Dissertations / Theses on the topic 'Modello ischemia in vitro'

Obiettivi Il COVID-19 è una malattia principalmente respiratoria nell’età adulta; nei bambini, al contrario, i sintomi gastrointestinali sono più frequenti. Inoltre, non si conoscono i meccanismi di infezione in epoca fetale, epoca in cui i pazienti sono raramente colpiti da COVID-19. Gli organoidi sono uno strumento relativamente nuovo per stabilire in vitro colture di lunga durata che assomigliano tridimensionalmente al tessuto di origine e possono sia mantenere la staminalità che differenziarsi completamente in tutti i tipi di cellule. Abbiamo quindi mirato a sviluppare un sistema di coltura per organoidi gastrointestinali (GIO) per studiare l'infezione da SARS-CoV-2 nell'epitelio gastrico lungo tutta la durata della vita. Metodi I GIO sono stati derivati da feti di 8-21 settimane e da tessuti pediatrici e adulti. Sono stati coltivati utilizzando un terreno chimicamente definito, per testare la loro capacità di mantenere la forma e di differenziarsi completamente. GIO sono stati analizzati in correlazione all'ECM circostante. Gli organoidi sono stati quindi indotti a differenziarsi nella forma a polarità cellulare inversa (RP-GO) e quindi incubati con SARS-CoV-2. Tutti gli esperimenti sono stati analizzati mediante qPCR, immunofluorescenza e analisi qualitativa a seconda dei casi. Risultati Gli organoidi gastrointestinali possono essere isolati da tutte le età gestazionali, dimostrando la normale morfologia dell'epitelio gastrico ed esprimendo tipi di cellule mature, comprese le cellule della nicchia, del villo/ghiandola e di tipo enteroendocrino. Queste colture possono essere mantenute indefinitamente in vitro e coltivate in condizioni conformi alle indicazioni GMP. Gli RP-GO mostrano polarità apicale, esponendo ACE2 sulla superficie esterna, ottimizzando le condizioni per l'infezione virale. La nucleoproteina virale è stata dimostrata nelle cellule in fase di apoptosi, con gli RP-GO pediatrici più suscettibili ed infettati in modo efficiente rispetto agli organoidi fetali e adulti. Conclusioni Abbiamo stabilito con successo un efficiente sistema di coltura di organoidi gastrointestinali per tutte le età, dalla vita fetale all'età adulta. La tecnologia basata sugli organoidi può essere utilizzata per modelli di malattie in vitro, test sui farmaci o terapia cellulare. L'applicazione di regole conformi alle GMP rende la traduzione clinica più vicina.<br>Aims Adult COVID-19 is mainly respiratory illness, but in children GI symptoms are more frequent. Furthermore, fetuses are rarely affected by COVID-19. Organoids are a relatively new tool to in vitro establish long-living culture that three-dimensionally resemble the tissue of origin and may both maintain the stemness and fully differentiate in all cell types. As a proof of concept, we aimed to develop a culture system for gastrointestinal organoids (GIOs) to investigate SARS-CoV-2 infection in gastric epithelium across the lifespan. Methods GIO were derived from 8-21 week fetuses and from pediatric and adult tissues. They were cultured using chemically-defined medium, to test their ability to maintain stemness and to fully differentiate. GIO were analyzed in correlation to the surrounding ECM. Reverse cellular polarity Organoids (RP-GOs) were induced and incubated with SARS-CoV-2. All experiments were analyzed by qPCR, immunofluorescence and qualitative analysis as appropriate. Results Gastrointestinal organoids can be isolated from all gestational ages, demonstrating normal gastric epithelial morphology and expressing mature gastric cell types including, the niche, secretive, and enteroendocrine cells. These cultures may be maintained indefinitely in vitro and cultured in GMP-compliant conditions. RP-GOs exhibit apical-out polarity, exposing ACE2 on the external surface, optimizing conditions for viral infection. Viral nucleoprotein was demonstrated in cells undergoing apoptosis, with pediatric RP-GOs most susceptible and efficiently infected compared to fetal and adult organoids. Conclusions We have successfully established an efficient gastrointestinal organoid culture systems for all ages, from fetal life to adulthood. Organoid-based technology can be used for in vitro disease modelling, drug testing or cell therapy. The application of GMP compliant rules makes the clinical translation closer.

Successful kidney transplantation is a life-saving procedure to patients with irreversible chronic renal failure. Despite the presence of various obstacles facing this surgery, preserving donor kidney and consequent ischemia reperfusion injury (IRI) are still major challenges affecting renal function as well as prognosis of transplant surgery. This study pursued two main aims: firstly, characterising changes in damage associated inflammatory gene expressions through developing, and analysis of an in vitro model of proximal tubular epithelial cells (PTEC) of normal human kidney mimicking renal IRI in vivo. The second aim was to simulate the concurrence of factors relevant to human intervention (renoprotective anaesthesia, peri- and postoperative analgesia, volume substitution) in mice deficient of properdin and congenic controls and to allow long-term observation of renal outcome after IRI. In this study, a reproducible and standardisable in vitro model was developed. It demonstrated the complexity of signalling where a multitude of factors affects the target cells. Secondly, the use of congenic properdin deficient mice showed that properdin has a significant role to play in renal injury (and recovery). There was significant impairment in renal function (and structure) compared to wildtype mice after IRI.

An interruption of the blood supply to the brain, as occurs during ischemic stroke, results in a rapid decline of ATP levels and a subsequent loss of neuronal function and viability. Under physiological conditions the brain reuses ATP degradation metabolites, such as hypoxanthine, via the purine salvage pathway, to restore its ATP pool. However, the massive degradation of ATP during ischemia results in the accumulation and loss of diffusible purine metabolites and thereby leads to a reduction in the post-ischemic ATP pool size, leaving the brain more vulnerable to secondary ischemic insults (recurrent strokes) and less able to deploy reparative mechanisms. The aim of this study was to improve the recovery of post-ischemic ATP levels by enhancing the purine salvage pathway, with substances that are already known to be tolerated in humans. Using acute hippocampal rat brain slices, I found that 1 mM Ribose (Rib) and 50 μM Adenine (Ade), two main metabolites of the purine salvage pathway, significantly increased the tissue ATP levels under basal conditions. Rib/Ade pre-treatment results in accelerated decline of synaptic transmission after onset of oxygen/glucose deprivation (OGD), due to increased adenosine release. However, this intervention does not delay the onset of anoxic depolarisation, or improve the recovery of synaptic transmission after prolonged ischemic periods. Pre-treatment of brain slices with 1 mM creatine, which increases phosphocreatine levels and thereby buffers the rapid decline of ATP levels upon energy shortage, significantly delays the onset of AD and helps to improve the recovery of synaptic transmission. By using cultured cerebellar granule cells, for more protracted studies on cell viability after OGD, I show that addition of Rib/Ade after ischemia helps to improves cell viability. Therefore my results suggest that both, delaying the decline of ATP upon onset of OGD (pre-treatment with creatine), or enhancing the post-ischemic recovery of ATP (post-treatment with Rib/Ade) are useful strategies to improve cell survival and function after in vitro ischemia.

Hearing impairment is an increasingly common disease. In Italy deaf people are about seven million, including half a million adults with disabling hearing loss and over one thousand births per year with congenital deafness. This causes difficulty in integration in society for adults and prevents the language acquisition for children (Fekete, 1999). As hearing loss has high social costs, the expectation for the development of new treatments is extensive and diseases leading to hearing damage are increasingly studied from clinic and base research. Hearing loss (HL) can have genetic causes, can be associated with aging or exposure to noise or ototoxic substances, and the aetiology can be attributed to vascular injury, trauma, tumours, infections or autoimmune response. All of these factors could be ascribed to alterations in cochlear microcirculation resulting in hypoxia. This condition can damage cochlear hair cells and neurons possibly leading to HL. Hypoxia and ischemia can then be identified as possible factors contributing to the aetiology of deafness, but they have not been experimentally studied yet. The purpose of this work is to develop animal models of ischemia and infarction suitable for the study of cochlear vascular damage, and to characterize them with electrophysiology and gene/protein expression analyses. For this reason it was decided to monitor the effects of ischemia in thrombosis mimicked by complete temporary carotid occlusion, and in stroke mimicked by incomplete permanent left coronary artery. In particular this study focused on the analysis of: organ of Corti and spiral ganglion structures, coagulation, oxidative stress and apoptosis. A further aim was to compare these models with other models of ototoxic damage, such as noise and cisplatin. These models are both characterized for electrophysiology, oxidative stress and apoptosis, but the possible involvement of vascular damage has not been investigated yet. This comparison helped us to characterize the new models of vascular injury in the oxidation and apoptosis expression patterns. In our models, both infarction and ischemia cause a small but significant hearing loss, localized at the cochlear apex. Furthermore, there is a slight induction of the coagulation cascade, both in procoagulant and anticoagulant part, and an activation of JNK, that may lead to cell survival. In addition, only in the carotid ischemia the cuticular plate of outer hair cells is damaged. Even noise and cisplatin cause vascular damage, but while in noise-treated animals the coagulation genes show only an mRNA expression increase, after cisplatin administration an mRNA and protein increase of the tissue factor is detected, which leads to the coagulation cascade activation. In the ischemic models we did not detect any apoptosis activation, while in the other models we noticed opposite reactions: in noise there is an increased transcription of the anti-apoptotic genes that leads to cell survival, while cisplatin activates pro-apoptotic factors. The activation of apoptosis is documented in literature and is confirmed in both conditions by OHC loss detected in histological sections, which leads to a more severe deafness than in the ischemia models. In conclusion, the two models of ischemia developed are suitable for the study of cochlear vascular damage, as they produce a slight hearing loss and give modifications in coagulative, oxidative and apoptotic factors gene expression. Furthermore, the comparison with two other widely used models allowed us to specify the pathways involved. We can therefore say that all types of damage taken into consideration act on the inner ear with vascular damage and oxidative mechanisms.

Inhibition of microglia-mediated neuroinflammation is an important terapeuthic target in order to avoid cognitive and motor impairment in brain ischemia . Reportedly, neural stem cell (NSC) brain grafts have neuroprotective effecs 1. It has been proposed that these positive effects are not caused only by NSC proliferation and generation of new neurons, but also by a modulation of the brain lesion environment 2. Our primary aim was to ascertain whether NSC were capable of modifying microglial activation in vitro. We used ATP as inflammatory stimuli, since it is massively released from damaged neurons and is responsible of activation of microglia during ischemia3. We demonstrated that N9 murine microglia cells incubated with conditioned media (CM) from NSC culture have a blunted response to ATP. In fact, ATP stimulation of N9 cells preincubated with CM at different passages induced a reduced release of intracellular calcium compared to controls (Fig.1). Moreover, CM preincubation significantly inhibited the expression of inflammatory cytokines like TNF-alfa, COX-2, and IL-10 that are up-regulated after ATP stimulation (Fig.2) Reportedly, high-dose ATP (>1mM) exposure is detrimental both for neurons and microglial cells4. We tested CM action of survival of N9 microglia treated with 3mM ATP for 24 hours. CM preincubation for 24 hours was capable of significantly reducing N9 mortality induced by ATP treatment (Fig.3). In conclusion NSC release soluble factors that have an antinfiammatory action blunting N9 response to ATP stimulation.

A isquemia cerebral causada pela parada cardíaca leva ao desapareciemnto neuronal. studamos os mecanismos de morte celular envolvidos na isquemia in vitro em linhagem de neuroblastoma.O insulto isquêmicao foi reproduzido cultivando as células sem fatores de crescimento, sem glicose e em embiente hipóxico produzido por um sistema de anaerobiose. Os resultados sugerem que a privação de oxigênio, glicose e fatores de cresciemtno do meio de cultura reproduzem o fenômeno semelhante a isquemia. INvestigamos ainda a participação de processo apoptótico e sua modulação térmica. Observams que a hipotermia produz neuroproteção, enquanto a hipertermia agrava o processo de morte celular por apoptose.<br>Cardiac arrest causes cerebral ischemia and neuronal disappearance. We investigate celular death mechanisms elucidated by a model of ischemia in neuroblastoma cell line. The ischemic insult was reproduced by deprivation of growth factors and glucose in a hypoxic environment produced by an anaerobiosis system. Our results validate the experimental model and revel the participation of an apoptotic process in the celular loss induced by ischemia. We also demonstrated that hypothermia can be used as a neuroprotector agent whereas hyperthermia aggavates celular damage.

In questo lavoro ci siamo concentrati sullo studio di un pane arricchito col 4% (p/p) di sansa sgrassata di oliva (DOP) prodotto nell’ambito del progetto europeo H2020 EcoProlive. La sansa di oliva sgrassata è un sottoprodotto derivante dalla produzione dell’olio di oliva ed è caratterizzata da un elevato contenuto di polifenoli, risulta interessante valutarne il potenziale prebiotico e l’impatto sul microbiota del colon umano. Per questo, i prodotti studiati sono stati sottoposti a digestione gastro duodenale e successivamente a fermentazione colonica distale nel modello intestinale in vitro MICODE (Multi-Unit In vitro Colon Model). Il campionamento è stato effettuato prima, durante e dopo 24 ore di fermentazione, al fine di eseguire analisi metabolomiche tramite SPME GCMS, sequenziamento genomico 16S attraverso MiSeq e quantificazione assoluta delle principali specie batteriche tramite qPCR. Dai risultati di questo studio è possibile affermare che il prototipo di pane Eco 4% possiede un potenziale prebiotico più debole rispetto ai frutto-oligosaccaridi dell’inulina, ma più forte del suo pane di controllo privo di polifenoli aggiunti. Durante il periodo di fermentazione Eco4% non ha influenzato l’eubiosi e non ha indotto la disbiosi. Inoltre sono stati registrati l’aumento dell’abbondanza di specie probiotiche o benefiche, come Lactobacillales e Bifidobacteriaceae e la diminuzione di specie opportunistiche o patogene. Grazie all’analisi del volatiloma è stata riscontrata la produzione di composti bioattivi come gli acidi grassi a corta e media catena. Composti dannosi come gli acidi grassi a catena ramificata ed indolo e scatolo, invece, sono stati ridotti. Questi risultati sono supportati da un approccio statistico multivariato in grado di combinare dati di genomica e di metabolomica microbica, in una vetrina multi-omica, a dimostrazione della visibile relazione causa-effetto generata da una certa fibra che possiede un potenziale prebiotico.

Le cellule CD4(+)CD25(+) T regulatorie (Treg) sono una sottopopolazione delle cellule T CD4(+) coinvolte nel controllo della risposta immunitaria. In vitro, le cellule CD4(+)CD25(+) Treg inibiscono la proliferazione delle cellule CD4(+)CD25(-) Th indotta mediante lo stimolo policlonale anti-CD3 mAb in presenza di APC. L’aggiunta di IL-4 alla coltura cellulare inibisce la soppressione mediata dalle cellule CD4(+)CD25(+) Treg. Partendo dal presupposto che tutti i tipi cellulari usati nella cotura esprimono la catena alpha del recettore per IL-4, noi abbiamo usato diverse combinazioni di cellule CD4(+)CD25(-) Th , cellule CD4(+)CD25(+) Treg, e APC da topi wild-type IL-4Ralpha(+/+) o topi knockout IL-4Ralpha(-/-). I risultati mostrano che la presenza della catena IL-4Ralpha sulle celluleCD4(+)CD25(-) Th le rende resistenti alla soppressione. In più, l’aggiunta di IL-4 alla coltura promuove la proliferazione delle cellule IL-4Ralpha(+/+)CD4(+)CD25(+) Treg , che comunque mantengono a pieno la capacità di sopprimere. Questi dati indicano che IL-4 è fondamentale per l’attiazione delle cellule CD4(+)CD25(-) Th e indicano che che la proliferazione delle cellule CD4(+)CD25(+) Treg indotta da IL-4 è compatibile con la loro attività soppressoria. Il trasferimento di cellule CD4(+)CD45RB(high) T cells da milze di topi normali a topi SCID recipienti induce lo sviluppo di una forma di colite Th1-mediata. E’ dimostrato che le cellule CD4(+)CD45RB(low) Treg prevengono lo sviluppo della colite. Il trasferimento di cellule CD4(+)CD45RB(high) T da milze di topi IL-4Ralfa-/-inducono lo sviluppo della colite in modo meno drammatico rispetto a quella indotta da cellule CD4(+)CD45RB(high) T provenienti da topi IL-4Ralfa+/+.<br>CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity. The transfert of CD4(+)CD45RB(high) T cells from the spleen of normal mice to SCID recipient leads to the development of Th1-mediated colitis. CD4(+)CD45RB(low) Treg cells show to prevent the development of colitis. The transfert of CD4(+)CD45RB(high) T cells from spleen of IL-4Ralpha-/-mice leads to the development colitis less seriously than CD4(+)CD45RB(high) T cells from spleen of IL-4Ralpha+/+ mice.

