Dissertations / Theses on the topic 'In cellulo and in vivo models'
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1
Fischer, Jared Michael. "Mouse Models of Mutation in vivo." University of Cincinnati / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1227214862.
2
Passeri, Elodie. "Use of nanoliposomes rich in omega-3 for the prevention of neurodegenerative diseases : bioavailability in vivo and in cortical neurons." Electronic Thesis or Diss., Université de Lorraine, 2021. https://docnum.univ-lorraine.fr/ulprive/DDOC_T_2021_0337_PASSERI.pdf.
Abstract:
La maladie d’Alzheimer (MA) est la cause d’affection neurodégénérative la plus fréquente et représente un enjeu majeur de santé publique au niveau mondial. De nombreuses stratégies thérapeutiques sont explorées depuis plusieurs décennies, néanmoins, il n'existe toujours aucun traitement curatif et la priorité reste la prévention. Dans ce travail de thèse, nous nous sommes focalisés sur l’approche préventive basée sur les lipides, en particulier les acides gras polyinsaturés oméga-3 (AGPI n-3). Les AGPI n-3 jouent un rôle important dans le développement, le maintien et le fonctionnement du cerveau et ils ont fait l'objet d'un intérêt particulier dans la prévention des déficits cognitifs liés aux maladies neurodégénératives. L’objectif de ce travail est d’étudier la fonctionnalité et la biodisponibilité des nanoliposomes (NL) riches en AGPI n-3, afin d’évaluer leur potentiel neuroprotecteur pour développer de nouvelles stratégies préventives dans les maladies liées au vieillissement comme la MA. Pour ce faire, une technique d'extraction verte a été utilisée pour préparer des NL, provenant de ressources naturelles à partir de lécithine de saumon riche en AGPI n-3. Cette thèse repose sur trois parties. La première partie est consacrée à une revue bibliographique des nouvelles techniques pour faciliter l’accès des molécules au cerveau. Les NL montrent un rôle prometteur, ils sont capables d’améliorer le transport de molécules à travers la barrière hémato-encéphalique et atteindre les régions cérébrales ciblées. Dans la deuxième partie de ce travail, la biodisponibilité des NL riches en AGPI n-3 a été étudiée dans une culture primaire de cellules neuronales corticales d’embryons de rat et dans un modèle de souris. Cette étude montre pour la première fois la biodisponibilité cérébrale des NL riches en AGPI n-3 dans des modèles in vitro et in vivo. Et enfin, dans la troisième partie de cette thèse, les propriétés physico-chimiques et des mécanismes de transfert des NL ont été étudiés dans une culture primaire de neurones corticaux. Ces résultats apportent de nouvelles informations sur l'interaction entre les NL et les neurones et sont prometteurs quant à l'utilisation des NL riches en AGPI n-3, ouvrant de nouvelles possibilités dans le développement de stratégies thérapeutiques préventives et neuroprotectrices pour les maladies neurodégénératives telles que la MAAlzheimer's disease (AD) is the most common cause of neurodegenerative disease and represents a major public health issue worldwide. Many therapeutic strategies have been explored for several decades, however, there is still no curative treatment and the priority remains prevention. In this thesis work, we focused on the preventive approach based on lipids, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs). N-3 PUFAs play an important role in the development, maintenance and function of the brain, and they have been the subject of particular interest in the prevention of cognitive deficits associated with neurodegenerative diseases. The objective of this work is to study the functionality and bioavailability of nanoliposomes (NL) rich in n-3 PUFAs, in order to assess their neuroprotective potential to develop new preventive strategies in aging-related diseases such as AD. To do this, a green extraction technique was used to prepare NL, from natural resources from salmon lecithin rich in n-3 PUFA. This thesis is based on three parts. The first part is devoted to a bibliographical review of new techniques to facilitate the access of molecules to the brain. NL shows a promising role, being able to improve the transport of molecules across the blood-brain barrier and reach relevant brain regions. In the second part of this work, the bioavailability of NL rich in n-3 PUFA was studied in a primary culture of cortical neuronal cells from rat embryos and in a mouse model. This study shows for the first time the brain bioavailability of NL rich in n-3 PUFA in in vitro and in vivo models. Finally, in the third part of this thesis, the physicochemical properties and transfer mechanisms of NL were studied in a primary culture of cortical neurons. These results provide new information on the interaction between NL and neurons and are promising with regards to the use of NL rich in n-3 PUFA, opening up new possibilities in the development of preventive and neuroprotective therapeutic strategies for neurodegenerative diseases such as AD
3
Rose, Nicolas. "New ex vivo models to study the mechanical interplay between muscle cells and their microenvironment." Electronic Thesis or Diss., Sorbonne université, 2021. http://www.theses.fr/2021SORUS440.
Abstract:
Un intérêt croissant est porté aux systèmes ex vivo pour des raisons bioéthiques, légales et économiques. Afin d'étudier la fonction et la régénération musculaire, la recherche médicale nécessite la mise au point de plateformes d’analyse fonctionnelle de myotubes matures et d’outils de culture de cellules souches musculaires (MuSCs) non conventionnelle intégrant les contraintes mécaniques. Dans le cadre de mes travaux, une puce de culture de myotubes en 3D avec capacité de mesure de la contraction a été développée. La combinaison d'une technologie de photo-patterning et de micro-substrats a permis d'obtenir des rendements de culture élevés dans des conditions physico-chimiques contrôlées. Des contractions de myotubes 3D dérivés de myoblastes humains primaires ont été observées, la force générée a été mesurée et le schéma de contraction caractérisé. L'impact de la culture 3D sur la morphologie nucléaire a été analysé, confirmant la similitude d'organisation entre les myotubes 3D obtenus et les myofibres in vivo. De plus, le système a permis de modéliser la dystrophie musculaire congénitale due à une mutation de LMNA avec la culture de myotubes 3D mutants présentant un phénotype de laminopathie typique. Enfin, des MuSCs ont été cultivées sur des hydrogels, démontrant l’effet des variations d'élasticité de la niche sur l'activation des MuSCs au cours de la régénération musculaireEx vivo systems are under increasing interest for bioethical, legal and financial stakes. To study muscle function and regeneration, medical research requires functional analysis platforms for mature myotubes and unconventional muscle stem cells (MuSCs) culture tools integrating mechanical consraints. During my doctoral works, a 3D myotube culture chip with contraction monitoring capacity has been developed. Combining photopatterning technology and microsubstrate design allowed to obtain high culture yield in controlled physical and chemical microenvironment. Spontaneous contractions of human primary myoblast-derived 3D myotubes has been observed, their generated forces measured and their contraction pattern characterised. The impact of 3D culture on nuclear morphology has been analysed, confirming organizational similarities betaween obtained 3 myotubes and in vivo myofibers. Moreover, LMNA-related Congenital Muscular Dystrophy has been modelled in 3D mutants myotubes displaying typical laminopathy phenotype. Finally, MuSCs has ben cultured on hydrogels, demonstrating the effect of niche elasticity variations on MuSCs activation during muscle regeneration
4
BAZZINI, CHIARA. "STUDY OF MOLECULAR MECHANISMS AND NEW STRATEGIES AGAINST A CYTOTOXICITY AND NEUROINFLAMMATION IN EX VIVO CELLULAR MODELS FROM ALZHEIMER’S DISEASE PATIENTS." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/306480.
Abstract:
La malattia di Alzheimer (AD) rappresenta una delle principali problematiche per la salute pubblica ed è stata identificata come una priorità per la ricerca. Le due caratteristiche patologiche fondamentali della malattia sono le placche amiloidi e i grovigli neuro fibrillari che sono alla base della neuroinfiammazione e del deterioramento cognitivo.Le forme solubili degli oligomeri sono la specie più tossica della β-amiloide (Aβ) e interagiscono con diverse chinasi proteiche coinvolte nella trasduzione del segnale intracellulare come Ras/MAPK e PI3K/AKT che regolano molti processi cellulari e funzioni cognitive, e alcuni meccanismi molecolari coinvolti nella degenerazione neuronale, come l'iperfosforilazione di tau e l'eccitotossicità del glutammato. Negli ultimi anni molta attenzione è stata focalizzata sull'utilizzo di composti naturali come agenti neuroprotettivi. Il luppolo (Humulus Lupulus) contiene flavonoidi, molecole aromatiche che hanno proprietà antiossidanti e antinfiammatorie. È stato dimostrato che l'estratto di luppolo ha effetti antiaggreganti sull’Aβ e sembra impedire la sua produzione nelle cellule in coltura. L'accumulo di Aβ induce anche l'attivazione della proteina 3 del recettore Nod-like receptor 3 (NLRP3) dell’inflammosoma e il conseguente rilascio di citochine proinfiammatorie, il quale svolge un ruolo fondamentale nella neuroinfiammazione associata all'AD. NLRP3 attivato induce la produzione e il rilascio di mediatori infiammatori, tra cui i complessi proteici ASC (ASC specks), IL-1β e IL-18, che facilitano la deposizione di Aβ in un ciclo che si auto alimenta. Impedire l’assemblaggio e l'attivazione del complesso dell’inflammosoma potrebbe essere una possibile strategia per la terapia dell'AD.
L'obiettivo generale di questo studio è quello di indagare i meccanismi molecolari coinvolti nelle malattie neurodegenerative e nella neuroinfiammazione utilizzando modelli cellulari periferici ex vivo di AD.Al fine di caratterizzare le interazioni Aβ e vie di trasduzione del segnale MAPK e AKT, abbiamo utilizzato fibroblasti di pazienti AD sporadici con diversa gravità della malattia. Per valutare i meccanismi molecolari che potrebbero prevenire o modulare la tossicità indotta da Aβ, sono stati studiati anche i potenziali effetti citoprotettivi dell'estratto di luppolo e il relativo signaling intracellulare. Inoltre, è stato dato particolare interesse alla via di attivazione del NLRP3-infiammasoma. Abbiamo studiato il coinvolgimento dell'attivazione di NLRP3 sulle vie MAPK e AKT e sui loro bersagli a valle, utilizzando una combinazione di studi in vitro e di campioni ottenuti dai pazienti. In particolare, abbiamo utilizzato monociti umani THP-1 di derivazione macrofagica e monociti derivati da cellule mononucleate del sangue periferico (PBMC) di soggetti sani (HC) e pazienti affetti da AD, per analizzare la modulazione autofagica e gli effetti della Stavudina (D4T), un inibitore nucleosidico della trascrittasi inversa, che riduce l'attivazione dell'inflammosoma bloccando il recettore purinergico P2X7R. Inoltre, abbiamo analizzato il pathway di attivazione dell'inflammosoma NLRP3 e il ruolo di CRID3 un inibitore selettivo, per confrontare gli effetti dell’inibizione dell’inflammosoma attraverso due pathway differenti. I monociti derivati da HC e AD sono stati differenziati in cellule microglia-like (MDMIs) e caratterizzati per l'espressione di proteine intracellulari e di superficie tipiche delle cellule mieloidi. Funzioni tipiche della microglia come il rilascio di citochine infiammatorie, la fagocitosi e la degradazione sono state valutate anche in seguito all'esposizione di attivatori dell'inflammosoma con o senza CRID3. MDMIs riflettono molte caratteristiche della microglia e sono un modello cellulare utile per comprendere la patogenesi dell'AD, identificare i target terapeutici e consentire lo screening farmacologico su larga scala dei nuovi composti per uso terapeutico.Alzheimer's disease (AD) is a major public health concern and has been identified as a priority for research in Life Science. The two core pathological hallmarks of AD are extracellular amyloid plaques and intracellular neurofibrillary tangles which underlie microglial and neuronal damage, neuroinflammation and cognitive impairment. Soluble oligomers are the most toxic species of β-amyloid (Aβ) and interact with several protein kinases such as Ras/MAPK and PI3K/AKT pathways, which regulate many cellular processes and cognitive functions. These pathways mediate Aβ toxicity, regulating some molecular mechanisms involved in neuronal degeneration such as cytoskeletal impairment, glutamate excitotoxicity and neuroinflammation. In the last years much attention has been focused on the potential role of natural compounds as neuroprotective agents. Hop (Humulus Lupulus) contains flavonoids, aromatic molecules which have antioxidant, anti-inflammatory and anti-atherogenic properties. In fact, hop extract has anti-aggregating effects on Aβ, and it seems to prevent its production in cultured cells. Aβ induces also the activation of the pattern recognition receptor Nod-like receptor protein 3 (NLRP3) inflammasome complex in microglia and the consequent release of proinflammatory cytokines, playing a pivotal role in AD-associated neuroinflammation. NLRP3 activation results in the release of inflammatory mediators, including ASC protein complexes (ASC specks), IL-1β and IL-18, that facilitate Aβ deposition and neuroinflammation in a self-feeding pathogenic loop. Since specific therapeutical strategies are still lacking, the dampening of the inflammasome assembly and activation could be a new strategy for AD. The overall focus of this study is to investigate molecular mechanisms involved in neurodegenerative diseases and in neuroinflammation, using peripheral ex vivo cellular models from AD, to check new potential therapeutical targets. In order to characterize the complex interactions among Aβ, MAPK and AKT signaling, we used fibroblasts from sporadic AD patients with different disease severity. To evaluate any molecular mechanisms that could prevent or modulate Aβ-induced toxicity, the potential cytoprotective effects of Hop extract and related intracellular signaling were also investigated. Fibroblasts provide a useful cellular model for studying AD, since they could be differentiated into patient-specific neural cell lines, using iPSC technologies. Moreover, particular interest was given to NLRP3-inflammasome activation pathway. We investigated the involvement of NLRP3 inflammasome activation on intracellular pathways and their downstream targets, using a combination of in vitro studies and patient-derived samples. In particular, we used macrophage-derived THP-1 human monocytes and peripheral blood mononuclear cells (PBMC)-derived monocytes from healthy control (HC) subjects and AD patients, to analyse phagocytosis, autophagy and apoptosis modulation and the effects of the nucleoside reverse transcriptase inhibitor Stavudine (D4T), that reduces NLRP3 inflammasome activation blocking the purinergic receptor P2X7R. Furthermore, we analyzed the NLRP3 inflammasome pathway and the role of the selective NLRP3 inhibitor CRID3, to compare the effects of inflammasome inhibition through two different mechanisms. At this purpose, HC and AD-derived monocytes were differentiated into microglia-like cells (MDMIs) and characterized for myeloid surface and intracellular proteins expression. Key microglia functions such as inflammatory cytokines release, Aβ phagocytosis and degradation were evaluated upon exposure to NLRP3 inflammasome activators with or without CRID3. MDMIs reflected many features of microglia and, as fibroblasts-derived iPSCs, they are attractive cellular models helpful to understand AD pathogenesis, identify therapeutic targets and allow large-scale drug screening of the novel therapeutic candidates.
