O-GEM nanoparticle could be used to eliminate metastatic cancer stem cells and it might induce greater antitumor effects than the standard treatment with Gemcitabine, reducing the side effects of standard chemotheratic treatments.
Universitat Autònoma de Barcelona. Programa de Doctorat en Cirurgia i Ciències Morfològiques

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DE, PONTI GIADA. "Exploring early therapeutic approaches in a Mucopolysaccharidosis type I (MPS I) mouse model." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/382061.

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In questo progetto di dottorato, sono stati presi in considerazione alcuni punti cruciali relativi alla malattia Mucopolisaccaridosi di tipo I (MPSI) e ai limiti delle attuali terapie, per migliorarle concentrandosi su approcci in epoca neonatale, a livello di combinazione terapeutiche (HSCT e ERT) e di terapia genica, e su riduzione delle tossicità associate ai regimi di condizionamento. Complessivamente, le problematiche più significative riguardo MPSI rimangono la necessità di un intervento precoce e rapido, l’incompleta correzione della malattia in seguito agli attuali approcci terapeutici e gli effetti collaterali dovuti al regime di precondizionamento. La prima parte del progetto si è concentrata sul testare un approccio terapeutico che combinasse i trattamenti canonici per MPSI. È stata testata l'efficacia della combinazione di HSCT ed ERT nel modello murino di MPSI come intervento neonatale, per valutare i benefici aggiuntivi della terapia enzimatica effettuata in modo continuativo a seguito del trapianto di cellule da donatore. Tre opzioni di trattamento sono state confrontate dalla nascita, considerando la mancante attività enzimatica (IDUA), l’accumulo di GAG e di vacuoli nei principali organi viscerali, la risposta nei confronti di IDUA ricombinante e i miglioramenti di tipo scheletrico e cerebrale. Pertanto, un approccio combinato di HSCT ed ERT nel periodo neonatale potrebbe essere applicato per migliorare alcune manifestazioni cliniche di MPSI, soprattutto evitando danni irreversibili. La seconda parte del progetto di dottorato è stata svolta in collaborazione con TIGET-SR e il Prof. Alessandro Aiuti. Si è concentrato sulla sperimentazione di un approccio di terapia genica neonatale in un modello murino di MPSI, considerando l'importanza di correggere precocemente la malattia. In particolare, abbiamo valutato se questo trattamento terapeutico potesse essere applicato nei neonati MPSI e potesse essere una strategia efficace per superare i principali problemi clinici che permangono dopo il trattamento canonico. Abbiamo valutato l'effetto della terapia genica somministrata nei neonati affetti da MPSI, monitorando i valori di IDUA e VCN nel sangue periferico e considerando infine la mancante attività enzimatica (IDUA), l’accumulo di GAG e di vacuoli nei principali organi viscerali, la risposta nei confronti di IDUA ricombinante e i miglioramenti di tipo scheletrico e cerebrale. Contemporaneamente, abbiamo cercato di ridurre gli effetti collaterali causati dal regime di condizionamento nel contesto delle terapie neonatali per MPSI, come progetto parallelo. L’obiettivo principale è stato trasporre l'applicazione di ADC, molecole congiunte di anticorpo-farmaco, capaci di agire specificatamente sulla cellula, come condizionamento per il trattamento neonatale di MPSI, in cui un intervento precoce è fondamentale. Poiché nessuna delle opzioni testate è stata in grado di indurre un sufficiente attecchimento di cellule del donatore da essere rilevante per il trattamento precoce dell'MPSI, ne abbiamo ricercato le cause, dimostrando la necessità di ulteriori studi prima dell'applicazione degli ADC nel modello studiato, in quelli umanizzati e nei cuccioli di MPSI NSG. Analisi preliminari sono state effettuate relativamente all’aumento dei livelli di citochine dopo CD117-SAP nei topi NSG adulti, confrontandoli agli altri regimi di condizionamento, per valutare una possibile applicazione di ADC con farmaci antinfiammatori prima della terapia precoce nei cuccioli MPSI.
The present PhD project has taken into account critical issues around Mucopolysaccharidosis type I (MPSI) and limitations of current therapies to further improve them, by generally focusing on neonatal therapeutic approaches, both in terms of combined HSCT and ERT and of gene therapy, and on trying to reduce the overall toxicities associated with pre-conditioning settings. Overall, the most important open issues regarding this rare life-threatening disorder are the need for a precocious and rapid intervention, the lack of complete disease correction after current therapeutic approaches and side effects due to pre-conditioning regiment. My PhD project partially focused on testing a combined approach of the current standard-of-cares for treating MPSI. HSCT and ERT combination efficacy was tested in a mouse model of MPSI as neonatal intervention, for evaluating additional benefits of continuous enzyme therapy after transplant of donor’s cells. We compared three treatment options starting from MPSI pups’ birth, considering IDUA deficient activity, GAGs storage and vacuoles in visceral organs, the immune response against the recombinant IDUA and skeletal and CNS ameliorations. Therefore, performing a combined approach of HSCT and ERT in the neonatal period could help improving some hard-to-treat MPSI manifestations. The second part of this PhD project was carried out in collaboration with TIGET-SR and Prof. Alessandro Aiuti. It was focused on testing a neonatal gene therapy approach in a mouse model of MPSI, considering the importance of an early phenotype correction. In particular, we evaluated if this early treatment could be applied in MPSI neonates and could be a successful strategy for overcoming the main clinical issues that still remain after current canonical HSCT treatment. We monitored peripheral blood of treated mice for 8 months in terms of enzymatic activity and VCN, and we evaluated the effect of GT performed in MPSI pups at endpoint, considering IDUA deficient activity, vector copies/genome, GAGs storage and vacuoles in visceral organs, the immune response against the recombinant IDUA and skeletal and CNS ameliorations. The last part of this PhD project was centered on trying to reduce the high morbidity and mortality due to the severe conditioning regimen in the context of neonatal MPSI therapies, as a side project. The main objective was to translate the application of hematopoietic cell–specific antibody-drug conjugates (ADCs) as conditioning for early MPSI treatment, in which a precocious intervention is crucial. Since none of the tested setting was able to induce enough engraftment of donor’s cells to be relevant for MPSI early treatment, we tried to understand what could interfere, but we demonstrated the need for further studies prior to ADCs application in humanised models and in MPSI NSG pups. Preliminary results on increased cytokines levels after CD117-SAP in adult NSG compared to other conditioning settings were performed to evaluate impairment of inflammation and possible ADC application with anti-inflammatory drugs prior to early therapy in MPSI pups.

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Agbulut, Onnik. "Modeles murins mimant des maladies neuromusculaires." Paris 7, 2001. http://www.theses.fr/2001PA077002.

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Les mutants de souris presentant une modification genetique procurent des renseignements essentiels pour comprendre le role joue par les proteines codees par le gene, et les mecanismes physiopathologiques impliques dans des maladies chez l'homme. L'objectif de mon travail est de participer a l'elaboration de nouveaux mutants correspondant a certaines atteintes neuromusculaires. Les deux modeles animaux presentes dans ce travail sont le mutant dystrophique sans desmine, et le mutant de la dystrophie myotonique. La desmine est un constituant des filaments intermediaires et un marqueur musculaire precoce. Afin de mieux comprendre le role joue par la desmine dans le developpement et la regeneration du muscle squelettique, une lignee de souris mutantes sans desmine a ete etablie (li et coll. , 1996). Nos resultats montrent que la desmine ne modifie pas la differenciation et la fusion des myoblastes mais qu'elle joue un role essentiel pour maintenir la structure de la fibre musculaire et ainsi la force musculaire. La dystrophie myotonique est une maladie genetique autosomique dominante associant des atteintes musculaires et des anomalies multisystemiques. Le defaut moleculaire de la dystrophie myotonique correspond a l'amplification d'un trinucleotide ctg repete situe dans le gene de la dmpk. Plus cette sequence est repetee, plus la maladie est grave. Des mutants de souris ont ete mis au point par l'equipe du pr c. Junien. En fonction du nombre de repetitions de ctg inserees chez les souris, nous avons analyse les perturbations du programme musculaire. Les souris portant une amplification de 300 ctg donnent les resultats les plus interessants : une atrophie des fibres lentes, des noyaux centraux et une myotonie. La comparaison

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Lancerotto, Luca. "Development of a Murine Model for the Exploration of the Biological Effects of External Volume Expansion. Sviluppo di un modello murino per l'esplorazione degli effetti biologici dell'espansione volumetrica esterna." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3422288.

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Background: External Volume Expansion (EVE) refers to a class of devices that non-invasively stretch and expand tissue compartments by external application of suction. EVE has been suggested to increase compartments volume and stimulate the formation of a more developed vascular network, leading to less stiff and better vascularized tissues. It is proposed to patients as a method to prepare recipient sites, in particular breasts, in view of a fat grafting procedure, basing on the theory that fat grafts will better survive and retain volume if the recipient site is more vascularized and provides less compression. However, the method requires high patient compliance and no experimental validation for it has been attempted. Aims: basing on our group's previous experience in downsizing and testing in animal models clinical devices for wound healing, in particular in settings requiring the application of mechanical forces to soft tissues, we proposed to design an animal model for EVE in which to test the validity of the hypothesis of its being beneficial to fat grafting and explore its mechanisms and potentials. Methods: we designed and built a miniaturized EVE device to be applied to the dorsum of mice. We then designed a series of stepwise incremental studies. We tested the capacity of EVE of inducing angiogenesis and cell proliferation with 28 days long continuous stimulation. We analyzed its effects on tissues in terms of mechanical stretch, hypoxia and ischemia, edema, inflammation, cell proliferation and angiogenesis after a single 2 hours stimulation. We produced a mathematical modeling for the effects of EVE on tissues in relation to fat grafting. We tested if EVE is beneficial to fat grafting and if beneficial effects are maintained also in the setting of chronic radiation damage. We tested if EVE can stimulate adipogenesis and what role inflammation can play in it. Results: in our series of studies, we successfully designed a miniaturized animal model in which to test External Volume Expansion. We demonstrated that the hypotheses of stimulation of cell proliferation, angiogenesis, and expansion of tissue compartments on which it is proposed as a preparatory method to fat grafting is confirmed in experimental settings. We showed how mechanical stretch of tissues, hypoxia and ischemia, edema, and inflammation are all intervening factors that can contribute to these effects. Our results suggest that pre-stimulation with EVE is successful in achieving increased fat graft weight and volume retention, and that its beneficial effects are maintained also in the setting of recipient sites having sustained radiation injury. We also demonstrated that EVE has a potential for direct stimulation of adipogenesis, and gathered supportive results to a role for macrophages in this. Discussion: our results validate the technique for its use in the preparatory phase to fat grafting, and can help moving towards making fat grafting a more effective and reliable procedure with improved outcomes for patients. We gathered evidence that help increasing our understanding of how EVE works and what it implies for tissues. This is the basis for optimizing the technique, make it safer, and increase patients' compliance. For example, stimulation patterns can be improved, duration of treatment can be reduced, and practices such as continuation of EVE after fat grafting should be abandoned as detrimental. Our unexpected observations on adipogenesis also open interesting opportunities, such as that of re-starting EVE after fat grafting when this is at the peak of its remodeling phase. And linking this effect with the understanding of the similarity to other conditions in which adipogenesis is seen and desired, such as tissue engineering, or pathological, such as lymphedema, can expand the potential of our animal model to alternative broader fields.
Introduzione: Espansione Volumetrica Esterna (EVE) si riferisce ad una classe di dispositivi che distendono ed espandono compartimenti tissutali in modo non invasivo attraverso l'applicazione dall'esterno di suzione. E' stato suggerito che EVE aumenti il volume dei compartimenti tissutali cui viene applicata e che stimoli un arricchimento della rete vascolare, portando a tessuti meno rigidi e meglio vascolarizzati. Viene proposta a pazienti come metodo per preparare un sito ricevente, soprattutto il seno, in vista di una procedura di innesto di tessuto adiposo con la tecnica del lipofilling, sulla base della teoria che l'innesto sopravvivrà e manterrà il suo volume meglio se il sito ricevente è più vascolarizzato e vi è sottoposto a meno compressione. Tuttavia, il metodo è impegnativo per le pazienti e proposto in assenza di validazione sperimentale delle ipotesi. Scopi: sulla scorta delle precedenti esperienze del nostro gruppo nel realizzare modelli sperimentali animali per dispositivi clinici nel campo della guarigione delle ferite, e in particolare di dispositivi per l'applicazione di forze meccaniche a tessuti molli in vivo, ci siamo proposti di realizzare un modello animale per EVE in cui testare la validità dell'ipotesi che possa offrire benefici all'innesto di tessuto adiposo ed esplorarne i meccanismi e il potenziale. Metodi: abbiamo realizzato un dispositivo per EVE miniaturizzato da applicare al dorso di topi. Abbiamo quindi disegnato una serie di studi con ipotesi e obiettivi incrementali. Abbiamo testato la capacità di EVE di stimolare angiogenesi e proliferazione cellulare applicandola in modo continuo per 28 giorni. Abbiamo analizzato i suoi effetti sui tessuti in termini di stiramento meccanico, ipossia, ischemia, edema, infiammazione, proliferazione cellulare e angiogenesi dopo una singola stimolazione da due ore. Abbiamo elaborato una teorizzazione matematica per gli effetti di EVE sui tessuti in relazione all'innesto di tessuto adiposo. Abbiamo testato se abbia un effetto positivo sull'innesto e se i suoi effetti positivi siano mantenuti nel caso di sito ricevente che abbia sostenuto danno da radiazioni. Abbiamo testato se EVE può timolare l'adipogenesi e che ruolo possano avere in questo i fenomeni infiammatori. Risultati: nella nostra serie di studi, è stato realizzato con successo un modello miniaturizzato con cui testare EVE. Abbiamo confermato sperimentalmente le ipotesi che induca proliferazione cellulare, angiogenesi e espansione dei compartimenti tessutali sulla base delle quali viene proposta come metodo preparatorio all'innesto di tessuto adiposo. Lo stiramento dei tessuti, l'induzione di ipossia e ischemia, edema e infiammazione sono tutti fattori che intervengono e possono contribuire a questi effetti. I nostri risultati suggeriscono che la pre-stimolazione con EVE possa essere efficace nell'ottenere un superiore mantenimento di massa degli innesti di tessuto adiposo, e che gli effetti positivi siano mantenuti anche in tessuti che abbiano subito danno da radiazione. Abbiamo inoltre dimostrato che EVE ha il potenziale di stimolare direttamente fenomeni adipogenici, e raccolto evidenze in favore di un ruolo primario dei macrofagi per questo effetto. Discussione: I nostri risultati offrono supporto sperimentale all'uso di EVE nella fase preparatoria agli innesti di tessuto adiposo, e possono contribuire a rendere l'innesto di tessuto adiposo una procedura più efficiente e prevedibile con maggiori garanzie per i pazienti. Abbiamo raccolto osservazioni che aiutano a comprendere come EVE funzioni e cosa implichi per i tessuti, il che è la base per ottimizzarla, renderla una tecnica più sicura, e aumentare la compliance dei pazienti. Ad esempio, i pattern di stimolazione possono essere migliorati, la durata del trattamento può essere ridotta, e pratiche come quella di riapplicare EVE subito dopo l'innesto di tessuto adiposo dovrebbero essere abbandonate in quanto dannose. La nostra inaspettata osservazione di effetti adipogenici diretti prospetta interessanti alternative, come quella di riapplicare EVE quando un innesto di tessuto adiposo è al picco della sua fase di rimodellamento. E collegare questo effetto con altre condizioni in cui adipogenesi è desiderata, ad esempio in ingegneria tissutale, o patologica, come nel linfedema, può ulteriormente espandere le potenzialità del nostro modello animale ad applicazioni in campi più vasti.

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Tiozzo, Fasiolo Roberta. "STUDI SPERIMENTALI SUL MECCANISMO DELLA DISTROFIA DA MUTAZIONE A CARICO DEI GENI CODIFICANTI PER IL COLLAGENE VI." Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3427564.