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.

Negli ultimi secoli, l'aspettativa di vita è aumentata grazie a uno stile di vita migliore, ma di conseguenza sono aumentate le patologie legate all'invecchiamento. L'invecchiamento è un processo fisiologico complesso e modificazioni legate all'età sono evidenti anatomicamente e fisiologicamente nella BEE. L'accumulo di danno ossidativo alle macromolecole da parte di RONS e ROS in BEE può essere cruciale nello sviluppo e nella progressione di diverse patologie del SNC. In questa situazione l'ischemia cerebrale potrebbe alterare ulteriormente l'equilibrio ossidante/antiossidante a favore degli ossidanti. In questo scenario, l'alimentazione può contrastare gli impatti ossidativi e le diete arricchite di polifenoli possono fornire effetti benefici. Il caffè è stato descritto come una fonte molto importante di composti antiossidanti (Ricci A. et al., 2018), ma la sua lavorazione produce ogni anno grosse quantità di scarto (Mussatto et al., 2011). Seguendo queste linee guida, lo scopo di questo studio era di valutare le proprietà antiossidanti di diversi composti correlati al caffè, da soli e combinati insieme, in un modello in vitro di ischemia. I composti utilizzati sono stati: fitoestratti derivanti dagli scarti della produzione del caffè e arricchiti in specifici componenti polifenolici; e metaboliti del caffè individuati nel plasma di persone che bevono caffè. Il momento successivo alla riossigenazione provoca un aumento dei ROS, raggiungendo un picco massimo entro 1h dal ripristino delle normali condizioni di coltura (Adibhatla RM et al., 2001). Pertanto, per la valutazione delle proprietà antiossidanti dopo OGD, come condizione di maggior stress è stato scelto l'intervallo 0-1h immediatamente successivo al recupero. Quindi, al fine di valutare le proprietà antiossidanti dei composti del caffè in OGD, sono state effettuate valutazioni sullo stato di fosforilazione delle chinasi Erk e Akt, che se attive promuovono la migrazione di Nrf2 nel nucleo, sui livelli della proteina Nrf2 e sulla sua distribuzione intracellulare, ed infine sui livelli proteici di HO-1, come una dei suoi geni bersaglio. Inoltre, è stata valutata anche la proteina Hsp70, che è coinvolta nel controllo del ripiegamento delle proteine ed infine, è stata misurata la produzione di malondialdeide (MDA) 24 ore dopo il recupero come marker di perossidazione lipidica. I risultati hanno dimostrato la capacità dei composti correlati al caffè di riuscire ad attivare la via di segnalazione Nrf2 in modo diverso e che solo i metaboliti modulavano positivamente Hsp70. I risultati dell'MDA hanno suggerito che la presenza dei composti antiossidanti, testati da soli o combinati, aveva avuto un effetto positivo sulla sua modulazione. I risultati, quindi, hanno dimostrato le proprietà antiossidanti dei fitoestratti e dei metaboliti specifici del caffè, suggerendo che le sostanze stimolano la risposta antiossidante attivando diverse vie, che combinate insieme potrebbero potenziare la difesa antiossidante. L'effetto antiossidante dei metaboliti potrebbe indicare che l'assunzione moderata di caffè giornaliera in soggetti anziani esposti all'invecchiamento e maggior rischio di insulto ischemico, potrebbe contribuire alla riduzione dello stress ossidativo limitando il danno da riperfusione in caso di attacchi ischemici. Queste difese potrebbero essere aumentate attraverso i fitoestratti derivati dal caffè ingeriti come integratori alimentari. Il riutilizzo di questa biomassa di scarto, avrebbe un impatto positivo sia sull'economia che sull'ecosistema, in quanto ridurrebbe notevolmente l'inquinamento.<br>Nowadays, the people get older and older thanks to a better life-style, but consequently, carrying on pathologies typical of the old age, included aging. The aging is a complex physiological process and age-related changes are evident anatomically and physiologically in the BBB. The accumulation of oxidative damage to macromolecules by RONS and ROS in BBB can be crucial in the development and progression of different CNS pathologies. In this situation, cerebral ischemia could further alter the oxidant/antioxidant balance in favour of oxidants. In this scenario, nutrition can counteract the oxidative impacts, polyphenol-enriched diets can provide beneficial effects, preventing cognitive decline and degenerative disorders. More recently, coffee has been described as a very important source of antioxidant compounds (Ricci A. et al., 2018) but its production generates large amount of waste. According to these guidelines, the aim of this study was to evaluate the antioxidant properties of several coffee-related compounds alone and combined together in an in vitro model of ischemia. The compounds used were: phytoextracts deriving from the waste of coffee production and enriched in specific polyphenolic components; and coffee metabolites found in plasma of people drinking coffee. The moment after reoxygenation causes a considerable increase in ROS, reaching a maximum peak within 1 hour of the restoration of normal culture conditions (Adibhatla RM et al., 2001). Therefore, for the evaluation of the antioxidant properties after OGD, the time span 0-1h immediately following recovery was chosen as the condition of greatest stress. Therefore, in order to evaluate the antioxidant properties of the coffee compound under OGD, the antioxidant pathway Nrf2 was analyzed within 0-1h, immediately following recovery. Evaluations were performed on the state of phosphorylation of Erk and Akt kinases, which if active promote Nrf2 migration in the nucleus, on the levels of the Nrf2 protein and on its intracellular distribution, and finally on the protein levels of HO-1, as one of its genes target. Furthermore, the protein Hsp70, which is involved in the control of protein folding, was also evaluated. Finally, malondialdehyde (MDA) production was measured as a marker of lipid peroxidation 24 hours after recovery. The results suggested the ability of coffee-related compounds to activate the Nrf2 signaling pathway differently and only the pre-treatment with metabolites modulated positively Hsp70. MDA results suggested that the presence of the antioxidant compounds, tested alone or combined, had a positive effect on its modulation. The results showed the antioxidant properties of phytoextracts and specific coffee metabolites, suggesting that the substances stimulate the antioxidant response by activating different pathways, which combined together in the mix, could enhance antioxidant defense. The antioxidant effect of coffee metabolites could indicate that the moderate intake of coffee every day in elderly subjects exposed to aging and greater risk of ischemic insult, could contribute to the reduction of oxidative stress by limiting reperfusion damage in the case of ischemic attacks. These defenses could be increased through the phytoextracts derived from coffee ingested as food supplements. The reuse of this waste biomass, would have a positive impact on both the economy and the ecosystem, as it would significantly reduce pollution.

La malattia di Johne o paratubercolosi è un’enterite cronica granulomatosa provocata dal Mycobacterium avium subsp paratubercolosis (MAP), che colpisce i ruminanti ed in particolare i bovini da latte ed ha un grande impatto economico a livello mondiale. Il MAP sembra anche avere un ruolo nella malattia umana di Crohn. Tale patogeno è capace di sopravvivere molto bene all’interno dei macrofagi dell’ospite dove previene la loro attivazione, blocca l’acidificazione e la maturazione del fagosoma, e interferisce con la presentazione degli antigeni al sistema immunitario. Al fine di analizzare la complessa interazione tra l’ospite e il patogeno, è stata valutata la risposta dopo 2h, 6h, e 24h di macrofagi derivati da monociti bovini (MDM), coltivati in vitro e infettati con il ceppo L1 di MAP utilizzando un approccio di RNA-Seq. L’analisi statistica dei dati di sequenza ha mostrato un aumento del numero di geni differenzialmente espressi durante l’esperimento in risposta all’infezione. Inoltre i geni sottoespressi negli MDM infettati sono stati individuati solo a 24h post-infezione. L’analisi dei pathway ha evidenziato tre network che sono associati alla risposta immunitaria e al processo infiammatorio. Inoltre lo studio dei geni sottoespressi a 24h ha mostrato il ruolo centrale del complemento e del complesso maggiore di istocompatibilità nella patogenesi della malattia.<br>Johne’s disease (paratuberculosis) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp paratubercolosis (MAP), affecting ruminants worldwide with a significant economic impact. MAP has also been speculated as a cause of human Crohn’s disease. MAP is a pathogen highly adapted for survival within host macrophages due to the organism's capacity to prevent macrophage activation, block phagosome acidification and maturation, and attenuate presentation of antigens to the immune system. The consequence is a very long silent infection and subclinical phases. To decipher the complex interaction between host and MAP, the response of in vitro bovine monocyte-derived macrophages (MDM) after 2h, 6h and 24h of infection with L1 strain of MAP was explored using RNA-Seq approach. Statistical analysis of sequence data revealed an increasing number of differentially expressed genes in MDM following infection through the three time points analysed. Furthermore down-regulated genes were only found at 24 h post-infection. Ingenuity Pathways Analysis of differentially expressed genes showed that “cell-mediated immune response” was the most significant network related to 2hpi dataset, “immune cell trafficking” for 6hpi, and “inflammatory response” for 24hpi. Finally the analysis of down-regulated genes at 24hpi confirmed the role of complement and major histocompatibility complex (MHC) in the pathogenesis of MAP in cattle.

Gli astrociti, principali cellule gliali del sistema nervoso centrale, sono coinvolti in diverse funzioni cerebrali tra cui trasmissione sinaptica, sintesi e riciclo di neurotrasmettitori, controllo dell’assorbimento di nutrienti e sopravvivenza neuronale. L’omeostasi del calcio è essenziale per le funzioni degli astrociti e per una corretta comunicazione bidirezionale tra astrociti e neuroni. Le diverse funzioni degli astrociti sono confinate in determinati compartimenti a livello cellulare, in particolare le oscillazioni di calcio possono avvenire in specifici microdomini. Un cambiamento dei transienti di calcio nei microdomini influenza la regolazione del rilascio di gliotrasmettitori che è alla base della comunicazione tra astrociti e neuroni. In questo contesto, i mitocondri giocano un ruolo cruciale nella modulazione dei segnali calcio e nella regolazione del metabolismo. Tuttavia, non è stato ancora completamente chiarito il ruolo svolto dal calcio mitocondriale nella fisiologia gliale. L’obiettivo finale di questo progetto è stato quello di studiare una possibile relazione tra calcio mitocondriale e metabolismo. I nostri risultati mostrano che gli astrociti radiali rappresentano un buon modello in vitro per studiare il ruolo del calcio e i segnali metabolici. In questa condizione, gli astrociti sono metabolicamente flessibili, poiché sono in grado di ossidare carboidrati, acidi grassi e amminoacidi. Questo risultato supporta il ruolo centrale degli astrociti nel soddisfare le richieste energetiche neuronali. Infatti, dal punto di vista del segnale calcio, ATP e glutammato generano simili mobilizzazioni di calcio a livello citosolico, tuttavia solo l’ATP causa un percettibile incremento nelle concentrazioni di calcio a livello della matrice mitocondriale. Inoltre, questi stimoli sono codificati in maniera diversa a livello metabolico. Da una parte, la stimolazione cellulare indotta dall’ATP incrementa il metabolismo glicolitico a livello citosolico. D’altra parte, la stimolazione cellulare dovuta al glutammato sostiene la respirazione mitocondriale, anche in assenza di un accumulo di calcio mitocondriale. Per studiare i meccanismi che sono alla base di questo diverso accoppiamento metabolico, abbiamo valutato il contributo dei trasportatori del glutammato GLT-1 e GLAST. In particolare, abbiamo dimostrato che la loro inibizione farmacologica previene parzialmente l’incremento della respirazione mitocondriale, ma influenzando solo limitatamente le dinamiche del calcio. Per analizzare ulteriormente il contributo del calcio mitocondriale sul metabolismo, abbiamo utilizzato due diverse strategie: una basata sull’ inibizione farmacologica del complesso MCU (Uniporto Mitocondriale del Calcio), e l’altra sull’utilizzo di un modello animale caratterizzato della delezione mono-allelica di Mcu. Esperimenti eseguiti in astrociti corticali isolati da topi Mcu+/- hanno mostrato, come atteso, un decremento nell’accumulo di calcio mitocondriale, ma senza causare alterazioni significative nel metabolismo ossidativo, suggerendo che l’aumento di calcio nella matrice mitocondriale giochi un ruolo marginale in questo contesto. In conclusione, i nostri risultati indicano che gli astrociti sono cellule con un profilo metabolico complesso e flessibile. Tuttavia, le dinamiche di calcio citosolico e mitocondriale svolgono una funzione secondaria in questa regolazione, almeno nelle nostre condizioni sperimentali.<br>Astrocytes are glial cells located in the central nervous system. They play several important roles, including synaptic signalling, neurotransmitter synthesis and recycling, control of nutrient uptake and neuronal survival. Calcium homeostasis is essential for astrocytes functions and for the correct bidirectional communication between astrocytes and neurons. Astrocytes are functionally compartmentalized, and calcium oscillations can occur in specific local microdomains. A change in astrocytic calcium microdomain activity influences the regulation of gliotransmitter release, a first crucial step in neuron to astrocytes signalling. In this context, mitochondria could play a pivotal role in shaping calcium waves and regulating cellular metabolism, at least in principle. However, the genuine contribution of mitochondrial calcium to astrocytes physiology is poorly investigated. Here, we study a possible link between mitochondrial calcium and metabolism. Our results show that star-shaped astrocytes represent a reasonable in vitro model for studying Ca2+ signalling and metabolic pathways. In our culture condition, astrocytes are metabolically flexible, being able to oxidize carbohydrates, fatty acids and amino acids. Thus, this supports the central role played by astrocytes in satisfying the brain energy demands. Indeed, in terms of Ca2+ signalling, ATP and glutamate cause similar cytosolic Ca2+ mobilization, but only ATP stimulates a consintent rise in [Ca2+] in mitochondrial matrix. Moreover, these stimuli are decoded differently at metabolic level. On the one hand, cellular stimulation with ATP selectively increases cytosolic glycolytic metabolism. On the other hand, cellular stimulation with glutamate boosts mitochondrial respiration, even in the absence of substantial mitochondrial Ca2+ uptake. To investigate the mechanisms underlying this different metabolic coupling, we evaluated the contribution of the glutamate transporters GLT-1 and GLAST, and showed that their pharmacological inhibition partially prevents the increase in mitochondrial respiration, but with limited impact on calcium dynamics. To further dissect the contribution of mitochondrial Ca2+ uptake to astrocytic metabolism, we devised two different strategies, one based on the pharmacological inhibition of the MCU (Mitochondrial Calcium Uniporter) complex, and the other based on the use of a mouse model carrying the monoallelic deletion of Mcu gene. Experiments performed in cortical astrocytes from Mcu+/- mice showed, as expected, lower mitochondrial calcium transients, but without major alterations in oxidative metabolism, suggesting a marginal role for matrix calcium elevations in this context. Overall, our results suggest that the astrocytes are cells with a complex and flexible metabolic profile. However, cellular and mitochondrial calcium dynamics play a minor role in this regulation, at least in our experimental settings.