5
Zangrossi, Manuela. "Study of the extra-telomeric functions of telomerase in in vitro and in vivo models." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3426233.
Abstract:
Maintenance of telomere length, required for the unlimited cell proliferation displayed by cancer cells, is provided by telomerase, a ribonucleoprotein complex containing a specialized reverse transcriptase, encoded by TERT gene, that uses an internal RNA template to maintain telomeres length, thus playing a critical role in tumor formation and progression. TERT is usually repressed in normal somatic cells, but is detectable in the vast majority of tumors.
Recent studies have suggested that TERT, besides maintaining telomere, is involved in other cellular functions, and it may contribute to carcinogenesis also via telomere length-independent mechanisms; therefore its inhibition could represent a promising strategy to improve cancer treatment, regardless of telomere length. The possible therapeutic effects of BIBR1532 (BIBR), a specific TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein-Barr virus (EBV)-driven and virus-unrelated B-cell malignancies.
The aim of this study was to characterize the biological effects of short-term TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt’s lymphoma (BL) cell lines; in addition, we investigated the effects of short-term BIBR treatment in vivo in wild type zebrafish embryos.
We found that short-term inhibition of TERT by BIBR, in in vitro models of B-cell malignancies, led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately increased apoptosis. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone.
In agreement with in vitro results, short-term inhibition of Tert by BIBR in wild type zebrafish embryos reduced cell proliferation, induced an accumulation of cells in S-phase, increased apoptosis, and triggered the activation of DDR. These effects were telomere length-unrelated, since the range of telomere length was not affected by the short-term BIBR treatment and the DNA damage foci were distributed randomly, rather than specifically located at telomeres. All these effects were specifically related to Tert inhibition since BIBR treatment showed no effect in Tert-negative zebrafish embryos.
Taken together these data demonstrate that TERT inhibition impairs cell proliferation and induces pro-apoptotic effects unrelated to telomere dysfunction, enforcing the concept that TERT per se exerts telomere length-independent tumor-promoting effects, and thus supporting the introduction of TERT inhibitors to complement current anticancer treatment modalities.Il mantenimento dei telomeri, necessario per la proliferazione illimitata delle cellule tumorali, è esercitato dalla telomerasi, un complesso ribonucleoproteico contenente una trascrittasi inversa specializzata, codificata dal gene TERT, che utilizza un templato ad RNA per sintetizzare nuove sequenze telomeriche, svolgendo quindi un ruolo critico nella formazione e nella progressione dei tumori. TERT viene infatti solitamente represso in normali cellule somatiche, mentre è rilevabile nella maggior parte dei tumori. Studi recenti hanno suggerito che TERT è coinvolto in altre funzioni cellulari e può contribuire alla carcinogenesi anche attraverso meccanismi indipendenti dal mantenimento dei telomeri, quindi la sua inibizione potrebbe rappresentare una strategia promettente per migliorare il trattamento antitumorale, al di là dell’effetto sui telomeri. I possibili effetti terapeutici di BIBR1532 (BIBR), un inibitore specifico del TERT, sono stati valutati in diversi contesti cellulari, ma non sono attualmente disponibili dati ottenuti su modelli di neoplasie delle cellule B sia associate al virus di Epstein-Barr (EBV) che virus-indipendenti. Lo scopo di questo studio era di caratterizzare gli effetti biologici dell'inibizione di TERT a breve termine da parte del BIBR su linee cellulari linfoblastoidi immortalizzate da EBV (LCL) e su modelli in vitro di linfoma di Burkitt (BL); inoltre, sono stati studiati gli effetti del trattamento con BIBR a breve termine in vivo negli embrioni di zebrafish. I risultati ottenuti hanno dimostrato che l'inibizione a breve termine di TERT da parte di BIBR, in modelli in vitro di tumori delle cellule B, ha portato a una diminuzione della proliferazione cellulare, all'accumulo di cellule nella fase S e infine all'aumento dell'apoptosi. L'arresto del ciclo cellulare e l'apoptosi, conseguenti all'inibizione di TERT a breve termine, erano associati e probabilmente dipendenti dall'attivazione della risposta al danno del DNA, come evidenziato dall’aumento dei livelli di γH2AX e dall'attivazione dei pathway di ATM e ATR. L’analisi della media e del range di lunghezza dei telomeri e dei foci di danno al DNA ha indicato che la risposta al danno attivata in seguito all’inibizione TERT a breve termine non era legata a disfunzioni telomeriche, suggerendo quindi che TERT, oltre a stabilizzare il telomero, può proteggere il DNA tramite meccanismi telomero-indipendenti. In particolare, LCL-TERT positive trattate con BIBR in combinazione con fludarabina o ciclofosfamide hanno mostrato un aumento significativo del numero di cellule apoptotiche rispetto a quelle trattate con agenti chemioterapici da soli. In accordo con i risultati in vitro, l'inibizione a breve termine di Tert da parte del BIBR in embrioni di zebrafish ha ridotto la proliferazione cellulare, indotto un accumulo di cellule nella fase S, aumentato il tasso di apoptosi e innescato l'attivazione della risposta al danno al DNA. Questi effetti non erano legati a disfunzioni telomeriche, poiché il range di lunghezza dei telomeri non era influenzato dal trattamento a breve termine con BIBR e i foci di danno al DNA erano distribuiti casualmente, piuttosto che localizzati in modo specifico sui telomeri. Tutti questi effetti erano specificamente associati all'inibizione di Tert poiché il trattamento con BIBR non mostrava alcun effetto negli embrioni di zebrafish Tert-negativi. Nel complesso questi dati dimostrano che l'inibizione del TERT compromette la proliferazione cellulare e induce effetti pro-apoptotici non associati a disfunzioni telomeriche, rafforzando il concetto che TERT esercita di per sé funzioni pro-tumorali indipendenti dalla lunghezza del telomero e quindi supportando l'introduzione di inibitori di TERT per integrare le attuali modalità di trattamento antitumorale.
6
Ronayne, Rachel E. "Human Ependymin-1 Neurotrophic Factor Mimetics Reduce Tau Phosphorylation and Cellular Apoptosis in Vitro and in Vivo in Alzheimer’s Disease Models." Digital WPI, 2008. https://digitalcommons.wpi.edu/etd-theses/1018.
Abstract:
"Alzheimer’s disease (AD) is the most widespread neurodegenerative disorder, affecting approximately 20 million people worldwide. AD pathology is primarily characterized by the formation of extracellular amyloid plaques resulting from the aggregation of insoluble amyloid-beta 1-42 (A-beta), and neurofibrillary tangles (NFT’s) resulting from intracellular aggregation of hyperphosphorylated tau protein. The current FDA-approved AD treatments do not stop or reverse neurodegeneration, but only treat the symptoms by increasing acetylcholine neurotransmitter. Our laboratory is attempting to provide an additional therapeutic approach by using neurotrophic factors to block apoptosis or to restore neurons. We previously demonstrated that, in an in vitro model for AD, hEPN-1 neurotrophic factor mimetics can block synthetic A-beta-induced neuronal cell death when added to cultures, presumably by blocking caspase activation. In this thesis, we extended these findings to study the effect of A-beta and hEPN-1 on tau hyperphosphorylation (as measured by immunoblots with phospho-specific antibodies) and nuclear DNA fragmentation (as measured by TUNEL staining), both in vitro and in vivo in AD transgenic mice. We found that A-beta induces the hyperphosphorylation of tau in both mouse N2a and human SHSY neuronal cells, and that hEPN-1 may lower this phosphorylation in N2a cells. Furthermore, we discovered that hEPN-1 can reduce nuclear DNA fragmentation when added both simultaneously to A-beta and 3 and 6 hours post A-beta addition. Finally, in vivo hEPN-1 may lower both tau hyperphosphorylation and caspase-7 related protein (C7RP) in AD transgenic (Tg) mice. The overall results validate our in vitro AD model, show the efficacy of hEPN-1 at blocking A-beta-induced DNA fragmentation even when added post-insult, and show that hEPN-1 may work in an AD mouse model. However, more studies must be conducted to confirm these findings. "
7
Lu, Yen-Zhen. "The effects of 670nm light on retinal Müller cell gliosis following retinal stress or injury: exploring the underlying cellular mechanisms using in vivo and in vitro models." Phd thesis, Canberra, ACT : The Australian National University, 2018. http://hdl.handle.net/1885/154729.
Abstract:
Photobiomodulation (PBM) describes a process whereby light
wavelengths of 600-
1000nm are used to initiate biological responses. PBM has been
shown to attenuate
inflammation and accelerate wound healing in skin and mucosal
tissues. In the nervous
system, it promotes recovery of injured spinal cord and optic
nerve. Our laboratory has
found that irradiation with 670nm light, applied prior to retinal
insult, reduced
photoreceptor death in retinal degeneration in vivo. However,
very little attention was
paid to the non-neuronal component of the retina, the macroglia
of the retina.
Müller cells (MCs), the principal macroglia of the retina, are
involved in supporting
retinal structure and maintaining its homeostasis. MCs react to
retinal stress or injuries,
described as gliosis, aiming to protect neurons. However when it
enters into a
progressive state, it becomes detrimental to the retina.
Activated MCs, if uncontrolled,
release a large amount of proinflammatory cytokines and
chemokines, recruiting
microglias (MGs) and monocytes, which lead to further retinal
inflammation. While
the retinal damage is extensive, MCs undergo mitosis and
thickening of their processes,
which reach the subretinal space to form glial scars that inhibit
nutrient delivery,
leading to further neuronal death. Thus, the aim of this thesis
is to investigate the effects
of 670nm irradiation on activated MCs using in vivo and in vitro
stress models,
exploring a new avenue that may prevent irreversible retinal
degeneration.
In Chapter 3, the effects of 670nm light on activated MCs using
in vitro and in vivo
stress models of retinal injury were investigated. Our results
demonstrated that 670nm
modified MC activation, both its proinflammatory and
proliferative processes. This
chapter additionally draws attention to the importance of
appropriate timing of
treatment, as there is a finite therapeutic window to effectively
mitigate gliotic changes.
In Chapter 4 investigated the effects of 670nm light on
interaction between MCs and
photoreceptors, and on subsequent MC-derived MG activation in
vitro. Results
confirmed that 670nm light mitigated MC gliosis induced by
photo-oxidative damage
(PD), subsequently reducing MG activation. This protective
mechanism of action of
670nm light in MCs was associated with increased mitochondrial
activity. Chapter 5
explored the cell communication between MCs and MGs in vitro,
focusing on the role
of exosomes. Exosomes have been discovered to carry microRNAs
(miRNAs), which
allows the transfer of genetic information between cells. In this
chapter, IL-1β
stimulation of MCs led to the release of exosomes, which
stimulated MGs to upregulate
expression of proinflammatory cytokines. Several miRNAs
implicated in regulating
inflammatory processes were identified in MC-derived exosomes in
stressed MCs.
