Dissertations / Theses on the topic 'Primary Human Tumor Cultures'

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The plant derived indoloquinoline alkaloid cryptolepine was investigated for its cytotoxic properties in 12 human tumour cell lines and in primary cultures of tumour cells from patients. The fluorometric microculture cytotoxicity assay was used to assess cytotoxicity and DNA mocro-array analysis to evaluate gene expression. Cryptolepine mean IC50 in the cell line panel was 0.9 microM compared with 1.0 and 2.8 microM in haemaotological and solid tumour malignancies respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as sensitive as haematological malignancies, respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as senstive as haematological malignancies. Cryptolepine activity showed highest correlations to topoisomerase II and microtubule targetting drugs. In the cell lines cryptolepine activity was essentially unaffected by established mechanisms of drug resistance. A number of genes were identified as associated with cryptolepine activity. In conclusion, cryptolepine shows interesting in vitro cytotoxic properties and its further evaluation as an anticancer drug seems warranted.

Mestrado em Biologia Aplicada - Molecular e Celular
Prostate cancer (Pca) is one of the most frequent diagnosed neoplasies and the second cause of cancer-related death in the world, in men. Between others risk factors, obesity has been associated to Pca although the innerent mechanisms to this association remain to be clear. With this work, throuhg in vitro studies, we wish to contribute to the understanding of the impact of white adipose tissue and its sub-fractions (adipocytes and stromal vascular fraction), from visceral and periprostatic anatomic regions, in celular proliferation, apoptosis and invasion of castration sensitivity (LNCaP) and castration resistant (PC-3) prostate cells. With the purpose of obtaining answers directly from humam studies, were performed visceral and periprostatic adipose tissue primary cultures obtained during urologic surgeries (radical prostatectomy and prostatic adenomectomy) (n=16). Adipose tissue was used to make primary organotipical cultures (WAT) and after collagenase digestion adipocytes and SVF primary cultures. Sobrenatants and infranatants of each culture were collect and used as conditioned medium representing adipokines production. LNCaP and PC-3 cell lines were stimulated with these mediums and apoptosis, proliferation and invasion were evaluated, in vitro. This study model represent a potential form for analyze the impact of adipose tissue in tumor cells, allowing to evaluate adipose tissue-tumor interactions. The results show that adipose tissue promotes tumor cells proliferation, that periprostatic adipose tissue increase apoptosis in obese individuals and that SVF subfraction suppresses invasion of PC-3 cells through a direct effect in tumor cells.
O Cancro da próstata (CaP) é uma das neoplasias mais frequentemente diagnosticadas e a segunda causa de morte por cancro no mundo nos homens. Entre outros factores de risco, a obesidade tem sido frequentemente associada a CaP, embora permaneçam por esclarecer os mecanismos subjacentes a esta associação. Com o presente trabalho pretendeu-se através de estudos in vitro, contribuir para a compreensão do impacto do tecido adiposo branco e suas sub-fracções (adipócitos e fracção vascular estromal), com origens anatómicas periprostática e viceral, na proliferação, apoptose, e invasão celular de células de cancro da próstata sensíveis à castração (LNCaP) e resistentes à castração (PC-3). Com o propósito de obter respostas directamente através de estudos em humanos, foram efectuadas culturas primárias de tecido adiposo periprostático e visceral obtido durante cirurgias urológicas (prostatectomia radical e adenomectomia prostática) (n=16). O tecido adiposo foi utilizado para realizar culturas primárias organotípicas (tecido adiposo total fraccionado) e após digestão com colagenase culturas primárias de adipócitos e de células da fracção vascular estromal do tecido adiposo. Foram colhidos sobrenadantes e infranadantes destas culturas de tecido adiposo e utilizados como meios condicionados representativos da produção de adipocinas. As linhas celulares LNCaP e PC-3 foram estimuladas com estes meios e avaliados a apoptose, proliferação celular e invasividade tumoral in vitro. Este modelo de estudo representa um potencial meio para análise do impacto do tecido adiposo nas células tumorais, permitindo avaliar as interacções tecido adiposo-tumor. Os resultados evidenciam que o tecido adiposo promove a proliferação das células tumorais, que o tecido adiposo periprostático aumenta a apoptose em indivíduos obesos e que os SVF suprimem a invasão das PC-3 através de um efeito directo nas células tumorais.