L’effet neuroprotecteur des agents anesthésiques est maintenant établi depuis plus de 30ans. Cependant, les mécanismes impliqués restent imparfaitement élucidés. A cours de cetravail nous avons étudié deux volets de leur protection :La première partie porte sur l’implication de la transmission glutaminergique dans leurseffets neuroprotecteurs directs, c'est-à-dire lorsqu’ils sont utilisés au cours d’une l’ischémiecérébrale. Nous avons étudié deux agents anesthésiques de classe distincte: le propofol et lesévoflurane sur des co-cultures de neurones et d’astrocytes corticaux de rat soumis à uneprivation en oxygène et en glucose transitoire (POG). Nous avons ainsi observé que laprésence de propofol ou de sévoflurane pendant la POG prévenait la mort neuronale,l’accumulation de glutamate extracellulaire et la diminution de la capture du glutamateinduites par l’ischémie. Nous avons également montré que cette restauration partielle del’activité de capture du glutamate impliquait des transporteurs distincts entre le propofol et lesévoflurane.La deuxième partie a porté sur la neuroprotection obtenue par un préconditionnement (PC)pharmacologique liée à l’utilisation avant l’ischémie d’agents anesthésique volatils. Nousavons tout d’abord confirmé in vitro l’existence d’une telle protection avec le sévoflurane etmis en évidence le rôle primordial, au cours de cette protection, des canaux potassiques ATPdépendantset des radicaux libres. Puis sur un modèle in vivo d’occlusion transitoire del’artère cérébrale moyenne, le PC par sévoflurane a induit une neuroprotection supérieure àcelle obtenue avec l’utilisation de sévoflurane uniquement pendant l’ischémie. Cependantcette protection est transitoire et ne perdure pas dans le temps. Le sévoflurane ne fait queretarder, sans l’empêcher, la mort neuronale liée à l’apoptose. Il offre cependant une fenêtrethérapeutique intéressante<br>The neuroprotective effect of anesthetic agents is now established for over 30 years.However, the mechanisms involved remains to be fully explored. This work focuses on twoneuroprotective strategies:The first part is on the involvement of glutamatergic transmission in their directneuroprotective effects. We studied the effect of two separate classes of anesthetic agents:propofol and sevoflurane on co-cultures of cortical neurons and astrocytes from rats subjectedto a transient oxygen and glucose deprivation (OGD) mimicking cerebral ischemia. Weobserved that the presence of propofol or sevoflurane during OGD prevented neuronal death,accumulation of extracellular glutamate and decreased uptake of glutamate induced byischemia. We also demonstrated that this partial restoration of glutamate uptake mediated bypropofol and sevoflurane involved differential transporters.The second part deals with the neuroprotection achieved by pharmacologicalpreconditioning with regard to the use of volatile anesthetic agents before ischemia. We firstconfirmed in vitro the existence of such protection with sevoflurane. We also highlighted therole of ATP-dependent potassium channels and reactive oxygen species in sevofluranepreconditioning-induced neuroprotection. Then, using an in vivo model of focal transientischemia, we showed that sevoflurane preconditioning significantly improved functionaloutcome and reduced infarct volume. However, this protection was transient. Sevofluraneonly delayed the neuronal death associated with apoptosis but offers an interesting therapeuticwindow

Opioid are the most commonly used analgesics drugs in medicine. Beside their effect on the nervous system additional peripheral properties are also being discovered. Recently, opioid pre-conditioning and anti-apoptotic functions are some of the most studied secondary effects of these drugs. Ischemia and reperfusion injury are unavoidable insults occurring to the graft as a consequence of transplantation. As opioid receptors are expressed in the kidney, the aim of this study is to assess the effect of this class of analgesics on viability, apoptosis and necrosis in a kidney proximal tubular cell line before and after undergoes an ischemic event. OK cells (Opossum Kidney tubular cells) expressing κ opioid receptor were exposed to 4 different opioid analgesic solutions (morphine, fentanyl, butorphanol and buprenorphine) containing 10-10, 10-8 and 10-5 M of each drug. Cells were tested in different conditions: 1) opioids were added to culture medium for 48 hours; 2) cells were pre-treated and recovered with an opioid or were exposed before or 2 hours after a simulated ischemia (SI) which was performed by ATP depletion with antimycin A and 2-deoxi-D-glucose. Colorimetric cell viability assay, luminescent ATP assay and caspase-3 and -7 activity were performed. Apoptosis and necrosis were also evaluated by annexine-V/propidium iodide staining and flow cytometric analysis. At a high concentration fentanyl and buprenorphine decreased OK cells survival after 48 hours of exposure but the effect was limited and not significant. In ATP depletion studies, morphine and fentanyl exhibited a positive effects in preserving celluar ATP content and in decreasing caspases activities and apoptotic and necrotic ratios. Fentanyl preserved the ATP content also when administered before the SI. The present study showed no effect by butorphanol and buprenorphine on improving theATP content nor decreasing caspases activity or apoptosis. Pure agonists of κ opioid receptors decrease the cellular damage due to ischemia/reperfusion injury mainly by maintaining intracellular ATP content but by also suppressing apoptosis. Therefore this class of drugs should be as prefered analgesics during kidney transplantation surgery.<br>Gli analgesici oppioidi sono ampiamente usati in medicina. Questa classe di farmaci, oltre ad una azione sul sistema nervoso, ha effetti anche a livello di tessuti periferici dove i recettori oppioidi vengono espressi. Negli ultimi anni diversi studi mostrano come il precondizionamento con oppioidi abbia effetti protettivi contro i danni dovuti a ischemia e riperfusione che si presentano inevitabilmente durante un trapianto d’organo. Lo scopo del lavoro è quello di valutare l’effetto su una linea cellulare derivante da tubulo renale prossimale che esprime i recettori κ degli oppioidi, sottoposta o meno ad un evento ischemico. Le cellule OK (Opossum Kidney tubular cells) sono state esposte a 4 oppioidi (morfina, fentanyl, butorfanolo e buprenorfina) alle concentrazioni di 10-10, 10-8, 10-5 M. Le cellule erano: 1) esposte ai farmaci per 48 ore; 2) esposte agli analgesici prima e dopo un evento ischemico indotto con antimicina A e 2-Deossi-D-glucosio od ancora trattate con i farmaci solo prima o solamente dopo. Sono state eseguite prove colorimetriche e di luminescenza per valutare la vitalità cellulare, il contenuto di ATP e attivazione delle caspasi-3 e -7. Prove citofluorimetriche erano impiegate per valutare l’apoptosi e la necrosi. Ad alte dosi fentanyl e buprenorfina dimiuiscono la sopravvivenza delle cellule OK dopo 48 ore di esposizione ma l’effetto è limitato e non rilevante. La morfina e il fentanyl hanno un effetto positivo nel preservare il contenuto di ATP e nel diminuire l’attivazione delle caspasi e l’apoptosi. anche la necrosi diminuisce quando le cellule sono esposte a questi oppioidi prima e dopo l’evento ischemico. Il fentanyl mantiene elevato l’ATP anche quando somministrato prima dell’evento ischemico. Butorfanolo e buprenorfina non mostrano alcun effetto positivo sul contenuto di ATP o sull’apoptosi. Gli agonisti puri dei recettori degli oppioidi κ prevengono la comparsa di apoptosi e necrosi preservando il contenuto cellulare di ATP dopo ischemia. Il loro impiego potrebbe dimostrare dei vantaggi nel prevenire i danni da ischemia e riperfusione durante interventi di trapianto renale.

The purpose of this PhD project is the development of in-vitro culture models to study different aspects of Osteoarthritis (OA), an entire joint degenerative disease that involved articular cartilage, synovium and subchondral bone. The global incidence of knee OA is 203 per 10,000 person-year that grows with the increase of age, reaching a peak between 70-79 years old, thus dragging the peak of prevalence in old age. The main OA disabling symptom is pain that is typically intermittent and weight-bearing (mechanical) and can lead to psychological stress. Contrary to the common description of a wear-related pathology, OA is the consequence of an active and unbalanced process of repair and destruction. The actual causes of OA are still unidentified and there is still debating on the precise order of events that trigger its onset. There is currently no predetermined in-vitro model of OA disease. This is essentially due to the defective knowledge on the onset of this pathology and to the numerous tissue modifications involved that make difficult to in-vitro reproduce OA. A clear comprehension of the various mechanisms engaged in the pathology is necessary to understand its progression, as well as the targets to restore joint function. Here, two different in-vitro culture models to study different aspects of OA were applied to investigate: (i) the involvement of adult stem/stromal cells of the synovial membrane in bone remodeling, through an indirect 2D co-culture approach and (ii) the crosstalk between chondrocytes and subchondral bone in normal and pathological condition, developing an engineered 3D scaffold and simulating an inflamed microenvironment. These two models allowed the investigation of the cell behaviours from the three tissues involved in the pathogenesis of OA, and the development of a possible in-vitro platform for future studies encompassing the three components simultaneously.<br>Lo scopo di questo progetto di dottorato è lo sviluppo di modelli di coltura in-vitro per studiare diversi aspetti dell'osteoartrosi (OA), una malattia degenerativa che coinvolge tutti i tessuti dell’articolazione tra cui la cartilagine articolare, la membrana sinoviale e l'osso subcondrale. L'incidenza globale dell'OA del ginocchio è di 203 per 10.000 persone all’anno e cresce con l'aumentare dell'età, raggiungendo un picco tra i 70-79 anni. Il principale sintomo disabilitante dell'OA è il dolore, tipicamente intermittente e portante (meccanico) che può, in alcuni casi, a stress psicologico. Contrariamente alla comune descrizione di una patologia correlata all'usura, l'OA è la conseguenza di un processo attivo e sbilanciato di riparazione e distruzione. Le cause che portano all’insorgenza di tale patologia non sono ancora state del tutto identificate e si dibatte ancora sull'ordine preciso degli eventi coinvolti nella sua progressione. Attualmente, la scarsa conoscenza della patogenesi dell’OA e le numerose modificazioni tissutali che la caratterizzano hanno reso complicato lo sviluppo di un modello in-vitro. È quindi necessario comprendere i meccanismi coinvolti nella progressione della patologia, nonché i target di nuovi trattamenti per il ripristino della funzionalità articolare. Qui, due diversi modelli di coltura in-vitro per indagare diversi aspetti dell'OA: (i) il coinvolgimento di cellule staminali / stromali adulte della membrana sinoviale nel rimodellamento osseo, attraverso un approccio di co-coltura 2D indiretta e (ii) il crosstalk tra condrociti e osso subcondrale in condizioni normali e patologiche, sviluppando uno scaffold 3D ingegnerizzato e simulando un microambiente infiammato. Questi due modelli hanno permesso lo studio dei comportamenti cellulari di tre tessuti coinvolti nella patogenesi dell'OA e lo sviluppo di una possibile piattaforma in-vitro per futuri studi che potranno comprendere simultaneamente queste tre componenti.

As doenças neurodegenerativas tais como as doenças de Alzheimer, Parkinson e desordens cerebrovasculares constituem-se em uma das principais causas de morbidade e de mortalidade na vida adulta. Além disso, o desequilíbrio entre os sistemas de geração e de proteção antioxidante celulares, chamado de estresse oxidativo, desempenha um papel importante nos danos neuronais causados pelos processos isquêmicos, provocando alterações funcionais em macromoléculas e promovendo a lipoperoxidação de membranas. Substâncias com dupla atividade anticolinesterásica e antioxidante vêm sendo consideradas como uma nova abordagem terapêutica para o tratamento farmacológico da doença de Alzheimer, incentivando a investigação e o estudo de produtos naturais para o desenvolvimento de fármacos novos e eficientes. Nesse estudo empregamos um modelo in vitro de fatias hipocampais de ratos, submetidas à privação de oxigênio e glicose (POG) e os métodos de avaliação da toxicidade dos alcalóides empregados foram a liberação da enzima lactato desidrogenase (LDH) citosólica e redução do MTT (viabilidade mitocondrial). Os alcalóides boldina e vincamina promoveram um aumento de 40 % na liberação de LDH nas fatias que sofreram POG na concentração de 100 μM, além de aumentos significativos na liberação desta enzima também nas fatias controles. Psicolatina e reserpina também tiveram efeitos neurotóxicos. Foi verificado que a POG em fatias hipocampais promove uma diminuição nas medidas do potencial antioxidante total (TRAP) e reatividade antioxidante total (TAR), de 63 % e 16,5 %, respectivamente, além de causar um aumento nos níveis de malonodialdeído liberado pelas fatias, detectado pelo ensaio de espécies reativas ao ácido tiobarbitúrico (TBA-RS). Entretanto, este efeito foi revertido pela presença de boldina nas concentrações de 10 μM e de 50 μM. Este mesmo alcalóide, com reconhecida atividade antioxidante, também demonstrou ser um seqüestrador de radicais peroxila mais potente que o padrão Trolox. Além disso, os alcalóides indólicos monoterpênicos coronaridina, venalstonina, andrangina, vincadiformina e voacristina, além da boldina, exibiram potentes atividades antioxidante e anticolinesterásica em ensaios autobiográficos in vitro.<br>Neurodegenerative disorders, such as Alzheimer, Parkinson and cerebrovascular diseases are one of the major causes of morbidity and mortality in the middle aged and the elderly. Also, the imbalance between the activity of free radicals generation and scavenging systems, called oxidative stress, plays a important role in the neuronal damages caused by ischemia, leading to functional alterations in macromolecules and promoting lipoperoxidation in membranes. Acetylcholinesterase inhibitors and antioxidant compounds have been extensively investigated as new pharmacological strategies for the symptomatic treatment of Alzheimer disease. In this way, natural products are potentially important in an attempt to develope newer and safer drugs. In the present study, we selected the in vitro model of oxygen and glucose deprivation (OGD) in hippocampal slices and the methods used to assess the neurotoxicity of the alkaloids were cellular lactate dehydrogenase (LDH) release and reduction of MTT salt (mitochondrial activity). Both alkaloids boldine and vincamine 100 μM promoted a 40 % increase in LDH release in POG slices, as well as significant increases in the activity of this enzyme in control slices. Psychollatine and reserpine had also neurotoxic effects. It was also verified that OGD decreased the measurements of total antioxidant potential (TRAP) in 63 % and the total antioxidant reactivity (TAR) levels in 16.5 %, as well as an increase in the malondialdehyde levels by slices, which was detected by thiobarbituric acid-reactive substances (TBA-RS). However, this effect was prevented by the presence of boldine 10 μM and 50 μM. This alkaloid is a known antioxidant and it displayed a potent scavenger activity for peroxyl radicals, when compared with Trolox. Another finding is that the monoterpene indole alkaloids coronaridine, venalstonine, andrangine, vincadifformine, voacristine and also boldine exhibited both potent antioxidant and acetylcholinesterase inhibitor activities in in vitro autobiographic assays.