Treatment with 670nm significantly reduced the expression of some
of the proinflammatory
genes.
The results from this thesis collectively indicate that following
MC activation in retinal
damage, 670nm treatment post-damage can mitigate MC gliosis and
subsequently
ameliorate retinal inflammation. Furthermore, this effect may be
achieved through
down-regulation of proinflammatory cytokine production in the
retina and modification
of exosome contents. Therefore, targeting activated MCs using
670nm light may be a
potential therapeutic strategy in mitigating inflammation
associated with the
irreversible retinal degeneration.
8
Toutain, Hervé. "Développement et caractérisation de modèles expérimentaux pour l'étude ex-vivo et in-vitro de la cellule tubulaire proximale de rein de lapin." Rouen, 1989. http://www.theses.fr/1989ROUES036.
Abstract:
Une suspension de cellules tubulaires proximales de rein de lapin est isolée par une nouvelle méthode qui ne fait pas intervenir d'enzymes protéolytiques. Ces cellules, après une caractérisation biochimique, morphologique, et métabolique, sont séparées en deux populations hautement purifiées par une technique d'électrophorèse ou flux libre en veine liquide. Présentation d'un modèle de culture primaire de cellules tubulaires proximales de rein de lapin
9
Sibeko, Sengeziwe. "The impact of macrophage inflammatory protein-3 alpha and other innate immune markers on susceptibility/resistance to HIV infection in the female genital tract mucosa using cellular and ex vivo tissue models." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:a7ecf529-b94e-492f-8374-675cb495ef05.
Abstract:
The distinctive feature of the Human Immunodeficiency Virus (HIV) epidemic in the 21st century is the burden it places on women. Scientists believe that the best opportunities for successful interventions to prevent sexual HIV transmission lie in the initial stages of infection at the portal of entry, the genital tract (GT), which offers the greatest host advantages and viral vulnerabilities. However, understanding of the correlates of protection/vulnerability and innate immunity at the portal of entry is poor. First and foremost, there is no agreement about which GT sub-compartment is the primary site of HIV/SIV infection. Second, the epithelium, previously studied solely for its function as a barrier, has hardly been investigated for its role in innate immunity in the context of SIV/HIV infection. MIP-3α, a chemokine secreted by epithelial cells, was previously proposed to have a role in amplifying the early Simian Immunodeficiency Virus (SIV) infection events in the GT of female macaques. Specifically, MIP-3α was shown to be secreted by epithelial cells of the endocervix, accumulating subepithelially within the first 24 hours post exposure, following deposition of an intravaginal inoculum of SIV. Similar studies in humans have not been reported. We hence undertook to study MIP-3α for its role in early HIV infection events in the endocervix of humans. In order to achieve this, we first characterised MIP-3α constitutive secretion patterns in different sub-compartments of the GT before proceeding to determine its induced secretion patterns, stimulating with HIV-1 and various Toll-like receptor ligands. For completeness we determined constitutive and induced secretion patterns of multiple soluble proteins (SPs) and antimicrobial peptides (AMPs) in the endocervices of humans and macaques. The GT being an immunohormonal system, we further studied the influence of endogenous hormonal changes on the stability of MIP-3α and that of other innate immune markers. We quantified MIP-3α with a sandwich Elisa, and SPs and AMPs with the Luminex multiplex bead assay. Our results showed that the GT is a rich source of MIP-3α with its levels being among those of the highest SPs in the GT. Constitutive levels were highest in the endocervical sub-compartment of all the sub-compartments studied. Further, the GT is an inflammatory environment, which would explain the high levels of MIP-3α. The primary driver of MIP-3α levels appears to be inflammation rather than hormonal levels. MIP-3α levels are significantly higher in the GT of humans than in macaques. There was no evidence that MIP-3α levels are elevated on exposure to HIV and SIV in humans and macaques, respectively. We therefore concluded that since the endocervix is unlikely to respond to HIV/SIV by secreting MIP-3α in vivo, contrary to the previous reports, MIP-3α is hence not a key player in amplifying early events in infection. And as such, it should not be a prime target for preventive therapy. Further, the human GT having a pre-existing inflammatory profile may explain the high rates of HIV sexual transmission. Lastly, we concluded that the infection mechanisms described in the macaque model (i.e. the 'outside-in' signaling) are likely not required for human infection.
10
Body, Simon. "Physiopathologie du lymphome à cellules du manteau : de la mécanistique aux modèles précliniques." Thesis, Normandie, 2017. http://www.theses.fr/2017NORMC419/document.
Abstract:
Le lymphome à cellules du manteau (LCM) est une hémopathie maligne B mature, appartenant à la famille des lymphomes non hodgkiniens. Le LCM est caractérisé par la translocation t(11;14)(q13;q32) qui provoque une expression aberrante de cycline D1. C’est une pathologie rare mais à haut risque de rechute, et qui reste le plus souvent incurable suite à l’apparition de clones chimiorésistants. L’acquisition de résistance est intimement liée aux interactions entre les cellules tumorales et leur microenvironnement. Afin de mimer de la manière la plus pertinente possible ces interactions, nous avons mis en place un modèle murin de xénogreffe en utilisant les lignées cellulaires de LCM JeKo1, REC1, Z138 et Granta-519 que nous avons modifiées afin qu’elles expriment un fluorophore (GFP ou m-cherry) et/ou le gène codant pour la luciférase. Après injection aux souris du substrat de la luciférase, la luciférine, nous sommes en mesure de suivre au cours du temps la progression tumorale. Nous pouvons également évaluer le degré d’infiltration tumorale dans la moelle osseuse, la rate, le cerveau et le sang après euthanasie des animaux, par des techniques de cytométrie en flux et d’immunocytochimie. Ce modèle nous a permis de montrer l’intérêt thérapeutique d’un inhibiteur de l’exportine 1 (XPO1) : le KPT 330 (ou selinexor) qui est capable de contenir cycline D1 uniquement au niveau nucléaire. Nous avons montré que la localisation subcellulaire de cycline D1, est retrouvée majoritairement cytoplasmique dans certaines lignées cellulaires de LCM (2/7) et chez un certain nombre de patients (6/42, 14%), et est associée à un fort potentiel d’invasion, de migration et à un phénotype agressif. Par ailleurs, grâce à ce modèle, nous avons pu objectiver le manque d’efficacité in vivo d’agonistes aux récepteurs aux œstrogènes de type β (ER β). Ces récepteurs, présents sur les lymphocytes B étaient supposés inhiber la prolifération cellulaire et provoquer la mort des cellules par apoptose. L’utilisation de deux agonistes des ER β, le diarylpropionitrile (DPN) et l’ERB-041 a montré une absence d’effet de ces molécules, lorsque les cellules tumorales sont au contact de leur microenvironnement. D’autre part, afin de mieux comprendre les mécanismes de résistance aux chimiothérapies, nous avons étudié la résistance de la lignée cellulaire REC-1 traitée par des agents génotoxiques. Nous avons montré que cette lignée présentait une anomalie de dégradation de cycline D1 associée à une activité diminuée du protéasome 26S. Enfin, nous avons montré dans des travaux préliminaires que la protéine fused in sarcoma (FUS) pourrait, lorsqu’elle est associée à cycline D1, être capable de réguler les voies de réparation des dommages à l’ADN. Des anomalies de ces voies induisent une grande instabilité génétique responsable de l’échappement des tumeurs aux traitements, le ciblage de FUS pourrait par conséquent présenter un intérêt thérapeutique.Pris dans leur ensemble, ces résultats permettent de renforcer ou d’infirmer l’intérêt de certaines cibles thérapeutiques dans l’espoir de pouvoir continuer à améliorer la prise en charge des patients. Ils fournissent également un outil pour l’évaluation de nouvelles molécules dans un modèle murin prenant en compte les interactions entre la cellule tumorale et son microenvironnementMantle cell lymphoma (MCL) is a mature malignant hemopathy, belonging to the non-Hodgkin's lymphoma family. The MCL is characterized by the translocation t(11;14)(q13;q32) which causes an aberrant expression of cyclin D1. It is a rare disease but at high risk of relapse, and it is most often incurable due to the appearance of chemoresistant clones. The acquisition of resistance is intimately linked to the interactions between the tumor cells and their microenvironment. In order to mimic, in the most relevant way, these interactions, we have implemented a mouse xenograft model using the MCL cell lines JeKo1, REC1, Z138 and Granta-519 which we have modified so that they express a fluorophore (GFP or m-cherry) and / or the gene encoding the luciferase. After injection to the mice of the luciferase substrate, luciferin, we are able to follow over time the tumor progression. We can also assess the degree of tumor infiltration in bone marrow, spleen, brain and blood after euthanasia of animals, by flow cytometry and immunocytochemistry. This model allowed us to show the therapeutic interest of an inhibitor of exportin 1 (XPO1): the KPT 330 (or selinexor) which is able to contain cyclin D1 only on the nuclear level. We have shown that the subcellular localization of cyclin D1 is mainly cytoplasmic in some LCM (2/7) cell lines and in a number of patients (6/42, 14%), and is associated with a high potential Invasion, migration and an aggressive phenotype. Moreover, thanks to this model, we have been able to objectify the in vivo lack of efficacy of agonists to β-type estrogen receptors (ER β). These receptors, present on B lymphocytes, were thought to inhibit cell proliferation and cause cell death by apoptosis. The use of two ER β agonists, diarylpropionitrile (DPN) and ERB-041 showed an absence of effect of these molecules, when the tumor cells are in contact with their microenvironment. On the other hand, in order to better understand the mechanisms of resistance to chemotherapies, we studied the resistance of the REC-1 cell line treated with genotoxic agents. We have shown that this line has an abnormality of cyclin D1 degradation associated with decreased activity of the 26S proteasome. Finally, we have shown in preliminary work that the fused in sarcoma protein (FUS) could, when associated with cyclin D1, be able to regulate the repair pathways of DNA damage. Abnormalities of these pathways induce a great genetic instability responsible for the escape of tumors to treatments, the targeting of FUS could therefore be of therapeutic interest.Taken as a whole, these results reinforce or invalidate the interest of certain therapeutic targets in the hope of continuing to improve the management of patients. They also provide a tool for evaluating new molecules in a murine model that takes into account the interactions between the tumor cell and its microenvironment
11
Obermaier, Carolin Dominique [Verfasser], and Rejko [Akademischer Betreuer] Krüger. "Identification of the underlying mechanism of the c.192G>C mutation in the DJ-1 gene and functional characterisation in patient-based cellular models of Parkinson’s disease ex vivo / Carolin Dominique Obermaier ; Betreuer: Rejko Krüger." Tübingen : Universitätsbibliothek Tübingen, 2015. http://d-nb.info/116523498X/34.
12
Obermaier, Carolin [Verfasser], and Rejko [Akademischer Betreuer] Krüger. "Identification of the underlying mechanism of the c.192G>C mutation in the DJ-1 gene and functional characterisation in patient-based cellular models of Parkinson’s disease ex vivo / Carolin Dominique Obermaier ; Betreuer: Rejko Krüger." Tübingen : Universitätsbibliothek Tübingen, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-649120.
13
Alaa, El Din Ferdos. "Le syndrome de Rendu-Osler-Weber : aspects génétiques, moléculaires et épidémiologiques." Thesis, Poitiers, 2015. http://www.theses.fr/2015POIT2260/document.