Respiratory syncytial virus (RSV) is the primary cause of severe lower respiratory tract infections in children. We exploited well-differentiated primary paediatric airway epithelial cells (WD-PAECs) to investigate innate immune responses to RSV. We demonstrated that RSV-conditioned medium (CMRSV) treated WD-PAECs were highly resistant to subsequent RSV or Sendai virus infection. IL-29 significantly contributes to the antiviral activity of CMRSV. Apical pre-treatment, but not basolateral pre-treatment, of WD-PBECs with IL-29 decreased RSV growth kinetics. We found that IL28RA, a component of the type III IFN receptor, was located exclusively on the apical surface of WD-PBECs. We demonstrated that type IIllFNs modulate the innate immune responses to RSV infection. Pre-treatment of WD-PBECs with a TLR4 signalling inhibitor resulted in massive reductions in RSV growth kinetics, and IL-29 and pro-inflammatory cytokine secretion. To elucidate the role of TLR4 in RSV infection we used inhibitors of TLR4 pathway intermediates. Our results showed it is likely that RSV-induced IL-29 is mediated, in part, by TLR4 signalling through NF-KB and/or p38 MAPK. We identified ten genes that were significantly upregulated in RSV-infected WD-PAEC cultures. Furthermore we identified nine genes that are differentially expressed between two patient cohorts: history of mild or severe RSV disease. The proteins encoded by isg15, rsad2 and tnfsf13b, were assessed for direct antiviral properties. However, they failed to reduce viral replication. The gene ptn was found to be differentially expressed in WD-PNECs from the two cohorts irrespective of RSV infection. Pre-treatment of WD-PBECs at 40C with PTN significantly reduced RSV replication. In summary, we demonstrated that RSV induced antiviral responses are mediated, in part, by IL-29. TLR4 signalling plays a significant role in RSV replication and induction of IL-29 and pro-inflammatory chemokines. We identified three potential anti-RSV molecules: IL-29, CLI-095 and PTN, which may be beneficial in the battle against RSV.

L'adenocarcinoma ductal pancreàtic (PDAC) és un dels càncers més letals. Per tal de millorar el diagnòstic precoç, s'estan investigant les etapes inicials de la formació del càncer, com és el cas de les lesions preneoplàstiques, i es vol desxifrar l'origen cel·lular de la malaltia. Les proteïnes Polycomb constitueixen una família de silenciadors epigenètics que es troben en una varietat de tumors sòlids. La hipòtesi principal és que Polycomb pot estar participant en els processos preneoplàstics del pàncreas i en l'aparició i progressió del tumor. La expressió de Bmi1 i Ring1B fou analitzada durant el desenvolupament del pàncreas, en teixit pancreàtic de diferents models murins de la malaltia i en mostres humans de teixit pancreàtic. Es va dur a terme l'anàlisi del mecanisme de Bmi1 mitjançant models in vitro i induint la depleció de Bmi1. Bmi1 i Ring1B s'expressaren en precursors pancreàtics durant etapes primerenques del desenvolupament i en cèl·lules ductals i dels illots,
però no en els acins, en el pàncrees adult. Bmi1 s'induí en cèl·lules acinars durant lesió aguda, en lesions metaplàstiques acinoductals, en neoplàsies intraepitelials pancreàtiques (PanIN) i en PDAC. Ring1B s'incrementà significativament en PanINs de grau alt i en PDAC. La disminució dels nivells de Bmi1 en la línia cel·lular acinar canvià l'expressió dels enzims digestius pancreàtics. Aquests resultats suggereixen que Bmi1 i Ring1B podrien estar contribuint de diferent manera en la progressió tumoral.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. To improve early diagnosis, research efforts are focused in characterising early events of cancer formation like preneoplastic lesions and deciphering the cell origin of the malignancy. Polycomb proteins constitute a family of epigenetic silencers found in a variety of solid tumours. The main hypothesis is that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development and progression. The expression of Bmi1 and RingB was analysed during pancreatic development, in pancreatic tissue from mouse models of disease and in human pancreatic tissue samples. Mechanistic insights of Bmi1 were performed using in vitro models and with induced Bmi1 depletion. Bmi1 and Ring1B were expressed in pancreatic exocrine precursors during early development and in ductal and islet cells, but not in acinar cells, in the adult pancreas. Bmi1 was induced in acinar cells during acute injury, in acinar-ductal metaplastic lesions, in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B was significantly increased in high-grade PanINs and in PDAC. Bmi1 knockdown in acinar cell line changed the expression of pancreatic digestive enzymes. These results suggest that Bmi1 and Ring1B could contribute differently to tumour development.