Charcot-Marie-Tooth disease type 2A (CMT2A) is a sensory-motor polyneuropathy, characterized by the involvement of motor and sensitive neurons, resulting in progressive weakness, limb muscle atrophy and loss of sensitivity. Mitofusin2 (MFN2) gene has been identified as causative of the disease. The MFN2 protein, located in the outer mitochondrial membrane, is involved in the mitochondrial network. Defects in this network are features of several neurodegenerative diseases. A treatment for CMT2A is not currently available. The development of an effective therapy needs a better understanding of the molecular mechanisms of the disease, not only to identify new therapeutic targets, but also to define biomarkers of the disease phenotype. The first aim of this study was the generation of an in vitro disease model, not currently available. The reprogramming of mature somatic cells into induced pluripotent stem cells (iPSCs) provides the derivation of disease-specific cell types, such as motor and sensitive neurons, affected in the disease. Based on this method, we successfully generated human iPSCs from a CMT2A patient and demonstrated their differentiation into motor neurons. We analyzed mitochondrial phenotypes (i.e mitochondrial localization, content and stability of mitochondrial DNA, and respiratory capacity) associated with the disease in CMT2A motor neurons as well as in fibroblasts. In particular, we observed an alteration in mitochondria localization, a reduction in the amount of mitochondrial DNA and a dysfunction of the mitochondrial respiratory chain, identifying specific hallmarks of the disease phenotype. These defects are more evident in neuronal cells compared to fibroblasts, in agreement with neuronal specificity of the disease. In addition in vitro models, the analysis of these aspects has been conducted in the only currently available murine model of CMT2A (MitoCharc 1) to extend its characterization, searching for biomarkers of disease phenotype. The second aim of this work was the development of a therapeutic approach for this disorder. We silenced endogenous MFN2 gene by short harpin RNA (shRNA) in CMT2A fibroblasts. At the same time, in order to restore correct MFN2 protein levels, we transfected a MFN2 c-DNA modified to be resistant to shRNA-mediated silencing. The results of this strategy were very promising in CMT2A fibroblasts, and preliminary data were also obtained in the CMT2A mouse model. In conclusion, this study contributed to deepen the knowledge about disease molecular mechanisms, generating an in vitro model of CMT2A by patient-specific iPSCs, and to identify a possible therapeutic strategy for CMT2A.

Introduction. Since our knowledge on the replication and differentiating capability of the respiratory tract is still limited, the therapeutic potential of this system is quite unexplored. The respiratory disease in pediatric age concerns several pathologies among which broncopulmonary dysplasia (BPD) in neonate and premature newborns, asthma and cistyc fibrosys are the most studied because of their diffusion among children and their infaust prognosis.. In the last few years some studies have shown the possibility of deriving progenitors with various potential from the amniotic fluid. Amniocentesis is a widely accepted method for prenatal diagnosis; it is associated with low risk both for the mother and the fetus and overcomes the ethical problems commonly associated to other sources. Recently, it has been described that amniotic fluid stem (hAFS) cells, for their ability to differentiate to various lineages, could represent a good candidate for therapeutic applications. The recent characterization of hAFS and the consolidation of the techniques for intratracheal transplantation have shown new perspectives for gene and cell therapy applications. In particular, for these purposes hAFS cells should be genetically modified with a therapeutic gene and delivered systematically or injected directly into the tissue of interest. Materials and methods The in vitro phase has evaluated for the first time the possibility to infect hAFS with first generation E1-deleted adenoviral vectors, and the mantainence of the stemness and differentiating capability even after transduction with foreign gene sequences. In the in vivo phase of the project we verified the pulmonary homing and the eventual engraftment of hAFS cells, after intratracheal administration, in a 60% O2 rat model presenting a respiratory disease similar to the one observed in human patients affected by broncopulmonary dysplasia (BPD) and cystic fibrosis. The symptoms were reproduced by using OXYCYCLER, with two pressurized rooms in which animals are exposed at controlled percentages of O2 and CO2. In this trial, hAFS cells have been infected AdHCMVsp1LacZ, a first generation E1 deleted viral vector transducing LacZ, the β-gal specific gene used as marker. Results At first, we investigated the feasibility of transducing hAFS cells with adenoviral vectors and to determine whether transduced stem cells retain the ability to differentiate into different lineages. Herein, we showed that hAFS cells could be efficiently infected by first generation adenovirus vectors. In addition, we demonstrated that infection and expression of two different marker genes, LacZ and EGFP, have no effect on cells phenotype and differentiation potential. In particular, on undifferentiated status, hAFS cells continued to express both the transgenes and stemness cell markers OCT4 and SSEA4 (stage-specific embryonic antigen 4). When cultured under mesenchymal conditions, infected cells could still differentiate into osteocytes and adipocytes expressing lineage specific genes. Differently to what observed in embryonic stem cells, the amniotic fluid stem cells easily infect very efficiently. This could represent an excellent starting point for gene therapy studies in which a transient expression would be a necessary condition to the therapeutic approach. In the in vivo phase we transplanted hAFS cells with an intratracheal administration in a rat model generated exposing newborns at 60% O2 for two weeks, reproducing in this way the chronic damage that can be seen in human patients affected by BPD. The results show that the model for chronic lung damage has been properly implemented; specific staining for lacZ performed three weeks post-transplant confirmed for hAFS cells a bronchiolar homing. After four weeks transplantation LacZ positive cells have been detected inside alveolis. Finally, an important phenomenon of damage repair was observed in the treated animals as compared to untreated controls.

L’ictus o accident cerebrovascular és la segona causa de mort en els països industrialitzats i constitueix la primera causa de discapacitat en adults. L’únic tractament aprovat en l’actualitat és el trombolític activador del plasminògen tissular (tPA), el qual només es pot aplicar en un nombre molt reduït de pacients i dintre d’una estreta finestra terapèutica. Els mecanismes de mort cel·lular en la isquèmia cerebral són amplis i venen provocats per l’interrupció del flux sanguini al cervell, el qual provoca una mort ràpida i eminentment necròtica en el nucli de la zona afectada i una mort de caràcter apoptòtic i més lenta al voltant, en la zona de penombra. El gran impacte socio-econòmic de la malaltia i l’existència d’una mort programada dil·latada en el temps han fet que es posin grans esforços en la búsqueda de mecanismes per salvar la zona de penombra. Tenint en compte aquests fets, el present treball s’ha centrat en l’estudi dels mecanismes de mort cel·lular involucrats en la isquèmia cerebral. Per fer-ho, s’ha utilitzat un model de privació d’oxigen i glucosa (OGD) en cultius corticals mixtes d’embrions de rata. Utilitzant aquest model d’isquèmia s’ha observat neuroprotecció pel bloqueig dels receptors NMDA, principal responsable de l’entrada massiva de calci, i l’activació de la caspasa-3, una proteasa encarregada del desmantellament cel·lular durant l’apoptosis. A més, s’ha estudiat el paper dels antagonistes dels receptors de mort en l’OGD. Aquests receptors són responsables de l’activació de la via apoptòtica extrínseca. S’ha observat que l’OGD indueix la degradació dels antagonistes FLIPL i IAP2 i modula l’expressió de FAIML a través de la via de les MAP cinases. Per altre banda, s’ha observat que el silenciament o la sobrexpressió de FAIML mitjançant vectors lentivirals no afecta la viabilitat dels cultius així com tampoc la morfologia nuclear apoptòtica ni els nivells de caspasa-3 activa en les neurones sotmeses a l’insult isquèmic. En conjunt aquests resultats han servit per aprofundir en els mecanismes moleculars implicats en la isquèmia cerebral i poden servir de base per futurs estudis que ajudin al disseny de noves estrategies terapèutiques.<br>Stroke is the second cause of death in industrialized countries and is the leading cause of disability in adults. The only currently approved treatment is the thrombolytic tissue plasminogen activator (tPA), which can be applied only in a very small number of patients and within a narrow therapeutic window. The mechanisms of cell death in brain ischemia are numerous and are caused by the interruption of the blood flow to the brain, which causes a quick necrotic death in the core of the affected area and a slow apoptotic-like death around, in the ischemic penumbra. The major socio-economic impact of the disease and the existence of a programmed cell death that stretches through time explain the effort that is being done to find new strategies to save the penumbra. Given these facts, the present work has focused on studying the mechanisms of cell death involved in brain ischemia. To do this, we used a model of oxygen and glucose deprivation (OGD) in mixed cortical cultures from rat embryos. Using this model of ischemia we observed neuroprotection by blocking NMDA receptor, the primarily responsible for the massive influx of calcium during ischemia, and activation of caspase-3, a protease responsible for dismantling the cell during apoptosis. In addition, we studied the role of death receptor antagonists in OGD. These receptors are responsible for the activation of the extrinsic apoptotic pathway. It has been observed that OGD induces degradation of the antagonists FLIPL and IAP2 and modulate the expression of FAIML through the MAP kinase pathway. On the other hand, we observed that the overexpression or silencing of FAIML using lentiviral vectors did not affect the viability of the cultures nor the apoptotic nuclear morphology or the levels of active caspase-3 in the neurons subjected to the ischemic insult. Together these results have served to study the molecular mechanisms involved in brain ischemia and may provide the basis for future studies that will help to design new therapeutic strategies.

Die subklinische Klauenrehe oder claw horn disruption (CHD) ist von großer wirtschaftlicher Bedeutung für die Rinderhaltung, da sie zu Lahmheiten, Beeinträchtigungen des Allgemeinbefindens sowie einer eingeschränkten Leistungsfähigkeit der Tiere führt. Trotz zahlreicher Untersuchungen sind die pathophysiologischen Grundlagen der CHD noch immer nicht vollständig geklärt. Die derzeitigen Hypothesen weisen auf eine Ischämie in den noch lebensfähigen Epidermisschichten infolge einer veränderten dermalen Mikrozirkulation. Diese hat pathophysiologische Veränderungen zur Folge, die eine Störung der epidermalen Zellproliferation, eine Schädigung der dermo-epidermalen Verbindung sowie eine veränderte Keratinisierung und Hornproduktion umfassen. Von Bedeutung sind daher In-vitro-Ischämiemodelle, um die epidermale Reaktionsmechanismen auf die pathologischen Veränderungen der Dermis zu untersuchen. Ziel in der vorliegenden Arbeit war die Etablierung eines In-vitro-Ischämiemodells auf der Grundlage boviner Keratinozyten aus der Klauenepidermis. Mithilfe dieses Modells sollten die zellulären Pathomechanismen infolge einer Ischämie, einer Hypoxie sowie eines Glukoseentzugs untersucht werden. Des Weiteren stand die Analyse des Differenzierungsverhaltens der Keratinozyten infolge ischämischer, hypoxischer und hypoglykämischer Konditionen im Mittelpunkt. Für die Etablierung des In-vitro-Ischämiemodells diente als Grundlage das oxygen glucose deprivation (OGD)-Modell, das die Untersuchung eines gleichzeitigen Sauerstoff- und Glukosemangels sowie lediglich einer Hypoxie und eines Glukoseentzugs bei bovinen Keratinozyten ermöglichte. Die Versuche wurden in eine Kurzzeitanalyse über 96 Stunden sowie eine Langzeitanalyse über drei Wochen geteilt. Nach erfolgter Exposition wurde die Zellviabilität mittels LDH(Lactatdehydrogenase)- und MTT(3-(4,5-Dimethylhiazol-2-yl)-2,5-diphenyltetrazoliumbromid)-Assay untersucht. Des Weiteren wurde das veränderte Differenzierungsverhalten der Keratinozyten infolge der veränderten Kultivierungsbedingungen mittels Western Blot-Analyse anhand der Involukrin- und Lorikrin-Expression untersucht. Die Keratinozyten zeigten infolge einer OGD nach kurzer Expositionsdauer die höchsten zytotoxischen Effekte, die von einer zeitabhängigen Abnahme der Zellviabilität sowie massiven morphologischen Veränderungen gefolgt wurde. Hypoxische Bedingungen bewirkten eine zeitabhängige Abnahme der Zellviabilität, die erst nach zweiwöchiger Inkubation die größte Zytotoxizität aufwies, sowie eine geringgradig veränderte Zellmorphologie bei Erhaltung des Zellverbands. Der Glukoseentzug bewirkte eine stark verminderte Zellviabilität sowie starke morphologische Zellveränderungen. In der Western Blot-Analyse konnte eine gesteigerte Involukrin- und Lorikrin-Expression infolge einer OGD, einer Hypoxie und eines Glukoseentzugs nachgewiesen werden. In der vorliegenden Arbeit konnte erstmalig ein auf bovinen Keratinozyten basierendes In-vitro-Ischämiemodell etabliert werden, das die Untersuchung zellulärer Mechanismen der Epidermis ermöglichte. Die OGD zeigte den stärksten Einfluss auf die Zellviabilität sowie eine veränderte Zelldifferenzierung der Keratinozyten, was die pathophysiologischen Veränderungen im Rahmen der CHD reflektiert. Die ebenfalls starken Zellveränderungen infolge eines Glukoseentzugs verdeutlichen die Rolle der Glukose im Zellmetabolismus der Keratinozyten. Solch ein epidermaler Glukosemangel ist in Verbindung mit der negativen Energiebilanz der Rinder im peripartalen Zeitraum denkbar. Die Ergebnisse infolge einer Hypoxie verweisen auf vielfältige Adaptationsmechanismen der Keratinozyten an hypoxische Bedingungen, denen sie in der Epidermis in vivo während der Zelldifferenzierung ausgesetzt werden. Damit besitzt das In-vitro-Ischämiemodell ein großes Potenzial für den Einsatz in der Klauenreheforschung, um einerseits die mit einer Ischämie einhergehenden pathologischen Veränderungen der CHD untersuchen zu können. Andererseits liefert das Modell wertvolle Informationen zu den physiologischen Grundlagenmechanismen der Epidermis, die mit der Zelldifferenzierung einhergehen<br>The subclinical laminitis or claw horn disruption (CHD) is of great economic importance in the dairy industry as it causes lameness, poor general condition and reduced performance. Despite extensive research efforts, the pathomechanism of CHD remains widely unclear. The current hypotheses on CHD pathogenesis include ischemic alterations of the epidermal keratinocytes resulting from an impaired blood supply. This causes an alteration of cell proliferation, a dermo-epidermal separation and an impaired keratinization and horn production. Therefore, in vitro ischemia models are of critical importance in clarification of the epidermal responses to an altered microcirculation. The aim of this study was the establishment of an in vitro ischemia model based on bovine claw keratinocytes. This in vitro model should enable the investigation of cellular pathomechanisms following exposure to ischemia, hypoxia and glucose deprivation. An additional aim was the analysis of the differentiation pattern of keratinocytes under ischemic, hypoxic and hypoglycaemic conditions. To establish the in vitro ischemia model, the keratinocytes were exposed to oxygen-glucose deprivation (OGD). In addition, this model allowed the parallel examination of hypoxic and hypoglycaemic conditions on bovine claw keratinocytes. The experiments were divided into a short-term analysis over 96h and a long-term analysis over three weeks. Measurement of cell viability was performed by LDH(lactatedehydrogenase) and MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide) assays. Furthermore, the differentiation pattern of the keratinocytes after exposure to ischemia, hypoxia and glucose deprivation was detected by western blot analysis of the focus on expression of involucrin and loricrin. The highest cytotoxic effect was measured after short exposure to OGD followed by a time-dependent decrease of cell viability and extensive morphological changes of the keratinocytes. Hypoxic conditions lead to a time-dependent decrease of cell viability with the highest cytotoxicity after two weeks. The keratinocytes showed slight changes in cell morphology while maintaining a confluent cell layer. Exposure of keratinocytes to glucose deprivation showed a high decrease of cell viability and strong morphological changes. Furthermore, western blot analysis showed an altered expression pattern with increased involucrin and loricrin levels after exposure to OGD, hypoxia and glucose deprivation. The present study established for the first time an in vitro ischemia model based on bovine claw keratinocytes to study the cellular mechanisms of the epidermis. After exposure to OGD, keratinocytes showed the highest loss in cell viability and an altered cell differentiation. This reflects the pathophysiological changes following epidermal ischemia occurring during the pathogenesis of CHD. The massive cellular alterations after glucose deprivation provide good evidence for the importance of glucose in the cellular metabolism of keratinocytes. An epidermal glucose deficiency may occur in combination with a negative energy balance during peripartal period in cattle. The results of hypoxia show the different adaptive mechanisms of keratinocytes to hypoxic conditions which are present in the epidermis during cell differentiation. Thus, the in vitro ischemia model has a great potential for use in research into CHD pathogenesis and pathomechanisms associated with ischemia. On one side, it is possible to investigate the pathological changes following ischemia during CHD. On the other side, the model offers useful information on physiological response mechanisms of the epidermis that correlate with cell differentiation