Abstract:
La télangiectasie hémorragique héréditaire (HHT) est une maladie rare (1/10.000). Son incidence est plus élevée (pouvant atteindre 1/1000) dans certaines zones géographiques dont la région Poitou-Charentes. Cette maladie autosomique dominante est causée par des mutations d'un des trois gènes identifiés ENG, ACVRL1 et SMAD4 codant pour des protéines de la voie BMP spécifiquement exprimés dans les cellules endothéliales. Le nombre croissant de mutations détectées chez les patients et l'expressivité variable de certaines mutations nous a ammené à déterminer les conséquences de mutations afin d'établir une corrélation génotype/phénotype. Cette corrélation est importante pour le conseil génétique et évidemment le diagnostic prénatal. Dans ce contexte, nous avons étudié aux niveaux cellulaire et moléculaire les effets de plusieurs mutations. L'effet délétère de ces mutations sur la protéine et/ou l'épissage de l'ARN a été évalué. Nous avons montré que sur les 23 mutations d'ACVRL1 : 1) 18 mutations faux-sens affectent la fonctionnalité de la protéine en réponse à BMP9 et 3 mutations sont de simples polymorphismes, 2) la mutation exonique c.733A>G (p.Ile245Val) affecte l'épissage de l'exon 6, 3) La mutation c.1048+5G>A de l'intron 7 en dehors du site consensus induit un épissage aberrant de l'exon 7. En ce qui concerne l'ENG, nous avons analysé 4 mutations et nous avons montré que la mutation c.1088G>A (p.Cys363Tyr) a un impact sur l'activité du récepteur et que les mutations c.1134G>A (p.Ala378Ala) et c.1060C>T (p.Leu364Leu) altèrent l'épissage de l'exon 8. Ce travail montre l'importance de l'étude approfondie de toute nouvelle mutation par des études in silico, in vitro et in cellulo à différents niveaux cellulaires. Des études in vivo ultérieures peuvent compléter et appuyer la stratégie expérimentale que nous avons suivieHereditary hemorrhagic telangiectasia (HHT) is a rare disease (1/10.000). Its incidence is higher in certain geographic areas including the Poitou-Charentes region (1/1000). This autosomal dominant disease is caused by mutations in one of three identified genes ENG, ACVRL1 and SMAD4 encoding BMP pathway proteins specially expressed in endothelial cells. The increasing number of mutations detected in patients and the variable expressivity of certain mutations has taken us to determine the consequences of mutations to establish a genotype/phenotype correlation. This correlation is important for genetic counseling and obviously for prenatal diagnosis. In this context, we investigated the effects of several mutations at the cellular and molecular levels. The deleterious impact of these mutations on the protein and/or RNA splicing was evaluated. We have shown that for the 23 mutations of ACVRL1: 1) 18 missense mutations affect the functionality of the protein in response to BMP9 and 3 are polymorphisms, 2) exonic mutation c.733A>G (p. Ile245Val) affects the splicing of the exon 6, 3) mutation c.1048+5G>A in intron 7 off the consensus site induces an aberrant splicing of exon 7. Concerning the ENG, we analyzed 4 mutations and we showed that the mutation c.1088G> A (p.Cys363Tyr) has an impact on the activity of the receptor and that the mutations c.1134G> A (p.Ala378Ala) and c.1060C> T (p.Leu364Leu) inhibit the splicing of exon 8. This work shows the importance of the comprehensive study of any new mutation by in silico, in vitro and in cellulo studies at different cellular levels. The in vivo studies can further complement and support the experimental strategy that we followed
14
Groult, Jessica. "Expansion ex vivo des Cellules Tumorales Circulantes comme modele de pharmacologie predictive des cancers." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS236/document.
Abstract:
L'émergence des thérapies ciblées dans le traitement des cancers a rendu indispensable la mise au point de marqueurs plus spécifiques et sensibles pour la surveillance des patients. Dès le stade invasif, des cellules tumorales peuvent passer dans le sang où elles constituent les Cellules Tumorales Circulantes (CTC). Les CTC sont accessibles par une simple prise de sang, évitant les biopsies invasives. De plus, elles représentent le seul matériel tumoral résiduel après traitement. C'est la raison pour laquelle les CTC constituent un axe de recherche très actif avec plus de 400 essais cliniques incluant ces cellules comme biomarqueurs. Ces essais apportent des renseignements importants sur le risque de récidive ou de progression métastatique, et ont pour objectif de pouvoir gérer en temps réel la conduite thérapeutique. Cependant, les CTC potentiellement métastatiques ne représentent qu'une fraction très minoritaire de ces cellules circulantes. Les technologies existantes, essentiellement basées sur une simple numération, ne suffisent pas pour guider efficacement la stratégie thérapeutique. Ce projet a évalué un ensemble de critères pouvant être utile pour la prise de décisions thérapeutiques pertinentes, adaptées à chaque patient, et la mesure de l'efficacité des traitements. Ce projet sera centre sur le mélanome. Les stades d'évolution de ce cancer sont bien définis, et dans les stades avances, le risque de développer des métastases est très élevé et la détection précoce de celles-ci est un enjeu important. Par ailleurs, ce cancer bénéficie de rapides progrès thérapeutiques, les CTC constituent donc un outil intéressant pour tester l'efficacité de ces nouveaux traitementsThe emergence of targeted therapies in cancer treatment has made essential the development of more specific and sensitive markers for monitoring patients. At the invasive stage, tumor cells can pass to blood. These cells are called Circulating Tumor Cells (CTC). CTCs are accessible through a simple blood test, avoiding invasive biopsies. Moreover, they represent the only residual tumor after treatment. It is why CTCs are a very active center of research with more than 400 clinical trials involving these cells as biomarkers. These tests provide important information on the risk of recurrence or metastatic progression and aim to manage in real time the therapeutic conduct. But the CTC potentially metastatic represents only a fraction very minority of these circulating cells. Existing technologies, mainly based on simple enumeration, are not enough to effectively guide therapeutic strategy. This project has evaluated a set of criteria to make appropriate therapeutic decisions, adapted to each patient, and able to measure the effectiveness of treatments. This project will focus on melanoma. Evolution stages of this cancer are well defined, and in advanced stages, the risk of developing metastases is very high and the early detection is an important issue. Moreover, CTC could be is an interesting tool to test the effectiveness of these new treatments
15
Belcher, Joyce Yvonne. "Bone Cell Autonomous Effects of Osteoactivin In Vivo." Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/183061.
Abstract:
Cell BiologyPh.D.
Osteoactivin (OA) is a type I transmembrane glycoprotein initially identified in bone in 2002. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. To determine the role of OA in skeletal homeostasis in vivo. we utilized a mouse model with a natural mutation in the osteoactivin gene. This mutation is due to a premature stop codon, which results in the generation of a truncated 150 amino acid OA protein. This animal, which we will refer to as OA mutant, was shown by ìCT and histomorphometric analysis to have increased bone volume, trabecular thickness, and trabecular number compared to wild-type (WT) mice at 4 weeks of age, which is a time at which bone formation is most active. Histological analysis of long bones stained with TRAP (tartrate resistant acid phosphatase) and colorimetric analysis of serum TRAP 5b levels indicated that the numbers of osteoclasts are significantly increased in OA mutant samples. Interestingly, although the numbers of osteoclasts as compared to WT were higher in OA mutant mice, serum levels of C-telopeptide of type I collagen (CTX) and osteocalcin, biomarkers for bone resorption and bone formation respectively, were significantly decreased. These data suggested that in mice the presence of truncated OA protein results in increased osteoclast number, but that they are inefficient in resorbing bone and may in part contribute to the increase in bone volume in OA mutant mice in vivo. To further investigate the role of OA in osteoclast differentiation, osteoclasts were differentiated from hematopoietic stem cell progenitors ex vivo. HSCs were cultured in the presence of 50 ng/ml of M-CSF for two days and then with M-CSF and 100 ng/ml of RANKL in the presence or absence of 50 ng/ml recombinant OA. We observed a dramatic increase in multinucleated TRAP-positive osteoclasts and the number of nuclei per osteoclast in OA-treated cultures compared to control. Additionally, analysis of HSCs showed increased cell proliferation in response to exogenous OA treatment. When osteoclasts were differentiated in ex vivo cultures derived from OA mutant and WT mice, we observed decreased osteoclast number, size, and function in OA mutant compared to WT cultures. This decrease was abrogated when cultures were treated exogenously with recombinant OA. Quantitative PCR analysis of RNA isolated during osteoclast differentiation from WT and OA mutant mice reveal decreased gene expression of critical osteoclast differentiation and functional markers, which explains the osteoclast defect observed ex vivo. To investigate the role of OA in osteoblast differentiation, primary osteoblasts were derived from mesenchymal progenitors isolated from calvariae of WT and OA mutant neonatal pups. OA mutant osteoblasts were found to have decreased alkaline phosphatase (ALP) staining and activity at day 14 in culture. Furthermore when cultures were differentiated to 21 days to simulate matrix mineralization in vitro, OA mutant osteoblasts exhibited decreased Alizarin Red and Von Kossa staining. Quantitative measurement of calcium also showed decreased mineral deposition in OA mutant mice compared to WT. Electron microscopic and protein studies were able to eliminate the notion of ER stress or cell toxicity as a result of ER stress playing a role in the delayed osteoblast differentiation observed in OA mutant osteoblasts. Furthermore, OA mutant osteoblasts exhibited decreased proliferation and survival ex vivo. These data reveal an effect of osteoactivin in osteoblasts ex vivo. This study provided an in vivo tool to study the role of osteoactivin in bone cells and the regulation of bone formation and bone resorption by this molecule. Taken together, these findings suggest that the presence of truncated OA leads to increased bone volume due to defective interplay between bone-resorbing osteoclasts and bone-forming osteoblasts. Data presented here support the notion of osteoactivin as a novel molecule in modulating skeletal homeostasis in vivo.
Temple University--Theses
16
Kim, Jaeyeon. "Model Analysis of Adipose Tissue and Whole Body Metabolism In Vivo." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1216436630.
17
Jeitany, Maya. "Les mécanismes ALTernatifs de maintenance des télomères dans les cellules souches de gliome." Thesis, Paris 5, 2014. http://www.theses.fr/2014PA05T010/document.
Abstract:
Les cellules souches de gliomes (CSG), une sous-population de cellules tumorales, seraient en partie responsables de l’échec des traitements des gliomes de par leur résistance et leur capacité régénérative. Le mécanisme alternatif (ALT) de maintenance des télomères, basé sur la recombinaison homologue et non pas sur la télomérase, est détecté dans environ 30% des gliomes humains suggérant que des stratégies thérapeutiques spécifiquement dirigées contre ALT pourraient avoir un intérêt thérapeutique. Dans ce travail, nous avons poursuivi la caractérisation du premier exemple de CSG humaines ayant un phénotype ALT, les cellules TG20. Nous avons montré que malgré leur très fort taux de recombinaison, les télomères de ces cellules continuaient à assurer leur fonction de protection des chromosomes. Nous avons vérifié que les cellules TG20 conservaient leur capacité à générer des tumeurs intracérébrales après des transplantations sériées chez les souris immunodéprimées tout en gardant un phénotype ALT. Ces résultats confirment à la fois les propriétés de cellules souches cancéreuses des cellules TG20 et la capacité de ALT à assurer la maintenance des télomères nécessaire à l’autorenouvellement et au fort taux de prolifération des CSG in vivo. La greffe intracérébrale de cellules TG20 chez des souris immunodéprimées représente donc un bon modèle d’étude préclinique des gliomes ALT. Nous avons ainsi montré qu’un traitement précoce par un ligand des G-quadruplexes télomériques, 360B, juste après la greffe de cellules TG20, était capable d’inhiber le développement tumoral suggérant l’intérêt de l’utilisation de ligands des G-quadruplexes pour cibler spécifiquement les CSG ALT. Une étude des profils d'expression transcriptomique des cellules TG20 et de plusieurs lignées de CSG humaines exprimant la télomérase, nous a conduit à nous intéresser aux rôles de deux lysine acétyl transférases homologues, PCAF (P300/CBP Associated Factor) et GCN5 (General Control Nonderepressible 5) dans la régulation de la recombinaison télomérique des cellules ALT. Nous avons montré que les inhibitions de ces deux protéines ont des effets opposés sur le mécanisme ALT. Nous proposons qu’une balance d’expression de PCAF et GCN5 régule la maintenance des télomères dans les cellules ALT via le contrôle du turnover de TRF1 ce qui pourrait constituer la base d’une nouvelle stratégie thérapeutique vis-à-vis des gliomes ayant un phénotype ALTGlioma stem cells (GSC), a subpopulation of tumor cells, are partly responsible for the failure of treatment of gliomas because of their resistance and regenerative capacity. The mechanism of alternative lengthening of telomere (ALT), based on homologous recombination, is detected in approximately 30 % of human gliomas. Therefore, therapeutic strategies directed specifically against ALT may have a therapeutic value. In this work, we further characterized the first model of human ALT GSC, the TG20 cells. We showed that despite their very high rate of recombination, the telomeres were still capable of fulfilling their protective function of chromosomes. We verified that the TG20 cells retained their ability to generate intracerebral tumors after serial transplantations in immunocompromised mice, while preserving an ALT phenotype. These results confirm the cancer stem properties of TG20 cells and the ability of ALT to ensure telomeres maintenance, which is required for the self-renewal and the high proliferation rate of GSC in vivo. Intracerebral grafts of TG20 cells in immunocompromised mice represent thus a good preclinical model for studying ALT gliomas. We have shown that treatment with a ligand of telomeric G-quadruplexes, the 360B, at an early stage of TG20 tumor engraftment, was able to inhibit tumor growth, showing the interest of the use of G-quadruplex ligands to specifically target ALT GSC. Transcriptomic profiling of TG20 cells and several other GSC telomerase-positive lines, incited us to study the roles of two homologous lysine acetyl transferases, PCAF (p300/CBP Associated Factor) and GCN5 (General Control Nonderepressible 5), in the regulation of telomeric recombination in ALT cells. We showed that the inhibition of these two proteins has opposite effects on the ALT mechanism. We propose that a balance of expression of PCAF and GCN5 regulates the telomere maintenance in ALT cells by controlling the turnover of TRF1. This model could serve for the development of new therapeutic strategies targeting ALT gliomas
18
Jarjour, Meryem. "Plasticité des réseaux de cellules folliculaires dentritiques : Développement & remodelage." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4014.