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Mutations of genes coding for collagen VI are responsible, in humans, of congenital muscular dystrophies, giving rise to three syndromes Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD) and Congenital Myosclerosis (CM). Based on the high degree of heterogeneity and the overlap between them, it has been proposed that these disorders may represent a clinical continuum rather than strictly separated entities, and that there may be a wider spectrum of collagen VI related disorders. Despite these major advances in understanding their genetic bases, the molecular pathogenesis remained partially obscure. As it is the case for many genetic diseases, creation of animal models may be the key to understand the physiopathology, and to devise and test potential therapies. Several years ago (Bonaldo at al, 1998) a mutant mouse with targeted inactivation of COL6A1 gene, coding for the 1(VI) chain, was created. In the absence of a 1(VI) chain, collagen VI does not assemble and is not secreted in the extracellular matrix, and therefore homozygous null mice (Col6a1-/-) completely lack collagen VI in their tissues. These mice are affected by early onset myopathic disease with weakness and histological alterations of skeletal muscles. Col6a1-/- muscle have loss of contractile strength with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. There is a latent mitochondrial dysfunction in myofibers which can be revealed upon incubation with the selective F1F0-ATPase inhibitor oligomycin, which causes mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they can be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Collagen VI myopathies, in mice and humans, can be effectively treated with drugs acting downstream on pathogenic lesion. These observations lead to the hypothesis that the lack of collagen VI causes an increased probability of opening of the PTP, but we don’t know through which signalling pathway the lack of collagen VI in the extracellular matrix can have effects on mitochondria. One important partner of collagen VI is the proteoglycan NG2. In particular, there is evidence that NG2 binds to collagen VI via a protein-protein interaction (Tillet et al, 1997). NG2 may be considered an important receptor mediating collagen VI-sarcolemma interactions and this relationship may be disrupted in the pathogenesis of Bethlem and Ullrich dystrophies. We also know that the perturbation of NG2 distribution on the cell surface results in parallel change in collagen VI distribution (Nishiyama et al,1997). The NG2-collagen VI interaction may be important for organization of the extracellular matrix, for binding of cells to the matrix, for determination of cell morphology in relation to the matrix, and for a transduction of transmembrane signalling. To clarify the relevance of NG2 for muscle function and structure, muscles of mice carrying a null mutation of NG2 were studied. The aim of this study was a comparative characterization (in vivo, ex vivo and in vitro) of the phenotype of muscles from ColVI-/- and NG2-/- mice and the evaluation of the differences between the two models to understand in what way the absence of these proteins might lead to mitochondrial damage. The experimental program was aimed to the characterization of muscles from C57BL/6, Col6a1-/- and NG2-/- mice in vivo, ex vivo ed in vitro. The integrity of the membrane was tested with the blue Evans dye which allows to detect, pointed out with blue color, muscle fibers in which the sarcolemma has been damaged. At first the analysis of functional parameters of muscle contraction was carried out in vivo, with the following tests: - mouse force (Grip test) - force developed by the muscle gastrocnemius during isometric contraction. Then the analysis of functional parameters was developed ex vivo: - intact muscle diaphragm, EDL, soleus dissected and analysed in myograph - electrophoresis of proteins from diaphragm, EDL, soleus - histological and histochemical analysis of gastrocnemius and tibial. Finally, single muscle fibres were kept in culture and studied, in vitro: - recording Ca2+ transient in single fibres of FDB - electrophoresis of single fibres - immunocytochemistry analyses. The results of all the above listed tests have shown that the two phenotypes have only a partial overlap and, thus, NG2 does not represent the only structural/functional connection between Collagen VI in the ECM and the intracellular processes in muscle fibres. The role of integrin require to be explored.
Le mutazioni dei geni che codificano per le subunità del collagene VI sono una delle cause delle patologie muscolari ereditarie umane che si manifestano come la Miopatia di Bethlem (BM), la Distrofia Muscolare Congenita di Ullrich (UCMD) e la Miosclerosi Congenita (CM). Basandosi sull’alto grado di eterogeneità e di parziale sovrapposizione tra di loro, è stato proposto che questi disordini possano rappresentare un “continuum” clinico piuttosto che entità strettamente separate, e che ci possa essere un più ampio spettro di disordini connessi al collagene VI. Nonostante i grandi progressi nella comprensione delle loro basi genetiche, la patogenesi molecolare rimane ancora in parte sconosciuta. Come nel caso di molti difetti genetici, la creazione di animali transgenici può essere la chiave per comprendere la fisiopatologia e per mettere a punto, e testare, potenziali terapie. Molti anni fa (Bonaldo et al, 1998) è stato creato un topo mutante con inattivazione mirata del gene COL6A1, che codifica per la catena 1(VI). In assenza della catena 1(VI), il collagene non è assemblato e non è secreto nella matrice extracellulare, e pertanto il topo omozigote mutante (Col6a1-/-) perde il collagene VI nei suoi tessuti. I topi sono affetti da un disordine miopatico ad esordio precoce con debolezza e cambiamenti istologici del muscolo scheletrico. I muscoli del Col6a1-/- perdono forza contrattile e mostrano alterazioni ultrastrutturali a livello del reticolo sarcoplasmatico (SR), dei mitocondri e apoptosi spontanea. E’ presente una disfunzione mitocondriale latente nelle miofibre che si può evidenziare con incubazione con oligomicina, inibitore della F1F0-ATPasi, che causa depolarizzazione mitocondriale, de-regolazione del Ca2+ e aumento dell’apoptosi. Questi difetti sono reversibili, e possono essere normalizzati piastrando le fibre muscolari di Col6a1-/- su collagene VI o somministrando cisclosporina A (CsA), un inibitore del poro di transizione di permeabilità mitocondriale (PTP). Le miopatie dovute al collagene VI, sia umane che negli animali, possono essere efficacemente trattate con tale farmaco che agisce a valle della lesione patogenetica (Irwin et al, 2003 e Merlini et al, 2008). Così queste osservazioni portano ad ipotizzare che la mancanza del collagene VI causa un aumento dell’apertura del PTP ma non è ancora noto per mezzo di quale via di segnale. Un importante partner del collagene VI è il proteoglicano NG2. In particolare, ci sono prove che NG2 si leghi al collagene VI attraverso un interazione proteina-proteina (Tillet et al, 1997). NG2 può essere considerato un importante mediatore dell’interazione collagene VI-sarcolemma e il venir meno di questa relazione potrebbe avere un ruolo nella patogenesi delle distrofie di Bethlem e Ullrich. Sappiamo inoltre che modificazioni nella distribuzione superficiale di NG2 porta ad un parallelo cambiamento nella distribuzione del collagene VI (Nishiyama et al,1997). L’interazione NG2-collagene VI può essere importante nell’organizzazione della matrice, per il legame delle cellule alla matrice, per determinare la morfologia cellulare in risposta alla matrice, e per la trasduzione del segnale transmembrana. Per chiarire l’importanza di NG2 per la funzione e la struttura muscolare, sono stati studiati muscoli di topi con mutazione di NG2. Lo scopo dello studio è la caratterizzazione fenotipica comparativa (in vivo, ex vivo and in vitro) di Col6a1-/- and NG2-/- e la valutazione delle differenze tra i due modelli per comprendere se la mancanza di queste proteine porti ad un simile danno nella fibra muscolare. Si è proceduto dapprima a saggiare l’integrità della membrana con il colorante vitale blue Evans che permette di rilevare, evidenziandole con colorazione blu, le fibre muscolari il cui sarcolemma ha subito un danno. Poi, si è passati quindi all’analisi dei parametri funzionali della contrazione muscolare in vivo quali: - sviluppo di forza (Grip test) - sviluppo della forza massimale del gastrocnemio stimolato per via nervosa. Successivamente è stata effettuata una valutazione ex vivo: - meccanica dei muscoli interi diaframma, EDL e soleo - elettroforesi delle proteine muscolari - analisi istochimiche di gastrocnemio e tibiale. Infine per avere un quadro completo si è continuata l’ indagine in vitro : - transienti di calcio su singole fibre di FDB - elettroforesi su singole fibre di FDB - analisi immunocitochimiche. I risultati di tali analisi hanno mostrato che i due fenotipi hanno solamente una parziale sovrapposizione e quindi NG2 non rappresenta l’unica via di mediazione della presenza del collagene VI. Il ruolo delle integrine richiede di essere esplorato.

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8

Dautova, Yana. "Atrial arrhythmias in murine hearts modelling sodium channelopathies." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609529.

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9

Iemmolo, Maria. "Effetti protettivi della Timosina-beta4 in un modello murino di danno polmonare da Bleomicina." Doctoral thesis, Università di Catania, 2013. http://hdl.handle.net/10761/1435.

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EFFETTI PROTETTIVI DELLA TIMOSINA-beta4 IN UN MODELLO MURINO DI DANNO POLMONARE DA BLEOMICINA Background. La Timosina-beta4, isolata dal timo per la prima volta da Goldstain e collaboratori. nel 1966, è il membro più abbondante della famiglia delle beta-Timosine e risulta la principale proteina sequestrante l actina nella cellula. Ha attività biologica pleiotropica e le varie funzioni sono attribuite a particolari siti attivi contenuti nei peptidi prodotti da attività endoproteinasiche, come ad esempio nel frammento Ac-SDKP. Molti studi attribuiscono ad Ac-SDKP potere antinfiammatorio e alla forma ossidata della Timosina-beta4 (Timosina-beta4-sulfoxide) potere antiossidante. Inoltre, il trattamento di cellule cardiache con la Timosina-beta4 impedisce l espressione di geni profibrotici sia in vitro che in vivo. In un recente studio sono state descritte elevate concentrazioni di beta-Timosine (Timosina-beta4, Timosina-beta4-sulfoxide, Timosina-beta10) nel BAL di una coorte di soggetti con sclerodermia a coinvolgimento polmonare. L ipotesi che la Timosina-beta4 possa esercitare un effetto cito-protettivo nel danno polmonare nasce dall osservazione che quando la concentrazione della proteina si abbassa si verifica una più veloce progressione dell interstiziopatia polmonare. Scopo di questa tesi è stato verificare i possibili effetti protettivi della Timosina-beta4 in un modello in vivo di fibrosi polmonare. Tali effetti risulterebbero particolarmente utili nella FPI, in particolare per rallentare la fase precoce del processo fibrotico, dove danni tissutali da ROS e da cellule infiammatorie concorrono attivamente all espressione del fenotipo fibrotico. Materiali e Metodi. In questo studio è stato utilizzato il modello murino, molto noto ed utilizzato, di danno polmonare indotto da Bleomicina,, anche se per un periodo limitato a 7 giorni. I topi appartenenti al ceppo C57BL/6 sono stati trattati con Bleomicina (BLEO 1 mg/Kg) in assenza e/o in presenza di Timosina-beta4 (6 mg/kg per via intraperitoneale dal giorno di trattamento con BLEO e per ulteriori due dosi). Dopo il sacrificio avvenuto una settimana più tardi, sono stati eseguiti: a) la misurazione del fluido e il contenuto di collagene del polmone; b) la conta cellulare e l analisi citobiologica differenziale nel lavaggio broncoalveolare (BAL); c) saggio per la misurazione dell attività mieloperossidasica; d) istologia polmonare; e) immunoistochimica (IHC); f) analisi al citofluorimetro (FACS) di cellule T estratte dal sangue periferico e dalla milza per la valutazione della percentuale di cellule positive per IL-17A, CD4, CD25 e Fox-p3. Risultati. Nei topi co-trattati con Timosina-beta4 è stato osservato un calo del peso corporeo meno evidente e un tasso di mortalità più basso rispetto ai topi trattati con BLEO. Inoltre, l infiammazione e il danno polmonare indotti da BLEO sono stati ridotti grazie al co-trattamento con Timosina-beta4, cosi come dimostrato dalla riduzione significativa di: a) edema; b) contenuto di collagene totale, c) infiltrazione leucocitaria polmonare; d) attività mieloperossidasica; e) danno al polmone evidenziato dall' analisi istologica. Inoltre, i risultati dell' IHC mostrano una più forte reattività alla Timosina-beta4, con evidente localizzazione alveolare, in topi trattati con la proteina esogena rispetto ai topi trattati solo con BLEO. L analisi al FACS mostra una ridotta percentuale di cellule T positive per IL-17A ed un aumento della percentuale di cellule positive per CD4,CD25 e Fox-p3 nei topi trattati con Timosina-beta4 rispetto ai topi trattati con BLEO. Conclusioni. Questo studio mostra un ruolo protettivo della Timosina-beta4 nel modello murino di danno polmonare indotto da Bleomicina e individua alcuni meccanismi immunologici che potrebbero determinarlo. Ulteriori studi sono necessari per valutare la durata degli effetti protettivi della Timosina-beta4 e il suo eventuale ruolo terapeutico.

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Castelló, Carla Martí. "Ultrassonografia do tumor sólido de Ehrlich inoculado em camundongos." Universidade Federal de Goiás, 2017. http://repositorio.bc.ufg.br/tede/handle/tede/7130.

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The research to prevent cancer, diagnose early, and find new therapies is one of the main challenges of current medicine, and in vivo tumor models are essential for this aim. Imaging techniques, such as ultrasound, assists the research by helping to obtain data that are more accurate and to reduce the number of animals necessary to obtain statistically significant results. Ehrlich carcinoma is one of the most widely used models but it has not an ultrasonographic description. In this study, serial ultrasound examinations were performed, in B-mode and Doppler, on Ehrlich solid carcinomas (ESC) inoculated in mice. From the measurements obtained by ultrasound, the growth patterns were analyzed and the tumors were separated in two groups depending on the specific growth rate (SGR). Ultrasonographic characteristics of capsule, margins, echotexture, vascular flow, and Doppler indices of Resistivity Index (RI) and Pulsability Index (PI) were compared between groups. ESC presents variable growth patterns; a capsule detectable by ultrasound, which sometimes present discontinuity; detectable flow in most of the exams; and the possibility of a central focus of necrosis or several necrosis focuses separated by tissue. In conclusion, tumors with high vascularization tend to have high SGR, while tumors that presented homogeneous echotexture and absence of blood flow tend to have lower SGR. The study also showed that changes in tumor vessels are reflected in Doppler indices, with significantly lower RI and PI, than normal vessels
A pesquisa para o diagnóstico precoce, prevenção e novos tratamentos do câncer é um dos principais desafios da medicina atual e os modelos tumorais in vivo são imprescindíveis para esse fim. Técnicas de imagem, como a ultrassonografia, auxiliam a obter dados mais precisos e diminuir o número de animais para obter resultados estatisticamente significativos. O carcinoma de Ehrlich é um dos modelos mais usados, mas não existem descrições ultrassonográficas dele. Nesse trabalho, foram realizados exames ultrassonográficos seriados, em modo-B e Doppler, em carcinomas sólidos de Ehrlich (ESC) inoculados em camundongos. A partir das medidas obtidas por ultrassom, foram analisados os padrões de crescimento e os tumores foram separados em dois grupos dependendo da taxa específica de crescimento (SGR). Foram comparadas as características ultrassonográficas da capsula, margens, ecotextura, distribuição e quantidade de fluxo e os índices Doppler, Índice de Resistividade (IR) e Índice de Pulsabilidade (IP), entre os grupos. Os ESCs apresentaram-se como tumores com padrões de crescimento variáveis; uma capsula detectável por ultrassonografia, mas que pode apresentar descontinuidade em alguns casos; fluxo detectável na maioria dos exames; e que podem apresentam um foco de necrose central ou vários focos de necrose separados. Conclui-se que os tumores que apresentam alta vascularização tendem a ter alta SGR enquanto os que apresentam ausência de fluxo e ecotextura homogênea estão correlacionados com baixa SGR. O estudo também demonstrou que as alterações e mudanças nos vasos tumorais se encontram refletidas nos índices Doppler, apresentando IR e IP significativamente menores as dos vasos extratumorais.

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11

Senesi, P. "STUDIO DELL'EFFETTO PREVENTIVO DELLA METFORMINA SUI DANNI ASSOCIATI ALLA SEDENTARIETA' IN MODELLI MURINI." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/260285.

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Metformin (METF), historical antihyperglycemic drug, is a likely candidate for lifespan extension, treatment and prevention of sedentariness damages, insulin resistance and obesity. Skeletal muscle is a highly adaptable tissue, capable to increase its mass in response to exercise and to regenerate new fibrils after damage. Aims of this work were to investigate METF ability to prevent sedentariness injury and enhance skeletal muscle function. Sedentary 12-weeks old C57BL/6 mice were treated with METF (250 mg/kg per day, in drinking water) for 60 days. METF role on skeletal muscle differentiation was studied in vitro using murine C2C12 myoblasts. Muscular performance evaluation revealed that METF enhanced mice physical performance. Tissues analysis indicated that in liver METF increased AMPK and CAMKII signaling, while inactivated ERKs, principal kinases involved in hepatic stress conditions. In skeletal muscle, METF activated AKT, key kinase in skeletal muscle maintenance. In in vitro studies, Immunofluorescence and Western blot analysis showed that METF did not modify the C2C12 proliferation capacity, while positively influenced the differentiation process and myotube maturation. Together, our novel results suggest that METF may have a positive action not only on the promotion of healthy aging but also on the prevention of sedentariness damages.