Le proprietà meccaniche dei sistemi biologici hanno una grande importanza nel determinare il loro comportamento. Molti potenziali farmaci possono modificare le proprietà meccaniche della membrana biologica e indirettamente modulare la funzione di proteine di membrana. Analogamente, molti stati patologici a livello cellulare presentano un fenotipo con proprietà meccaniche alterate e la modifica di tali proprietà è tipicamente il risultato di una riorganizzazione del citoscheletro. Allo stesso tempo, le cellule sondano le proprietà reologiche della matrice extracellulare (ECM) attivando, a seconda della risposta ottenuta, diversi percorsi biochimici. Tali fenomeni sono spesso caratterizzati da una riorganizzazione citoscheletrica a seguito di stimoli periodici, come avviene ad esempio nel sistema cardiovascolare o nei polmoni. La scienza che tratta questi fenomeni è la meccanobiologia. Il lavoro di questa tesi di dottorato è dedicato all’analisi delle proprietà meccaniche di costituenti biologici, da semplici modelli di membrana, come doppi-strati-lipidici supportati (SLB) e vescicole giganti unilamellari (GUV), a sistemi quali colture cellulari in-vitro. Le tecniche di indagine usate hanno coinvolto: microscopia ottica in contrasto di fase, DIC e fluorescente; microscopia a forza atomica (AFM). Sono stati sviluppati, all’interno della tesi, metodi di analisi e dispositivi dedicati per specifiche applicazioni e misure di campioni. È stato progettato, testato e impiegato un incubatore per esperimenti di live-cell imaging da integrare direttamente sul tavolino (on-stage) di un microscopio ottico. Sono stati ottenuti simultaneamente parametri di migrazione di cellule esposte a diversi trattamenti, o poste su substrati aventi diversa rigidità meccanica. Lo stesso incubatore è stato ridisegnato per poter alloggiare uno stretcher uniassiale in grado di fornire al substrato specifiche funzioni periodiche di deformazione e valutare la conseguente risposta delle cellule in termini di migrazione e polarizzazione. Tra i metodi di indagine, è stata sviluppata l’analisi quantitativa di migrazione di singola cellula, ed è stato impiegato il modello “Persistence-Random-Walk”. Lo scopo era quello di analizzare l’effetto citostatico di un potenziale farmaco nelle cellule U87MG, usate come modello del glioblastoma multiforme. L’analisi effettuata ha infatti mostrato l’efficacia sia citostatica che antimitotica della molecola. Sono stati indagati inoltre i possibili meccanismi biochimici alla base di tali effetti. Nel contesto dei SLB e GUV è stata implementata rispettivamente l’analisi sulla tensione di linea di domini che simulano lipid-rafts e la costante di bending, basandosi sulla teoria delle fluttuazioni di membrana. Nel primo caso sono stati confrontati i risultati sulla misura della tensione di linea di miscele ternarie costituite da diverse componenti rilevanti nella formazione di lipid-rafts. Nel secondo caso, è stato valutato il ruolo di molecole esogene (peptidi antimicrobici e lipopeptidi) nella determinazione della costante di bending. Nella caratterizzazione visco-elastica del citoscheletro con AFM, è stato implementato un software basato sul modello di Ting, in grado di estrapolare i parametri viscoelastici dalle singole curve andata-ritorno. Si è studiato l’effetto del potenziale farmaco prima citato, sulle proprietà reologiche di cellule U87MG al fine di correlare migrazione e proprietà meccaniche cellulari. Sono stati sviluppati software dedicati alla ricerca di eventi Jump-Through-Force nell’analisi di SLB, e di eventi di estrazioni di tubi durante la retrazione della punta AFM sulla membrana plasmatica come possibile metodo per evidenziare variazioni di proprietà reologiche di membrane esposte a diversi trattamenti farmacologici.<br>Mechanical properties of biological systems play a crucial role for their own behavior. As an example, many potential drugs could modify mechanical properties of the biological membrane and indirectly modulate transmembrane protein functions. Similarly, many pathological conditions at the cellular level are characterized by a phenotype with altered mechanical properties, and these alterations are due to cytoskeleton reorganization. At the same time, cells continuously probe rheological properties of extracellular matrix (ECM) enabling, depending on response obtained by the substrate, different downstream signaling cascades. In many cases, cytoskeleton reorganization occurs also when cells are experiencing periodic mechanical stimuli, as it happens for example in the cardiovascular system or in lungs. All these aspects are treated by a recent branch of physic and biology sciences: “Mechano-biology”. This PhD thesis work has been devoted to study some specific aspects of mechanical properties of biological systems: from simple models of the biological-membrane, like supported-lipid-bilayer (SLB) or giant-unilamellar-vescicle (GUV), to in-vitro cells. Investigation techniques exploited in this work include: phase-contrast optical microscopy, DIC and fluorescence microscopy and atomic force microscopy (AFM). In the thesis we developed analysis-methods and devices dedicated to specific application and measurements of biological samples. It has been designed, tested and employed successfully an on-stage cell incubator for live cell imaging. From time-lapse microscopy experiments we obtained different quantitative migration parameters both for cell exposed to different drugs and for cells seeded on substrates with different mechanical rigidity. The same cell incubator has been modified to include an uniaxial stretcher, able to provide specific periodic deformation functions to the substrate on which cells are growing, and we studied the effect of the periodic stimulation on cell migration and polarization. Among the different analysis methods, a single cell migration analysis protocol has been developed, exploiting the “Persistence-Random-Walk” model. The ultimate goal was that of analyzing the cytostatic effect of a potential drug for U87MG cell line, employed as model of the glioblastoma multiforme disease. The analysis has in fact shown the efficiency of this molecule for both migration and replication of this cell line. Furthermore, possible biochemical mechanisms of action involved in these effects have been investigated. In the context of SLBs and GUVs a line tension analysis of domains recapitulating lipid-raft and a bending constant measurement have been implemented, both based on Flickering spectroscopy theory. In the former case, line tension results of ternary mixture containing different components relevant for lipid-rafts formation have been compared for different lipid compositions. In the latter case, the role of exogenous molecules (antimicrobial peptides and lipopeptides) on the bending constant has been investigated. In viscoelastic characterization of the cell cytoskeleton through AFM, a Ting model-based software has been implemented, allowing to extrapolate viscoelastic parameters from single indentation-retraction curves. Using this method, the effect of the previously mentioned potential drug has been investigated, trying to correlate rheological properties to migration capabilities of U87MG. Finally, software dedicated to Jump-Through-Force curves by AFM to identify specific events on SLB, and tether pulling events during AFM tip retraction on plasma-membrane have been developed; in order to find possible methods to highlight variations in rheological properties of membrane exposed to different drug treatments.

Recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for the treatment of acute ischaemic stroke. However, the application of this therapy is limited to <5-7% of patients due to the associated increased risk of haemorrhagic transformation (HT). Although it is known that HT is related to rt-PA-induced blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. The obtained results show that long-term studies are needed to elucidate time-dependent molecular mechanisms associated to BBB breakdown, and to explore protective BBB therapies after ischaemic stroke and rt-PA treatment. On the other hand, it has been demonstrated that OGD induces significant alterations to loading control proteins for Western Blot analysis proposing Stain-Free technology as an alternative normalization method to traditional housekeeping proteins. Finally, serum Cav-1 levels could represent a potential biomarker predicting the ischaemic outcome before rt-PA administration<br>L'rt-PA és l’únic fàrmac aprovat per tractar l’ictus isquèmic agut. No obstant, l’estreta finestra terapèutica, deguda al risc associat de transformació hemorràgica (TH) provoca que només s’apliqui a <5-7% dels pacients. La TH està relacionada amb la disrupció de la barrera hematoencefàlica (BHE) deguda a l’rt-PA però els mecanismes subjacents encara no estan del tot establerts. Els resultats obtinguts mostren que es requereixen estudis a llarg termini per tal de dilucidar els mecanismes dependents del temps associats a la disrupció de la BHE, i explorar noves teràpies protectores per al tractament de l’ictus isquèmic. S’ha demostrat que la POG provoca canvis significatius en els nivells proteics de controls de càrrega per “Western blot” i es presenta la tecnologia “Stain-Free” com a una alternativa a la normalització tradicional. Finalment, els nivells sèrics de Cav-1 podrien representar un potencial biomarcador predictor del pronòstic després d’una isquèmia en absència d’rt-PA<br>S'ha extret el capítol de resultats del pdf de la tesi fins a la seva publicació en forma d'article. Results chapter removed from pdf file until publication

Les ultrasons focalisés permettent d’effectuer des traitements thérapeutiques ciblés dans le corps humain. Dans le domaine des applications cardiovasculaires, ils permettent de détruire des caillots sanguins susceptibles de se former dans le système vasculaire. Dans ce cas, les mécanismes de thrombolyse sont largement liés à la cavitation ultrasonore, dont la dynamique complexe reste un obstacle à l’élaboration d’un dispositif thérapeutique. Dans le cadre de cette thèse, un système permettant le contrôle temporel de l’activité de cavitation en régime pulsé a été développé puis caractérisé. Ce dispositif utilise un transducteur focalisé et un hydrophone avec une boucle de rétroaction pour réguler l’activité de cavitation. Alors qu’en régime non régulé l’activité de cavitation a un caractère très aléatoire, le système de régulation mis au point permet d’atteindre un niveau de cavitation souhaité de manière très reproductible et avec une bonne stabilité temporelle. L’application de ce dispositif à la thrombolyse ultrasonore a été testée in vitro sur des caillots de sang humain. Au dispositif précédent a été ajouté un système permettant de déplacer le caillot sanguin au niveau du foyer, ainsi qu’un conduit permettant de compter le nombre de fragments libérés par la destruction du caillot. En comparaison des essais en régime non régulé, les essais en régime régulé ont montré une excellente efficacité thrombolytique et une très bonne reproductibilité, tout en diminuant les intensités acoustiques utilisées pour lyser les caillots sanguins. En parallèle des essais in vitro, une campagne de thrombolyse ultrasonore in vivo a été mise en place afin de réaliser des essais sur un modèle animal d’ischémie aiguë de membre inférieur. Un dispositif ultrasonore extracorporel in vivo guidé par échographie et monté sur un bras robotisé 6 axes a été développé. Un modèle ovin de thrombose artérielle a également été développé. Les tests ont permis de valider, d’une part, la faisabilité du modèle de caillot artériel et, d’autre part, le concept de thrombolyse extracorporelle purement ultrasonore basée sur la cavitation inertielle régulée<br>Focused ultrasound can be used for therapeutic applications in the human body. In cardiovascular applications, they can destroy blood clots formed in the vascular system. In this case, thrombolysis mechanisms are related to ultrasonic cavitation, but the complex dynamics remains an obstacle to the development of a therapeutic device. In this thesis, a system for the temporal control of the pulsed cavitation activity has been developed and characterized. This device uses a focused transducer and a hydrophone with a feedback loop for regulating the cavitation activity. While cavitation activity has a random behaviour in non-regulated conditions, the control system developed achieves a desired level of cavitation with very reproducibly and with good temporal stability. The application of this device to the ultrasound thrombolysis was tested in vitro on human blood clots. In the previous device was added a system for moving the blood clot at the focal point, and a tube for counting the number of fragments released by the destruction of the clot. In comparison to uncontrolled regime, tests showed an excellent thrombolytic efficacy and a very good reproducibility, with reduced acoustic intensities. In parallel to the in vitro tests, ultrasound thrombolysis was tested in vivo on an animal model of acute limb ischemia. An extracorporeal ultrasound device, guided by ultrasound and mounted on a robotic arm, has been developed for in vivo investigation. An ovine model of arterial thrombosis has also been developed. Tests were used to validate the feasibility of the model of arterial clots and to validate in vivo the concept of purely ultrasonic extracorporeal thrombolysis based on inertial cavitation regulation system

In questo lavoro di tesi viene affrontato il tema della fluidodinamica computazionale mediante un modello in 3D basato su geometria e motilità dello stomaco umano al fine di analizzare il comportamento del contenuto gastrico, le cui differenti proprietà reologiche influenzano non solo il flusso dei fluidi gastrici, ma anche la distribuzione delle particelle di cibo all’interno dello stomaco, organo cavo a forma di J: il fine ultimo è dunque quello di fornire uno sguardo introspettivo, con un punto di vista unico, sull’argomento, chiarendo i meccanismi insiti nel processo della digestione gastrica.

La pollution atmosphérique particulaire représente un facteur de risque potentiel même à de faibles concentrations. Quelle que soit leur composition, les nanoparticules (NP) sont parmi les plus nocives en raison de leur capacité à atteindre profondément les tissus pulmonaires, la circulation sanguine et les organes. Notre travail constitue une base solide dans la compréhension des effets induits par une sélection de polluants atmosphériques représentatifs, utilisés dans un contexte environnemental réaliste. Nos résultats in vitro fournissent l'évidence d'un effet génotoxique à dominance clastogène lors de l’exposition de cellules à des NP contenant du CeO2, qui d’une part exercent une action mécanique sur le noyau, et d’autre part stimulent la production de O2•- / H2O2 via la NADPH oxydase et la mitochondrie, favorisant la production d’HO•. En présence de 1-Nitropyrène, on observe selon la dose utilisée deux mécanismes génotoxiques successifs, passant d'une dominance aneugène à clastogène, cet effet étant relié à une surproduction de HO• détectable par RPE. L'étude in vivo chez le rat a mis en œuvre un système de génération d'aérosols de NP contenant des pesticides, réalisant une exposition quotidienne et chronique à de faibles doses. Un effet direct sur la fonction hémodynamique myocardique reflétant l'apparition de dommages cellulaires irréversibles a été observée, ainsi que des lésions rénales, hépatiques, et l'installation d'un stress oxydant et d’une inflammation systémiques. Sur le long terme, l'effet des NP modifie les profils lipidiques et favorise la survenue d’une intolérance au glucose. Des modes d'action spécifiques à chaque pesticide employé ont été proposés<br>Epidemiological studies have consistently reported that particulate matter in ambient air pollution is associated with increases in cardiopulmonary diseases and mortality. Because they can deeply penetrate lung tissue, reaching blood stream and organs, nanoparticles (NPs) are considered particularly harmful. Here, our foray into the specific mutagenicity and cytotoxicity of NPs focused on manufactured nano-CeO2 (a fuel additive) and NPs known to form in air from a variety of atmospheric toxicants (eg, combustion residues, pesticides). We first revealed a genotoxic effect of nanoCeO2 on human fibroblasts by a clastogenic mechanism following stimulation of the release of O2•-/H2O2 by NADPH oxidase and mitochondria. However, upon exposure of these cells to nM doses of 1-nitropyrene (a combustion byproduct) promotion of DNA damage involving an aneugenic mechanism occurred before a clastogenic effect was seen at µM doses. Second, using a home-made chamber equipped with an aerosol generator, we determined indices of oxidative stress and tissue damage in rats chronically inhaling toxicant NPs for 1-5 months at environmentally relevant doses. Long term exposure, even at low NPs doses, provoked systemic oxidative stress, lipid peroxidation, kidney damage, liver dysfunction, changes in lipid profile and occurrence of disorders of glucose tolerance. Moreover, a strong impairment of hemodynamic performance was evidenced in hearts from NP-exposed rats. By extending literature data of the in vitro toxicity of NPs to the in vivo situation, our study incriminates the nano-sized components of atmospheric particulate matter as a privileged vector of genotoxicity and cardiotoxicity