Abstract:
Les Cellules Folliculaires Dendritiques (FDC) régulent l'homéostasie des lymphocytes B et sont indispensables à la mise en place des réponses immunes humorales. Les FDC s'organisent, au sein des organes lymphoïdes secondaires, en réseaux tridimensionnels denses, nécessaires à leur fonctionnement. Les études s'intéressant aux FDCs, empruntent classiquement des approches in vitro ou ex vivo, peu adaptées à la nature de ce type cellulaire. Au cours de mon travail de thèse, nous avons utilisé plusieurs systèmes de 'multicolor fate mapping' dans le but de déchiffrer in situ les mécanismes à l'origine du développement initial, et du remodelage des réseaux de FDCs en contexte inflammatoire. Nous avons démontré que les FDCs provenaient de la prolifération clonale et de la différentiation des Cellules Marginales Réticulaires (MRC), un autre sous-type cellulaire stromal résidant près des sinus sous-capsulaires ganglionnaires, et dont les fonctions étaient à ce jour, inconnues. Lors des réponses immunes, nous avons prouvé que les FDCs nouvellement formées, ne dérivaient ni du recrutement de progéniteurs circulants ni de la prolifération de FDCs matures, mais plutôt de la prolifération clonale des MRCs, suivie de leur différentiation en FDCs. Au-delà de l'étude de la biologie des FDCs, notre travail a révélé une fonction importante des MRCs dans le soutien de la plasticité des réseaux de FDCsFollicular Dendritic Cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods may thus be of little interest to understand the genuine immunobiology of FDCs in their native habitat. Herein, we utilised various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of Marginal Reticular Cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. In contrast, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population
19
Chang, Cynthia J. "In vivo and in vitro models of distraction osteogenesis." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558214.
Abstract:
Distraction osteogenesis (DO) is a unique process of bone formation used for clinical correction of skeletal deformities. In DO, bone is osteotomised, and the cut ends are slowly pulled apart by mechanical means to induce de novo ossification. Despite the extensive current and historical use of DO, the .mechanisms involved are not well understood. A novel mouse model of DO featuring a custom-developed external fixator was validated and characterised by using radiography, immunohistochemistry, and micro array techniques. The in vivo model was subsequently studied in a whole-transcriptome time course micro array analysis of DO. Genes relevant to osteogenesis, angiogenesis, mechanotransduction, cytoskeletal signalling, and the Wnt pathway were highly expressed. Time course statistical methods applied to the micro array data enabled profiling of global gene expression throughout DO and identification of genes and functions that showed significant differential expression over time. Concurrently, a novel three-dimensional in vitro model of DO was developed to assess the mechanobiological effects of distraction. The system consists of two pieces of hard mineral scaffold held in a rigid distractor. A cell-seeded fibrin clot bridges the scaffold ends to simulate the in vivo distraction gap. Using this in vitro model, the effects of a single application of tensile strain on the model were assessed. Digital image correlation demonstrated that strain patterns in the stretched construct are similar to those in the distraction gap. Additionally, murine osteoblasts were viable and proliferated in the scaffold-fibrin construct. Following a single distraction, cells exhibited elongated morphology, greater alignment, and increased alkaline phosphatase activity. In conclusion, a new mouse model and a novel in vitro model were shown to be useful correlates to clinical DO. Additionally, time course statistical analyses and in situ 3D staining techniques provide new tools for understanding and improving surgical bone lengthening. These findings serve as a promising starting point for future investigations into the key mediators of the regenerative potential unleashed in DO.
20
Calabro, Lorenzo. "Improving in vivo models of fracture fixation associated osteomyelitis." Thesis, Queensland University of Technology, 2013. https://eprints.qut.edu.au/64112/1/Lorenzo_Calabro_Thesis.pdf.
Abstract:
This project’s aim was to create new experimental models in small animals for the investigation of infections related to bone fracture fixation implants. Animal models are essential in orthopaedic trauma research and this study evaluated new implants and surgical techniques designed to improve standardisation in these experiments, and ultimately to minimise the number of animals needed in future work. This study developed and assessed procedures using plates and inter-locked nails to stabilise fractures in rabbit thigh bones. Fracture healing was examined with mechanical testing and histology. The results of this work contribute to improvements in future small animal infection experiments.
21
Robin, Catherine. "Identification chez l'homme de cellules souches totipotentes lympho-myeloides transplantables in vivo dans un modele nod-scid." Paris 7, 2000. http://www.theses.fr/2000PA077205.
Abstract:
L'etude des cellules souches hematopoietiques (csh) a ete freinee pendant longtemps par l'absence de conditions experimentales appropriees a l'identification de tous les potentiels de differenciation lympho-myeloide de cellules primitives a l'echelon clonal. Dans ce travail, nous avons montre que la descendance lymphoide (t, b, nk) et myeloide (granulo-macrophagique) de cellules humaines de sang de cordon peut etre identifiee simultanement in vitro. Les potentiels myeloides, lymphoides b et nk ont ete testes dans un meme systeme de culture en presence de cellules stromales murines (ms-5) et de cytokines (il-2, il-7, il-15, scf, tpo, flt-3). Le potentiel lymphoide t a ete etudie dans un systeme a part de culture organotypique de lobes thymiques ftaux nod-scid (ftoc). En utilisant ces conditions, nous avons montre que des cellules de la population cd34+cd19- et des sous-populations thy-1+ ou cd38- purifiees de sang de cordon frais ou issues de la moelle osseuse de souris nod-scid greffees 4 mois auparavant avec des cellules cd34+ de sang de cordon, sont capables de generer a l'echelon clonal a la fois des cellules myeloides et des cellules lymphoides t, b et nk. Ces resultats ont permis, pour la premiere fois chez l'homme, de mettre en evidence des cellules totipotentes lympho-myeloides et capables de transplantation in vivo ce qui repond aux criteres de definition d'une cellule souche hematopoietique.
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DAVID, PASCALE. "Transplantation d'hepatocytes isoles : de la disponibilite en cellules humaines a l'application dans un modele rat in vivo." Université Louis Pasteur (Strasbourg) (1971-2008), 2000. http://www.theses.fr/2000STR13109.
Abstract:
L'objet de ce travail a ete dans un premier temps d'ameliorer la disponibilite en hepatocytes humains en evaluant la cryoconservation ou l'immortalisation des cellules afin de creer une banque hepatocytaire humaine et, dans un second temps d'evaluer l'efficacite de la thi dans un modele animal, le rat analbuminemique (nar) a partir de ces differentes sources cellulaires. Nous avons mis au point une technique d'isolement des hepatocytes humains par non perfusion. Cette technique, permettaient a partir de pieces d'exerese hepatique, d'utiliser des fragments de petite taille, presentant peu d'acces vasculaires et plusieurs tranches de section. Les essais d'immortalisation d'hepatocytes humains que nous avons realise se sont reveles infructueux faute de construction genique et de vecteur de transfection efficaces. Cependant, a partir de lignees hepatocytaires, nous avons verifie que des cellules presentant des capacites de proliferation etaient capables de presenter des fonctions metaboliques differenciees. Les essais de congelation d'hepatocytes humains realises a l'aide d'un protocole de congelation par descente en temperature programmee ont montre que du point de vue fonctionnel, en culture, les hepatocytes apres decongelation presentaient des capacites metaboliques similaires a celles d'hepatocytes fraichement isoles. En revanche, le rendement cellulaire apres decongelation etait faible (27% maximum). L'evaluation de l'efficacite de la thi a ete realisee in vivo dans une combinaison syngenique par la transplantation intrasplenique d'hepatocytes isoles normaux chez des rats analbuminemiques de souche sprague dawley mutante et a ete determinee par le suivi de la fonctionnalite (production d'albumine) et de l'implantation des cellules transplantees presentant un marquage albumine positif. Les resultats ont montre que la transplantation d'hepatocytes fraichement isoles se traduisait par une implantation et une fonctionnalite transitoire des cellules transplantees. La transplantation d'hepatocytes cryoconserves se traduisaient par une implantation deux fois moins importante comparee a celle d'hepatocytes fraichement isoles et par l'absence de fonctionnalite des cellules chez les animaux receveurs. Ces resultats refletaient a la fois la fragilite membranaire et les alterations membranaires liee a la congelation des cellules. La transplantation d'hepatocytes de rat immortalises, se traduisait par une implantation et une multiplication des cellules dans le parenchyme hepatique receveur mais par l'absence de fonctionnalite de ces cellules etant donne l'absence de secretion d'albumine par ces cellules in vitro. Nous avons par ailleurs montre que la thi en presence de signaux de regeneration hepatique induits par hepatectomie ou par ligature portale partielles ne permettaient qu'un doublement de l'implantation des hepatocytes transplantes et par la perte de fonctionnalite de ces cellules liee, selon le protocole, soit a l'apparition de processus de senescence rapide, soit a un etat peu differencies de ces cellules. Lors de notre etude, nous avons montre que les rats nar etaient resistants a l'induction chimique d'hepatite fulminante par la d galactosamine ou de cirrhose par le thioacetamide. De ce fait, nous n'avons pu juger de l'efficacite de la thi dans un modele d'insuffisance hepatique.
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PILLAI, Vinoshene. "Intravital two photon clcium imaging of glioblastoma mouse models." Doctoral thesis, Scuola Normale Superiore, 2021. http://hdl.handle.net/11384/109211.
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Legaz, Sophie. "Les nanoparticules de poly(acide lactique) comme plateforme d'imagerie et de vectorisation de molécules actives chez Drosophila Melanogaster : analyses in cellulo et in vivo du couple GAL4/UAS." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10293.
Abstract:
Les nanoparticules (NP) de poly(acide lactique) (PLA) sont des vecteurs biodégradables prometteurs pour la vaccination et la délivrance thérapeutique. Cependant leur évaluation in vivo n'est pas toujours couronnée de succès. Un des écueils réside dans la difficulté à suivre la prise en charge cellulaire de ces nanomatériaux à l'échelle d'un organisme, ces NP étant indécelables dans les tissus profonds. L'objectif de cette thèse est de valider l'utilisation des NP de PLA comme plateforme d'imagerie et de vectorisation d'actifs chez Drosophila Melanogaster et d'analyser ainsi le devenir de NP dans un corps entier. Le modèle drosophile a été choisi pour son faible encombrement, sa facilité d'élevage, la diversité des lignées transgéniques et la puissance des outils génétiques à disposition. Il permet également de mener des études mécanistiques in vivo dans un laps de temps restreint. Nous avons évalué in cellulo et in vivo la toxicité de ces NP afin d'établir des conditions optimales expérimentales. Ensuite le potentiel des NP de PLA a été évalué in cellulo sur des cellules de drosophile transfectées transitoirement par un plasmide porteur du gène GFP sous le contrôle du promoteur UAS. Les NP vectorisant le gène gal4 ou la protéine GAL4 permettent de confirmer par simple observation en microscopie à fluorescence l'efficacité de délivrance de molécules actives dans la cellule via la fixation de la protéine GAL4 sur le promoteur UAS. Enfin, ces formulations ont été administrées par voie orale à des drosophiles transgéniques UAS-RFP pour confirmer les résultats précédents in vivo. GAL4 est un outil prometteur pour le suivi indirect de NP dans des organismes transgéniquesThe biodegradable NanoParticles (NPs) of PolyLactic Acid) (PLA) are promising vectors for vaccination and therapeutic delivery. However, their in vivo evaluation is not always successful. NPs being undetectable in deep tissues, one of the challenges is the difficulty to follow the cellular uptake of these nanomaterials at the organism level. The aim of this thesis is to validate the use of PLA NPs as an imaging and drug vectorization platform in Drosophila melanogaster, and to analyze their fate in the whole fly body. The Drosophila model was chosen for its small footprint, the ease of breeding, the variety of transgenic lines, and the power of genetic tools available. Tt also allows to carry out in vivo mechanistic studies in a limited time window. We evaluated in cellulo and in vivo toxicity of these NP to determine optimal experimental conditions. Then the potential of PLA NPs was evaluated in cellulo on transiently transfected Drosophila cells by a plasmid carrying the GFP gene under the control of the UAS promoter. A simple observation by fluorescence microscopy of NPs vectorizing the gal4 gene or the GAL4 protein can confirm the effective delivery of active molecules into the cell through the binding of GAL4 protein to the UAS promoter. Finally, these formulations were orally administered to transgenic Drosophila UAS-RFP to confirm the previous in vivo results. GAL4 is a promising tool for indirect monitoring of NPs in transgenic organisms
25
Fenton, Mark. "The role of ageing in atherogenesis : two in vivo models." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445443/.