The natriuretic peptide system consists primarily of the peptides, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP) and c-type natriuretic peptide (CNP) and their actions are mediated via the natriuretic peptide receptors -A, -B and -C (NPR-A, -B, -C). Collectively, these peptides and receptors form an integrated hormonal system, which acts to regulate body fluid homeostasis and blood pressure control. This thesis aimed to confirm natriuretic peptide and NPR expression in freshly isolated and primary cultures of both rat and human proximal tubular (PT) cells, and to identify a possible role for local natriuretic peptide production. Local expression and production of the natriuretic peptides and NPRs was confirmed by RT-PCR, Northern analysis, cGMP response to ANF (NPR-A) and CNP (NPR-B) and RIA for the peptides A, B and C. This study demonstrated that PT cells in culture express both natriuretic peptides and NPRs, and that the process of culture results in increased natriuretic peptide expression and secretion. Furthermore, growth in culture results in a shift in NPR expression from the NPR-C in freshly isolated cells to the functional GC-linked NPR-A and -B at confleunce. Short-term incubation with exogenous natriuretic peptides suggested that natriuretic peptides could accelerate or induce changes in natriuretic peptide and NPR subtype expression, possibly via the NPR-C. The differential expression and secretion of natriuretic peptides and NPRs at different stages of culture strongly suggests a growth modulatory role for the natriuretic peptide system. These growth modulatory actions appear to be mediated by the NPR-C during the initial stages of culture and by the GC-linked NPR at later stages in culture. These results add further strength to the hypothesis that the natriuretic peptides and both the GC-linked NPR and the NPR-C, once thought to be a 'quiescent' receptor, act to modulate cell growth.

Le développement de méthodes in vitro fiables et prédictives représente à l’heure actuelle un véritable défi. En effet, la demande en méthodes alternatives à l’expérimentation animale n’a cessé de croître ces dernières années du fait de la mise en place de législations limitant par considérations éthiques l’utilisation de ces modèles in vivo. De plus, ce besoin a été renforcé par le règlement européen REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) imposant aux industriels de valider l’innocuité de nombreuses substances déjà commercialisées. Toutefois, les modèles in vitro classiques consistant en la culture simple de cellules en monocouche dans des boîtes de Petri ne permettent pas de conserver les propriétés initiales de ces cellules et de retranscrire les conditions et l’environnement cellulaire des organes in vivo. Le développement de méthodes alternatives in vitro prédictives s’avèrent donc crucial en particulier pour mimer le fonctionnement de deux organes : l’intestin et le foie. En effet, ces deux organes sont largement impliqués dans les processus d’Absorption, Distribution, Métabolisme et Excrétion (ADME) de la plupart des xénobiotiques ingérés. C’est pour ces raisons que nous avons testé la faisabilité de l’une de ces méthodes in vitro alternative permettant d’associer une barrière intestinale à la culture dynamique de cellules hépatiques au sein de microsystèmes dans le cadre de ce doctorat. Cette coculture est effectuée au sein du dispositif appelé IIDMP (Integrated Insert in a Dynamic Microfluidic Platform). Nous avons décidé de tester d’une part l’influence de la culture dynamique et d’autre part d’éventuelles interactions entre les cellules intestinales et hépatiques sur la fonctionnalité et l’activité métabolique de ces deux types cellulaires. Les résultats obtenus durant ce doctorat ont permis d’atteindre 4 objectifs :- Développer un dispositif fiable en termes de fonctionnalité (fluidique, robustesse…).- Mettre en évidence l’innocuité du dispositif lorsque des cellules de lignée et primaires y étaient cultivées.- Démontrer les avantages de l’utilisation de ce dispositif comparativement à l’utilisation de modèles classiques in vitro, en particulier avec des cellules de lignée.- Démontrer que l’utilisation de ce dispositif permettait de mettre en évidence des phénomènes d’interactions entre cellules intestinales et hépatiques notamment sur l’activité du CYP1A2 des hépatocytes qu’ils soient issus d’une lignée ou de cultures primaires
The development of reliable and predictive in vitro methods is a real challenge. Indeed, the demand for alternative methods to animal experimentation has been growing in recent years due to the introduction of legislation limiting the use of these models in vivo by ethical considerations. Moreover, this need was amplified by regulations such as the European REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) requiring the safety validation of many substances. However, the conventional in vitro model consisting in a simple cell culture monolayer in Petri dishes does not preserve the initial properties of these cells and does not mimic the conditions of the cellular environment and organs in vivo. The development of alternative in vitro predictive methods is crucial especially to mimic the working of two organs: the intestine and liver. Indeed, these two organs are involved in the process of Absorption, Distribution, Metabolism and Excretion (ADME) of most xenobiotics ingested.We propose in this thesis to test the feasibility of one of these in vitro alternative methods allowing the association between an intestinal barrier and the dynamic culture of hepatic cells in microsystems in a device called IIDMP (Integrated Dynamic Insert in a Microfluidic Platform). We tested the influence of the flow of culture and possible interactions between intestinal and liver cells on the function and metabolic activity of these two cell types.Then, we demonstrated that : - This device is reliable in terms of global functionality (fluid, robustness ...).