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12

Patrick, Claire Nicole. "Murine modelling of adoptive therapy for B cell lymphoma." Thesis, University of Southampton, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398592.

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Ugel, stefano. "Approcci di immunoterapia attiva e passiva basati sull'antigene telomerasi in modelli di carcinogenesi murina." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3426378.

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Aim of this work was to verify the therapeutic efficacy of active and passive vaccination against m-TERT in mice bearing transplantable tumors and in an autochthonous mouse model of prostate cancer: the TRAMP model. To choose the best vaccination protocol we developed DNA-based antigen-specific expression systems employing eukaryotic plasmid expression vectors and recombinant adenoviruses. We used splenocytes from vaccinated mice to set up mixed leukocyte peptide cultures (MLPC) with the mouse (m)-TERT198-205 peptide. This sequence of mouse TERT was identified as the immunodominant epitope by screening overlapping peptides covering the whole protein. MLPC were tested in IFN?-specific E.L.IS.A. assay against peptide-pulsed cells and unpulsed tumor cell lines of different histotypes, but with the same H-2b haplotype. A persistently high level of recognition was noted against peptidepulsed cell lines, whereas repeated in vitro stimulation with ?-irradiated syngeneic splenocytes pulsed with a low dose of m-TERT198-205 peptide (0.1 ?M) every week led to an increased recognition of unpulsed tumor cell lines. This enhanced recognition was observed only in MLPC from mice vaccinated with DNA plasmid vector and suggested a preferential proliferation of high-avidity CTL clones under these culture conditions. The augment in high-avidity CTLs is supported by TCR analysis by spectratyping, that proved a positive selection of V?-11 chain of TCR during in vitro passages together with TCR clonotype enrichment. We demonstrated an important therapeutic effect by m-TERT198-205 polyclonal CTLs transfer in a metastatic melanoma model, where mice treated with CTLs transfer presented a significant reduction of the lung metastases compared with untreated controls, and in trasplantable tumor models, where TERT198-205 CTLs induced a statistically significant delay in tumor growth and a survival increase in both melanoma- and prostate carcinoma-bearing mice. To identify and isolate high affinity CTL clones specific for m-TERT198-205 epitope we used the limiting dilution technique starting from the polyclonal CTLs. We obtained a large spectrum of clones, among which clone CTL-7 was the most intriguing. In fact it was able to recognize tumor cell lines of different histotypes (melanoma, prostate carcinoma, colon carcinoma and sarcoma) as well as peptidepulsed cell lines. Transfer of CTL-7 clone exerted a better therapeutic effect on lung metastases generated by melanoma cells. We also investigated the possibility to elicit a protective antitumor response through an active immunization in TRAMP mice. We performed a cycle of two biweekly i.m. injections of a plasmid encoding m-TERT repeated every 10 weeks for the entire life of the mouse. Mice vaccinated against m-TERT showed a reduction of tumor progression at week 24th and a survival prolongation compared to mock vector-treated groups. These data represent a good platform to translate more effective vaccines for the therapy of human cancers.

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Schmoeckel, Katrin [Verfasser]. "Entzündungs-induzierte Immunsuppression im murinen Modell / Katrin Schmoeckel." Greifswald : Universitätsbibliothek Greifswald, 2015. http://d-nb.info/107746617X/34.

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Raspa, A. "RECUPERO MOTORIO IN MODELLO MURINO DI MORBO DI PARKINSON DOPO TRAPIANTO DI CELLULE STAMINALI POST-MORTEM." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/217712.

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Abbiamo studiato una strategia per migliorare i sintomi motori di topi che avevano ricevuto lesioni con una neurotossina specifica, MPTP, un modello murino di morbo di Parkinson, attraverso il trapianto di precursori cellulari post mortem nello striato. I precursori cellulari neurali post-mortem (PM-NPC) sono stati isolati dalla zona subventricolare (SVZ) dei ventricoli laterali del topo, coltivati in vitro e la loro capacità di differenziarsi è stata studiata valutando l'espressione di diversi marcatori neurali. PM-NPC differenziano in neuroni e in gran parte a differenza di molti altri precursori, mantengono la capacità di migrazione e integrazione, quando trapiantate nel cervello adulto. Abbiamo eseguito il trapianto di PM-NPC nei gangli della base di topi MPTP lesionati. PM-NPC trapiantate sopravvivono, differenziano e si integrano nel circuito di accoglienza, e modificano il comportamento motorio nei topi MPTP lesionati. I nostri dati suggeriscono che il trapianto di PM-NPC nel corpo striato è un approccio promettente per investigare i potenziali benefici di rimodellamento dei circuiti dei gangli basali nelle malattie neurodegenerative.
We investigated a strategy to ameliorate the motor symptoms of mice that received MPTP lesions, a rodent model of Parkinson’s disease, through transplantation of adult precursor post-mortem cells into the striatum. Post mortem neural precursors (PM-NPCs) were isolated from the subventricular zone (SVZ), grown in vitro and their differentiation capability was investigated by evaluating the expression of different neuronal markers. PM-NPCs differentiate mostly in neurons and unlike most other precursor cells, retain the capacity for migration and integration when transplanted into the adult brain. We performed PM-NPCs transplantation into the basal ganglia of MPTP lesioned mice. Transplanted PM-NPCs survived, differentiated and integrated into host circuitry, and modified motor behavior in MPTP lesioned mice. Our data suggest that PM-NPCs transplantation into the the striatum is a promising approach to invenstigate the potential benefits of remodeling basal ganglia circuitry in neurodegenerative diseases.

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LIGUORI, ELEONORA. "MICROTROMBOSI SINUSOIDALE EPATICA INDOTTA DAL LIPOPOLISACCARIDE: RUOLO PROTETTIVO DELLA SIMVASTATINA IN UN MODELLO MURINO DI ENDOTOSSIEMIA." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/628418.

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Background:Liver dysfunction is one of the earliest high-risk markers for multi-organ failure (MOF) in patients with sepsis. In a recent study, using a murine model, we demonstrated that lipopolysaccharide (LPS) injection provokes hepatic microvascular dysfunction, associated with marked endothelitis in perisinusoidal areas. Furthermore, endothelial dysfunction drives vascular derangement and organ failure associated with sepsis. However, the consequences of sepsis on liver sinusoidal endothelial function are unknown. Statins might improve microvascular dysfunction in sepsis. The present study explores liver vascular abnormalities and the effects of statins in a rat model of endotoxemia. Methods: For this purpose, lipopolysaccharide (LPS) or saline was given to: (1) rats treated with placebo; (2) rats treated with simvastatin (25 mg/kg, orally), given at 3 and 23 hours after LPS/saline challenge; (3) rats treated with simvastatin (25 mg/kg/24 h, orally) from 3 days before LPS/saline injection. Liver microvascular function was assessed by molecular studies and liver function tests. Results: At 24 h, LPS induced liver endothelial dysfunction, as shown by a decreased of thrombomodulin and a increase of fibrin. Treatment with simvastatin from 3 days before LPS prevented the increase of fibrin. Conclusions: LPS administration induces intrahepatic endothelial dysfunction that might be prevented by simvastatin, suggesting that statins might have potential for liver protection during endotoxemia.

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Eberle, Fabian [Verfasser]. "Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells / Fabian Eberle." Marburg : Philipps-Universität Marburg, 2013. http://d-nb.info/1032315067/34.

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Eberle, Fabian B. [Verfasser]. "Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells / Fabian Eberle." Marburg : Philipps-Universität Marburg, 2013. http://nbn-resolving.de/urn:nbn:de:hebis:04-z2013-01383.

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PIERI, MASSIMO. "Alterazione funzionale dei canali ionici nel sistema nervoso centrale in un modello murino di sclerosi laterale amiotrofica." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/927.

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Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brainstem and motor cortex. The degeneration of motor neurons leads to skeletal muscle atrophy, paralysis and death. The mechanisms by which mutations in SOD1 lead to motor neuron degeneration remain unidentified. Several mechanisms have been suggested by which a mutation of SOD1 may lead to motor neuron toxicity and degeneration, including the formation of intracellular aggregates (Johnston et al., 2000), production of reactive oxidative species (Estevez et al., 1999), mitochondrial degeneration (Higgins et al., 2002), altered AMPA receptor permeability and subunit composition (Pieri et al., 2003; Spalloni et al., 2004), increase in intracellular Ca2+ concentration by Ca2+-permeable AMPA receptor channels (Van Den Bosch et al., 2000; Weiss et al., 2000) and protein nitration (Casoni et al., 2005). In the present work, the excitability of motor neurons and the physiological properties of the macroscopic voltage-dependent Na+, Ca++ and K+ currents have been tested in a transgenic mouse model of a familial form of ALS, associated with a mutation in Cu,Zn superoxide dismutase (Gly93 Ala). The results indicate that the passive membrane properties were not modified in G93A motor neurons compared to control neurons whereas the firing properties of single motor neurons in G93A are altered to induce in these neurons hyperexcitability. In addition, the voltage dependence of activation and of steady-state inactivation, the kinetics of fast inactivation and slow inactivation of the voltage-dependent Na+ channels were not modified in the mutated mice. Conversely, the recovery from fast inactivation was significantly faster in G93A motor neurons compared to control neurons. The recovery from fast inactivation was still significantly faster in G93A motor neurons exposed for different times (3-48 hours) and concentration (5-500 μM) to edaravone, a free radical scavenger. The analysis of the voltage-dependent calcium currents and potassium currents showed not significant differences in all the parameters studied in the three neuronal populations. Finally, to investigate which modifications at the ionic current level can produce the observed hyperexcitability in G93A motor neurons, we developed a numerical simulator of the electrical activity of mouse spinal motor neuron. We found that changes limited to ionic current conductances or to faster recovery from fast inactivation of the Na+ channels are not able to reproduce G93A firing alterations. We observed that the mutant motor neuron hyperexcitability can be reproduced by means of a faster kinetic of small conductance calcium-dependent potassium current. These results indicate for the first time that, in accordance with clinical data, the firing properties of single motor neurons in a genetic mouse model of ALS are altered to induce neuronal hyperexcitability and that they may contribute to the pathogenesis of the disease. The clarification of the importance of these changes in membrane ion channel functionality may have diagnostic and therapeutic implications in the pathogenesis of ALS. This alteration, demonstrated at the single-cell level, may affect the normal orchestrated activation and inactivation gating of the voltagedependent channels involved in the neuronal excitability and may be significant in the onset and progression of the pathology. For these reasons, an electrophysiological characterization of small conductance calcium-activated potassium current will be necessary in order to experimentally verify the simulation results.

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SCIAMANNA, GIUSEPPE. "La disfunzione del recettore striatale D2 induce un’alterata trasmissione GABAergica in un modello murino di distonia DYT1." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/849.

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La distonia DYT1 è una grave forma di distonia generalizzata causata da una mutazione del gene DYT1 che codifica per la proteina TorsinA. La funzione di tale proteina rimane ancora poco chiara anche se è stato proposto che possa svolgere importanti funzioni nel traffico proteico intracellulare e nei processi secretori. Lo striato, all'interno dei gangli della base svolge un importante ruolo nella regolazione dell'attività motoria, ed alterazioni a carico di tale struttura appaiono essere coinvolti nella patogenesi della distonia. Ho registrato pertanto le correnti sinaptiche spontanee sia di tipo GABAergico che glutamatergico in neuroni spinosi striatali (MSNs) da animali che sovraesprimevano la proteina umana mutata (hMT) confrontandoli poi con animali di controllo (CTRL) e con quelli che esprimevano la proteina umana non-mutata (hWT). Gli animali mutati presentavano un significativo aumento nella frequenza degli eventi sinaptici GABAergici (sIPSCs) non accompagnato però da variazioni nell'ampiezza di tali correnti. Al contrario l'attività spontanea di tipo glutamatergico (sEPSC) risultava essere del tutto normale. L'inibizione GABAergica striatale è di origine esclusivamente instrinseca e deriva da due distinte fonti. Una delle più importanti tuttavia fa capo agli interneuroni GABAergici Fast Spiking (FS). Ho pertanto verificato l'ipotesi che tali cellule potessero presentare alterazioni nella loro normale funzionalità. Sia gli sIPSCs che gli sEPSC registrati risultavano tuttavia essere invariati fra gli animali hMT, hWT e quelli di controllo. In condizioni fisiologiche l'attivazione del recettore dopaminergico D2 agisce presinapticamente inibendo il rilascio di GABA. Nei MSNs di animali di controllo e hWT, tale funzionalità risultava essere del tutto preservata. L'applicazione di quinpirolo (agonista D2-like) portava infatti ad una significativa riduzione della frequenza degli sIPSCs misurati. Tale effetto tuttavia era assente negli animali hMT. Inoltre sia MSNs sia FS di topi hMT non presentavano l'effetto inibitorio tipico del quinpirolo sulle correnti sinaptiche evocate tramite stimolazione elettrica (eIPSCs). In conclusione il mio lavoro dimostra la presenza di un'alterata attività del circuito GABAergico striatale in un modello animale di distonia DYT1, che può essere in parte giustificata da una disfunzione del recettore dopaminergico D2.
DYT1 dystonia is a severe form of inherited generalized dystonia, caused by a deletion in the DYT1 gene encoding the protein torsinA. The physiological function of torsinA is unclear, though it has been proposed to perform chaperone-like functions, assist in protein trafficking, membrane fusion and participate in secretory processing. Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. I recorded GABA- and glutamate-mediated synaptic currents from striatal neurons obtained from a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents (mIPSCs), whereas glutamatergic spontaneous excitatory synaptic potentials (sEPSCs) activity was normal. No alterations were found in mice overexpressing normal human torsinA (hWT). To identify the possible sources of the increased GABAergic tone, I recorded GABAergic Fast-Spiking (FS) interneurons that exert a feed-forward inhibition on MSNs. Both sEPSC and sIPSC recorded from hMT FS interneurons were comparable to hWT and controls.In physiological conditions, dopamine (DA) D2 receptor act presynaptically to reduce striatal GABA release. Notably, application of the D2-like receptor agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to hWT and controls. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in MSNs and FS interneurons from hMT mice. My findings demonstrate a disinhibition of GABAergic synaptic activity, that can be partially attributed to a D2 DA receptor dysregulation. A rise in GABA transmission would result in a profound alteration of striatal output, that might be relevant to the pathogenesis of dystonia.

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Seria, Elisa Libera. "Rigenerazione epatica mediante l'impiego di cellule staminali mesenchimali in un modello murino di danno epatico tetracloruro-indotto." Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1133.

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Bone marrow-derived mesenchymal stem cells (MSCs) have been investigated as marker of liver regeneration in an experimental model of acute liver failure (ALF). This model was created in Wistar rats through an intraperitoneal injection of carbon tetrachloride (CCl4). MSCs (3 million) were collected by long bones of 10 Wistar rats and then intravenously infused 24 hours after induction of ALF in 16 rats, Group A. In control Group B, 16 rats received a peritoneal injection of CCl4, and an intravenous infusion of normal saline. All rats were sacrificed at 2, 3, 4 and 7 days post-CCl4-injection. To assess the efficacy of the induction of the ALF, 4 rats Group (C) were sacrified 1 day after poisoning. Biochemical markers (BM) of liver function as platelets counts and pathology examination of the procured livers were staged on post-infusion-days 2, 3, 4 and 7. In Group A platelets count was higher than in the untreated control Group on post-CCl4-infusion day 2nd (p = 0.02) and 3rd (p=0.001). Also The trend of BM as transaminases was higher in the non-treated Group B, GOT (p=0.002), GPT (p<0.0001). Pathology examination showed greater grade of hepatocellular necrosis, and neutrophilic infiltration in Group B (p=0.02). MSCs infusion determined a less aggressive picture of hepatic damage.

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BASSI, Elena. "Utilizzo di Nanoparticelle per il trasporto di Molecole Antisenso nel modello murino di Distrofia Muscolare di Duchenne." Doctoral thesis, Università degli studi di Ferrara, 2011. http://hdl.handle.net/11392/2388828.