Abstract:
Most mammalian cells grown in culture undergo only a limited number of rounds of replicative activity. This exhaustion of proliferative capacity is termed replicative senescence. There is some evidence that replicative senescence may also occur in vivo, and it has been postulated that such cellular ageing may contribute to age-related pathologies such as atherosclerosis, and to organismic ageing itself. The aim of this thesis is to explore the associations between replicative senescence, organismic ageing and atherosclerosis. It was found that a cytochemical assay, senescence-associated P-galactosidase (SA-p- gal), could detect in vitro replicative senescence in human endothelial cells (ECs) and rabbit vascular smooth muscle cells (VSMCs). Endothelial denudation was then undertaken in rabbit carotid arteries, and in some experiments repeated six weeks later. Morphometric analysis of SA-p-gal activity demonstrated mat senescent ECs and VSMCs accumulated in the injured vessel wall, a second denudation augmenting mis accumulation. Further analysis suggested that these senescent cells showed no proliferative or apoptotic activity. An animal model of accelerated organismic ageing, the senescence-accelerated prone mouse (SAM-P), and mice from a related strain showing normal ageing (SAM-R), were fed a Western-type diet. Morphometric analysis of lipid deposition in their aortic roots demonstrated increased lipid deposition in SAM-P compared with SAM-R mice, despite lower serum cholesterol levels in SAM-P mice. Study of telomere lengths and SA-p-gal activity showed no evidence of accelerated cellular ageing in SAM-P mice. It is concluded that cellular ageing can occur in the vasculature, and that a murine strain which ages at an accelerated rate shows a greater susceptibility to atherogenesis. Since no evidence of accelerated cellular ageing was found in this strain, it is postulated that the increased susceptibility of SAM-P mice to atherogenesis, and perhaps also their ageing phenotype, may be attributable to other abnormalities in these mice, such as increased oxidative status.
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Ölschläger, Peter. "In vitro and in silico models for in vivo events." [S.l. : s.n.], 2002. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10252229.
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MILANI, CHIARA. "Air pollution in neurodegeneration: in-vitro and in-vivo models." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/158159.
Abstract:
Il particolato atmosferico (PM) è una miscela complessa di particelle solide e liquide sospese nell'atmosfera e questa sospensione può essere formata da una varietà di particelle di dimensione e composizione diversa a seconda della loro provenienza (Marconi, 2003). Tra le diverse frazioni di PM, si ritiene che le particelle ultrafini (UFP) provochino i maggiori effetti sulla salute a causa delle loro caratteristiche. Le UFP derivano principalmente da processi di combustione in ambienti urbani (Cassee et al., 2013) e, in Regione Lombardia, la combustione di biomassa solida (BC) per il riscaldamento residenziale e quella di diesel (DEP) per il trasporto pubblico e privato ne rappresentano le fonti principali (Longhin et al., 2016 ).
Evidenze emergenti provenienti da diversi studi suggeriscono che le malattie neurologiche possono essere fortemente associate al PM ambientale (Genc at al., 2012). È stato dimostrato che l'esposizione continua a livelli significativi di PM può comportare la traslocazione diretta degli inquinanti a diversi siti extra-polmonari, compreso il sistema nervoso centrale (CNS), o innescare il rilascio di mediatori solubili dell’infiammazione dagli organi di ingresso primari o dai siti di deposizione secondaria (Genc at al., 2012). L’infiammazione sistemica potrebbe poi attivare le cellule endoteliali cerebrali, alterare l'integrità della BBB, o innescare processi che portano all’attivazione delle vie di segnalazione di MAPK e NFκB (Calderón-Garcidueñas et al., 2008). In particolare, esami post-mortem di adulti residenti in aree urbane altamente inquinate mostrano livelli cerebrali di COX-2 e accumulo di Aβ42 significativamente più alti rispetto a soggetti che vivono in città a bassi livelli di inquinamento (Calderón-Garcidueñas et al., 2004).
Pertanto, l'obiettivo di questo progetto è stato quello di valutare su modelli in-vitro e in-vivo di CNS l'effetto dannoso dell'esposizione alle UFP e la sua possibile correlazione fisiopatologica con la neurodegenerazione tipica della malattia di Alzheimer (AD).
In primo luogo, abbiamo testato l'effetto della somministrazione di DEP su cellule gliali, che attualmente sono riconosciute come soggetti attivi nella regolazione della funzione sinaptica, nella riparazione neurale e nell’immunità del CNS (Lee e MacLean, 2015) e sono state recentemente collegate a vie neuroinfiammatorie e neurotossiche indotte dall’esposizione al PM (Li et al., 2016). Abbiamo dimostrato che dosi sub-letali di DEP inducono stress ossidativo in cellule C6 glioma, mentre l'infiammazione non è coinvolta. Inoltre, abbiamo dimostrato che le cellule C6 glioma attivano vie antiossidanti per contrastare lo stato ossidativo indotto dal DEP e che la via MEK-ERK1-2 sembra importante nel regolare tali strategie.
Successivamente, abbiamo dimostrato concentrazioni sub-letali di DEP inducono stress ossidativo e infiammazione in cellule neuronali HT22, supportando l’ipotesi che i neuroni siano più sensibili alla somministrazione rispetto alle cellule gliali. Inoltre, in seguito al trattamento abbiamo osservato una modulazione dei lipidi e un'alterazione dei livelli di espressione di APP e BACE1.
Infine, abbiamo sottoposto topi BALB/c ad instillazione intratracheale singola e ripetuta con BC e DEP, attraverso il sistema MicroSprayer® Aerosolizer. Questa analisi ha confermato il potenziale infiammatorio e ossidativo di BC e DEP sul cervello dei topi, accompagnato da induzione del metabolismo degli idrocarburi policiclici aromatici e alterazione del processamento di APP dopo esposizione ripetuta.
Per concludere, questi risultati contribuiscono a chiarire la relazione esistente tra esposizione al PM, generazione cronica di stress ossidativo e infiammazione e sviluppo di malattie neurodegenerative.Particulate matter (PM) is a complex mixture of solid and liquid particles suspended in the air, and this suspension could be formed by a variety of particles of different size and composition depending on their origin (Marconi, 2003). Among the different fractions, ultrafine particles (UFPs) are thought to have the greatest health effects because of their characteristics. UFPs derive primarily from combustion processes in urban settings (Cassee et al., 2013) and, in the Lombardy Region, solid biomass burning (BC) for residential heating and diesel (DEP) combustion used for private and public transport represent their major sources (Longhin et al., 2016). Interestingly, emerging evidences from different studies suggest that neurological diseases, such as AD, PD and stroke, may be strongly associated with ambient PM (Genc at al., 2012). It has been demonstrated that continuous exposure to significant levels of airborne PM may result in the direct translocation of pollutants to different extra pulmonary sites, including central nervous system (CNS), or trigger the release of soluble inflammatory mediators from primary entry organs or secondary deposition sites (Genc at al., 2012). Systemic inflammation could activate cerebral endothelial cells, alter BBB integrity, or trigger signalling cascades that lead to the activation of MAPK and NFκB pathways (Calderón-Garcidueñas et al., 2008). Notably, post-mortem examinations of adult humans resident in highly polluted urban areas exhibited significantly higher brain COX-2 expression and accumulation of Aβ42 when compared to subjects living in cities with low pollution levels (Calderón-Garcidueñas et al., 2004). Therefore, the aim of this project was to evaluate the detrimental effect of UFPs exposure, regarding oxidative stress and inflammation, on in-vitro and in-vivo models of CNS. Moreover, this work meant to investigate the possible physiopathological correlation between these two mechanisms and AD neurodegeneration. First, we tested the effect of DEP administration on C6 glioma cells, which have properties of both astrocytes and oligodendrocytes. In fact, glial cells are now recognized as active players in the regulation of synaptic function, neural repair, and CNS immunity (Lee and MacLean, 2015), and astrocytes have been recently linked to neuroinflammatory and neurotoxic pathways induced by PM exposure (Li et al., 2016). We demonstrated that DEP treatment at sub-lethal concentrations induced oxidative stress in glial cells, while inflammation was not involved. Moreover, we found that C6 glioma cells activate anti-oxidant pathways to contrast the oxidative status induced by DEP treatment and that the MEK-ERK1-2 pathway seems important in regulating these anti-oxidant strategies. Afterwards, we selected HT22 nerve cell line as a neuronal in-vitro model to study the effect of direct DEP administration. We demonstrated that DEP treatment at sub-lethal concentrations induced oxidative stress and inflammation in neuronal cells, supporting the idea that neurons are more sensitive to DEP administration than glial cells. Moreover, we extended the analysis of DEP detrimental effects and we found lipid reshaping and alteration of APP and BACE1 protein levels in HT22 cells. Finally, we exposed male BALB/c mice to single and repeated Intratracheal instillation of BC and DEP by means of a MicroSprayer® Aerosolizer system. This analysis confirmed the inflammatory and oxidative potential of BC and DEP exposure on mouse brain, which was accompanied by induction of PAHs metabolism and alteration of APP processing after sub-acute exposure. In conclusion, these findings may contribute to the knowledge of the interplay between PM exposure, the chronic oxidative stress and inflammation generation and the development of neurodegenerative diseases.
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Lesnicar-Pucko, Gaja 1981. "Cellular mechanisms behind vertebrate limp outgrowth." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/346928.
Abstract:
In this work we use a novel in vivo imaging technique and cell behaviour analysis to investigate the cellular basis of limb bud elongation. Recently, it has become clear that oriented cell activities must be involved in this process and cell migration and cell division orientation stood out as the most probable candidates. Here, we present a comprehensive study with a focus on cell orientations and active cell behaviour in the developing chick limb bud. We find that cells orient themselves toward the closest ectoderm and divide along their long axis and that, although the gross cell movement shows distal bias, active migration orientation of single cells seems to be random. As our results do not support the distalward migration and oriented division theory, we propose cell intercalation as the most probable cell behaviour involved in limb morphogenesis. We validate this new hypothesis with a Cellular Potts computer model that theoretically confirms our assumptionEn aquest projecte utilitzem una nova tècnica de microscòpia in vivo, així com l’anàlisi del comportament cel.lular per tal d’investigar les bases cel.lulars de l’elongació de l’extremitat durant el desenvolupament embrionari. Investigacions recents clarament indiquen que activitats cel.lulars orientades estan involucrades en aquest procès, i d’entre elles la migració cel.lular i la divisió cel.lular orientada semblen les candidates més probables. Aquí presentem un estudi exhaustiu enfocat principalment en l’orientació cel.lular i el comportament cel.lular actiu durant el desenvolupament de l’extremitat del pollet. Trobem que les cel.lules s’orienten cap a l’ectodermi més proper i es divideixen al llarg a l’axis més llarg, així com veiem que, malgrat que el moviment cel.lular global mostra una tendència distal, la migració activa de cel.lules individuals sembla ser aleatòria. Donat que els nostres resultats no recolzen la teoria d’una migració distalment orientada amb divisió cel.lular orientada, proposem la intercal.lació cel.lular com al comportament cel.lular més probable involucrat en la morfogènesi de l’extremitat. Aquesta nova hipòtesi es veu validada mitjançant un model computacional de Cellular Potts que confirma el nostre supòsit
29
Rowland, Jennifer Elizabeth. "Analysis of growth hormone receptor signalling in vivo - transgenic mouse models /." [St. Lucia, Qld.], 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17013.pdf.
30
Leoni, Francesca. "Hsp72 translocation and secretion in in vivo and in vitro models." Thesis, University of Chester, 2009. http://hdl.handle.net/10034/93835.
Abstract:
Evidence suggesting that Hsp72 is actively participating in cellular signalling as well interacting with immune system dynamics has been increasing. This is true in healthy, stressed and diseased cells but to different degrees. Modulation of the plasma membrane association and secretion in the extracellular environment by different types of stressors is the key event that leads to different degrees of immune system activation. Hence a better understanding of the mechanisms of Hsp72 secretion and association with plasma membrane is crucial. This thesis investigated the tissue source and mechanism of Hsp72 surface presentation to plasma membrane structures and release in relation with different cellular and physiological stressors. In vivo models confirmed that different tissue types determine specific Hsp72 responses following the same stress and increase serum Hsp72 dependant on intensity and duration of the stress. Diseases models confirm that Hsp72 responses in specific cell populations is related to disease progression, while in vitro models clearly showed that there are multiple mechanisms of secretion and surface presentation, dependent on the nature of the stressor as well as the intensity and duration. This observations clearly change the view of extracellular Hsp72 as a danger signal and lead to a revision of the original danger model. It also suggests that manipulation of Hsp72 translocation through the different pathways involved may prove effective therapeutically.
31
Thangavelu, Amudha. "Targeted therapies in endomental cancer - in vitro and in vivo models." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531515.