- This device did not injury the integrity of the cell line and primary cells.- The use of this device has many advantages when compared with the use of conventional in vitro models, especially with cells line.- The use of this device highlights phenomena of interaction between hepatic and intestinal cells as an increase of the CYP1A2 activity of HepG2 C3A and human primary hepatocytes

La sensibilité des cellules de mélanomes aux molécules de thérapies ciblées dépend du microenvironnement tumoral (interactions cellule-cellule et cellule-matrice extracellulaire). Les systèmes tridimensionnels (3D) de culture in vitro reflètent mieux l’architecture structurelle native des tissus et sont attrayants pour l’étude des interactions cellulaires. Nous avons développé et comparé plusieurs modèles de mélanome métastatique : les cellules de mélanomes (SK-MEL-28 et SK-MEL-3, mutées BRAF V600E et SK-MEL-2, BRAF sauvages) cultivées en monocouche (2D) et co-cultivées en 3D sur des équivalents de derme avec des fibroblastes, afin de mieux comprendre les facteurs modulant la sensibilité cellulaire à un inhibiteur de BRAF (BRAFi, Vémurafenib) et au Vémurafenib associé à un inhibiteur de MEK (MEKi, Cobimetinib). La sensibilité cellulaire aux traitements a été évaluée sous différents aspects : prolifération cellulaire (numération cellulaire, incorporation d'EdU, test MTS), analyse des voies de signalisation MAPK et PKB / Akt (Western-blot), apoptose (TUNEL), libération de cytokines et de facteurs de croissance (ELISA) et histologie (modèles 3D). Un effet cytostatique de BRAFi a été observé sur les cellules SK-MEL-28 et SK-MEL-3 cultivées dans les modèles 2D et 3D. La lignée cellulaire SK-MEL-2 était résistante au BRAFi lorsqu'elle a été cultivée en monocouche, mais sensible lorsqu'elle a été co-cultivée avec des fibroblastes incorporés dans une matrice de collagène de type I. Les milieux conditionnés par les fibroblastes 3D (équivalents de derme) ont sensibilisé les cellules SK-MEL-2 (2D) au BRAFi. L'analyse des surnageants de culture cellulaire a révélé que les équivalents de derme libéraient certains facteurs solubles (IL-6, IL-8, HGF, TGF-β) : ces sécrétions ont été modifiées au cours du traitement par Vémurafenib. La combinaison du traitement avec MEKi a renforcé l'action du Vémurafenib sur les cellules de mélanomes métastatiques tout en diminuant la capacité de prolifération des fibroblastes. Des populations de cellules contenant des cellules de mélanomes ou des fibroblastes associés au cancer (CAFs) ont été isolées à partir d'une biopsie de métastase cutanée provenant d'une patiente atteinte d'un mélanome métastatique. Ces cellules ont permis de réaliser des modèles de mélanome métastatique patient-spécifique afin d’étudier in vitro la sensibilité des cellules de la patiente aux traitements dans un microenvironnement tumoral (sécrétion paracrine de cellules stromales et matrice de collagène). Ces modèles prédictifs 3D patient-spécifique pourront être utilisés pour déterminer des stratégies de thérapies personnalisées, ainsi que pour comprendre les phénomènes de résistance des cellules de mélanomes aux traitements
Melanoma cell sensitivity to targeted therapy molecules is dependent on the tumor microenvironment (cell-cell and cell-extracellular matrix interactions). Three dimensional (3D) in vitro cell culture systems better reflect the native structural architecture of tissues and are attractive to investigate cellular interactions. We have developed and compared several metastatic melanoma models: melanoma cells (SK-MEL-28 and SK-MEL-3, BRAF V600E mutant and SK-MEL-2 BRAF wt) cultured as a monolayer (2D) and co-cultured on 3D dermal equivalents with fibroblasts to better unravel factors modulating cell sensitivity to a BRAF inhibitor (BRAFi, Vemurafenib) and a BRAFi combined with a MEK inhibitor (MEKi, Cobimetinib). Cell sensitivity to treatments was evaluated under various aspects: cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK and PKB/Akt signaling pathway analysis (Western-blotting), apoptosis (TUNEL), cytokine and growth factor release (ELISA) and histology (3D models). A cytostatic effect of BRAFi was observed on SK-MEL-28 and SK-MEL-3 cells in both models. SK-MEL-2 cell line was clearly resistant to BRAFi when cultured as a monolayer but not when co-cultured with 3D fibroblasts embedded in a type I collagen matrix. Conditioned media provided by 3D fibroblasts (dermal equivalents) underlined 2D SK-MEL-2 sensitivity to BRAFi. Cell culture supernatant analysis revealed that dermal