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Duchenne Muscular Dystrophy (DMD) is an X-linked severe neuromuscular disease, mainly caused by mutations that disrupt the mRNA reading frame resulting in the absence of dystrophin protein in skeletal and cardiac muscles. Currently the most promising therapeutic approach, defined as exon-skipping, is based on the use of antisense oligonucleotides (AONs) to recognize specific RNA sequences that induce the exclusion of target exon from the mRNA and the restoration of the frame, allowing the synthesis of a functional protein. One of the main difficulties of this therapeutic approach is to obtain protection from degradation and transport of the AONs to the target tissues. Therefore, to obtain consistent results, high doses of AONs are needed, which are, however, a potential source of adverse reactions to life-long therapies as in DMD. Our research group is testing nanoparticles as a new delivery system for AONs with phosphorothioate backbone (2'-O-methyl-phosphorothioate 2'OMePs) in the mouse model of muscular dystrophy (mdx mice). These nanoparticles consist of an inert biocompatible material (polymethylmethacrylate) with covalently linked cationic groups, which allow the electrostatic bond with the antisense molecules. In an initial pilot experiment, we used nanoparticles of about 500 nm of diameter and we have proved that they are able to bind and release the AONs 2'OMePs, leading to the restoration of dystrophin expression in mdx mouse muscles. Despite the positive results obtained, these nanoparticles have a low capacity to adsorb on the surface AONs, and their diameter does not allow for intravenous administration. In subsequent experiments we therefore used nanoparticles of smaller size (about 130 nm of diameter) but with a 5 times higher adsorption capacity. We treated systemically mdx mice with low doses (52,5 mg AON / kg) of nanoparticle-AON complexes, and we analyzed the mice 1 and 12 weeks after the end of the treatment. At 1 week after the end of the treatment analysis of mRNA (Nested RT-PCR and Real Time-RT-PCR) has allowed us to identify in skeletal muscles and heart the transcript missing the target exon. Protein analysis (Western blot and immunofluorescence) also showed the correct molecular weight of restored dystrophin and proper localization in the sarcolemma of skeletal muscle fibers, cardiac, and arrector pili smooth muscle. At a distance of 12 weeks after treatment the correct transcript was still present in the tissues analyzed, although in lesser quantities, and the protein also detectable by immunofluorescence and Western blot. These results demonstrate that these nanoparticles represent a promising system for AONs delivery, as they are able to: i) ensure the protection of RNA molecules from nucleases, allowing lower doses to produce a measurable effect, ii) spread in target tissues (muscles) where release the adsorbed molecules in a slow and controlled manner with a longer treatment effect.

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Cigognini, D. "ISOLAMENTO, CARATTERIZZAZIONE E TRAPIANTO IN UN MODELLO MURINO DI LESIONE SPINALE DI CELLULE UMANE DEL LIQUIDO AMNIOTICO." Doctoral thesis, Università degli Studi di Milano, 2009. http://hdl.handle.net/2434/62128.

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Nicora, E. "ISOLAMENTO, CARATTERIZZAZIONE E TRAPIANTO IN UN MODELLO MURINO DI LESIONE SPINALE DI CELLULE UMANE DI LIQUIDO AMNIOTICO." Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/151780.

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Since its devastating consequences, spinal cord injury (SCI) has polarized the efforts of many research groups all around the world. Pharmacological approaches aimed at preventing secondary degeneration in spinal cord injury have been recently flanked by cellular methods and stem cells have been studied as a new tool for spinal cord therapy. Latest evidences indicate that stem cells could be a good tool to reduce secondary degeneration in spinal cord injury. Multiple cell types derived from the developing fetus compose the cellular compartment of the amniotic fluid. For instance, epithelioid cells derive from fetal skin and urinary tract, amniotic fluid specific cells come from fetal membranes and trophoblast and fibroblastic cells derivate from fibrous connective tissue and dermal fibroblasts. These cells are able to differentiate in adipogenic, osteogenic, myogenic, endothelial, neurogenic and hepatic lineages, indicating their multipotency. The main goal of this study was the characterization of the cells from the third trimester amniotic fluid obtained from programmed caesarean births (instead of cells usually retrieved from amniocentesis) and test their therapeutic potential in a mouse model of SCI. Different populations of adherent cells were isolated from eleven human amniotic fluids and they were characterized for in vitro proliferation and differentiation potential. The antigenic profile was performed either by immunocitochemistry and citofluorimetric analysis. In particular, four cultures were deeply investigated and, by immunoistochemical analysis, all of them showed the expression of neural markers such as nestin,  tubulin III and GFAP. After citofluorimetric analysis, the samples showed a noticeable expression of adult mesenchymal markers (CD146+, CD73+, CD105+, CD90+) directed to the muscle-neural lineage (CD146+, NG2+, CD56+) (#3.5, #3.6 and #9.1); one of them also expressed CD117 (#3.6); culture #1.1, instead, showed a mesenchymal phenotype directed to the perivascular lineage (CD146+, CD90+, CD73+). From morphological point of view we were able to identify a new sub population of small cell spindle like shaped which were highly representative in #9.1 culture. We decided to use four populations (#3.6, #3.5, #1.1, #9.1) to transplant spinal cord injured mice. One week after transplantation immunosuppressed animals were intravenously injected with cells or PBS (controls) and motor recovery of the transplanted animals was studied for other 28 days by open field analysis. The animals transplanted with culture 3.5, 3.6 showed a significant motor recovery than animals treated with PBS only; animals transplanted with cultures 1.1 and #9.1, instead, didn’t show any significant enhanced performance than PBS treated animals. We tried then to investigate the reasons of these different results and, after histological analysis, we noticed that cultures 3.6 and #3.5 (the “therapeutic” lines) induced a better preservation of the myelin in the ventral white matter within the lesion site than PBS animals. Moreover, in these animals we could appreciate an increased angiogenesis (by lectin staining) in the peri-injured area, one month after lesion, in transplanted animals compared to the controls. It was also possible to find transplanted cells at the lesion site and in a region of 4 mm rostrally to the injured area. An additional intriguing element of the story is that the therapeutic lines all showed the highest expression levels of the common marker NG2, that seems to play a crucial role in the developing and mature central nervous system but also in the process of angiogenesis. For this reason, we are now investigating the expression levels of HIF and VEGF at the site of injury by Real Time PCR and we are now planning to depict the hypoxia cascade modifications in our model to understand if transplanted cells could, by this pathway, play a role in the myelin preservation. This could be the physiological phenomenon responsible of the behavioral improvements observed.

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Focarelli, M. L. "GENERAZIONE DI CELLULE STAMINALI PLURIPOTENTI INDOTTE E LORO CORREZIONE IN VITRO IN UN MODELLO MURINO DI OSTEOPETROSI." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/231159.

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ABSTRACT The induced pluripotent stem cells (iPSc) entrance in the stem cell landscape has given the scientific community a novel approach for studying human diseases and a new promising tool for regenerative medicine. iPSc generation from patients affected by genetic diseases could allow their site-specific genetic correction followed by differentiation and autologous transplantation for disease cure. Infantile malignant osteopetrosis is a life-threatening recessive bone disease caused by a mutation in the TCIRG1 gene, which severely affects osteoclasts resorbing activity. The resulting increased bone density causes severe growth retardation, thickened bones, and reduced medullary cavity, symptoms recapitulated by the oc/oc mouse. Hematopoietic stem cell (HSC) transplantation is the unique possible treatment, however the chance of cure is strongly limited by the need for a matched donor. Therefore, patients should benefit from the generation of corrected autologous HSCs for a novel approach to therapy. The aim of the present thesis was to generate iPSc from murine wt and affected fibroblasts, to correct the TCIRG1 genetic mutation, to differentiate iPSc into the hematopoietic lineage including HSCs, and to transplant them in vivo to revert the oc/oc phenotype. To generate iPSc lines, as delivery system for the reprogramming genes Oct4, Sox2 and Klf4 we employed a third generation polycistronic lentiviral vector, excisable from the host genome by the Cre recombinase. After reprogramming, iPS clones with low vector copy number and normal numerical distribution of chromosomes were chosen, treated with Cre recombinase and sub-cloned to select lines without integrated vectors. Pluripotency of the obtained iPSc was tested by teratoma formation assay, embryonic germ layers in vitro differentiation, and expression of pluripotency markers through immunocytochemistry and real time PCR. Karyotype analyses showed the presence of normal sets of chromosomes. Importantly, iPSc were successfully derived from oc/oc fibroblasts, and subsequently corrected through homologous recombination upon transfection with a BAC containing wt TCIRG1. In conclusion, with our studies we will provide a proof of principle for the future clinical use of a new tool to treat osteopetrosis and potentially other genetic blood disorders.

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Horne, Jacqueline Avril. "Mathematical modelling of soft callus formation in early murine bone repair." Thesis, Queensland University of Technology, 2013. https://eprints.qut.edu.au/63823/1/Jacqueline_Avril_Horne_Thesis.pdf.

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Fracture healing is a complicated coupling of many processes. Yet despite the apparent complexity, fracture repair is usually effective. There is, however, no comprehensive mathematical model addressing the multiple interactions of cells, cytokines and oxygen that includes extra-cellular matrix production and that results in the formation of the early stage soft callus. This thesis develops a one dimensional continuum transport model in the context of early fracture healing. Although fracture healing is a complex interplay of many local factors, critical components are identified and used to construct an hypothesis about regulation of the evolution of early callus formation. Multiple cell lines, cellular differentiation, oxygen levels and cytokine concentrations are examined as factors affecting this model of early bone repair. The model presumes diffusive and chemotactic cell migration mechanisms. It is proposed that the initial signalling regime and oxygen availability arising as consequences of bone fracture, are sufficient to determine the quantity and quality of early soft callus formation. Readily available software and purpose written algorithms have been used to obtain numerical solutions representative of various initial conditions. These numerical distributions of cellular populations reflect available histology obtained from murine osteotomies. The behaviour of the numerical system in response to differing initial conditions can be described by alternative in vivo healing pathways. An experimental basis, as illustrated in murine fracture histology, has been utilised to validate the mathematical model outcomes. The model developed in this thesis has potential for future extension, to incorporate processes leading to woven bone deposition, while maintaining the characteristics that regulate early callus formation.

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ZOCCHI, LOREDANA. "Studio funzionale della transglutaminasi 3 e di p63 nel differenziamento epidermico mediante l'uso di modelli murini." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2007. http://hdl.handle.net/2108/382.

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RIASSUNTO BREVE Il differenziamento epidermico prevede la migrazione dei cheratinociti dallo strato basale dell’epidermide a quelli sopra basali fino al raggiungimento dello strato più superficiale dove formano l’involucro corneo. Durante questo processo i futuri corneociti subiscono una serie di cambiamenti nella loro struttura e tali modifiche sono mediate dagli enzimi transglutaminasi. Le transglutaminasi (TGasi) rappresentano una famiglia di enzimi funzionalmente e strutturalmente correlati che catalizzano la reazione di trasferimento acilico Ca2+-dipendente tra il gruppo γ- carbossiamidico di residui di glutammina e il gruppo amminico di varie ammine primarie. In questo modo le transglutaminasi catalizzano la formazione di legami crociati (cross-linking) fra proteine che compongono l’involucro corneo. Quattro sono le TGasi espresse a livello epidermico (TGasi 1, TGasi 2, TGasi 3 e TGasi 5) e tutte, esclusa la TGasi 1, mostrano anche un’attività GTPasica. Nello strato basale dell’epidermide svolge invece una funzione molto importante il gene p63. Quest’ultimo è un membro della famiglia di p53, la cui trascrizione da due differenti promotori origina due diverse proteine:TAp63 che contiene un dominio di transattivazione all’N-terminale e ∆Np63che non lo possiede. Tale dominio è importante per l’attivazione dei geni bersaglio che presentano un motivo di legame per p63 di conseguenza TAp63 è considerato l’attivatore trascrizionale dei geni indotti durante il differenziamento, mentre l’espressione di ∆Np63 potrebbe essere correlata con il mantenimento dell’attività proliferante delle cellule staminali epidermiche localizzate nella membrana basale. Ognuna di queste due isoforme va inoltre incontro a splicing alternativo al C-terminale originando tre differenti varianti di splicing:α, β, γ. Topi p63-/- presentano severe anomalie nella pelle e negli arti. Lo scopo di questo progetto di ricerca è sia quello di fornire nuove conoscenze sul ruolo svolto dalla TGasi 3 durante il differenziamento epidermico che quello di individuare nuovi geni la cui attività risulta essere regolata da p63. Per quanto riguarda la TGasi 3 abbiamo deciso di realizzare topi knock-out condizionali attraverso l’utilizzo della ricombinasi Cre e dei siti loxP. Tale sistema consente infatti l’eliminazione tessuto-specifica dell’espressione del gene bersaglio. Durante gli esperimenti preliminari per la creazione di tali topi abbiamo inoltre scoperto due nuove varianti di splicing per il gene TGM3. La prima, denominata ∆6∆7, isolata nel topo manca della cisteina catalitica e sia l’attività di cross-linking che quella GTPasica risultano essere compromesse. La variante ∆9∆10, isolata nell’uomo, pur conservando intatta la triade catalitica presenta una bassa attività transamidasica, mentre l’attività GTPasica risulta essere persa. Nella seconda parte di questo lavoro di tesi abbiamo invece identificato un nuovo gene bersaglio denominato Scotin, la cui espressione risulta essere regolata positivamente in vitro dall’isoforma TA, ma non da ∆N. L’espressione di Scotin è localizzata nel reticolo endoplasmatico e nell’involucro nucleare. Questo gene è inoltre in grado di indurre apoptosi attraverso l’induzione di stress del reticolo endoplasmatico. Esperimenti condotti su colture epiteliali primarie (murine ed umane) hanno evidenziato come l’espressione di Scotin durante il differenziamento sia accompagnata, come atteso, dall’incremento dell’isoforma TA e dalla dimuzione di ∆N. In conclusione i nostri dati hanno portato all’isolamento di Scotin come nuovo gene bersaglio indotto da p63 durante il differenziamento epidermico. Tale meccanismo attiva la risposta allo “stress” da parte del reticolo endoplasmatico,di cui Scotin è un induttore, correlando così per la prima volta queste due differenti vie.
SHORT ABSTRACT Epidermal differentiation begins with the migration of keratinocytes from the basal layer and ends with the formation of the cornified layer. Cell proliferation, differentiation and death occur sequentially and each process is characterized by the expression of specific proteins. Transglutaminases (TGases) play an important role during the differentiation program. These enzymes catalyse post-translational modifications of proteins by the formation of isopeptide bonds (a cross-linking reaction). The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation. In mammals, nine distinct TGase isoenzymes have been identified at the genomic level. Four TGases are expressed in the epidermis (TGase 1, TGase 2, TGase 3 and TGase 5) and the last three TGases show also a GTPase activity. TGM1-/- knock-out mice show severe epidermal abnormalities and they dye soon after birth, while TGM2 -/- knock-out mice dont’show any epidermal abnormality. During the complex mechanism that leads to epidermis formation an important role is played by the action of p63. This transcription factor, belonging to the p53 gene family, is transcribed from two different promoters, generating two classes of proteins: one containing an N-terminal transactivation domain (TA) and another that lacks this domain (∆N). The first isoform is considered the transcriptional inducer whereas ∆N could exert a role of dominant negative. In addition alternative splicing at the 3’ end of both transcripts (TA and ∆N) generates three different C-terminal splicing variants: α, β and γ. Mice p63-/- show severe limb, cranofacial and skin defects and die soon after birth. The aim of this work is to acquire new knowledges in the complex mechanism that leads to epidermal differentiation thanks to the investigation of TGase 3 and p63 function during the skin process formation. Regarding TGase 3 we plan to create conditional knock-out mice for this ezyme using the Cre/loxP system. During our work we isolated also two novel splicing variants for the TGM3 gene. The first one, isolated in mouse, lacks exons six and seven (∆6∆7), while the second one, isolated in NHEK cells, lacks of exons nine and ten (∆9∆10). The experiment demonstrated that the ∆9∆10 is at least 4 times less active than the wild type one, while ∆6∆7 is inactive because it lacks of the catalytic cysteine Regarding to the GTPase, it is lost in both splicing variants because several residues, that are important for the creation of the GTP pocket, are missed. In the second part of this work, we isolated a new p63-target gene: Scotin. This gene was previously isolated as a p53-inducible proapoptotic gene and the protein is located in the endoplasmic reticulum and in the nuclear membrane. Scotin expression is induced in response to endoplasmic reticulum (ER) stress in a p53 dependent or independent manner and this event lead to apoptosis. RT-qPCR showed that Scotin transcript is positively regulated by TAp63α in vitro, while ∆N isoform inhibits its expression. Western blot experiments, performed on differentiating mouse and human primary keratinocytes, revealed Scotin upregulation following the differentiation program. Immunofluorescence performed on mouse new born skin sections demontrated that Scotin expression is located in the suprabasal layer of epidermis where TAp63, but not ∆Np63 is expressed. In conclusion our data lead to isolation of a new p63 target gene induced during the epithelial differentiation program, a complex process that also involves endoplasmic reticulum stress induction.