32
Aston, Nicola Susan. "In vivo and in vitro models for infantile copper-associated cirrhosis." Thesis, University of Sheffield, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264442.
33
Zwaini, Zinah Dheyaa Razzaq. "In vitro and in vivo models of renal ischemia reperfusion injury." Thesis, University of Leicester, 2017. http://hdl.handle.net/2381/39344.
Abstract:
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.
34
Shahidipour, H. "Development of in vitro and in vivo models of uveal melanoma." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004584/.
Abstract:
Uveal melanoma (UM) is the most common primary intraocular cancer of the eye in adults. Despite successful treatment of the primary tumour, approximately half of patients diagnosed with UM will develop metastases. To date, treatments for metastatic UM have had only limited effect, hence prognosis remains poor. Metastatic UM occurs predominantly in the liver. The mechanisms responsible for this remain largely unknown. Investigations into the molecular and cellular processes underlying UM development and metastasis are essential to develop therapeutic approaches directed against dissemination of this disease. In vitro and in vivo models play a crucial role in basic and translational oncology research. The scope of the work presented in this thesis was to develop innovative and clinically relevant in vitro and in vivo preclinical models that reproduce various aspects of metastatic processes in UM. In Chapter 2, primary UM cells were used to establish 3D tumour spheroids which retained many morphological and molecular characteristics of the original tumour. These 3D tumour spheroids more accurately mimic the 3D in vivo environment in which cancer cells reside and as a result can be used in co-culture assays, drug assays, and further transferred to an animal model to examine metastatic progression. Chapter 3 outlined one such in vivo model; the chick embryo. This model represents an accessible and economical in vivo model that has long been used in developmental biology. A panel of UM cell lines that represent the characteristic genetic and morphological profile in UM were used to form tumour nodules on the chorioallantoic membrane (CAM) and metastatic colonisation in internal organs following intravenous injection. Cells predominantly homed to the chick embryo eye spreading widely within the uveal tract, and to the liver, representative of the disease in patients allowing for a better understanding in the spread of this disease. Furthermore, cell lines forming tumour nodules on the CAM can be further used to assess candidate therapeutic drugs in an in vivo setting. Chapter 4 aimed to investigate such drug candidates by assessing the effects of targeted clinical inhibitors on UM cell lines. One particular inhibitor, the bromodomain PPI inhibitor JQ1, caused cell cycle arrest in two UM cell lines paving the way for many further insightful investigations. The models established in this thesis represent valuable, inexpensive and rapid in vitro and in vivo systems that can improve our understanding of the metastatic process in UM and provide a platform for the preliminary testing of potential new agents to aid molecular-based cancer therapy.
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Culver, Hannah. "Modulation of leukocyte recruitment in animal models of inflammation in vivo." Thesis, Griffith University, 2009. http://hdl.handle.net/10072/365779.
Abstract:
Inflammation is an important underlying cause of many chronic diseases, for example in allergic asthma. Glucocorticosteroids provide a powerful anti-inflammatory treatment but are associated with severe side effects when used at high doses for prolonged periods of time; new alternative or adjunct drugs therapies are therefore required. The recruitment of leukocytes to sites of inflammation represents both a key feature of the inflammatory response and a principal source of inflammatory mediators to perpetuate disease. Novel drugs which inhibit leukocyte recruitment or activation may offer a basis for new anti-inflammatory treatments, while early evidence of pharmacological activity in vivo can help prioritise the selection and development of compounds. The present study employed, in controlled experiments, animal models to ascertain in vivo the action of several naturally occurring or synthetic compounds to modulate leukocyte recruitment and to measure any effect they may exert by counting the number of recruited leukocytes (neutrophils or eosinophils) in response to an inflammatory stimuli (used as an objective measure of drug action). Three series of experiments were conducted: one, in a mouse pouch model to test the reaction of differently pre-treated animals to cytokine stimulus; the second, in a guinea-pig model to assess the potentiating effect of pre-treatment to allergen challenge in sensitised animals; thirdly, in the same sensitised guinea-pig model to determine the anti-inflammatory action of a commercially available complementary therapy.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Angioi, Duprez Karine. "Le melanome malin oculaire et le retinoblastome : deux modeles d'etudes cliniques et experimentales in vivo de la proliferation cancereuse humaine ; a propos de 12 observations." Nancy 1, 1991. http://www.theses.fr/1991NAN11189.
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Joly, Candie. "Dynamique des réponses lymphocytaires T locales et systémiques à l'injection d'un vaccin dans la peau." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS317.
Abstract:
La vaccination est considérée comme l’un des plus grandes découvertes de l’histoire des maladies infectieuses, ayant permis le déclin et l’éradication de plusieurs pathogènes. Cependant, nous ignorons encore tous les mécanismes impliqués dans la protection contre les pathogènes. Cette méconnaissance est la cause de notre incapacité à formuler des nouveaux vaccins contre le VIH, la tuberculose, le paludisme et les pathogènes émergents. Récemment, on note des efforts pour induire une réponse cellulaire efficace après une vaccination, qui joue un rôle crucial dans la clairance des pathogènes.Cette thèse s’appuie sur un modèle de vaccin vivant atténue issu du virus de la vaccine : le MVA (Modified Vaccinia Ankara) et sur le modèle de primate non-humain. Nous avons caractérisé la réponse cellulaire après une immunisation intradermique suivant un schéma en prime-boost homologue, avec un boost à 2, suivi d’un boost à 9 mois. Le MVA a induit une infiltration massive de Lymphocytes T CD8 au niveau du site d’injection, 7 jours après l’immunisation. La réponse cellulaire systémique était modérée et ne reflétait pas l’amplitude de la réponse locale. Les injections du prime et du boost ont orienté la réponse cellulaire de façon différente, ce qui a mené à une importante induction de cellules T CD4 et CD8, persistantes, spécifiques de l’antigène et polyfonctionnelles après l’injection du boost à 9 mois.Cette étude souligne la différence entre les réponses systémiques et locales, démontrant l’importance de se focaliser sur la réponse tissulaire. Elle a également mis en lumière l’impact du schéma d’immunisation sur la qualité de la réponse cellulaireVaccination has been considered as one of the greatest discoveries in the history of infectious diseases by allowing pathogens decline or eradication. However, we still ignore all the mechanism that lead to protection and therefore, fail to elaborate new vaccines against HIV, tuberculosis, malaria and emergent pathogens. Recently, efforts have been made to elicit effective cellular response after vaccination, which is crucial for pathogen clearance.This thesis relied on live-attenuated vaccine model derived from the vaccinia virus: the MVA (Modified Vaccinia Ankara) and a non-human primate model. We characterized the cellular immune response triggered by a homologous prime-boost intradermal injection of MVA, with a 2 months and 9 months boost. The MVA induced a massive infiltration of CD8 T cells at the injection site 7 days post immunization. In comparison, the systemic cellular response was mild and did not reflect the magnitude of the local response. The prime and boost injections elicited distinct orientation of the systemic and local T cells, which led to an important induction of a persistent, antigen-specific and polyfunctional CD8 and CD4 T cell responses after the 9 months boost.This work emphasizes the difference between local and systemic response, demonstrating the importance of the focus on tissue immunity. It also highlights the impact of the immunization schedule on the quality of the cellular response
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Hooley, Robert P. "Studies on Sertoli cell function using in vivo and in vitro models." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/24705.
Abstract:
SK11 cells had detectable expression of ERβ mRNA, and activated an ERE-luciferase reporter construct in response to a range of oestrogenic ligands. In contrast endogenous Ar expression was low, but transfection with mouse Ar cDNA resulted in sufficient Ar expression to activate a luciferase reporter under control of the Rhox5 proximal promoter in response to androgen treatment. However transfected SK11 cells could not stimulate endogenous Rhox5 mRNA expression under similar conditions. Infection of SK11 cells with an advanced construct in vitro resulted in efficient transgenic expression in 80-100% of cells, and was associated with excellent cell survival. In vivo infection in mouse testes with an adenoviral vector containing a GFP transgene, performed by injection via the efferent ductules, resulted in SC-only transgene expression. When a dose of 4x108 pfu was introduced the seminiferous tubule’s architecture was severely damaged, invasion by macrophages and neutrophils occurred, plus expression of markers of hypoxia and apoptosis was detected. Infection with lower doses (1x105 – 1x107 pfu) resulted in disruption to the seminiferous epithelium consisting of loss of pachytene germ cells and formation of intra-epithelial vacuoles. Formation of vacuoles may be due to interaction of adenovirus with the coxsackie/adenoviral receptor (CAR) in SC-SC junctions. Androgen-depletion in rats using a single dose of EDS to ablate the Lc population that synthesise testosterone caused reduced expression of Ar, Rhox5, espin and β3-tubulin mRNA and Ar protein 6 days after treatment, but the expression and distribution of the junctional proteins espin, Cx43, zona occludins 1, and N-cadherin were unaffected. We can conclude that SK11 cells lack vital factor/s required for activation of response elements by androgens in their endogenous promoter regions, which raises the possibility that association with other testicular cell types (GC or PTM) may be required for normal SC function to be maintained. Adenoviral vectors appear good for efficient introduction of transgenes into SC in vitro but not for use in vivo. Finally, testosterone depletion using the EDS-rat model revealed reduced mRNA expression of putative androgen responsive genes identified in previous array studies, and provides a valuable model for validation of other putative targets identified in other studies.
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McDonald, Lita E. C. "The establishment of in vivo and in vitro models for myoclonus dystonia." Thesis, University of Ottawa (Canada), 2007. http://hdl.handle.net/10393/27889.
Abstract:
Myoclonus dystonia (DYT11, OMIM 159900) (MD) is a movement disorder characterized by bilateral alcohol responsive myoclonic jerks often seen in combination with dystonia. MD is inherited as an autosomal dominant trait with reduced penetrance upon maternal transmission. Patients with this disorder are not diminished in their intellectual capacity and have a normal life span. In 2001, mutations in the epsilon-sarcoglycan gene on human chromosome 7q21 were implicated in causing this disorder. Our laboratory identified a 2nd locus on chromosome 18p11 that co-segregates with this disorder, however, a disease causing mutation has not been identified. To establish the function of epsilon-sarcoglycan within the mammalian brain, I generated a conditional knock-out (CKO) mouse of Sgce and utilized bacterial recombineering to generate a cassette that contained a "floxed" exon 1 of epsilon-sarcoglycan. ES cells that had correctly incorporated the CKO targeting cassette were selected and used to generate 3 chimeric male mice by blastocyst injection or morula aggregation. Once germline transmission of the CKO allele has been established, these mice will be bred to Cre expressing mice to generate an sgcenull, and recapitulate the phenotype of MD patients. In addition, I also developed an in vitro model of MD using RNAi directed against epsilon-sarcoglycan in the mouse neuroblastoma cell line, N1E-115. Silencing of epsilon-sarcoglycan in this cell line resulted in a decrease in the neuronal nitric oxide synthase (nNos) and an increase in cell proliferation. This data will provide insight and direction during the characterization of the sgcenull mice.
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Kalber, Tammy Louise. "In vivo susceptibility-contrast MRI studies of mouse models of liver metastasis." Thesis, St George's, University of London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440446.
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Emerson, Michael. "Endogenous nitric oxide and platelet function in in vivo models of thromboembolism." Thesis, King's College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300152.
42
Immonen, Taina Tuulia. "Computational Models of Ex Vivo HIV-1 Dynamics and Fitness Across Scales." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1365173809.
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Ahmed, Jamileh. "Receptor-Associated Protein (RAP) Models In Vivo Reelin Haploinsufficiency: Implications in Schizophrenia." Scholar Commons, 2017. http://scholarcommons.usf.edu/etd/6670.
Abstract:
The “two-hit” schizophrenia hypothesis suggests genetic and environmental abnormalities interrupt early CNS function. This increases vulnerability of a “second hit” and schizophrenia onset. Chronic stress and decreased Reelin signaling are reportedly associated with schizophrenia. Heterozygous Reeler Mice (HRM) show a 50% reduction in Reelin and display major schizophrenia phenotypes. Receptor-Associated Protein (RAP) blocks ligand-association to Reelin receptor Apolipoprotein E receptor 2 (ApoER2). In this study, we sought to replicate major heterozygous reeler mouse (HRM) phenotypes using in vivo RAP studies to establish an experimental in vitro model. Using an in vitro model, we investigated the effects of chronic stress and decreased Reelin signaling on AMPAR subunit expression.
Implantable Alzet osmotic pumps allowed bilateral ventricular 7nM RAP perfusion in 12-14 week-old mice. An assay revealed significant Dab-1 phosphorylation reduction in RAP-perfused animals. These results correspond with learning and memory and associative-fear conditioning abnormalities. Overall activity, sensory perception, and nociception remained unaltered. RAP-perfused mice displayed deficits in pre-pulse inhibition to acoustic startle, and therefore sensory-gating deficits. A significant decrease in Glur1 and Glur2/3 expression was observed in primary hippocampal/cortical neurons following chronic RAP and CORT exposure.