equivalents released some soluble factors (IL-6, IL-8, HGF, TGF-β): these secretions were modified during vemurafenib treatment. The combination of treatment with MEKi enhances the action of Vemurafenib on metastatic melanoma cells while decreasing the proliferation capacity of fibroblasts. Cell populations containing melanoma cells or fibroblasts associated with cancer (CAFs) were isolated from a cutaneous metastasis biopsy of a patient with metastatic melanoma. These cells allowed the realization of patient-specific models of metastatic melanoma in order to study in vitro the sensitivity of the patient’s melanoma cells to treatments in a tumor microenvironment (paracrine secretion of stromal cells and collagen matrix). These 3D predictive patient-specific models could be used to determine personalized therapy strategies, as well as to understand the resistance phenomena of melanoma cells to treatments

Cinquante à 80% des patients atteints d'une lésion médullaire traumatique (LM) présente des d'épisodes d'incontinence urinaire liés à une hyperactivité détrusorienne neurogène (HDN). L’HDN est caractérisée par des contractions non inhibées du détrusor pendant la phase de remplissage vésical, qui conduit à une augmentation des pressions détrusoriennes, particulièrement lorsque l’HDN est associée à une dyssynergie vésico-sphinctérienne. L'objectif principal de la prise en charge de l’HDN est d'obtenir une vidange vésicale régulière, complète et à basse pression, ainsi que de maintenir la continence urinaire, afin d'améliorer la qualité de vie des patients et de prévenir les complications uro-néphrologiques dont l’insuffisance rénale. La prise en charge actuelle repose sur une pharmacothérapie agissant principalement au niveau de la branche efférente, motrice, du réflexe mictionnel, permettant ainsi un remplissage vésical à basse pression. Le traitement de première intention repose sur des antimuscariniques oraux, le plus souvent associés à la réalisation d’autosondages pluriquotidiens. En cas d’échec de cette thérapeutique, l’injection intradétrusorienne de toxine botulique A est proposée. Cependant, malgré leur efficacité, ces traitements induisent des effets secondaires et souffrent d’un échappement thérapeutique, conduisant à un traitement chirurgical de troisième ligne. La technique de Brindley, qui consiste en une désafférentation des racines postérieures sacrées innervant la vessie associée à une stimulation électrique, à la demande, des racines antérieures est une alternative prometteuse, mais reste peu proposée en raison de la complexité de la procédure chirurgicale requise. L'HDN résulte de l'émergence d'un réflexe spinal anormal médié par une plasticité des afférences vésicales de type-C dans les suites de la LM. Le projet de mon équipe est de réaliser une déafférentation vésicale par thérapie génique afin d'abolir le réflexe de miction spinale altérée. Dans un second temps, la miction sera déclenchée par une stimulation électrique à la demande, des neurones efférents de la vessie. Mon travail de thèse consistait à développer les outils nécessaires à une telle désafférentation moléculaire. En conséquence, j'ai construit des vecteurs défectifs HSV-1 délivrant comme transgène thérapeutique la chaine légère d’un toxine botulique (BoNT-LC), sous le contrôle du promoteur du gène codant pour la protéine liée au gène de la calcitonine (hCGRP), permettant une expression sélective au sein des neurones sensoriels. La cassette de transcription a été insérée dans le locus LAT du génome HSV-1, la seule région du génome du virus qui reste active sur le plan transcriptionnel pendant une infection latente. Ces vecteurs ont été évalués (i) in vitro, sur des lignées cellulaires d'origine neurale et sur des cultures primaires de neurones sensoriels embryonnaires et adultes de rats, ainsi que sur des cultures primaires de neurones sensoriels et sympathiques humains adultes, (ii) ex vivo, sur des cultures organotypiques de ganglions sensoriels, sympathiques et parasympathiques de rats adultes, et (iii) in vivo, post inoculation au niveau du coussinet plantaire de rats adultes. Nos résultats indiquent que (i) les vecteurs expriment des BoNT-LC fonctionnelles, clivant ainsi les protéines SNARE post infection de cultures primaires de neurones sensoriels de rats et d’être humain, et inhibant la libération du neuromédiateur CGRP dans les neurones sensoriels de rat, (ii) la sélectivité d’expression de ces vecteurs dans des neurones sensoriels humains, par rapport aux neurones sympathiques humains, et (iii) une expression transgénique prolongée in-vivo au sein de ganglions sensoriels (DRG), au moins pour trois mois, après injection. Par conséquent, ces vecteurs semblent présenter les trois principales spécifications requises pour le développement d’une future stratégie de thérapie génique visant à traiter l’HDN