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Ferrari, L. L. "STUDIO DEL RUOLO DELLA PROTEINA PRIONICA NELLA REGOLAZIONE DEL SONNO MEDIANTE L'UTILIZZO DI MODELLI MURINI TRANSGENICI." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/229547.

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Introduction: an inherited form of Creutzfeldt-Jakob disease (CJD) is linked to the D178N/V129 mutation in the prion protein (PrP) gene. CJD is usually characterized by motor disorders, cognitive impairment and electroencephalographic alterations but recently sleep modification have been described. Moreover a role for PrP in sleep has been proposed on the basis of data collected in Knock-out mice. We generated a new mouse model of CJD (Tg(CJD)) that express the murine homologue of the D178N/V129 human mutation. We used as control mice Knock-out (KO) for the PrP, mice that over-express the normal form of the PrP Tg(WT) and Wt mice. Goals: 1) the characterization of Tg(CJD) mice 2) the study of the evolution during aging of sleep and EEG patterns in our CJD model and in KO and Tg(WT) mice 3) the study of response to sleep deprivation in all our strains. Materials: mice were anesthetized and instrumented for chronic EEG recordings, using standard techniques. Body movements were detected by means of an infrared sensor. Mice were individually housed in soundproof rooms, and maintained on a 12 hours light:dark cycle, at an ambient temperature between 23 and 24 °C. The study of the evolution of sleep patterns over time was conducted using animals of 3 different ages for each strain (6, 12 and 18 months). Sleep deprivation was carried out by means of gentle handling and lasted 6 hours starting from the beginning of light phase. Results: 18 months old Tg(CJD) mice showed a striking reduction in the amount of REM sleep compared to control strains. They also showed abundance of EEG alterations that remind those described in humans and motor alterations typical of CJD. The decrease in REM sleep is already present at 12 months of age in Tg(CJD) mice. Tg(CJD) and KO mice showed a flattening of the circadianity of sleep/wake cycle at 12 and 18 months of age. Sleep deprivation analysis showed a loss of rebound of REM sleep in mice with alterations of PrP. Conclusions: We propose that Tg(CJD) mice establish the first transgenic animal model of a genetic prion disease recapitulating cognitive, motor and neurophysiological abnormalities of the human disorder. We also propose that REM sleep decrease in Tg(CJD) mice is an early sign of CJD and could be a new tool for rapid diagnosis of this disease.

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Mantovani, S. "Modelli murini transgenici di malattie da prioni per lo studio del ruolo fisiopatologico della proteina prionica." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/150216.

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Inherited prion diseases are linked to mutations in the prion protein (PrP) gene that are thought to favor the conformational conversion of PrP into a pathogenic misfolded isoform. Each mutation is associated with a distinct disease phenotype, which is profoundly affected by the Met/Val polymorphism at codon 129 in the PrP gene. The most clear example of the phenotypic heterogeneity determined by the Met/Val polymorphism is represented by fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD178), two clinically and neuropathologically distinct diseases linked to the D178N mutation in the gene encoding PrP; D178N/M129 segregates with FFI, while D178N/V129 is associated with CJD. We have engineered transgenic (Tg) mice to express mouse PrP (moPrP) homologues of the human D178N/V129 and D178N/M129 mutations. cDNAs encoding D177N/V128 and D177N/M128 moPrP were cloned under the moPrP promoter. After microinjection into fertilized eggs from C57BL/6J X CBA/J parental mice (random transgenesis), we identified Tg mice which were backcrossed to PrP knockout (C57BL/6J/Prnp0/0) mice. We have obtained vary Tg founders: carrying D177N/V128 or D177/M128 PrP. The Tg copy number of the founders was determined by quantitative PCR, and the expression level of Tg PrP in the brain of hemizygous mice was determined by quantitative Western blotting. We found that all CJD mice expressing the transgene at a level higher than the physiological one develop motor dysfunction; mice expressing the FFI mutation showed motor abnormalities only when the expression of the transgene was higher than 1X. We have analyzed biochemical characteristics of mutated PrPs finding that they are similar to those observed in humans carrying the homologous mutations. Ultrastructural analysis showed enlarged ER in CJD neurons and an abnormal onion-shaped Golgi in the FFI neurons. This characteristic could be the basis of the phenotypic heterogeneity between CJD and FFI and will be an important feature to study. The transgenic mice, good models of the human pathologies, could be use as a tool to study the physiological role of PrP and to test possible therapeutical approaches.

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30

Levison, Scott. "Characterization of a murine model of colitis." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/characterization-of-a-murine-model-of-colitis(4b966667-a763-4bcc-8896-7a157cf3d3dd).html.

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Inflammatory bowel disease (IBD) represents a complex spectrum of gastrointestinal diseases. Incorporating Crohn's disease (CD) and ulcerative colitis (UC), IBD is characterised by recurrent and chronic inflammation, significant morbidity, and an increasing global prevalence. Scientific advances regarding the aetiology, pathogenesis and treatment of these relapsing immune-mediated diseases have developed in parallel to the study of experimental models of intestinal inflammation. The correlation of phenotype, histology and immune response with mucosal gene expression, permits the investigation of induced pathology for human translation. Trichuris muris (mouse whipworm) infection induces chronic colitis in susceptible strains (e.g. AKR). Chronic disease displays both a polarised CD4+ T-helper1 (TH1) immune response and histological, transmural colonic inflammation. Conversely, resistant mouse strains (e.g. BALB/c) exhibit transient infection and inflammation which quickly resolves under a presiding TH2 response. Work presented in this thesis investigates differential gene expression and biological pathways central to colonic outcome, and the genetic basis of chronic T. muris-induced colitis. This thesis demonstrates that the phenotypic and transcriptional profile of the T. muris model shared many similarities to widely used experimental models of colonic inflammation and to human IBD. Mice susceptible to chronic colonic inflammation displayed functional gene expression differences to those of resistant mice, including the up-regulation of pro-inflammatory, apoptosis and chemokine signalling pathway genes. Cellular homeostasis pathways and tight junction molecules were conversely down-regulated. Infected AKR demonstrated predominant TH1/ TH17 transcriptional activity, presenting this model as a platform to examine biological commonalities among chronic colitides. A Quantitative Trait Locus (QTL) study, performed by crossbreeding resistant and susceptible strains to T. muris infection, then identified key autosomal loci linked to chronic disease. Genes associated with known biological pathways, differential gene expression, and parental strain single nucleotide polymorphisms provided a novel and powerful strategy to reduce the number of candidate genes for further analysis. Of 7 T. muris (TM) QTL identified, 3 displayed overlap with other murine studies of parasite susceptibility. A separate locus, TM3, demonstrated overlap with published QTL in 3 unrelated experimental models of colitis and overlaid the Cdcs1 locus. TM3 possessed 33 significantly transcribed polymorphic genes (e.g. Ptpn22, Fcgr1, Rorc, Vcam1 and Vav3). Phenotypic pathway analysis, text mining and time-course qPCR all highlighted Vav3 (Human 1p13.3, murine Chr3 101.9 MB) as a key biological candidate in colitis susceptibility. As a final test of relevance to human disease, clinically proven IBD medications were administered to AKR mice post-T. muris infection, at a time-point when chronic disease was well established. Anti-TNFα Ab and corticosteroid therapy were shown to suppress TH1-driven experimental colitis, without affecting parasitic infection. Additionally, anti-TNFα Ab treatment was found to reduce pro-inflammatory macrophages yet preserve regulatory alternatively activated macrophage numbers within the colon. A previously unreported finding. Sharing biological commonalities with human CD, Trichuris muris colitis represents an advanced and tractable murine model for understanding the pathobiological mechanisms of chronic inflammatory disease. The key facet of this model is that despite identical injury some inbred mouse strains develop chronic colitis whilst others recover quickly and fully. The identification of differential mechanisms which govern outcome and a new platform to investigate novel targets of disease and disease therapy has implications for gastrointestinal inflammatory diseases and mucosal immunology.

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31

Gültner, Sandra [Verfasser]. "Mechanismen der Neurodegeneration in murinen Modellen / Sandra Gültner." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2011. http://d-nb.info/1026264723/34.

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32

COSTANTINI, ROSSELLA. "generazione e caratterizzazione di due differenti modelli murini di condrodisplasie causate da difetti nella biosintesi dei proteoglicani." Doctoral thesis, Università degli studi di Pavia, 2019. http://hdl.handle.net/11571/1248469.

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Among the recent classification of genetic skeletal disorders, there is a cluster of diseases such as, Desbuquois dysplasia, diastrophic dysplasia that display common features including short stature, dysplasia of skeletal elements, and congenital joint dislocations. At biochemical level all these disorders are characterized by defects in proteoglycans (PGs) that represent one of the most important components of the cartilage extracellular matrix. alterations of PG synthesis, due to mutations in genes encoding for enzymes or proteins involved in the process, lead to the onset of genetic diseases affecting cartilage.The molecular basis of many skeletal disorders are poorly understood, thus mechanistic studies using an in vivo approach is necessary to investigate the role of the disease gene in the disorder.Among this scenario, this work has been focused, using in vivo models, on the study of two chondrodysplasias in which proteoglycan synthesis is impaired: Chondrodysplasia with joint dislocation gPAPP type and Desbuquois dysplasia type 1. Chondrodysplasia with joint dislocation gPAPP type is a recessive osteochondrodysplasia caused by mutations in IMPAD1 gene that encodes for a Golgi resident adenosine 3‟,5‟-bisphosphate phosphatase crucial for proteoglycan sulfation. Desbuquois dysplasia type 1 is a rare chondrodysplasia caused by mutations in the CANT1 gene encoding for calcium-activated nucleotidase 1, a Golgi protein that preferentially hydrolyzes UDP.The molecular knowledge of the two disorders is different; thus, in this thesis the two models have been used to pursue different objectives: I) the generation and characterization of a conditional knock-in mouse as model for the study of the chondrodysplasia with joint dislocations gPAPP type, II) the validation of a Cant1 knock-out mouse (dbqd mouse) as an animal model of Desbuquois dysplasia type 1 and the study of Cant1 role in PG biosynthesis. In this work, we have tested an innovative strategy called “the Cre-mediated genetic switch” in vivo, with the aim to generate a Impad1 conditional knock-in mouse for a missense mutation.The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* containing the point mutations, were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon.The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate Impad1 knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation.Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Expression studies demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. the skeletal phenotypes were not caused by the missense mutations, but by aberrant splicing.The dbqd mouse, an animal model of human Desbuquois dysplasia type I, was previously generated by our research group. The clinical phenotype of Cant1 knock-out mice showed the same typical features of patients with Desbuquois dysplasia type 1 confirming the dbqd mouse as an animal model of the human disorder. Biochemical studies highlighted the contribution of CANT1 in PG synthesis. GAG synthesis was reduced and chains were shorter and oversulfated

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33

COSTANTINI, ROSSELLA. "generazione e caratterizzazione di due differenti modelli murini di condrodisplasie causate da difetti nella biosintesi dei proteoglicani." Doctoral thesis, Università degli studi di Pavia, 2019. http://hdl.handle.net/11571/1248449.

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Abstract:

Among the recent classification of genetic skeletal disorders, there is a cluster of diseases such as, Desbuquois dysplasia, diastrophic dysplasia that display common features including short stature, dysplasia of skeletal elements, and congenital joint dislocations. At biochemical level all these disorders are characterized by defects in proteoglycans (PGs) that represent one of the most important components of the cartilage extracellular matrix. alterations of PG synthesis, due to mutations in genes encoding for enzymes or proteins involved in the process, lead to the onset of genetic diseases affecting cartilage.The molecular basis of many skeletal disorders are poorly understood, thus mechanistic studies using an in vivo approach is necessary to investigate the role of the disease gene in the disorder.Among this scenario, this work has been focused, using in vivo models, on the study of two chondrodysplasias in which proteoglycan synthesis is impaired: Chondrodysplasia with joint dislocation gPAPP type and Desbuquois dysplasia type 1. Chondrodysplasia with joint dislocation gPAPP type is a recessive osteochondrodysplasia caused by mutations in IMPAD1 gene that encodes for a Golgi resident adenosine 3‟,5‟-bisphosphate phosphatase crucial for proteoglycan sulfation. Desbuquois dysplasia type 1 is a rare chondrodysplasia caused by mutations in the CANT1 gene encoding for calcium-activated nucleotidase 1, a Golgi protein that preferentially hydrolyzes UDP.The molecular knowledge of the two disorders is different; thus, in this thesis the two models have been used to pursue different objectives: I) the generation and characterization of a conditional knock-in mouse as model for the study of the chondrodysplasia with joint dislocations gPAPP type, II) the validation of a Cant1 knock-out mouse (dbqd mouse) as an animal model of Desbuquois dysplasia type 1 and the study of Cant1 role in PG biosynthesis. In this work, we have tested an innovative strategy called “the Cre-mediated genetic switch” in vivo, with the aim to generate a Impad1 conditional knock-in mouse for a missense mutation.The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* containing the point mutations, were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon.The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate Impad1 knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation.Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Expression studies demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. the skeletal phenotypes were not caused by the missense mutations, but by aberrant splicing.The dbqd mouse, an animal model of human Desbuquois dysplasia type I, was previously generated by our research group. The clinical phenotype of Cant1 knock-out mice showed the same typical features of patients with Desbuquois dysplasia type 1 confirming the dbqd mouse as an animal model of the human disorder. Biochemical studies highlighted the contribution of CANT1 in PG synthesis. GAG synthesis was reduced and chains were shorter and oversulfated

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34

COSTANTINI, ROSSELLA. "generazione e caratterizzazione di due differenti modelli murini di condrodisplasie causate da difetti nella biosintesi dei proteoglicani." Doctoral thesis, Università degli studi di Pavia, 2019. http://hdl.handle.net/11571/1248390.

Full text

Abstract:

Among the recent classification of genetic skeletal disorders, there is a cluster of diseases such as, Desbuquois dysplasia, diastrophic dysplasia that display common features including short stature, dysplasia of skeletal elements, and congenital joint dislocations. At biochemical level all these disorders are characterized by defects in proteoglycans (PGs) that represent one of the most important components of the cartilage extracellular matrix. alterations of PG synthesis, due to mutations in genes encoding for enzymes or proteins involved in the process, lead to the onset of genetic diseases affecting cartilage.The molecular basis of many skeletal disorders are poorly understood, thus mechanistic studies using an in vivo approach is necessary to investigate the role of the disease gene in the disorder.Among this scenario, this work has been focused, using in vivo models, on the study of two chondrodysplasias in which proteoglycan synthesis is impaired: Chondrodysplasia with joint dislocation gPAPP type and Desbuquois dysplasia type 1. Chondrodysplasia with joint dislocation gPAPP type is a recessive osteochondrodysplasia caused by mutations in IMPAD1 gene that encodes for a Golgi resident adenosine 3‟,5‟-bisphosphate phosphatase crucial for proteoglycan sulfation. Desbuquois dysplasia type 1 is a rare chondrodysplasia caused by mutations in the CANT1 gene encoding for calcium-activated nucleotidase 1, a Golgi protein that preferentially hydrolyzes UDP.The molecular knowledge of the two disorders is different; thus, in this thesis the two models have been used to pursue different objectives: I) the generation and characterization of a conditional knock-in mouse as model for the study of the chondrodysplasia with joint dislocations gPAPP type, II) the validation of a Cant1 knock-out mouse (dbqd mouse) as an animal model of Desbuquois dysplasia type 1 and the study of Cant1 role in PG biosynthesis. In this work, we have tested an innovative strategy called “the Cre-mediated genetic switch” in vivo, with the aim to generate a Impad1 conditional knock-in mouse for a missense mutation.The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* containing the point mutations, were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon.The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate Impad1 knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation.Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Expression studies demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. the skeletal phenotypes were not caused by the missense mutations, but by aberrant splicing.The dbqd mouse, an animal model of human Desbuquois dysplasia type I, was previously generated by our research group. The clinical phenotype of Cant1 knock-out mice showed the same typical features of patients with Desbuquois dysplasia type 1 confirming the dbqd mouse as an animal model of the human disorder. Biochemical studies highlighted the contribution of CANT1 in PG synthesis. GAG synthesis was reduced and chains were shorter and oversulfated

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35

CAVAGNINI, MIRIAM. "STUDIO IN VIVO DEL RUOLO DEGLI iMSN NELLA CODIFICA DELL’ATTIVITÀ MOTORIA IN MODELLI MURINI MEDIANTE CALCIUM IMAGING." Doctoral thesis, Università degli studi di Modena e Reggio Emilia, 2022. http://hdl.handle.net/11380/1273445.