Collectively, our results show postnatal Reelin signaling disruption produces physiological, biochemical, and behavioral phenotypes similar to the HRM model. The exact mechanism of Reelin-dependent AMPAR insertion remains unclear. Glur1 and Glur2/3 appear to be inserted by differing mechanisms. Glur1 is reported to be inserted with Reelin activation of phosphoinositol-3-kinase (PI3K) signaling. Glur2/3, whose mechanism of insertion is unknown, has not been shown to be inserted via PI3K. Our findings also demonstrate the usefulness of in vitro RAP use, in which apolipoprotein E receptor 2 (ApoER2) expression is predominant compared to other lipoprotein receptors that may be affected with RAP application.
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Lattanzio, Francesca <1982>. "Biomolecular studies in Alzheimer’s Disease models: investigations in vitro and in vivo." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5507/1/Lattanzio_Francesca_tesi.pdf.
Abstract:
The Alzheimer’s disease (AD), the most prevalent form of age-related dementia, is a multifactorial and heterogeneous neurodegenerative disease. The molecular mechanisms underlying the pathogenesis of AD are yet largely unknown. However, the etiopathogenesis of AD likely resides in the interaction between genetic and environmental risk factors. Among the different factors that contribute to the pathogenesis of AD, amyloid-beta peptides and the genetic risk factor apoE4 are prominent on the basis of genetic evidence and experimental data. ApoE4 transgenic mice have deficits in spatial learning and memory associated with inflammation and brain atrophy. Evidences suggest that apoE4 is implicated in amyloid-beta accumulation, imbalance of cellular antioxidant system and in apoptotic phenomena. The mechanisms by which apoE4 interacts with other AD risk factors leading to an increased susceptibility to the dementia are still unknown. The aim of this research was to provide new insights into molecular mechanisms of AD neurodegeneration, investigating the effect of amyloid-beta peptides and apoE4 genotype on the modulation of genes and proteins differently involved in cellular processes related to aging and oxidative balance such as PIN1, SIRT1, PSEN1, BDNF, TRX1 and GRX1. In particular, we used human neuroblastoma cells exposed to amyloid-beta or apoE3 and apoE4 proteins at different time-points, and selected brain regions of human apoE3 and apoE4 targeted replacement mice, as in vitro and in vivo models, respectively. All genes and proteins studied in the present investigation are modulated by amyloid-beta and apoE4 in different ways, suggesting their involvement in the neurodegenerative mechanisms underlying the AD. Finally, these proteins might represent novel potential diagnostic and therapeutic targets in AD.
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Lattanzio, Francesca <1982>. "Biomolecular studies in Alzheimer’s Disease models: investigations in vitro and in vivo." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5507/.
Abstract:
The Alzheimer’s disease (AD), the most prevalent form of age-related dementia, is a multifactorial and heterogeneous neurodegenerative disease. The molecular mechanisms underlying the pathogenesis of AD are yet largely unknown. However, the etiopathogenesis of AD likely resides in the interaction between genetic and environmental risk factors. Among the different factors that contribute to the pathogenesis of AD, amyloid-beta peptides and the genetic risk factor apoE4 are prominent on the basis of genetic evidence and experimental data. ApoE4 transgenic mice have deficits in spatial learning and memory associated with inflammation and brain atrophy. Evidences suggest that apoE4 is implicated in amyloid-beta accumulation, imbalance of cellular antioxidant system and in apoptotic phenomena. The mechanisms by which apoE4 interacts with other AD risk factors leading to an increased susceptibility to the dementia are still unknown. The aim of this research was to provide new insights into molecular mechanisms of AD neurodegeneration, investigating the effect of amyloid-beta peptides and apoE4 genotype on the modulation of genes and proteins differently involved in cellular processes related to aging and oxidative balance such as PIN1, SIRT1, PSEN1, BDNF, TRX1 and GRX1. In particular, we used human neuroblastoma cells exposed to amyloid-beta or apoE3 and apoE4 proteins at different time-points, and selected brain regions of human apoE3 and apoE4 targeted replacement mice, as in vitro and in vivo models, respectively. All genes and proteins studied in the present investigation are modulated by amyloid-beta and apoE4 in different ways, suggesting their involvement in the neurodegenerative mechanisms underlying the AD. Finally, these proteins might represent novel potential diagnostic and therapeutic targets in AD.
46
Renaud, Matthieu. "Évaluation d'un substitut osseux résorbable porteur de cellules souches : approche cellulaire pour la régénération osseuse in vivo." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT081.
Abstract:
Malgré le développement de biomatériaux de plus en plus nombreux dans le domaine des greffes osseuses et de la préservation alvéolaire, les résultats sont toujours insuffisants pour assurer des reconstructions ad integrum du tissu osseux. Les techniques d’ingénieries osseuses semblent être la piste à privilégier pour améliorer nos techniques chirurgicales. Le silicium poreux est un matériau prometteur pour l’ingénierie tissulaire et notamment pour la régénération osseuse. En effet, son état de surface permet une adhésion cellulaire importante et ses propriétés non toxique et résorbable en fond un matériau porteur de cellules souches intéressant. Les cellules souches de la pulpe dentaire (DPSC) sont des cellules facilement accessibles dans la cavité buccale. Leurs capacités de prolifération et de différenciation associées au silicium poreux semblent être un atout pour les applications thérapeutique pour la régénération osseuse. Des résultats d’études ultérieures in vitro ont montré leur fort intérêt à une application in vivo. Dans ce travail thèse, nous avons tester l’association silicium poreux et cellules souches de la pulpe dentaire, aux même caractéristiques énoncées dans l’étude de référence in vitro, chez l’animal. Pour cela, le matériau a été produit sous forme de particules de manière a être utilisé comme moyen de comblement osseux, associé ou non à des DPSC. Le modèle de queue de rat a été développé et tester pour diminuer le nombre d’animaux nécessaire à l’étude tout en conservant la puissance statistique des résultats. Les études ont montré la possibilité d’utiliser ce modèle pour la régénération de défauts osseux crées chirurgicalement. De plus, il semblerait que ce modèle puisse également être utile pour les études sur l’ostéointégration de système implantables et sur la régénération osseuse autour de ces implants. Le silicium poreux a ensuite été testé dans ces conditions, associé ou non aux DPSC, en comparaison avec un témoin positif et un témoin négatif. Cette association est apparue comme une piste prometteuse pour la régénération osseuse in vivoDespite the development of biomaterials in the field of bone grafts and alveolar preservation, the results are no sufficient to made reconstructions ad integrum of bone tissue. Bone engineering techniques seem to be the preferred way to improve our surgical techniques. Porous silicon is a promising material for tissue engineering and especially for bone regeneration. Indeed, its surface allows cell adhesion. And then, it’s a non-toxic and bioresorbable interesting material properties carrying stem cells. Dental pulp stem cells (DPSC) are easily accessible cells in the oral cavity. Their proliferation and differentiation capacities associated with porous silicon appear to be attractive for therapeutic applications in bone regeneration. The results of the in vitro studies have shown the interest for in vivo application. In this thesis, we have tested the combination of porous silicon and dental pulp stem cells in vivo experimentation, using the same characteristics of the in vitro reference study. For this, the material was produced in particle form to be used as bone filling material, associated or not with DPSC. The rat-tail model was developed and tested to reduce the number of animals needed for the study while maintaining the statistical power of the results. Studies have shown the possibility of using this model for bone regeneration defects surgically created. In addition, it seems that this model can also be useful for studies on osseointegration of implantable systems and bone regeneration around these implants. Then, the porous silicon was tested under these conditions, with or without DPSC, in comparison with a positive control and a negative control. This association has emerged as a promising approach for bone regeneration in vivo
47
Wall, Lucy Rosalind. "The proliferative activity of interferon on ovarian cancer : in vitro and vivo models." Thesis, Queen Mary, University of London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398948.
48
Ljungdahl, Ståhle Ewa. "In vivo and in vitro models for determination of antiviral activity and resistance /." Stockholm, 1997. http://www.kibic.ki.se/ki/diss/971212ljun.html.
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Griffin, Michael. "Use of in vitro metabolism models for in vivo drug metabolic clearance prediction." Thesis, University of Manchester, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487998.
Abstract:
Recently human liver microsomes have been found to underpredict in vivo human intrinsic clearance (CLint); this has led to an increase in the use of fresh and cryopreserved human hepatocytes as an alternative in vitro system for the prediction of in vivo human CLint. The aim of the work in this thesis was to assess the utility of human hepatocytes for the prediction of in vivo drug metabolic clearance. It was found that fresh and cryopreserved human hepatocytes also underpredict in vivo human CLint (6 and 6 fold respectively). The effect of including the binding to the in vitro system in the clearance prediction was assessed and even though the prediction improved (from 6 fold to 4 fold) the underprediction remained. Investigations were undertaken to explain this underprediction. These involved investigating the metabolism of the probe substrates, tolbutamide and dextromethorphan in different incubation vessels. It was found that there was no significant difference in the CLint of Tolbutamide between 96 well and 24 well plates. The effect of using a preservation solution for the transport of fresh liver tissue was also investigated. Two probe substrates were investigated, namely Midazolam and Clonazepam. It was found that the CLmax of Midazolam in 2 out of the 3 rat livers preserved for one hour in a preservation solution was reduced 2 fold when compared to the CLmax freshly isolated rat hepatocytes. The activity of the CYP3A4 enzyme over the course of a typical metabolism incubation period was also assessed. Studies in cryopreserved human hepatocytes indicated that there was an exponential loss of the CYP3A4 enzyme over the incubation period. The activity of the CYP3A4 enzyme in human liver microsomes remained constant for the first 60 minutes of the incubation. In the case of terfenadine the clearance when measured by the substrate depletion approach was observed to have a biphasic profile in cryopreserved human hepatocytes. This biphasic profile was explained by the loss of the activity of the CYP3A4 enzyme over the incubation period of the assay in cryopreserved hepatocytes. When the CLint was adjusted for the loss of enzyme activity the underprediction was reduced 2 fold. The rate of enzyme activity loss was not apparent in cryopreserved hepatocytes incubated in WME. This may be due to the presence of essential amino acids in WME that are essential for glutathione maintenance in the cell and subsequent protection of the enzyme from reactive oxygen species attack. The underprediction observed in human hepatocytes may be explained by one or a number of the above factors but it most likely that the underprediction is due to a combination of each of the above factors thus efforts to improve the underprediction requires further investigation.
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Pugh, Perdita Lucy. "Development of rodent in vivo models : neuroinflammation and neurodegeneration relevant to Alzheimer's disease." Thesis, Cardiff University, 2007. http://orca.cf.ac.uk/56148/.
Abstract:
Alzheimer's disease (AD) is characterised by Ap plaque formation, neuroinflammation and neurodegeneration. Current therapies for AD do not modify disease progression therefore, putative anti-inflammatory and neuroprotective agents need to be assessed in rodent in vivo models that demonstrate robust and reproducible markers of neuroinflammation and neurodegeneration. This thesis interrogated the development of in vivo models comprising markers of neuroinflammation and neurodegeneration in rodent brain. The overview describes innate immunity focusing on lipopolysaccharide (LPS) as a standard immunostimulant followed by a review of AD and beta amyloid (Ap). Current rodent in vivo models of LPS or AP-induced neuroinflammation or neurodegeneration are also examined. Chapters 2 and 3 describe the novel application of Luminex suspension bead array systems for the detection of LPS-induced cytokine and other intracellular proteins in brain tissue. Intraperitoneal LPS modulated interleukin (IL)-lp, phosphorylated (p)-IicBa, p-p38 kinase and p-JNK protein and intracerebroventricular LPS increased IL-ip, IL-la and tumour necrosis factor (TNF) -a protein in rat brain. Cytokine protein in rat brain was abrogated by dexamethasone and the a2-adrenoceptor antagonist, fluparoxan. Subsequent chapters investigate more disease relevant models of Ap-induced neuroinflammation and neurodegeneration detected by immunohistochemistry, Taqman or Luminex techniques. Chapter 4 discussed the assessment, by western blotting, of Ap forms expelled from apparatus commonly used to inject solutions into rodent brain tissue and identifying the most consistent method of Ap delivery. Subsequent studies revealing inconsistent neurotoxicity but robust neuroinflammation following intra-hippocampal injection of Ap were described. Final chapters focus on neuroinflammation and neurodegeneration following peripheral insult (LPS or the noradrenergic neurotoxin, DSP-4) to amyloid precursor protein (APP) / presenilin 1 (PS1) transgenic mice. Peripheral administration of LPS or DSP-4 modulated markers of neuroinflammation and did not initiate neurodegeneration. The implications of the current data on the future development of in vivo models are discussed in the final chapter.