Dottorato di ricerca in Farmacologia e Biochimica della morte della cellulare, XIX Ciclo, a.a. 2006-2007<br>In questo studio sono stati determinati i livelli endogeni dell’endocannabinoide finora meglio caratterizzato, anandamide (AEA) e l’attività degli enzimi responsabili, rispettivamente, della sua sintesi e degradazione, la NAPE-PLD e la FAAH, nella corteccia e nello striato di ratti sottoposti ad occlusione dell’arteria cerebrale media (MCAo) di 2 ore. È stato osservato che il contenuto di AEA nello striato di ratti sottoposti a MCAo, ma non nella corteccia, era significativamente incrementato (all’incirca di 3 volte rispetto ai ratti controllo, P < 0.01) e questo incremento era accompagnato parallelamente da un aumentata attività della NAPE-PLD (di circa 1.7 volte rispetto ai ratti controllo, P < 0.01) e da una ridotta attività (~ 0.6 volte; P<0.05) ed espressione della FAAH (~0.7 volte; P< 0.05). Questi effetti indotti dalla MCAo venivano ulteriormente potenziati da un ora di riperfusione, mentre il legame dell’AEA al recettore cannabinoide CB1 e al recettore vanilloide TRPV1 non erano influenzati in maniera significativa dall’insulto ischemico. Inoltre, il trattamento con l’antagonista del recettore CB1, SR141716, e non quello con l’agonista R-(+)- WIN55,212-2, ha dimostrato di ridurre significativamente (33%; P<0.05) il volume cerebrale d’infarto dopo 22 ore di riperfusione; mentre la somministrazione di una dose neuroprotettiva di 17β-estradiolo (0.20 mg/kg, i.p.), che era in grado di ridurre il volume d’infarto del 43%, si dimostrava capace anche di ridurre l’effetto dell’ischemia cerebrale sul sistema endocannabinoide in maniera recettore estrogenico dipendente. In conclusione, abbiamo dimostrato che il sistema endocannabinoide è implicato nella fisiopatologia del danno cerebrale tMCAo indotto e che la modulazione farmacologica di questo sistema endogeno da parte dell’estradiolo conferisce neuroprotezione.<br>Università della calabria

Dottorato di Ricerca in Biochimica Cellulare ed attività dei Farmaci in Oncologia XXIV Ciclo,,a.a. 2010-2011<br>La leptina, oltre ad avere effetti sull’ipotalamo per il controllo del peso corporeo, è coinvolta nella regolazione della funzionalità, dello sviluppo e della sopravvivenza neuronale. Studi recenti hanno evidenziato i suoi effetti neuroprotettivi nel danno ischemico cerebrale, ma fino ad oggi il ruolo del fattore di trasduzione ed attivatore trascrizionale (STAT)-3, il principale mediatore della via di trasduzione del segnale di ObR nel cervello, non è stato chiarito. I nostri dati dimostrano che la somministrazione sistemica acuta di leptina è neuroprotettiva in ratti sottoposti ad occlusione permanente dell’arteria cerebrale media (MCAo), come documentato dalla riduzione significativa del volume di infarto cerebrale e del deficit neurologico fino a 7 giorni dopo l’induzione di ischemia. Mediante analisi di immunofluorescenza e tecniche di frazionamento subcellulare abbiamo osservato che la neuroprotezione da leptina è associata con la modulazione dei livelli di fosforilazione di STAT-3 in differenti tipi cellulari nella corteccia cerebrale ischemica. Infatti, poche ore dopo l’insulto la leptina aumenta i livelli di p-STAT3 nel nucleo degli astrociti della penombra ischemica contribuendo così agli effetti benefici di queste cellule sull’evoluzione del danno. L’aumentata espressione di homer-1a che osserviamo negli astrociti fino a 7 giorni dopo l’induzione di ischemia, sottolinea ulteriormente il loro ruolo benefico. Mediante ricostruzione 3D di immagini di microscopia elettronica, combinata con analisi morfometrica, abbiamo osservato che gli astrociti reattivi mostrano un ridotto coverage bilaterale, mentre la percentuale di contatto con le sinapsi glutammatergiche rimane invariata. Inoltre, l’aumento di p-STAT3 indotto dalla leptina nei neuroni dopo 24h di MCAo è associato con un aumento dell’espressione dell’inibitore tissutale delle metalloproteasi della matrice (TIMP)-1 nella corteccia, suggerendo un suo coinvolgimento nella neuroprotezione indotta dall’adipochina<br>Università degli Studi della Calabria

Tese de doutoramento em Ciências e Tecnologias da Saúde (Pré-Bolonha), especialidade de Biologia Celular e Molecular, apresentadas à Faculdade de Farmácia da Universidade de Coimbra<br>A isquémia cerebral global devido à interrupção do fluxo sanguíneo no cérebro leva à privação de oxigénio e glicose nas células, reduzindo a energia disponível para manutenção do funcionamento celular. Os neurónios são especialmente sensíveis a esta falha energética, o que pode levar à activação de vias moleculares de morte celular. A região CA1 do hipocampo é particularmente vulnerável à isquémia global. Contudo, os sinais de morte celular surgem apenas horas ou dias após o insulto, criando uma janela temporal na qual podem ser aplicadas estratégias terapêuticas com vista à diminuição dos danos neurológicos causados pelo insulto. Pensa-se que este intervalo se deve a modificações transcripcionais que podem promover a sobrevivência ou a morte neuronal. No entanto, apesar do trabalho desenvolvido na identificação de genes cuja expressão está alterada após a isquémia global, os mecanismos responsáveis pela vulnerabilidade dos neurónios do hipocampo continuam por esclarecer. Assim, este trabalho teve como objectivo a identificação de mecanismos moleculares diferentemente regulados em culturas primárias de neurónios do hipocampo submetidos a um modelo in vitro para a isquémia global, a privação de oxigénio e glicose (do inglês, oxygen and glucose deprivation – OGD). Começámos por estabelecer o protocolo para análise da expressão genética usando este modelo. Verificámos que o estímulo de OGD induz um aumento na morte celular em função da duração do insulto, e que esta é significativa 24h após o estímulo, mostrando que este modelo induz morte celular retardada. Utilizando antagonistas selectivos para os receptores de glutamato do tipo AMPA e NMDA vimos que o bloqueio destes receptores teve um efeito neuroprotector, confirmando o seu papel na componente excitotóxica da morte por OGD. Vimos ainda a activação de calpaínas, proteases ligadas a vias de morte celular. De seguida investigámos as alterações no transcriptoma dos neurónios do hipocampo submetidos a OGD utilizando microarrays. Para isso, o RNA total das células foi extraído 7h e 24h após o estímulo com o objectivo de identificar genes envolvidos na resposta imediata e mais tardia à isquémia. Vimos que às 7h após OGD há uma maior repressão, enquanto 24h após o insulto há uma maior indução, na expressão de genes. A análise da ontologia genética mostrou que genes relacionados com stress oxidativo, metabolismo, apoptose, sinapse e actividade de canais iónicos apresentam diferentes níveis de expressão após OGD. Tanto quanto sabemos, este é o primeiro estudo a combinar microarrays e OGD como ferramenta para estudar alterações no perfil genético de neurónios do hipocampo a diferentes tempos de recuperação. Os resultados obtidos nos microarrays foram validados através da análise de qPCR para genes selecionados, pertencentes a diferentes grupos ontológicos. Observámos também que vários genes codificantes para proteínas da sinapse apresentaram diminuição na sua expressão em neurónios submetidos a OGD, bem como um aumento dos níveis de REST, um factor de transcrição que reprime a expressão de genes codificantes de proteínas neuronais sinápticas, como alguns dos que apresentam diminuição da expressão após OGD. Vimos ainda que este estímulo provoca a diminuição nos níveis de mRNA e proteína da subunidade GluA1 dos receptores AMPA, bem como das subunidades GluN2A e GluN2B e o aumento dos níveis de mRNA da subunidade GluN3A dos receptores NMDA, o que pode levar à alteração da composição destes receptores em neurónios do hipocampo. Estes resultados sugerem que o estímulo de OGD leva à activação de um programa transcripcional que causa uma repressão da expressão de proteínas sinápticas. Por fim, investigámos os níveis de expressão de genes codificantes para canais iónicos e, nomeadamente, a contribuição do canal de cloreto CLIC1 para a morte induzida por OGD, uma vez que o mRNA do CLIC1 está aumentado em neurónios submetidos a OGD. O papel do CLIC1 em neurónios é desconhecido, embora se pense que a activação deste canal em micróglia leva à morte de neurónios em condições tóxicas. Contudo, os nossos resultados mostram que a sobre-expressão do CLIC1 torna os neurónios menos vulneráveis ao dano induzido por OGD, e resultados preliminares sugerem que a expressão do CLIC1 pode estar aumentada em regiões do hipocampo mais resistentes à isquémia global. Os nossos resultados sugerem que o CLIC1 faz parte de um mecanismo intrínseco de protecção activado em neurónios após o insulto isquémico. Os resultados obtidos caracterizam o perfil global de expressão genética induzido por OGD em neurónios do hipocampo, permitindo o estudo de dois mecanismos distintos que podem contribuir para a sobrevivência neuronal: a diminuição da expressão de componentes sinápticos, nomeadamente dos que estão envolvidos na excitotoxicidade, e o aumento na expressão do CLIC1. Estes mecanismos podem ser explorados no sentido de desenvolver estratégias terapêuticas para o tratamento da isquémia cerebral.<br>Cerebral global ischemia due to interruption of blood supply to the brain results in oxygen and glucose deprivation of brain cells, reducing the energy available to maintain normal cellular functions. Neurons are especially sensitive to this energetic insufficiency and consequently fail to maintain the ionic gradients necessary for cellular function and homeostasis, which ultimately leads to the activation of several molecular pathways that impair neuronal function or may lead to cell death. The neurons of the CA1 region of the hippocampus are particularly vulnerable to global ischemia. However, signs of cell death arise only hours to days after the insult, providing a temporal window in which therapeutical approaches to prevent the delayed neurological and cognitive deficits triggered by ischemia can be employed. This delay is thought to include transcriptional changes that can either prime cell survival or enhance neuronal death. However, despite the effort to identify genes differently expressed after ischemia, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain elusive. As such, the present work was aimed at investigating which transcription-dependent molecular mechanisms are differently regulated in hippocampal neuronal cultures subjected to oxygen and glucose deprivation (OGD), an in vitro model for cerebral global ischemia. Initially, we established the experimental set-up for the analysis of gene expression in this model. We observed an increase in OGD-induced neuronal death as a function of OGD time length, but neuronal death was only significant 24h after the stimulus, showing that OGD induced delayed neuronal death. Selective antagonists of the AMPA and NMDA glutamate receptors were neuroprotective, confirming the contribution of these receptors to the excitotoxic component of this in vitro ischemic model. OGD also triggered the activation of calpains, proteases known to mediate many deleterious effects in post-ischemic neurons. We then used microarray technology to study changes in the transcriptome of rat hippocampal neurons submitted to OGD. For that purpose, total RNA was extracted 7h and 24h after OGD and used in a whole-genome RNA microarray to identify genes related to an early and a delayed ischemic response. At 7h of recovery there is a general repression of genes, while at 24h there is a general induction of gene expression. Analysis of the gene ontology showed that genes related with a variety of cellular functions, such as the response to oxidative stress, metabolism, apoptosis, synapse and ion channel activity, were differently regulated after OGD. As far as we know, no previous study has used microarray technology and the OGD model as a tool to investigate ischemia-induced changes in the transcriptome of hippocampal neurons at different periods of recovery. The validity of the microarray data was confirmed by qPCR analysis of selected genes from different functional groups. According to the microarray data, several synaptic protein genes were down-regulated after OGD. We also observed that OGD triggers the up-regulation of REST, a transcription factor that represses the expression of genes related with the synaptic function. Additionally, OGD decreased the mRNA and protein levels of the GluA1 AMPAR subunit as well as the GluN2A and GluN2B subunits of NMDARs, but increased the mRNA expression of the NMDAR subunit GluN3A, which might lead to a change in the composition of ionotropic glutamate receptors in hippocampal neurons. These results suggest that OGD activates a transcriptional program leading to a general repression of proteins present in the synapse. Moreover, we analyzed OGD-induced changes in the expression levels of ion channel protein genes. In particular, we pursued the contribution of the chloride channel CLIC1 to the OGD-mediated neuronal response. The role of CLIC1 in neurons is largely unknown but it has been suggested to contribute to neuronal death when activated in microglia under toxic conditions. However, we observed that neurons over-expressing CLIC1 are less vulnerable to OGD-induced cell death, and preliminary results suggest that CLIC1 may be up-regulated in the hippocampal regions that are more resistant to ischemia. Overall, these results suggest that CLIC1 can be part of an intrinsic protective mechanism activated in neurons after an ischemic insult. The results obtained in this work present the global expression profile elicited by in vitro ischemia in hippocampal neurons, and help to investigate two different mechanisms that may contribute to neuronal survival in neurons submitted to OGD: decrease in the expression of synaptic components, namely those involved in excitotoxicity, and up-regulation of the chloride channel CLIC1. The targets that we identified may be explored to develop attractive therapeutic strategies for the treatment of cerebral ischemia.