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Lo striato, il principale nucleo dei gangli della base, è costituito per il 95% da Medium Spiny Neurons (MSN) GABAergici. Gli MSN si suddividono in neuroni della via diretta e della via indiretta. Gli MSN della via diretta (dMSN), esprimono i recettori per la dopamina D1 (D1R), mentre gli MSNs della via indiretta (iMSN) esprimono i recettori dopaminergici D2 (D2R) e per l’adenosina A2A (A2AR) [Gerfen 1990; Gerfen 1992; Gong 2003]. Secondo l’interpretazione classica, queste due vie esercitano un effetto opposto nella regolazione dell’attività motoria: la via diretta promuove l’attivazione della corteccia coinvolta nella codifica dei piani motori, e di conseguenza promuove l’avvio del movimento, mentre la via indiretta inibisce l’attività corticale e il movimento [Albin 1989; Alexander and Crutcher, 1990; DeLong 1990]. Studi più recenti hanno modificato questa interpretazione e dimostrato che entrambe le vie risultano essere attive durante l’avvio del movimento, sebbene con alcune differenze [Bonnavion 2019; Tecuapetla 2016]. Ad oggi, mentre il ruolo di queste due vie è globalmente compreso, gli specifici meccanismi cellulari e la loro interazione in relazione al controllo motorio rimangono parzialmente sconosciuti. Tecnologie moderne, fra cui il calcium (Ca2+) imaging, possono essere applicate per meglio comprendere il coinvolgimento di queste due vie. Quando vi è un’intensa attività cellulare, il Ca2+ entra nelle diramazioni dendritiche e nei corpi cellulari, incrementando la concentrazione intracellulare. Queste fluttuazioni del Ca2+ possono essere monitorate mediante indicatori del Ca2+, come il GCaMP. Lo scopo di questo lavoro di tesi è stato quello di investigare il ruolo dello striato nella locomozione murina, in particolare il coinvolgimento degli iMSN mediante tecnica di Ca2+ imaging in vivo. L’attività motoria è stata studiata mediante test comportamentali, mentre l’attività GABAergica degli iMSN è stata investigata dopo somministrazione di anfetamina, una sostanza psicostimolante, a diverse dosi. In questo studio, il circuito della via indiretta è stato visualizzato mediante calcium imaging in vivo attraverso un microscopio associato ad un endoscopio impiantato cronicamente. Come modelli murini sono stati utilizzati topi A2AGCaMP6S esprimenti GCaMP negli iMSN. L’analisi comportamentale ha rivelato che l’iniezione acuta intraperitoneale di anfetamina alla dose di 3 mg/kg incrementa significativamente la locomozione, con un picco a 15 min dopo l’iniezione, mentre la dose di 1 mg/kg riduce lievemente l’attività motoria, probabilmente a seguito dell’induzione di stereotipie. L’analisi dei transienti del calcio negli iMSN ha evidenziato una decrescita marcata del numero di iMSN attivi e un incremento della durata degli spike a seguito della somministrazione di anfetamina a 3 mg/Kg. La stessa tendenza è stata mantenuta a seguito dell’iniezione ad 1 mg/kg. Inoltre, comparando i dati ricavati dal Ca2+imaging e i dati comportamentali, è emerso come gli iMSN codifichino il movimento, confermando precedenti studi [Parker 2018]. L’analisi dimostra che l’attività degli iMSN aumenta circa 0,5-0,7 secondi prima dell’avvio del movimento e decresce circa 1-0,5 secondi prima dell’arresto del movimento. Tuttavia, non è ancora chiaro se il movimento o l’assenza di movimento siano codificati da cambiamenti di attività distribuiti casualmente nella popolazione di iMSN o se determinate sottopopolazioni di iMSN modifichino con regolarità la loro attività specificatamente durante uno dei due stati di attività motoria. Questo studio ha portato nuovi elementi interpretativi relativi alla complessità dell’attività degli iMSN, portando supporto all’ipotesi che sostiene la necessità di un’attivazione della via indiretta all’avvio del movimento.
Striatum is the main input nucleus of the basal ganglia and 95% of its neurons are GABAergic Medium Spiny Neurons (MSNs). MSNs are subdivided into neurons of the direct and indirect pathway. The direct pathway consists of MSNs (direct MSNs, dMSNs) mainly expressing dopamine D1 receptors (D1R), whereas MSNs of the indirect pathway (iMSNs) express dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AR) [Gerfen et al., 1990; Gerfen et al., 1992; Gong et al. 2003]. According to the classical model, these two pathways exert an opposite effect on movement regulation: the direct pathway promotes activity of the cortex that codes a motor plan, and therefore movement initiation, while the indirect pathway inhibits cortical activity and movement [Albin et al., 1989; Alexander and Crutcher, 1990; DeLong 1990]. Recent studies have challenged this interpretation and demonstrated that both pathways are co-activated during movement initiation but with differential activities [Bonnavion et al., 2019; Tecuapetla et al., 2016]. Yet, while the role of these two pathways is globally understood, cell-specific mechanisms and their interaction in relation to movement control are only partially known. Modern technological advances such as calcium (Ca2+) imaging techniques can be applied to directly observe the neuronal activity in these two pathways. During periods of heightened neural activity, Ca2+ flows into the dendritic branches and cell bodies of neurons, increasing its intracellular concentrations. These activity-dependent fluctuations in intracellular Ca2+ can be monitored by expressing a Ca2+ indicator, such as GCaMP, into the neuronal population of interest. The aim of this study was to investigate the role of murine striatum in locomotion, and in particular the involvement of iMSNs, using recent in vivo Ca2+ imaging technologies. Locomotor activity was tested by behavioural experiments and the GABAergic iMSN activity was stimulated by a psychostimulant drug, d-Amphetamine, at different doses. In this study, in vivo calcium imaging was used to visualize striatal neural circuit dynamics of the indirect pathway during mouse behaviour with head-mounted microscopes and chronically implanted endoscopes. A2AGCaMPs mice expressing GCaMP in iMSNs were used as an animal model. Behavioural analysis showed that acute intraperitoneal injection of d-Amphetamine at 3 mg/kg dose markedly increases locomotor activity with a peak 15 minutes after injection, while a dose of d-Amphetamine at 1 mg/kg modestly decreases locomotor activity, possibly due to the induction of stereotyped behaviour. Traces of iMSN calcium activity were extracted from the imaging data and analysed with custom developed software. The results showed a significant decrease in the average number of active iMSNs and a significant increase in the average spike duration after d-Amphetamine 3 mg/kg injection. At 1mg/kg dose, there was a trend for a significant decrease in the average number of active cells and a significant increase in the average spike duration. Analysis of the acquired datasets showed how iMSNs encode movement, confirming previous results reported by Parker and colleagues [Parker et al, 2018]. In detail, the analysis revealed that iMSN activity rises around 0.5-0.7 seconds before motion onset and falls around 1-0.5 seconds before motion offset, suggesting that increased firing of iMSNs encodes locomotion. However, it is not clear whether movement or rest are encoded by changes in the activity of randomly distributed iMSNs or specific sub-sets of iMSNs change their activity during rest or movement. This study allows us to better understand the complexity of MSN activity, challenging the classical view where the direct and indirect pathway show an opposite pattern of activity during movement.

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CARUSO, Lorenzo. "Nuovi approcci basati su TRAIL ed MSC per la terapia delle malattie oncoematologiche." Doctoral thesis, Università degli studi di Ferrara, 2012. http://hdl.handle.net/11392/2389424.

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TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines, including 18 genes which code 19 transmembrane proteins, strictly correlated, and playing important roles in: cell death regulation, immune response and inflammation. In humans at least 5 different TRAIL receptors have been described belonging to the TNF-R family, each one expressed in different cellular lines. TRAIL-R1 (DR4) and TRAIL-R2 (DR5) transduce apoptotic signals, while TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and osteoprotegerina (OPG) are decoy receptors unable to induce cellular death. Significant levels of TRAIL have been detected in many human tissues, including spleen, prostate, lungs, thymus, kidney, intestine, ovary and, at lower levels, heart, skeletal muscle cells, pancreas, liver, brain and testicle. One of the primary functions of TRAIL is to induce apoptosis in numerous transformed cell lines and cancer cells in vivo, without significant cytotoxicity on normal cells or tissues. With the aim to identify antitumor agents, we have focused our attention on the therapeutic potential of TRAIL. During the last three years, we tested new strategies based on TRAIL in order to induce death selectively in neoplastic cells which have developed resistance to conventional treatment. For this purpose we conducted a set of studies in vitro and in vivo employing recombinant TRAIL. Furthermore our research has concentrated on the therapeutic efficacy of human bone marrow (BM) mesenchymal stem cells (MSC), which are considered the stromal progenitor stem cells within the bone marrow. Our objective was to explore the antitumor activity of these cells and to describe the mechanisms causing this effect. The development of an animal model in SCID (Severe Combined Immunodeficiency) mice of Non-Hodgkin lymphoma (NHL) was necessary in order to investigate the in vivo dissemination of cancer cells and to assess the therapeutic efficacy of recombinant TRAIL and BM-MSC. These models were established by intraperitoneal (i.p.) injection of EBV- Burkitt-type BJAB and EBV+ B lymphoblastoid SKW6.4 cell lines. Xenografts mice were then i.p. injected on the opposite site with either human recombinant TRAIL or BM-MSC at a lymphoma:MSC assessing mice survival after the treatment. The results reported in this paper demonstrate that both treatments reduce the growth of the tumoral masses, with a consequent significantly higher rate of mice survival. Furthermore, similar results were obtained when mesenchimal stem cells, previously embedded in hyaluronan scaffolds to avoid the integration of mesenchymal cells in the fibrovascular cancer network, were implanted in mice. Interestingly, this hyaluronan-embedded MSC exert anti-lymphoma activity by ameliorating hepatic functionality, as demonstrated by measurement of serum ALT/AST levels. In conclusion, our research suggests that both strategies, i.e. rTRAIL and mesenchymal stem cells can exert antitumoral activity and are good candidate for development of new therapies.

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Qesari, Marsela. "Neuropatia enterica in un modello murino di infezione del sistema nervoso enterico con Herpes simplex virus di tipo 1." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423394.

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Herpes simplex virus type 1 infection of murine enteric nervous system leads to gut neuropathy. The functional gastrointestinal disorders (FGIDs) are a heterogeneous group of chronic conditions characterized by disabling symptoms and decreased patients’ quality of life (Corazziari et al., 2004; Talley et al., 2008). Among these diseases, the Irritable Bowel Syndrome (IBS) is the most relevant for the severe prognosis and the high social and economic burden (Drossman, 2006; Fichna and Storr, 2012; Quigley et al., 2012). In clinical practice, IBS is characterized by visceral hypersensitivity and altered bowel function (Larauche et al., 2012). Since the etiology is still poorly understood and no curative treatments are available, nowadays therapy for IBS is palliative and supportive but is notoriously unsatisfactory (Halland and Talley, 2012). Although the underlying pathophysiology of FGIDs is still undefined, there are several evidences suggesting that functional or anatomical alterations of the enteric nervous system (ENS) disrupt gastrointestinal homeostasis determining FGIDs (De Giorgio and Camilleri, 2004; Furness, 2012). Infectious agents, such as neurotropic viruses, have been proposed to infect and disrupt ENS integrity either directly or indirectly eliciting harmful inflammatory responses by the innate and adaptive immune system (Chen et al., 2003; Facco et al., 2008; Selgrad et al., 2009). Among common pathogens the Herpes simplex virus type 1 (HSV-1), shows several interesting features as candidate pathogen involved in FGIDs, being highly prevalent in human populations (Knipe and Cliffe, 2008) and able to infect ENS neurons (Gesser and Koo, 1996; Brun et al., 2010). Our research group has recently established a novel animal model in rodents where a persistent HSV-1 infection in ENS leads to intestinal motor abnormalities with no macroscopic histological damage or clinical signs of disease (Brun et al., 2010). The aim of my Ph.D. research project was to study the mechanisms responsible of the altered intestinal function secondary to HSV-1 infection of mice ENS. At 1-10 weeks after viral administration the presence of HSV-1 infection in the ENS determined time-dependent alterations of enteric neural network architecture, shown by an altered distribution and/or expression of specific neural proteins, such as HuC/D, peripherin e βIII-tubulin, and glial proteins, such as S100β and glial fibrillary acidic protein (GFAP; Qesari et al., 2011). These structural modifications in myenteric plexus were linked to changes in the equilibrium between excitatory and inhibitory neurotransmission, revealed by an increased expression of both enzymes, nitric oxide synthase (nNOS), acetylcholinetransferase (ChAT) and by a modified distribution of the vasoactive intestinal polypeptide (VIP), substance P (SP) and acetylcholinesterase (AChE). The presence of an unbalanced inhibitory/excitatory activity was further confirmed by experiments on tissue cultures from longitudinal muscle with myenteric plexus (LMMP) and on gut contractility of ileum segments, mounted vertically in organ baths. Depolarization-evoked release of [3H]acetylcholine was significantly reduced for most of viral infection time course and in the same time neurally-mediated cholinergic responses to electric field stimulation were significantly reduced in the early and late phases of infection. All these data suggest that HSV-1 infection induces structural alterations and changes in neural excitatory activity and/or neurotransmitters release, all signs of an underlying enteric neuropathy (Qesari et al., 2011). To study the role of innate immunity and of viral replication in the onset of HSV-1 infection-induced gut neurodysfunction, mice deficient for Toll-like receptor 2 (TLR2 KO) and ICP27-null HSV-1 (ICP27 KO), a mutant virus unable to replicate (Uprichard and Knipe, 1996), have subsequently been used. ICP27 KO administration induced anomalies in the ENS only in the early phases of infection to indicate an involvement of viral replication in the onset of enteric neuropathy only in the latest times of infection (Qesari et al., 2012). Recent studies have shown that an interaction between TLR2 and HSV-1 can trigger excessive signaling TLR2-dependent, leading to an excessive inflammation and tissue damage (Soberman et al., 2012). HSV-1 infection in the ENS of TLR2 KO mice induced only a reduction in nNOS positive neurons with no effects on myenteric plexus anatomy and intestinal contractile function. In conclusion, this study demonstrated that HSV-1 infection of mice ENS triggers TLR2-mediated immune responses which are consequently responsible of myenteric plexus structural abnormaities and changes in ileal contractile function. My results also highlight TLR2 as an attractive therapeutic target for an effective modulation of pathogenic immune responses and possibly for the treatment of viral-associated neuropathies.
I disordini funzionali gastrointestinali (DFGI) comprendono un gruppo eterogeneo di malattie croniche i cui sintomi possono essere invalidanti e compromettere la qualità della vita dei pazienti che ne sono affetti (Corazziari et al., 2004; Talley et al., 2008). Tra queste, la sindrome dell'intestino irritabile (Irritable Bowel Syndrome, IBS) è considerata la più rilevante per la gravità della prognosi ed il forte impatto economico-sociale nella popolazione mondiale (Drossman, 2006; Fichna and Storr, 2012; Quigley et al., 2012). Le manifestazioni cliniche dell'IBS includono aumento della sensibilità viscerale ed alterazioni della motilità intestinale (Larauche et al., 2012). Essendo l'eziologia ancora poco conosciuta, a tutt'oggi le terapie disponibili sono per lo più palliative e poco soddisfacenti (Halland and Talley, 2012). Sebbene la patofisiologia dei DFGI non sia ancora stata definita, numerose evidenze scientifiche suggeriscono che alterazioni permanenti o transitorie a carico del sistema nervoso enterico (SNE) possono determinare anomalie funzionali con compromissione dell'omeostasi del tratto gastrointestinale (De Giorgio and Camilleri, 2004; Furness, 2012). Agenti infettivi, come virus neurotropi, sono stati a lungo sospettati di rappresentare uno dei fattori in grado di interferire con l'integrità del SNE determinando alterazioni della rete neuronale sia direttamente che mediante una risposta immunitaria di tipo innato e/o adattativo spesso dannosa per l'ospite (Chen et al., 2003; Facco et al., 2008; Selgrad et al., 2009). L'Herpes simplex virus di tipo I (HSV-1), un virus neurotropo altamente diffuso nella popolazione (Knipe and Cliffe, 2008) e capace di infettare il SNE (Gesser and Koo, 1996; Brun et al., 2010), è stato proposto come un possibile agente eziopatologico coinvolto nell'insorgenza di DFGI. In questo contesto, il nostro gruppo di ricerca ha di recente descritto un nuovo modello sperimentale in roditori nel quale un'infezione persistente nel SNE ad opera dell'HSV-1 causa delle complesse anomalie funzionali in assenza di macroscopici danni istologici o segni di malattia (Brun et al., 2010). Il mio progetto di dottorato ha avuto come obiettivo lo studio dei meccanismi responsabili dell'alterata funzione intestinale secondaria all'infezione del SNE murino da parte dell'HSV-1. L'infezione con HSV-1 ha determinato a 1-10 settimane dalla somministrazione del virus alterazioni tempo-dipendenti dell'architettura della rete nervosa enterica evidenziate da un'alterata distribuzione e/o espressione di specifiche proteine neuronali, quali HuC/D, periferina e βIII-tubulina, e di specifiche proteine gliali, quali S100β e proteina acidica fibrillare gliale (GFAP; Qesari et al., 2011). Tali cambiamenti strutturali a livello del plesso mienterico sono stati accompagnati da variazioni nell'equilibrio tra la neurotrasmissione eccitatoria ed inibitoria, rilevate da un aumento dell'espressione degli enzimi ossido nitrico sintetasi (nNOS), acetilcolintrasferasi (ChAT) e da cambiamenti nella distribuzione del polipetide intestinale vasoattivo (VIP), della sostanza P (SP) e dell'enzima acetilcolinesterasi (AChE). La presenza di uno sbilanciamento dell'attività eccitatoria/inibitoria è stata ulteriormente confermata da studi su culture tissutali di cellule derivate dalla muscolatura longitudinale con annesso il plesso mienterico (LMMP) e di contrattilità su preparati di ileo, montati verticalmente in bagni per organo isolato. La liberazione di acetilcolina triziata in seguito a depolarizzazione è risultata ridotta in quasi tutti i tempi di infezione virale. In parallelo, la risposta colinergica neuronale evocata dalla stimolazione elettrica è risultata significativamente ridotta sia in una fase iniziale che tardiva dell'infezione. Tali risultati suggeriscono che il decorso dell'infezione virale ha indotto sia alterazioni strutturali che di eccitabilità neuronale e/o di rilascio di neurotrasmettitori, segni della presenza di una neuropatia a livello enterico (Qesari et al., 2011). Allo scopo di studiare il ruolo dell'immunità innata e della replicazione virale nell'insorgenza della neurodisfunzione enterica indotta dall'infezione da HSV-1, si sono impiegati topi deficienti del gene che codifica per il recettore Toll-like 2 (TLR2 KO) ed un ceppo di HSV-1 deficiente del gene immediatamente precoce che codifica per la proteina ICP27 (ICP27 KO) essenziale per la replicazione virale (Uprichard and Knipe, 1996). L’infezione con ICP27 KO ha determinato alterazioni del SNE solamente nel primo periodo di infezione, a suggerire un coinvolgimento dei meccanismi di replicazione virale nell'insorgenza della neuropatia nelle fasi tardive dell’infezione (Qesari et al., 2012). Studi recenti hanno evidenziato che l'interazione fra TLR2 e HSV-1 porta allo sviluppo di una risposta immunitaria innata con produzione eccessiva di citochine pro-infiammatorie e conseguente danno tissutale di natura neuroimmunitaria (Soberman et al., 2012). L'infezione del SNE con HSV-1 nei topi TLR2 KO ha prodotto solo una riduzione dei neuroni nitrergici nNOS positivi senza determinare anomalie strutturali del plesso mienterico ed alterazioni della contrattilità intestinale. In conclusione, questa ricerca ha dimostrato che l'infezione del SNE in topi ad opera di HSV-1 determina alterazioni strutturali nel plesso mienterico e funzionali dell'attività contrattile dell'ileo che insorgono in seguito all'attivazione della risposta immunitaria mediata dai TLR2. Alla luce di questi risultati il mio lavoro di ricerca evidenzia i TLR2 come un interessante bersaglio molecolare per modulare le risposte immunitarie innescate da patogeni e possibilmente per il trattamento di neuropatie di origine virale.