Fifty to 80% of patients with traumatic spinal cord injury (SCI) undergo urinary incontinence episodes, mostly related to neurogenic detrusor overactivity (NDO). NDO is characterized by uninhibited detrusor contractions during the bladder-filling phase which could lead to a significant increase in bladder pressures, especially when associated to sphincter-destrusor-dyssynergia, leading to uro-nephrological complications. The main goal of NDO management following SCI is to achieve regular and complete bladder emptying, avoiding high intra-detrusor pressure and maintaining continence, in order to improve patients’ quality of life and to prevent renal failure. The current management is well characterized and relies on pharmacotherapy acting primarily at the level of efferent motor micturition reflex branch, thus allowing bladder filling at low pressure. First line treatment relies on oral antimuscarinics, often associated to clean intermittent bladder self-catheterization. When patients are refractory to antimuscarinics, injection of botulinum toxin A into the detrusor is proposed. However, despite their efficacy, these treatments fail to persist in the long term, leading to a third-line surgical treatment, which consists in cystoplasty augmentation or sacral neuromodulation. The Brindley technique, which consist in a sacral deafferentation of bladder posterior roots associated to an electrical stimulation, on demand, of anterior roots is a promising alternative, but remains seldom performed because of the complex surgical procedure required. NDO results from the emergence, secondary to neuronal plasticity following SCI, of an abnormal micturition reflex mediated by bladder afferent C-fibers, conveying aberrant sensory information to the spinal cord. The aim of the team where I developed my work is to silence these bladder afferent C-fibers in order to abolish the impaired spinal micturition reflex after SCI. In a second time, micturition would be fired, on demand, by electric stimulation of the bladder efferent neurons. My work consisted in developing the tools and methods required for such molecular deafferentation. Accordingly, I constructed replication-incompetent HSV-1 vectors conceived to deliver a therapeutic transcription cassette, consisting in the light chains of botulinum toxin (BoNT-LC) driven by the human version of the promoter of the gene encoding calcitonin gene-related protein (hCGRP), to achieve sensory neuron-selective transgenic expression. The transcription cassette was inserted into the LAT locus of the HSV-1 genome, the only region of the virus genome that remains transcriptionally active during latent infection. These vectors have been assessed (i) in vitro, on cell lines of neural origin and on primary cultures of rat embryonic and adult sensory neurons, and on primary cultures of adult human sensory and sympathetic neurons, (ii) ex vivo, on organotypic cultures of sensory, sympathetic and parasympathetic ganglia from adult rats, and (iii) in vivo, in sensory ganglia following infection at the hind footpad of adult rats.Our results indicate that (i) the vectors express functional BoNT-LC, thereby cleaving proteins of the SNARE complex in rat and human sensory neurons and inhibiting release of the neuromediator CGRP in rat sensory neurons, (ii) the transcription cassette delivered by the vectors display highly selectively expression towards human sensory neurons, as compared to human sympathetic neurons, and (iii) the vectors induced long-term transgenic expression in sensory (DRG) ganglia (at least for three months) following footpad injection. Therefore, the vectors seem to accomplish the three main specifications required for a future gene therapy strategy, allowing to restore urinary continence and micturition without catheterization and without any major surgery. This approach will represent a major breakthrough in the management of NDO in SCI patients with complete and incomplete lesion

Les maladies neurodégénératives liées à l'âge, telle celle de Parkinson, sont un problème majeur de santé publique. Cependant, la maladie de Parkinson reste incurable et les traitements sont très limités. En effet, les causes de la maladie restent encore mal comprises et la recherche se concentre sur ses mécanismes moléculaires. Dans cette étude, nous nous sommes intéressés à deux phénomènes anormaux se produisant dans la maladie de Parkinson : l'agrégation de l'α-synucléine et l'activation des cellules microgliales. Pour étudier la polymérisation de l'α-synucléine, nous avons établi de nouvelles méthodes permettant la production in vitro de différents types d'oligomères d'α-synucléine. Grâce à des méthodes biophysiques de pointe, nous avons caractérisé ces différents oligomères à l'échelle moléculaire. Puis nous avons étudié leurs effets toxiques sur les neurones. Ensuite, nous nous sommes intéressés à l'activation des microglies et en particulier à leurs canaux potassiques et aux changements liés au vieillissement. Nous avons identifié les canaux Kv1.3 et Kir2.1 et montré qu'ils étaient impliqués dans l'activation des microglies. En parallèle, nous avons établi une méthode originale qui permet l'isolation et la culture de microglies primaires issues de cerveaux adultes. En comparaison à celles de nouveaux-nés, les microglies adultes montrent des différences subtiles mais cruciales qui soutiennent l'hypothèse de changements liés au vieillissement. Globalement, nos résultats suggèrent qu'il est possible de développer de nouvelles approches thérapeutiques contre la maladie de Parkinson en modulant l'action des microglies ou en bloquant l'oligomérisation de l' α-synucléine.