Dissertação de Mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.<br>Cardiovascular diseases, including myocardial infarction, are a leading cause of morbidity and mortality worldwide. Ischemia is a major event during myocardial infarction and results from the deprivation of blood to the heart, caused by the obstruction of a coronary artery. Reperfusion, i.e. the restoration of blood flow following an ischemic event, is the routine clinical procedure. Although reperfusion is essential to preserve the hypoxic myocardium, the sudden reoxygenation of the hypoxic tissues has important adverse effects, including initiation of cell death programs. This phenomenon, known as ischemia-reperfusion injury, is responsible for a fraction of the myocyte death observed following myocardial infarction. Since the human heart has limited regenerative capacity, dead cardiomyocytes are not renewed and, instead, a reparative scarring mechanism occurs. Cardiac fibrosis promoted by cardiac fibroblasts is the principal event underlying scar formation. Upon ischemia-reperfusion injury, cardiac fibroblasts increase their proliferative rate and transform into a contractile and active form, called myofibroblasts. Myofibroblasts are hyper stimulated cells, that produce large amounts of ECM proteins such as collagens, contributing to ECM deposition. Although fibrosis is essential to mending a damaged heart, excessive scarring and persistence of myofibroblasts in the heart is a maladaptive event that leads to ventricle wall stiffness and impairment of heart function. To minimize the effects caused by myocardial infarction, two main therapeutic endpoints could be pursued: i) increase of cardiomyocyte proliferation, to promote replacement of cells lost upon injury, and ii) blocking of excessive scarring process and fibrosis, which contributes to heart dysfunction. MicroRNAs are endogenous small non-coding RNAs that play a major role in the posttranscriptional regulation of gene expression. Currently, 2,588 mature microRNAs are annotated in the human genome (miRBase Release 21, June 2014) and it is estimated that microRNAs might control the expression of ca. 60% of the human genes. MicroRNAs have been shown to be involved in diverse cellular functions such as proliferation, differentiation and apoptosis. The observation that each microRNA can regulate multiple target transcripts, together with the fact that microRNA expression can be modulated by synthetic molecules that mimic or prevent microRNA function (microRNA mimics and inhibitors, respectively), position microRNAs as attractive therapeutic tools. This project is focused on the two main pathological events of myocardial infarction, ischemia-reperfusion and fibrosis, and the potential role of microRNAs in regulating these processes. In the first stage of the project, we optimized experimental conditions to model cardiac ischemia-reperfusion injury in vitro and assessed the impact of injury to cardiomyocyte survival. For this purpose, we used two sources of human cardiomyocytes, both derived from induced pluripotent stem cells. Cells were first exposed to ischemia, achieved by incubation in a hypoxic environment for 24 or 48 hours in the absence of nutrients (except lactate in selected conditions) and subsequently returned to normal culturing conditions, mimicking the reperfusion step. We observed a strong decrease of cardiomyocyte number following ischemia-reperfusion injury, accompanied by changes in cell morphology. In addition, we also optimized a reverse transfection protocol that can be used for transfection of microRNAs in large-scale screenings aimed at clarifying the role of microRNAs in ischemia-reperfusion injury. From these experiments, we were able to demonstrate that hsa-miR-302d-3p strongly induces proliferation of human cardiomyocytes. The second stage of the project was focused on human cardiac fibroblasts. Cardiac fibroblasts were exposed to ischemia-reperfusion injury in vitro using a protocol similar to that applied for cardiomyocytes. Interestingly, our results suggested that ischemiareperfusion leads to a slight increase of cardiac fibroblast proliferation, which is triggered by reperfusion. A reverse transfection protocol for microRNA transfection was also optimized and we performed a “proof-of-principle” pilot screening with 36 microRNAs, aiming at identifying microRNAs controlling myofibroblast activation. Among those, we identified microRNAs that have a strong activity in promoting (hsa-miR-26b-5p, hsa-miR-19b-2-5p, hsamiR- 2052, hsa-miR-875-5p, hsa-miR-210-3p and hsa-miR-19a-5p) or preventing (hsa-miR- 1281, hsa-miR-130a-5p and hsa-miR-143-5p) myofibroblast activation. Additionally, it was also possible to establish a preliminary correlation between cardiac fibroblast activation and proliferation after microRNA treatment. Our findings indicate that cardiac fibroblasts transformation into myofibroblasts results in a significant decrease of their proliferation rate. Overall, this study has established the experimental conditions for performing largescale screening studies in human cardiomyocytes and cardiac fibroblasts, which will be important to identify novel microRNAs involved in ischemia-reperfusion injury.<br>As doenças cardiovasculares, nomeadamente o enfarte do miocárdio, são uma das principais causas de morbilidade e mortalidade a nível mundial. A isquémia é o principal acontecimento de um enfarte do miocárdio, e resulta da obstrução de uma artéria coronária, reduzindo o fluxo sanguíneo no coração. A reperfusão é uma intervenção médica na qual o fluxo sanguíneo é restabelecido após isquémia. Apesar de essencial à preservação do miocárdio isquémico, a rápida reoxigenação provocada pela reperfusão tem efeitos devastadores no tecido cardíaco, que incluem a ativação de mecanismos de morte celular. Este fenómeno, conhecido como lesão de isquémia-reperfusão, é em parte responsável pela morte de cardiomiócitos observada após o enfarte do miocárdio. Uma vez que o coração humano é incapaz de se regenerar, os cardiomiócitos danificados não são repostos e, em vez disso, é ativado um mecanismo reparador, conhecido como cicatrização. A fibrose cardíaca promovida por fibroblastos cardíacos é o principal evento subjacente à formação da cicatriz. Após exposição a isquémia-reperfusão, os fibroblastos cardíacos aumentam a sua taxa proliferativa e transformam-se numa forma ativa e contrátil denominada de miofibroblastos. Os miofibroblastos são células híper estimuladas, que produzem grandes quantidades de proteínas de matriz extracelular (MEC), nomeadamente colagénio, contribuindo assim para a deposição de MEC. Embora a fibrose seja essencial para reparar danos cardíacos, a excessiva cicatrização e a permanência de miofibroblastos no coração é um processo patológico que pode conduzir à rigidez da parede do ventrículo, deteriorando a função cardíaca. Para diminuir os efeitos provocados pelo enfarte do miocárdio, duas abordagens terapêuticas podem ser implementadas: i) aumentar a proliferação de cardiomiócitos para promover a substituição das células danificadas durante o enfarte e ii) bloquear a cicatrização excessiva e fibrose cardíaca, que contribuem para a disfunção cardíaca. MicroARNs são pequenos ARN endógenos não-codificantes, que desempenham um papel chave na regulação pós-transcricional da expressão de genes. Atualmente, estão descritos 2588 microARNs maduros no genoma humano (miRBase Release em 21, Junho 2014) e é estimado que estes microARNs possam controlar até 60% da expressão de genes humanos. Foi demonstrado que os microARNs estão envolvidos em diversas funções celulares como a proliferação, diferenciação celular e apoptose. A observação de que cada microARN é capaz de regular vários transcritos alvo, conjuntamente com o facto de que a sua expressão pode ser modulada por moléculas sintéticas que mimetizam ou antagonizam a função de cada microARN (mímicos ou inibidores de microARNs, respetivamente), posicionam os microARNs como atrativas ferramentas terapêuticas. Este projeto foca-se nos dois eventos patológicos associados ao enfarte do miocárdio, nomeadamente, a isquémia-reperfusão e a fibrose cardíaca, e no potencial papel dos microARNs na regulação destes processos. A primeira parte deste projeto visou a otimização das condições experimentais para mimetizar os danos provocados pela isquémia-reperfusão cardíaca, e posteriormente, a avaliação do impacto desses mesmos danos na sobrevivência dos cardiomiócitos. Para tal, foram usados cardiomiócitos humanos de duas origens diferentes, mas ambos derivados de células estaminais pluripotentes induzidas. As células foram expostas a isquémia, obtida através de incubação das células num ambiente hipóxico, durante 24 ou 48 horas na ausência de nutrientes (exceto lactato, em condições específicas), e subsequentemente, devolvidas às condições normais de cultura, simulando o passo da reperfusão. Após a lesão de isquémiareperfusão observou-se um forte decréscimo no número de cardiomiócitos, acompanhado por alterações na morfologia celular. Adicionalmente, foi também possível otimizar o protocolo de transfeção reversa de microARNs que poderá ser usado para a realização de um screening de larga escala com o objetivo de clarificar o papel dos microARNs na lesão de isquémia-reperfusão. Através destas experiências foi possível demonstrar que o hsa-miR- 302d-3p induz um forte aumento da capacidade proliferativa de cardiomiócitos humanos. A segunda fase do projeto focou-se em fibroblastos cardíacos humanos. Os fibroblastos cardíacos foram expostos a lesão de isquémia-reperfusão in vitro usando um protocolo semelhante ao utilizado em cardiomiócitos. Curiosamente, os resultados obtidos sugerem que a isquémia-reperfusão leva a um ligeiro aumento da proliferação de fibroblastos cardíacos, que é estimulado pela reperfusão. O protocolo de transfeção reversa para microARNs foi também otimizado, e foi realizado ainda um screen piloto com 36 microRNAs, que serviu de “prova de princípio” para a validação da tecnologia e identificação de microARNs que controlam a ativação de miofibroblastos. De entre os microRNAs testados, identificámos microARNs com um efeito marcante na estimulação (hsa-miR-26b-5p, hsa-miR- 19b-2-5p, hsa-miR-2052, hsa-miR-875-5p, hsa-miR-210-3p e hsa-miR-19a-5p) ou na repressão (hsa-miR-1281, hsa-miR-130a-5p e hsa-miR-143-5p) da ativação de miofibroblastos. Adicionalmente, foi também possível estabelecer uma correlação preliminar entre a ativação e a proliferação dos fibroblastos cardíacos após tratamento com microARNs. Os nossos resultados indicam que a transformação de fibroblastos cardíacos em miofibroblastos resulta num significativo decréscimo da sua taxa de proliferação. Em geral, este estudo estabeleceu as condições experimentais necessárias à realização de um screen em larga escala em cardiomiócitos e fibroblastos cardíacos humanos, que será importante para a identificação de novos microARNs envolvidos na lesão de isquémia-reperfusão.

Tese de doutoramento em Ciências e Tecnologias da Saúde, especialidade de Biologia Celular e Molecular, apresentada à Faculdade de Farmácia da Universidade de Coimbra<br>Cerebral global ischemia induces selective neurodegeneration of specific subsets of neurons throughout the brain, namely in the CA1 region of the hippocampus. Despite its high prevalence and intensive research, there is still need of effective treatments to reduce the neurodegeneration associated with global ischemia. This pathology can be studied in vitro by depriving neurons of oxygen and glucose (OGD). Our main goal was to study the molecular mechanisms of neuronal death activated by OGD in primary cultures of hippocampal neurons. For this purpose we targeted distinct aspects of cell death. We studied the excitotoxic component of cell death mediated by NMDARs, we addressed the activation of a novel mechanism of programmed cell death, necroptosis, and we analyzed the activation of effector signaling cascades, in particular MAPKs. We started by studying the influence of the GluN2B subunit of NMDARs to OGD-induced neuronal demise. NMDARs are major contributors to the overload of intracellular Ca2+ characteristic of excitotoxicity and the role of GluN2 subunits has remained controversial. This is due to a variety of conflicting evidence showing that either both GluN2A and GluN2B contribute to neuronal death or that GluN2B is mostly pro-death and GluN2A pro-survival. To clarify this question we used cultured cortical neurons from GluN2B-/- mice and wild-type littermates, and observed that GluN2B is determinant for induction of excitotoxic neuronal death following OGD. We observed that the absence of this subunit blocked neuronal death induced by OGD and that the toxicity was rescued when we reintroduced the subunit in the KO neurons. Moreover, we demonstrated that the C-terminal domain (CTD) had a preponderant role in GluN2B-induced toxicity, and we identified molecular determinants in the CTD of GluN2B responsible for this function. We confirmed that the PDZ-binding domain was partly responsible for NMDAR toxicity. This domain is responsible for the interaction with PSD95 that couples to nNOS, and interfering with this interaction was neuroprotective. Additionally, we identified two other regions on the GluN2B CTD that are required for OGD-induced cell death, the AP2- and the CaMKII-binding domains. Mutations in either of these sites blocked GluN2B-mediated toxicity. These findings confirmed the crucial role of GluN2B-containing NMDARs in a context of in vitro ischemia, and our study is particularly relevant since most previous work was performed under excitotoxic conditions. Next, we investigated whether OGD induced necroptosis, a novel type of programmed necrosis, in hippocampal neurons. This type of cell death has been recently described to occur following death receptor (DR) signaling. In certain conditions a complex called DISC is formed. DISC induces apoptosis and downregulates necroptosis via caspase8-mediated cleavage of the proteins RIP1/RIP3. However, when caspase8 is inhibited, RIP3 is recruited to RIP1 and together they form a complex called the necrosome, and activate necroptosis. We observed that OGD induced a component of cell death that was reversed by the necroptotic inhibitor Nec-1 but not by zVAD.fmk, an apoptotic inhibitor. Notably, we observed that Nec-1 had no effect on the apoptotic component of neuronal death. Additionally, OGD induced the expression of RIP1 and RIP3. We confirmed the toxic role of RIP3 by performing overexpression and knock-down experiments. We observed that overexpression of both RIP1 and RIP3 exacerbated neuronal death induced by OGD whereas knockdown of RIP3 significantly reduced OGD-mediated toxicity. The damaging effect of the OGD challenge was rescued by reintroducing RIP3 in neurons where endogenous RIP3 was knocked-down, confirming the specificity of the requirement for RIP3. Finally, we correlated these in vitro events with the in vivo challenge, by confirming that global cerebral ischemia in the rat also induces RIP3 expression in the CA1 area of the hippocampus. Lastly, we studied the activation of MAPKs in hippocampal neurons submitted to OGD. These kinases are responsible for the majority of the cellular response to stress and are involved in several paradigms of cell death, including in neurons. We determined that both p38 and JNK are activated following OGD, at 2h and 6h of reoxygenation, respectively. Furthermore, inhibition of the activity of these MAPKs has a neuroprotective effect, suggesting a cytotoxic function. Interestingly, JNK seems to have biphasic function since neuroprotection was only achieved when we inhibited JNK at 4h reoxygenation. Overall, our results demonstrate that OGD induces a variety of changes in neurons, including several mechanisms that contribute to neurodegeneration. This in vitro model is thus a powerful tool to address the molecular mechanisms underlying cerebral ischemia, which may provide useful insights into the development of therapeutic strategies to this pathology.<br>A isquemia cerebral induz neurodegeneração de populações específicas de neurónios, nomeadamente na área CA1 do hipocampo. Apesar da sua elevada prevalência e dos esforços na investigação, não existem actualmente tratamentos eficazes para prevenir a neurodegeneração associada à isquemia global. Esta patologia pode ser estudada in vitro submetendo os neurónios a privação de oxigénio e glicose (OGD, do inglês “oxygen and glucose deprivation”). O principal objectivo deste trabalho foi o de estudar os mecanismos moleculares associados à morte neuronal iniciada pela OGD em culturas primárias de neurónios de hipocampo. Para este propósito pesquisámos diferentes aspectos da morte celular. Assim, estudámos o fenómeno de excitotoxicidade mediada por receptores de glutamato do tipo NMDA (NMDARs), analisámos a activação de um novo mecanismo de morte celular, necroptose, e verificámos a activação de vias efectoras da morte celular, em particular, as vias das MAPKs. Começámos por estudar a influência da subunidade GluN2B dos NMDARs na morte neuronal induzida por OGD. Os NMDARs têm um papel de relevo no excesso de Ca2+ intracelular característico da excitotoxicidade; contudo, o papel das subunidades GluN2 tem permanecido controverso. Há evidências que mostram o envolvimento das subunidades GluN2A e GluN2B no processo de morte celular enquanto outras mostram o papel tóxico do GluN2B e uma função neuroprotectora do GluN2A. Para clarificar esta questão recorremos a um sistema de cultura neuronal de murganhos GluN2B-/- e observámos um papel determinante da subunidade GluN2B na indução de morte após OGD em neurónios de córtex. Verificámos que a ausência do GluN2B eliminou a toxicidade induzida por OGD, que foi recuperada com a reintrodução da subunidade nos neurónios GluN2B-/-. Demonstrámos ainda o papel preponderante do domínio C-terminal (CTD) da subunidade na toxicidade mediada por GluN2B e mapeámos alguns locais de interacção no CTD de GluN2B responsáveis por esta função tóxica. O domínio PDZ é responsável pela interacção com a PSD95, que faz o acoplamento à nNOS. A interferência com esta interacção teve um efeito neuroprotector. Identificámos também dois determinantes moleculares no CTD de GluN2B relevantes para este processo, o local de ligação às proteínas AP2 e CaMKII. A mutação destes locais eliminou a toxicidade induzida pela subunidade GluN2B. Estas evidências apoiam o papel determinante dos NMDARs contendo GluN2B num contexto de isquemia cerebral in vitro. O nosso estudo é particularmente relevante na medida em que os trabalhos anteriores utilizavam, na sua maioria, estímulos excitotóxicos. De seguida, investigámos a indução de morte neuronal por necroptose, um novo mecanismo de necrose programada, em neurónios de hipocampo submetidos a OGD. Este tipo de morte celular ocorre na sequência da activação de “death receptors” (DRs). Em determinadas condições, ocorre a formação de um complexo que medeia a apoptose e inibe a necroptose através da clivagem das proteínas RIP1/3. Contudo, se ocorrer inibição da caspase8, a RIP3 é recrutada para a RIP1 e juntas formam um complexo chamado necrosoma, que activa a necroptose. A OGD induziu um componente de morte celular que foi revertido pelo inibidor de necroptose Nec-1, mas não pelo inibidor da apoptose zVAD.fmk. A especificidade do efeito protector da Nec-1 sobre a necroptose foi comprovada pela ausência de efeito da Nec- 1 no componente apoptótico. Além disso, a OGD induziu a expressão das proteínas RIP1 e RIP3. Confirmámos o papel tóxico da RIP3 através de ensaios de sobreexpressão e silenciamento. A sobreexpressão das proteínas RIP1 e RIP3 aumentou a morte neuronal, enquanto o silenciamento da RIP3 reduziu a toxicidade induzida por OGD. O efeito tóxico da OGD foi recuperado com a reintrodução da proteína RIP3 em neurónios sem RIP3 endógena. Por fim, relacionámos os mecanismos observados in vitro com o modelo in vivo, ao observarmos que a isquemia global induz o aumento da expressão da RIP3 na área CA1 do hipocampo. Estudámos por fim a activação de MAPKs em neurónios de hipocampo submetidos a OGD. Estas cinases são responsáveis pela resposta ao stress celular e estão envolvidas em diversos paradigmas de morte neuronal. Verificámos a activação das cinases p38 e JNK após OGD, às 2h e 6h de recuperação, respectivamente. Verificámos ainda que a inibição destas cinases teve um efeito protector, o que sugere um papel citotóxico. Curiosamente, a cinase JNK parece ter um duplo papel, já que a sua inibição só foi protectora quando efectuada às 4h após o estímulo de OGD. Concluindo, os nossos resultados demonstram que a OGD afecta os neurónios a vários níveis, incluindo na indução de mecanismos que contribuem para a neurodegeneração. Este modelo in vitro apresenta-se assim como uma ferramenta importante para a dissecção de mecanismos moleculares subjacentes à isquemia cerebral, o que poderá contribuir para o desenvolvimento de estratégias terapêuticas para esta patologia.