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Velazquez, Rogelio Ivan Ortiz. "Efeitos da sobrecarga hemodinâmica na bifurcação aórtica: desenvolvimento de um modelo murino de fadiga estrutural aneurismática." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5151/tde-05052011-141217/.

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INTRODUÇÃO: Evidencia experimental sugere que padrões alterados de fluxo vascular, associados às bifurcações, estão envolvidos no desenvolvimento de lesões aneurismáticas. Pesquisamos os efeitos que a sobrecarga hemodinâmica condiciona sobre a parede arterial do ápice da bifurcação aórtica de modelos murinos. MÉTODOS: Sessenta ratos Wistar, selecionados e designados mediante amostragem probabilística simples foram agrupados equitativamente em um grupo controle e três grupos experimentais. Os espécimes foram anestesiados e sob magnificação microscópica, foi realizada uma incisão abdominal média e a aorta e os vasos ilíacos abordados e isolados desde a porção infra-renal até a porção distal da bifurcação. Modificação da geometria da bifurcação aórtica foi realizada mediante tunelamento da porção distal da artéria ilíaca no músculo ílio-lombar, no nível da raiz do membro inferior nos grupos II e IV. Nefrectomia esquerda e ligação da artéria renal inferior direita foram completadas para reforçar o estresse hemodinâmico nos grupos III e IV. Os modelos mantiveram-se em condições de laboratório convencionais com dieta standard para a espécie e água ad libitum para os grupos I e II e solução de NaCl 0,9% para os grupos III e IV. Após seis meses de seguimento, a bifurcação aórtica e as artérias ilíacas foram inspecionadas e subseqüentemente removidas para sua análise histopatológica. Um espécime por cada grupo foi submetido à angiografia digital com reconstrução tridimensional da bifurcação aórtica. RESULTADOS: 1) A pressão arterial, a freqüência cardíaca e a pressão de pulso entre os grupos, com e sem nefrectomia, mostraram diferenças com significância estatística (p <0,05). Os espécimes reunidos nos grupos III e IV que receberam sobrecarga de sódio desenvolveram um padrão hemodinâmico caracterizado por incremento da freqüência cardíaca e da pressão de pulso. 2) Seis espécimes (60%) do grupo IV desenvolveram aneurismas do ápice da bifurcação aórtica. 3) A avaliação angiográfica demonstrou que a morfologia da bifurcação do grupo controle se mantém sem modificações aparentes durante o período de seguimento. Entretanto, o grupo II apresenta dados de remodelamento longitudinal com tortuosidade e alongamento do tronco e ramos que conformam a bifurcação. Já o grupo III apresenta estenose proximal e dilatação incipiente da região do ápice da bifurcação em um padrão descrito como blister-like. Finalmente, o grupo IV demonstra aneurismas e estenoses múltiplas da porção proximal e distal ao divisor de fluxo. CONCLUSÕES: Em modelos murinos, deformações da geometria arterial, introduzidas por mudanças do ângulo de bifurcação, induzem a formação de aneurismas e a associação com hipertensão arterial, pressão de pulso aumentada, freqüência cardíaca elevada e sobrecarga de sódio potencializam a dilatação sacular desses segmentos
BACKGROUND: Experimental evidence indicates that altered patterns of vascular flow associated with bifurcations are involved in the development of aneurysmatic lesions. The effects of the hemodynamic overload on the arterial wall of the aortic bifurcation in murine models were studied. METHODS: Sixty Wistar rats were selected and assigned by simple random sampling into a control group and three experimental groups. The specimens were anesthetized. Under microscopic magnification an abdominal incision was performed and the aortic and iliac vessels were isolated from the infra-renal portion until the distal bifurcation. The modification of the geometry of the aortic bifurcation was accomplished by tunneling of the distal iliac artery into ilio-lumbar muscle in groups II and IV. Left nephrectomy and ligation of inferior right renal artery were completed to enhance the hemodynamic stress in groups III and IV. The models were maintained in conventional laboratory conditions with standard diet for the species and water ad libitum for groups I and II, and NaCl 0.9% for groups III and IV. After six months of follow up, the aortic bifurcation and iliac arteries were inspected and subsequently removed to its histopathological evaluation. One specimen from each group underwent angiography with digital three-dimensional reconstruction of the aortic bifurcation prior to sacrifice. RESULTS: 1) Blood pressure, heart rate and pulse pressure between the groups with and without nephrectomy showed statistically significant differences (p <0.05). The specimens collected in groups III and IV who received sodium overload developed a hemodynamic pattern characterized by increased heart rate and pulse pressure. 2) Six specimens (60%) in group IV developed aneurysmatic dilatation of the apex of the aortic bifurcation. 3) The angiographic evaluation showed that the morphology of the bifurcation of the control group remains unchanged during the study period. However, group II presents data from longitudinal remodeling with tortuosity and lengthening of the trunk and branches that make up the fork. The Group III presents stenosis and proximal dilatation of the apex of the bifurcation in a pattern described as blister-like. Finally, Group IV shows multiple stenosis proximal and distal to the flow divider. CONCLUSIONS: In murine models, the geometry deformation introduced by changes in the angle of bifurcation, induce inflammation of the flow divider, whereas, high blood pressure, pulse pressure, heart rate and high sodium overload catalyze the aneurysmatic dilatation of these segments

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BRIEND, EMMANUEL. "Hgp92, une glycoproteine humaine a potentialites immunostimulantes : etude dans des modeles experimentaux murins." Paris 11, 1996. http://www.theses.fr/1996PA112381.

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L'hgp92, glycoproteine humaine (92 kda) purifiee a partir d'urine, possede des activites immunostimulantes dans des modeles experimentaux murins. Son sequencage partiel en aminoacides a revele une homologie totale avec le monomere de la proteine de tamm-horsfall, glycoproteine produite par les cellules epitheliales renales et ayant la capacite de s'auto-agreger. In vitro, des phagocytes mononuclees murins stimules par hgp92 exercent une activite cytostatique vis-a-vis des cellules du carcinome pulmonaire de lewis, et, en presence d'ifngamma, vis-a-vis des cellules mastocytaires p815, via des mecanismes impliquant respectivement la production d'h#2o#2 et de no. Cette activation macrophagique resulte egalement en une accumulation d'arnm codant pour des cytokines, dont l'il-6 et le tnfalpha qui sont effectivement produites. In vivo, des souris traitees par hgp92, en i. V. , 24 heures avant l'inoculation d'une dose letale de listeria monocytogenes survivent. Une accumulation d'arnm codant pour l'ifngamma, l'il-12p40, le tnfalpha, l'il-6, l'il-10 et l'inos est detectee dans la rate et le foie 1 a 6 heures apres l'administration d'hgp92. Les taux d'il-6 et de tnf dans ces organes et dans le serum sont egalement augmentes de facon transitoire. Vingt-quatre heures apres l'injection d'hgp92, un accroissement du nombre de cellules lymphomyeloides hepatiques extrasinusoidales cr3#+ et lgl-1#+ est observe, correspondant probablement a l'activation de cellules residentes ou a un recrutement (nk, phagocytes mononuclees et/ou neutrophiles). En augmentant les effecteurs solubles et cellulaires importants pendant la phase precoce, non specifique, de la reponse immune a l. Monocytogenes, l'hgp92 pourrait limiter la multiplication bacterienne. Apres 48 heures d'infection, les souris traitees par l'hgp92 presentent, dans le foie et la rate, 100 fois moins de bacteries que les souris temoins mais aussi des taux plus faibles d'il-6, de tnf et d'ifngamma. Ce dernier phenomene pourrait etre du a la charge bacterienne plus reduite, a une consommation plus importante de cytokines ou a la synthese de produits anti-inflammatoires en reponse a l'hgp92

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Newbury, Lucy Jade. "Targeting Ras GTPases in murine models of renal fibrosis." Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/targeting-ras-gtpases-in-murine-models-of-renal-fibrosis(01debcfb-6d17-4452-b142-e686eb9d059e).html.

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End stage kidney disease (ESKD) affects over 5300 people. The progression of chronic kidney disease (CKD) to ESKD is characterised by cytokine stimulation, leading to the activation of myofibroblasts, resulting in fibrosis. Kirsten rat sarcoma (KNRas) has a key role in the proliferation of renal fibroblasts in vitro and has been highlighted as a possible target for fibrosis. Previous research in our laboratory showed that inhibiting KNRas in the UUO model inhibited renal fibrosis. The aim of this thesis was to investigate the outcome of inhibiting KNRas using Antisense Oligonucleotide (ASO) in the novel Chronic Folic Acid Nephropathy (CFAN) model to study the effect on both fibrosis and renal function. The effect of inhibiting KNRas was also investigated in vitro, to try and elucidate the mechanism by which KNRas controls the progression of fibrosis. KNRas knockdown with ASO143 resulted in 50% reduction in KNRas mRNA which was associated with: 37%-50% reduction in total collagen and protection of renal function (BUN) in the 12 week CFAN model. TGFβ1 treated cells showed an upN regulation i KNRas, Jag1 and Collagen 1a mRNA. Treatment with KNRas ASO was associated with a 3.5 fold reduction in Jag 1 and a 55% reduction in collagen 1a. Jag 1 has been linked to the progression of renal fibrosis via biNdirectional signalling with Notch 1. In conclusion, ASO knockdown of KNRas inhibits fibrosis in vitro and in vivo, and in some instances protects renal function. These results support the hypothesis that KNRas targeting may be beneficial in the treatment of renal fibrosis. Further work is required to further understand the relationship between Jag 1,Notch 1 and KNRas, but this data suggests that KNRas affects renal fibrosis in a Jag 1 dependent pathway.

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Nizzardo, M. "UTILIZZO E CONFRONTO TRA CELLULE STAMINALI NEURONALI DI DIVERSA ORIGINE: EFFICACIA TERAPEUTICA IN UN MODELLO MURINO DI ATROFIA MUSCOLARE SPINALE." Doctoral thesis, Università degli Studi di Milano, 2009. http://hdl.handle.net/2434/157862.

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Spinal muscular atrophy (SMA), characterized by selective loss of lower motor neurons, is an incurable genetic neurodegenerative disease.and represents one of the most common genetic causes of infant mortality. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other motor neuronal diseases. In this study, we analized the therapeutic capacity of different stem cells sources in order to improve SMA phenotype in a SMA murine model. First of all we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein (GFP) only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function, increased life span, and improved motor unit pathology NSC transplantation positively affected the SMA disease phenotype, indicating that transplantation of NSCs may be a possible treatment for SMA. However primary NSC as stem cell source have limited translational value. Thus we used alternative stem cells sources, NSC derived from wild-type embryonic stem cells (wt-ESCs) and from a drug-selectable embryonic stem cell line (OSG-ESC. This cells have promise as an unlimited source of NSCs for transplantation. We found that ESC-derived NSCs can differentiated into motor neuron in vitro, and, when intrathecally transplanted into SMA mice survived, migrated, ameliorated behavioral and life-span and may confer neuroprotection in SMA mice. NSCs obtained using a drug-selectable ESC line (positively for neuroepithelial cells and negatively for undifferentiated cells) yielded the greatest improvements. As with cells originating from primary tissue, the ESC-derived NSCs integrated appropriately into the parenchyma, expressing neuron- and motor neuron-specific markers. Our results suggest translational potential for the use of pluripotent cells in NSC-mediated therapies and highlight potential safety improvements and benefits of drug-selection for neuroepithelial cells.

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42

Tagliavini, Francesca <1984&gt. "Studio di espressione e funzione della PLCβ1 nucleare in modelli murini ed umani di cellule ematopoietiche mieloidi e linfoidi." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4261/1/Tagliavini__Francesca_tesi.pdf.