Yes
A sub-population of cells named cancer stem cells (CSCs) that initiate and promote tumour growth have been demonstrated to exist in several malignancies including colon carcinoma. The objective of our pilot study was to isolate CD133+CD26+CD44+ CSCs from patient colon tumours, culture spheres or organoids and observe their proliferation in primary cultures. Parallel cultures of non-cancer controls from colon normal lining and nonadenomatous polyps were set up. Magnetic activated cell sorting was used to isolate CD133+CD26+CD44+ cell populations followed by primary cell culturing under stem cell culture conditions. Number, cells/organoid and daughter generations of organoids were calculated using phase contrast microscope. Trypan blue exclusion method was used to test the viability of the cells. Both colon tumour and colon non-adenomatous polyp formed floating organoids in suspension; however non-adenomatous polyp cultures did not show self-renewal properties for more than 1 passage. Normal colon singlecell suspension did not create organoids. Metastatic colon tumours rapidly produce cancer cell organoids in less than 24 hours in larger numbers compared to non-metastatic colon tumours (1-3 weeks). Metastatic colon tumour organoids have the ability for proliferation for upto five daughter generations in primary culture compared to three generations for those grown from non-metastatic tumours. This in vitro CSC organoid model will help study colon cancer biology, in particular providing a valuable source of primary cell-derived tissue for studying personalized molecular profiling using ‘omics strategies to direct therapeutic intervention.

碩士
台北醫學院
細胞及分子生物研究所
85
Esophageal Carcinoma is the tenth most common cancer in the Taiwanese population and the fifth most common cancer in the male population of Taiwan. Various genetic abnormalities have already been reported such as changes of tumor suppressor genes including p53, APC, MCC that genes are involved in esophageal carcinoma.The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation by modulating the activities of the transcriptional factors that control expression of S phase genes. D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, promote progression through the G1 phase of the cell cycle by phosphorylating the pRB. The activities of Cdk4 and Cdk6 are constrained by inhibitors such as p16, the product of the p16 gene on human chromosome 9p21. The tumor suppressor gene p16(CDKN2/INK4A/MTS1) has been found to be deleted or mutated in a variety of human cancers. Thirteen human esophageal carcinoma primary tissue samples and five human esophageal carcinoma cell lines were analyzed for Rb gene alteration by single-strand conformational polymorphism (SSCP), DNA sequence analyses, and western blot assay. p16 gene alteration were detected by a method combining reverse transcription and nested polymerase chain reaction to detect different RNA transcripts of the p16 gene, associate with western blot assay to detect p16 protein expression, and polymerase chain reaction-methylation assay to detect the methylation status of genomic p16 gene in human esophageal carcinoma.The results showed that 2 of 13 esophageal carcinoma primary tissue samples and none of 5 esophageal carcinoma cell lines had altered Rb tumor suppressor gene; 3 of 10 primary tissue samples didn''t express pRb protein, including 1 primary tissue sample with altered Rb gene, but all of 5 cell lines express pRb protein. 8 of 13 primary tissues samples and all of 5 cell lines had altered p16 gene, 4 of 10 primary tissue samples and all of 5 cell lines fail to express p16 protein.Our result revealed that inactivation of the p16 tumor suppressor gene may play an important role in the development of esophageal carcinoma. But since the Rb tumor suppressor gene transcription factors binding sites alteration is not a frequent event in esophageal carcinoma, it will require more studies to understand Rb''s role in esophageal carcinoma.