Ischaemic stroke has become one of the leading causes of death and disability in the world. Protease activated receptors (PARs, PAR-1 to PAR-4) belong to G protein coupled receptors that can be self-activated by tethered ligands (TL) revealed through proteolytic cleavage. Based on these TL, many activating peptides (APs) and antagonists have been synthesized to investigate PARs actions.<br>In the present study, the roles of PARs were examined in two models of ischaemic stroke. For the in vivo model, transient middle cerebral artery occlusion (tMCAO) was performed to establish cerebral ischaemia in rats. For the in vitro model, oxygen and glucose deprivation (OGD) was used to mimic an ischaemia insult in primary cultured rat embryonic cortical neurones.<br>Western blot studies showed that expressions of PAR-1 and PAR-2 were increased in the rat ischaemic brain cortex, whereas PAR-1 was reduced in the rat cortical neurones subjected to OGD. Pretreatments of PAR-1 AP (SFLLRN-NH₂) and PAR-2 AP (SLIGRL-NH₂) produced significant protection against ischaemia-induced damage. Pretreatment of PAR-3 AP (SFNGGP-NH₂) only improved ischaemic symptoms in in vivo but not in in vitro model. When treated after ischaemia, only PAR-1 AP produced significant reductions on ischaemia-induced damage. Protective actions of PAR-1 and PAR-2 APs were inhibited by PAR-1 antagonist (BMS-200261) and PAR-2 antagonist (ENMD-1068) respectively, but PAR-1 antagonist did not affect posttreatment effects of PAR-1 AP in in vitro model. Pre- and posttreatments of thrombin, and pretreatment of trypsin also protected ischaemia-induced damage in the two models.<br>PAR-1 AP produced marked increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and ratio of bcl-2/bax, but reduced contents of reactive oxygen species (ROS), nitric oxide (NO) and malondialdehyde (MDA) in both ipsilateral ischaemic brain cortices and in rat cortical neurones subjected to OGD. In the in vitro model, PAR-1 AP greatly decreased caspase-3 activity and TUNEL positive cells, while markedly increased mitochondrial membrane potential (MMP). All these protective actions were inhibited by its antagonist, which suggests it was mediated via activation of PAR-1.<br>In MCA isolated from normal and ischameic rats, PAR-2 AP and trypsin produced vasodilatation while PAR-3 AP elicited vasoconstriction. However, another PAR-3 AP had no effect in the two types of MCA. A high concentration of PAR-1 AP relaxed MCA isolated form ischaemic rats, and it was not inhibited by a PAR-1 antagonist. The vasodilator action of PAR-2 AP was inhibited by one of two PAR-2 antagonists tested. The vasodilator actions induced by PAR-1 and PAR-2 APs involved NO production since L-NAME was effective in inhibiting their actions.<br>In conclusion, PAR-1 AP was found to be the most efficacious in protecting the brain from ischaemia-induced damage when administered either before or after ischaemia insults. The protective actions were likely to be attributed to its anti-oxidant properties in the ischaemic brain that reduced apoptosis of brain cells. Therefore, PAR-1 was identified as a promising target for development of novel prophylactic and therapeutic treatments of ischaemic brain disease.<br>缺血性腦中風已經成為全世界導致死亡和殘疾的最主要的疾病之一。蛋白酶激活受體（PARs, PAR-1 to PAR-4）屬於G蛋白偶聯受體並且可以通過蛋白水解生成系鎖配體（TL）從而作用於受體本身而激活信號通路。根據TL的序列已經合成了很多激活肽和拮抗劑，它們可以作為有價值的工具藥進行PAR的作用研究。<br>當前，PAR的作用在兩個缺血性腦中風模型中進行研究。體內模型是通過大鼠大腦中動脈阻塞手術而建立；體外模型是通過對大鼠胚胎大腦皮層神經元進行氧糖剝奪模擬缺血性損傷。<br>蛋白質印跡法的實驗表明PAR-1和PAR-2的表達在缺血側大腦皮層中有所增多，而PAR-1在氧糖剝奪的大鼠皮層神經元中表達卻有所降低。預處理PAR-1（SFLLRN-NH₂）和PAR-2（SLIGRL-NH₂）的激活肽顯著改善了缺血導致的損傷。預處理PAR-3激活肽（SFNGGP-NH₂）僅僅改善了體內缺血症狀，卻對體外缺血模型沒有效果。然而，當這些激活肽在缺血后給予的時候，只有PAR-1的激活肽顯著改善了缺血損傷。PAR-1的拮抗劑（BMS-200261）和PAR-2的拮抗劑（ENMD-1068）抑制了PAR-1和PAR-2激活肽的保護作用，但是體外實驗後處理PAR-1激活肽的保護作用卻未收影響。預處理及後處理凝血酶，預處理胰酶都在這兩個模型中顯示出保護缺血性損傷的作用。<br>PAR-1激活肽在缺血同側大腦皮層以及經受氧糖剝奪的大鼠皮層神經元中，顯著提高了超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）的活力以及bcl-2/bax的比例，同時顯著降低了活性氧自由基（ROS）、一氧化氮（NO）以及丙二醛（MDA）的含量。在體外模型中，PAR-1激活肽還顯著降低了caspase-3的活力以及TUNEL陽性細胞的比例，同時顯著提高了線粒體膜電位（MMP）。所有這些作用都可以被拮抗劑抑制，說明PAR-1激活肽的保護作用是通過激活PAR-1介導的。<br>不管是從正常還是缺血的大鼠中分離出來的大腦中動脈，PAR-2激活肽和胰酶都可以使之舒張，PAR-3激活肽卻對其有收縮作用。然而，另外一種PAR-3激活肽卻未顯現出對血管活性的影響。高劑量的PAR-1激活肽只可以在分離于缺血大鼠的大腦中動脈中引起舒張，但此作用不能被其拮抗劑所抑制。PAR-2激活肽導致的血管舒張只可以被檢測的兩個拮抗劑中的其中一個所抑制。PAR-1和PAR-2激活肽引起的血管舒張與NO的產生有關，因為L-NAME可以有效抑制它們的作用。<br>總之，不管是預處理還是後處理的給藥方式，PAR-1的激活肽在保護大腦的缺血性損傷中都是最有效果的。保護作用可能可以歸因于其抗氧化以及抗凋亡的特性。所以，PAR-1是研究防治缺血性腦疾病的發展中富有希望的一個靶點。<br>Zhen, Xia.<br>Thesis Ph.D. Chinese University of Hong Kong 2014.<br>Includes bibliographical references (leaves 194-206).<br>Abstracts also in Chinese.<br>Title from PDF title page (viewed on 11, October, 2016).<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.

Chiu, Wui Man.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.<br>Includes bibliographical references (leaves 107-128).<br>Abstracts in English and Chinese.<br>Abstract --- p.i<br>中文摘要 --- p.iv<br>Acknowledgements --- p.vi<br>Publications --- p.viii<br>Table of Contents --- p.ix<br>List of Tables --- p.xiv<br>List of Figures --- p.xv<br>List of Abbreviations --- p.xviii<br>Chapter Chapter 1 --- Introduction --- p.1<br>Chapter 1.1 --- Hypoxic-ischemic encephalopathy in human infants --- p.1<br>Chapter 1.1.1 --- Incidence --- p.1<br>Chapter 1.1.2 --- Biphasic development of HI brain damage --- p.2<br>Chapter 1.1.2.1 --- Initiating mechanism: energy failure in immature brain --- p.3<br>Chapter 1.1.2.2 --- Biochemical cascades --- p.4<br>Chapter 1.1.2.2.1 --- Excitatory amino acid receptor activation by glutamate --- p.4<br>Chapter 1.1.2.2.2 --- Intracellular calcium accumulation --- p.4<br>Chapter 1.1.2.2.3 --- Formation of free radicals --- p.5<br>Chapter 1.1.2.2.3.1 --- Reactive oxygen species (ROS) --- p.5<br>Chapter 1.1.2.2.3.2 --- Nitric oxide (NO) --- p.6<br>Chapter 1.1.2.3 --- Release of inflammatory mediators --- p.6<br>Chapter 1.1.2.4 --- Mitochondrial dysfunction --- p.7<br>Chapter 1.1.2.5 --- Final path to death: necrosis or apoptosis --- p.8<br>Chapter 1.1.2.6 --- Ways to change: neuronal survival and proliferation signaling --- p.8<br>Chapter 1.1.3 --- Interventions for neonatal hypoxia-ischemia --- p.9<br>Chapter 1.2 --- Animal models mimicking hypoxia-ischemia brain injury --- p.12<br>Chapter 1.2.1 --- Comparisons of animal models of hypoxia-ischemia --- p.12<br>Chapter 1.2.2 --- Development of neonatal rat model with hypoxic-ischemic damage --- p.14<br>Chapter 1.3 --- Neural stem/progenitor cells --- p.15<br>Chapter 1.3.1 --- Effect of hypoxic-ischemia on neural stem/progenitor cells --- p.17<br>Chapter 1.4 --- Thrombopoietin --- p.18<br>Chapter Chapter 2 --- Objectives --- p.23<br>Chapter Chapter 3 --- Materials and Methodology --- p.24<br>Chapter 3.1 --- Establishment of neonatal rat model of HI brain damage and effects of TPO on neural protection --- p.24<br>Chapter 3.1.1 --- Animal protocols --- p.24<br>Chapter 3.1.2 --- Induction of HI brain damage in neonatal rats --- p.24<br>Chapter 3.1.3 --- Treatment with TPO --- p.25<br>Chapter 3.1.4 --- Sacrifice of rats --- p.25<br>Chapter 3.1.5 --- Read-out measurements --- p.26<br>Chapter 3.1.5.1 --- Brain weight --- p.26<br>Chapter 3.1.5.2 --- Gross injury assessment of the right hemisphere --- p.26<br>Chapter 3.1.5.3 --- Histology --- p.27<br>Chapter 3.1.5.4 --- Blood cell count --- p.27<br>Chapter 3.1.5.6 --- Functional assessments --- p.28<br>Chapter 3.1.5.6.1 --- Grip traction test --- p.28<br>Chapter 3.1.5.6.2 --- Elevated body swing test --- p.28<br>Chapter 3.1.5.7 --- Statistical analysis --- p.28<br>Chapter 3.2 --- Establishment of in vitro model of primary mouse NSPs and the effect of TPO on their proliferation --- p.29<br>Chapter 3.2.1 --- Mouse embryo dissection for the extraction of NSP --- p.29<br>Chapter 3.2.2 --- Culturing of NSP --- p.30<br>Chapter 3.2.3 --- Immunofluorescence staining for stem cell markers --- p.31<br>Chapter 3.2.4 --- Neurosphere assay with different combinations of mitogens --- p.31<br>Chapter 3.2.5 --- Neurosphere assay with different concentrations of TPO --- p.32<br>Chapter 3.2.6 --- Neurosphere assay under hypoxia --- p.32<br>Chapter 3.2.7 --- Statistical analysis --- p.33<br>Chapter Chapter 4 --- Effects of thrombopoietin on neonatal rat models of hypoxia-ischemia brain damage --- p.39<br>Chapter 4.1 --- Summary of experimental settings --- p.39<br>Chapter 4.2 --- Results --- p.39<br>Chapter 4.2.1 --- Mortality --- p.39<br>Chapter 4.2.2 --- Effects of TPO on p7 mild damage model 1 week post-surgery --- p.40<br>Chapter 4.2.2.1 --- Body and brain weights --- p.40<br>Chapter 4.2.2.2 --- Gross injury score --- p.41<br>Chapter 4.2.2.3 --- Cortex and hippocampus area --- p.41<br>Chapter 4.2.2.4 --- Blood cell counts --- p.42<br>Chapter 4.2.3 --- Effects of TPO on p7 severe damage model 1 week post-surgery --- p.43<br>Chapter 4.2.3.1 --- Body and brain weights --- p.43<br>Chapter 4.2.3.2 --- Gross injury score --- p.43<br>Chapter 4.2.3.3 --- Cortex area --- p.44<br>Chapter 4.2.3.4 --- Blood cell counts --- p.44<br>Chapter 4.2.4 --- Effects of TPO on p7 severe damage model 3 week post-surgery --- p.45<br>Chapter 4.2.4.1 --- Body and brain weights --- p.45<br>Chapter 4.2.4.2 --- Gross injury score --- p.46<br>Chapter 4.2.4.3 --- Blood cell counts --- p.46<br>Chapter 4.2.4.4 --- Functional outcomes --- p.46<br>Chapter 4.2.5 --- Effects of TPO on pl4 severe damage model 1 week post-surgery --- p.47<br>Chapter 4.2.5.1 --- Body and brain weights --- p.47<br>Chapter 4.2.5.2 --- Gross injury score --- p.48<br>Chapter 4.2.5.3 --- Cortex area --- p.48<br>Chapter 4.2.5.4 --- Blood cell counts --- p.49<br>Chapter 4.3 --- Discussion --- p.49<br>Chapter Chapter 5 --- Effects of thrombopoietin on the proliferation of primary mouse neural stem/ progenitor cells in culture --- p.83<br>Chapter 5.1 --- Summary of experimental settings --- p.83<br>Chapter 5.2 --- Results --- p.83<br>Chapter 5.2.1 --- Effect of EGF or bFGF withdrawal on NSP proliferation --- p.84<br>Chapter 5.2.2 --- Dose effect of TPO treatment on NSP proliferation --- p.85<br>Chapter 5.2.3 --- Effect of hypoxia --- p.85<br>Chapter 5.2.4 --- Effect of TPO treatment in combination with hypoxia --- p.86<br>Chapter 5.2.5 --- Detection of neural progenitor cell marker --- p.87<br>Chapter 5.3 --- Discussion --- p.88<br>Chapter Chapter 6 --- General discussion --- p.101<br>Bibliography --- p.106