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The aims of this work were to investigate the role of nuclear Phospholipase C beta 1 (PI-PLCβ1) in human and mouse cell lines and to identify new binding partners of nuclear PI-PLCβ1 to further understand the functional network in which the enzyme acts. The intracellular distribution of PI-PLCβ1 was further investigated in human leukaemia cell lines (NB4, HL60, THP1, CEM, Jurkat, K562). With the exception of HL60, a high endogenous level of PI-PLCβ1 was detected in purified nuclei in each of the cell lines. We found that also in Ba/F3 pro-B cells overexpressing PI-PLCβ1b the protein localize within the nucleus. Although our data demonstrated that PI-PLCβ1b was not involved in cell proliferation and IGF-1 response as shown in other cell lines (FELC and Swiss 3T3), there was an effect on apoptosis. Activation of early apoptotic markers caspase-3 and PARP was delayed in PI-PLCβ1b overexpressing Ba/F3 cells treated with 5 gr/ml mitomycin C for 24h. We performed an antibody-specific immunoprecipitation on nuclear lysates from FELC-PLCβ1b cells. Mass spectrometry analysis (nano-ESI-Q-TOF) of co-immunoprecipitated proteins allowed for identification of 92 potential nuclear PI-PLCβ1b interactors. Among these, several already documented PI-PLCβ1b interacting partners (Srp20, LaminB, EF1α2) were identified, further validating our data. All the identified proteins were nuclear, mostly localized within the nuclear speckles. This evidence is particularly relevant as PI-PLCβ1 is known to localize in the same domains. Many of the identified proteins are involved in cell cycle, proliferation and transcriptional control. In particular, many of the proteins are components of the spliceosome multi-complex, strengthening the idea that PI-PLCβ1b is involved in mRNA processing and maturation. Future work will aim to better characterize the regulatory role of PI-PLCβ1b in mRNA splicing.

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43

Tagliavini, Francesca <1984&gt. "Studio di espressione e funzione della PLCβ1 nucleare in modelli murini ed umani di cellule ematopoietiche mieloidi e linfoidi." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4261/.

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The aims of this work were to investigate the role of nuclear Phospholipase C beta 1 (PI-PLCβ1) in human and mouse cell lines and to identify new binding partners of nuclear PI-PLCβ1 to further understand the functional network in which the enzyme acts. The intracellular distribution of PI-PLCβ1 was further investigated in human leukaemia cell lines (NB4, HL60, THP1, CEM, Jurkat, K562). With the exception of HL60, a high endogenous level of PI-PLCβ1 was detected in purified nuclei in each of the cell lines. We found that also in Ba/F3 pro-B cells overexpressing PI-PLCβ1b the protein localize within the nucleus. Although our data demonstrated that PI-PLCβ1b was not involved in cell proliferation and IGF-1 response as shown in other cell lines (FELC and Swiss 3T3), there was an effect on apoptosis. Activation of early apoptotic markers caspase-3 and PARP was delayed in PI-PLCβ1b overexpressing Ba/F3 cells treated with 5 gr/ml mitomycin C for 24h. We performed an antibody-specific immunoprecipitation on nuclear lysates from FELC-PLCβ1b cells. Mass spectrometry analysis (nano-ESI-Q-TOF) of co-immunoprecipitated proteins allowed for identification of 92 potential nuclear PI-PLCβ1b interactors. Among these, several already documented PI-PLCβ1b interacting partners (Srp20, LaminB, EF1α2) were identified, further validating our data. All the identified proteins were nuclear, mostly localized within the nuclear speckles. This evidence is particularly relevant as PI-PLCβ1 is known to localize in the same domains. Many of the identified proteins are involved in cell cycle, proliferation and transcriptional control. In particular, many of the proteins are components of the spliceosome multi-complex, strengthening the idea that PI-PLCβ1b is involved in mRNA processing and maturation. Future work will aim to better characterize the regulatory role of PI-PLCβ1b in mRNA splicing.

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44

Lorenz, Daniel [Verfasser]. "Die Bedeutung des Nalp3-Inflammasoms im Modell der murinen Pneumokokkenpneumonie / Daniel Lorenz." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2013. http://d-nb.info/1035182394/34.

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45

Martucci, C. "PPADS un antagonista dei recettori purinergici modula le citochine IL-1 beta e IL-6 in un modello di neuropatia murino." Doctoral thesis, Università degli Studi di Milano, 2006. http://hdl.handle.net/2434/63222.

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46

Wagner, Martin [Verfasser]. "Zelltransplantation zur Förderung der Kollateralenbildung im murinen Modell der Hinterlauf-Ischämie / Martin Wagner." Magdeburg : Universitätsbibliothek, 2018. http://d-nb.info/1162189924/34.

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47

Otto, Bettina Regina [Verfasser]. "Charakterisierung von Komponenten des angeborenen Immunsystems im murinen Colitis-Modell / Bettina Regina Otto." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2011. http://d-nb.info/1026263735/34.

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48

Stokoe, Kate Sarah. "Arrhythmogenic properties of genetically modified murine hearts modelling clinical abnormalities of the cardiac Na⁺ channel." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611899.

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49

Salvalaio, Marika. "Valutazione del profilo di espressione genica cerebrale nel modello murino per la mucopolisaccaridosi di tipo II (sindrome di Hunter) effettuata mediante tecnologia RNA-Seq." Doctoral thesis, Università degli studi di Padova, 2011. http://hdl.handle.net/11577/3421640.

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Hunter Syndrome (mucopolysaccharidosis type II, MPS II) is an inherited metabolic disease belonging to the wide group of lysosomal storage disorders (LSDs), including nearly 50 different pathologies. Although individually rare, these pathologies have a collective incidence from 1:4000 to 1:7000 live births, depending on the analyzed population. Most LSDs are due to single, or more rarely multiple, deficit of lysosomal enzymes, responsible of macromolecules degradation. Unmetabolized substrates result in multiorgan and multisystem diseases, which in the vast majority of the patients seriously affect also the central nervous system. Very little is known on LSDs pathophysiology and even less on the determinants of their neurological impairment. Since ERT (enzymatic replacement therapy) is getting promising results only in treatment of the LSDs without neurological involvement, because the enzyme cannot efficiently cross the blood brain barrier, more attention should be put in handling the cognitive and behavioral components of these pathologies. In this scenario, the comprehension of the neurological pathogenesis of these diseases, and in this specific case, of Hunter syndrome, becomes mandatory. Such understanding, although quite complex, might allow, among others, the development of new specific therapeutic strategies targeted to the brain. In this pre-clinic investigation, the murine MPS II model was used for a complex molecular analysis through high-throughput technology. RNAs from two different brain areas, cortex and midbrain, have been analyzed with next generation sequencing, by using SOliD® (Sequencing by oligo ligation and detection) technology. Although this technology is considered the most specific for RNA sequencing, it has not been extensively used so far due to its very high costs and to the complexity of the data analysis requiring advanced bioinformatics competence and a good capacity of software handling. RNA sequencing is a very powerful technology that, in contrast to microarray, can point out every single cellular transcript indistinctly. This work is a comparative study between brain areas derived from the IDS-knock-out and the healthy control mice. Data obtained, after alignment and filtration, have been classified according to Gene Ontology Domains, and analyzed by functional categories. The analysis has clearly pointed out the involvement of a wide group of genes and pathways implicated in neurological processes. Altered structures and cellular functions have been highlighted both through the analysis of terms that have the same alteration in the two cerebral areas and, in a more specific way, through the analysis of terms related to each area. This approach can underline genes whose altered expression is directly related to the cell pathological condition and, at the same time, genes with differential expression, representing instead specific pathways for the function of the brain area considered. Also the detection of alterations in genes involved in some common neurodegenerative diseases (as Parkinson’s and Alzheimer’s) could be very interesting; common pathways could be hypothesized for the disease development or as a consequence of the pathological state. The last part of this work has focused on some selected pathways that have been chosen as the most interesting candidates for the pathogenesis of the disease: heparan sulphate binding protein, calcium homeostasis, oxidative stress, autophagy, axon guidance, neuroinflammation, correlation with other neurodegenerative diseases and the growth hormone. A significant endocellular alteration, due to progressive increase of glycosaminoglican deposits and also of secondary deposits as gangliosides, could justify the involvement of proteins related to calcium metabolism, detected by the present analysis; since calcium plays an important ubiquitous messenger function in different biological processes, its role of leitmotiv in many pathways emerging from this analysis is not surprising. In conclusion, although very complex, the analysis here presented has highlighted the great power of this technology, due to its ability to detect, not only pathways obviously related to this disease, but also non-suspected pathways, whose role in the determination of the pathological condition has not been yet clarified.
La Sindrome di Hunter (o Mucopolisaccaridosi di tipo II, MPS II) è una patologia ereditaria appartenente al più vasto gruppo delle malattie da accumulo lisosomiale (Lysosomal Storage Disorders, LSDs), comprendente quasi 50 diverse patologie. Tali patologie, sebbene individualmente molto rare, presentano un’incidenza complessiva che va da 1:4000 a 1:7000, a seconda della popolazione considerata. Le LSDs, che risultano per lo più da deficit singoli, e più raramente multipli, di enzimi lisosomiali deputati alla degradazione di molecole complesse, sono devastanti malattie multiorgano e multisistemiche che, in buona parte dei casi, comprendono un coinvolgimento neurologico grave. Poco è noto tuttora sulla patofisiologia di queste sindromi e, ancor meno, sulle cause del loro deficit neurologico. Nel momento in cui alcune di queste patologie trovano finalmente beneficio dall’applicazione della terapia enzimatica sostitutiva, risalta maggiormente la problematica del trattamento della componente cognitiva e comportamentale. Essa infatti non trova beneficio da questi nuovi approcci terapeutici, poiché gli enzimi impiegati non sono in grado di attraversare la barriera emato-encefalica. Nasce da qui la necessità di comprendere la patogenesi neurologica di queste sindromi e, nel caso specifico di questo lavoro, della sindrome di Hunter. Tale comprensione, seppure molto complessa, potrebbe consentire, tra le altre cose, lo sviluppo di specifiche strategie terapeutiche mirate al cervello. In questo lavoro di ricerca preclinica, il modello murino per la MPS II è stato impiegato per effettuare una valutazione molecolare complessa attraverso l’impiego di tecnologie high throughput. RNA derivati da 2 aree cerebrali, la corteccia e il mesencefalo, sono stati analizzati mediante next generation sequencing, impiegando la procedura SOLiD® (Sequencing by Oligo Ligation and Detection). Questa tecnologia, seppure estremamente indicata proprio per il sequenziamento dell’RNA, è stata finora poco utilizzata a questo scopo, a causa dei costi ancora piuttosto elevati, ma soprattutto, a causa della complessità dell’analisi che richiede notevoli competenze bioinformatiche e capacità di gestione dei software. Il sequenziamento dell’RNA è una tecnologia estremamente potente in grado di evidenziare, a differenza della tecnica del microarray, tutti i trascritti cellulari in maniera indistinta. Il lavoro qui presentato è un’analisi di tipo comparativo tra le aree cerebrali dell’animale knock-out per l’IDS e le corrispondenti aree dell’animale sano di controllo. I dati, dopo la fase di allineamento e di filtrazione, sono stati classificati secondo i domini della Gene Ontology e analizzati in base alle principali categorie funzionali. L’analisi ha chiaramente evidenziato il coinvolgimento di molti geni e di parecchie vie specificamente implicati in processi neurologici. L’alterata struttura e funzione cellulare sono state evidenziate sia in modo generico, attraverso analisi di termini ugualmente alterati nelle due diverse aree cerebrali, sia in modo specifico, all’interno di ciascuna area cerebrale. Ciò consente di mettere in risalto i geni la cui alterata espressione è direttamente correlata allo stato di accumulo patologico della cellula e i geni con espressione differenziale che, invece, rappresentano pathways specifici per le funzioni svolte da quell’area cerebrale. Molto interessante potrebbe risultare anche l’alterazione di geni implicati in alcune comuni patologie neurodegenerative croniche quali il morbo di Alzheimer e di Parkinson. Vie comuni potrebbero essere ipotizzate per l’instaurarsi delle malattie o come conseguenza dello stato patologico. La parte finale dell’analisi ha preso in considerazione le vie di segnale e le correlazioni più interessanti, alcune delle quali già precedentemente considerate come potenziali candidati nella patogenesi delle malattie da accumulo lisosomiale: l'eparan solfato binding protein, l'omeostasi del calcio, lo stress ossidativo, il processo dell’autofagia, l'axon guidance, la neuroinfiammazione, la correlazione con altre malattie neurodegenerative e l'ormone della crescita. Una forte alterazione del comparto endocellulare dovuta al progressivo, patologico aumento dei depositi primari di glicosaminoglicani, ma anche di quelli secondari quali i gangliosidi, potrebbe giustificare il coinvolgimento delle proteine implicate nel metabolismo del calcio cellulare, rilevato da questo lavoro. Essendo poi il calcio un messaggero ubiquitario coinvolto in differenti processi biologici non stupisce il ruolo di filo conduttore in molti pathways, evidenziato da questa analisi. In conclusione, seppure fortemente complessa, l’analisi intrapresa in questo studio ha evidenziato le enormi potenzialità della procedura, dovute alla sua caratteristica capacità di mettere in luce, oltre a processi correlati in modo sospetto alla patologia, anche pathways non sospetti o la cui implicazione nella determinazione dello stato patologico non sia ancora stata definita.

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CAPELLI, ROBERTA. "Analisi dei livelli d’espressione di fattori di natura epigenetica e marcatori molecolari nel processo di epatocarcinogenesi indotta da dieta su modello murino." Doctoral thesis, Università degli Studi dell'Aquila, 2022. http://hdl.handle.net/11697/192066.

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La steatosi epatica non alcolica (NAFLD) rappresenta una delle patologie epatiche croniche più diffuse. Essa può evolvere da steatosi, condizione in cui si riscontra un accumulo di acidi grassi nel citoplasma degli epatociti, alla più severa steato-epatite (NASH), caratterizzata da infiammazione e danno epatocitario. Inoltre, in un certo numero di casi, la NASH può progredire in fibrosi, cirrosi ed in ultimo in carcinoma epatocellulare (HCC). L’HCC è la tipologia di tumore epatico più comune e costituisce il quinto tumore più frequente al mondo e la terza causa di morte legata al cancro. I microRNA (miRNA, miR) sono dei piccoli RNA non codificanti di circa 18-25 nucleotidi che regolano l’espressione genica a livello post-trascrizionale, esercitando la loro azione sull’RNA messaggero target. Essi sono coinvolti in diversi processi biologici fondamentali sia fisiologici che patologici, ed in particolare, è stato descritto un loro coinvolgimento e ruolo anche nella progressione NAFL/NASH/HCC. Lo scopo di questo lavoro è stato analizzare alcuni microRNA e fattori molecolari coinvolti nel processo di epatocarcinogenesi NAFL-NASH-HCC indotta da dieta, ad alto contenuto di grassi (high fat, HF) o basso contenuto di grassi ed alto di carboidrati (low fat-high carbohydrate, LF-HC), sul modello di NAFLD murino C57BL/6J. Sono stati in particolare identificati e analizzati due miRNA (miR-125a-5p e miR-182), modulati già dopo 3 mesi di dieta nei tessuti epatici murini, e due geni target oncosoppressori (Cyld e FoxO1) che sono risultati ipoespressi nei tessuti tumorali. Gli stessi miRNA sono stati testati su sieri di pazienti affetti da NAFLD a vari stadi di progressione (NASH, cirrosi, HCC) e in soggetti sani di controllo (CTRL), mostrando differenze significative tra alcuni di questi gruppi (miR-125: NASH/cirrosi/HCC vs. CTRL; miR-182: NASH/cirrosi vs. CTRL, HCC vs. CTRL). Per una valutazione più ampia della progressione NAFLD, sui tessuti epatici murini sono stati inoltre analizzati i livelli di espressione di marker di stress ossidativo (SOD, CAT, GST), di citochine pro-infiammatorie (IL-1β, IL-6, TNF-α) e marker di fibrosi (TGF-β, CTGF, LepR), i cui livelli di espressione hanno indicato una progressione del danno epatico più rapida nel gruppo alimentato con dieta HF e una stessa tipologia di danno, più tardivo, nel gruppo LF-HC, evidenziando così che entrambi i tipi di regimi alimentari contribuiscono all’instaurarsi di condizioni coinvolte nel processo di epatocarcinogenesi indotta da dieta. In conclusione, lo studio ha portato all’identificazione di nuovi miRNA e, per la prima volta su modello in vivo, relativi geni target ad attività oncosoppressoria coinvolti nel processo di epatocarcinogenesi indotta da dieta su modello murino. L’analisi di marcatori di stress ossidativo, infiammazione e fibrosi ha inoltre messo in luce che non solo la dieta HF, ma anche quella LF-HC, seppure più tardivamente, è in grado di indurre un danno epatico con progressione ad HCC. Tali risultati aprono a nuovi approfondimenti sui meccanismi molecolari alla base della progressione del danno epatico correlato alla NAFLD. In particolare, dato il ruolo dei microRNA come biomarcatori circolanti non invasivi, lo studio, esteso a casistiche più ampie di pazienti, potrà potenzialmente portare all’identificazione di nuove molecole utili per una più accurata e precoce definizione della diagnosi e prognosi della malattia.

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Dissertations / Theses on the topic 'Modelli murino'

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