Yes
Recently a sub-population of cells with stem cell characteristics, reported to be associated with initiation, growth, spread and recurrence, has been identified in several solid tumors including oral tongue squamous cell carcinoma (OTSCC). The aim of our pilot study was to isolate CD44+ cancer stem cells from primary cultures of OTSCC and neck node Level I (node-I) biopsies, grow cell spheres and observe their characteristics in primary cultures. Parallel cultures of hyperplastic lesions of tongue (non-cancer) were set up as a control. Immunohistochemistry was used to detect CD44/CD24 expression and magnetic activated cell sorting to isolate CD44+ cell populations followed by primary cell culturing. Both OTSCC and node-I biopsies produced floating spheres in suspension, however those grown in hyperplastic and node-I primary cultures did not exhibit self-renewal properties. Lymph node metastatic OTSCC, express higher CD44/CD24 levels, produce cancer cell spheres in larger number and rapidly (24 hours) compared to node negative OTSCC (1 week) and non-cancer specimens (3 weeks). In addition, metastatic OTSCC have the capacity for proliferation for up to three generations in primary culture. This in vitro system will be used to study cancer stem cell behavior, therapeutic drug screening and optimization of radiation dose for elimination of resistant cancer cells.
SKMCH&RC, Yorkshire Cancer Research

Adriamycin (Adr), the single most active agent used in the treatment of breast cancer, may become ineffective as treatment progresses due to the development of multidrug resistant (MDR) tumors. A major mechanism associated with MDR is increased P-glycoprotein (Pgp) expression. This thesis examined the abilities of the antiestrogen tamoxifen (TAM) and the progestin medroxyprogesterone acetate (MPA) as well as cyclosporin A (CsA), a known resistance modifier, to enhance the cytotoxic effects of Adr on human breast epithelial cells (HBEC) in primary culture. Pgp and estrogen receptor (ER) expression were determined in each of the cultures by immunocytochemical assays using the monoclonal antibodies C219 and H222 Spy, respectively. The Adrsensitive, Pgp-, ER+ MCF-7 cell line and the Adr-resistant, Pgp+, ER- MCF7-AdrR cell line were used as controls. Primary cultures were categorized as HBEC from tissues with or without previous chemotherapy. Pgp was detected in 1 of the 15 cell cultures from tissues without previous chemotherapy and in 5 of the 6 cell cultures from tissues previously exposed to chemotherapy. Incubation with either CsA or MPA plus Adr enhanced Adr toxicity in Pgp+ but not Pgp- cell cultures, whereas T AM had no effect on the sensitivity of any of the cultures. Of the 21 primary cultures of HBEC, 3 were ER+. There was no correlation between the enhancement of Adr cytotoxicity and ER status. The data suggest that MPA as well as CsA may be useful as modifying agents in overcoming Pgp-associated multidrug resistance.

碩士
國立臺灣大學
食品科技研究所
91
Abstract The objectives of this study was to investigate the effects of twenty-four lines garlics included two kinds of type which were hard-neck and soft-neck from Taiwan. We extracted garlic oil (GO) by steam distillation method, and the average extraction were around 0.2%. The highest extraction (0.26%) was Chia-yi lines of hard-neck type. The components of twenty-four lines garlic oils were analyzed by GC-MS and used Cluster analysis method to analysis the five major organosulfur components (OSCs) which were diallyl sulfide (DAS), diallyl disulfide (DADS), methyl allyl disulfide (MADS), diallyl trisulfide (DATS), and methyl allyl trisulfide (MATS) to get four groups. The types of four groups were chosen hard-neck type of black leave, Chia-yi, white leave of Si-luo, and soft-neck type of An-nan flower to be sampling, and used the four samples to observe the effect of cell viability of the cell line HepG2 from human liver tumor cell and normal primary SD rat hepatocytes. The lowest IC50 effects of garlic oil concentraction for HepG2 from human liver tumor cell viability was black leave, and then were white leave of Si-luo and Chia-yi lines. The garlic oil from An-nan flower had the highest IC50 effects. The IC50 effects of garlic oil concentraction for normal primary SD rat hepatocytes, the garlic oil from white leave of Si-luo had the lowest effect, and then were black leave and Chia-yi lines. The garlic oil from An-nan flower had the highest IC50 effects. In a word garlic oil had lowet IC50 effects for HepG2 cell viability than normal primary SD rat hepatocytes.