Дисертації з теми "Intracellular accumulation"

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Pharmacy." Discipline: Pharmacy; Department/School: Pharmacy.

Down syndrome (DS) is caused by the presence of an extra-copy of chromosome 21 and is the most frequent genetic cause of mental retardation. DS cognitive disabilities primary arise from the triplication of dosage-sensitive genes on chromosome 21. However, a global dysregulation in the expression of extra-chromosome 21 genes greatly complicates the understanding of the underling pathological mechanisms. We have recently found that cognitive impairment in the Ts65Dn mouse model of DS depends on the upregulation of a non-triplicated gene encoding for the chloride importer NKCC1, which we found increased also in brain tissue from individuals with DS. Consequently, the intracellular chloride concentration is increased and GABAergic signaling, through chloride-permeable GABAA receptors, is depolarizing rather than hyperpolarizing. Here, we aimed at addressing the molecular mechanisms responsible for NKCC1 overexpression in DS. Real-time qPRC and Western Blot analysis showed that NKCC1 overexpression in trisomic neurons does not derive from greater mRNA transcription or decreased protein turnover but rather from a diminished translational repression exerted on the 3’ untranslated region (3'UTR) of the gene. As 3’ UTRs are the preferred sites of action of microRNAs (miRs), we applied a combination of bioinformatics prediction tools and gene expression screening to identify candidate miRs downregulated in trisomic samples that could mediate NKCC1 overexpression. Our results show that different candidates miRs interact with NKCC1 3’UTR and repress its expression. Additionally, overexpression of the same miRs can normalize NKCC1 levels and intracellular chloride concentration in trisomic neurons, restoring GABAergic inhibitory signaling at the network level as shown by Multi-Electrode Array recordings. Our findings will help to elucidate molecular pathways dysregulated in DS and suggest possible targets for future therapeutic intervention.

Extracellular accumulation of Aβ in β-amyloid plaques is thought to be associated with the neurodegeneration observed in Alzheimer’s disease (AD) patients, although a lack of correlation with cognitive decline raised doubts on this hypothesis. In different transgenic mouse models Aβ accumulates inside the cells and mice develop behavioral deficits well before visible extracellular β-amyloid accumulation. Here we show that intracellular Aβ accumulates in flotillin-1 positive endocytic vesicles. We also demonstrate that flotillin-1 is not only associated with intracellular Aβ in transgenic mice but also with extracellular β-amyloid plaques in AD patient brain sections
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Extracellular accumulation of Aβ in β-amyloid plaques is thought to be associated with the neurodegeneration observed in Alzheimer’s disease (AD) patients, although a lack of correlation with cognitive decline raised doubts on this hypothesis. In different transgenic mouse models Aβ accumulates inside the cells and mice develop behavioral deficits well before visible extracellular β-amyloid accumulation. Here we show that intracellular Aβ accumulates in flotillin-1 positive endocytic vesicles. We also demonstrate that flotillin-1 is not only associated with intracellular Aβ in transgenic mice but also with extracellular β-amyloid plaques in AD patient brain sections.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs. This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior. The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments. The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity. Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be active in cellular assays. In conclusion, the methodology can be used to predict if new drug candidates reach their intracellular targets in sufficient amounts. Furthermore, the methodology can improve in vitro predictions of drug clearance and drug-drug interactions, by measuring the drug available for intracellular enzymes. Finally, this work can be expanded to other xenobiotics, e.g., to predict their intracellular toxicity.

Les diatomées sont des microalgues unicellulaires omniprésentes sur Terre et utilisées comme bioindicateurs pour évaluer l'impact de contaminations sur les écosystèmes aquatiques. Elles font également l'objet d'une attention croissante en tant que matériel de décontamination de métaux lourds d’effluents contaminés ou à des fins de biorestauration in situ. Cependant, les interactions entre les diatomées et les radioéléments, notamment l'uranium (U) et le radium (Ra), restent peu documentées. Ce travail vise à étudier tant au niveau macroscopique que moléculaire, les interactions de U et du Ra avec une culture xénique de diatomées Achnanthidium saprophilum, dans laquelle une communauté bactérienne est naturellement associée de manière symbiotique. Des expériences de bio-association de U et du Ra sont faites en présence de diatomées afin d’évaluer les fractions en U/Ra adsorbées et incorporées. En parallèle, diverses techniques de microscopie et de spectroscopie sont appliquées pour étudier la localisation et la spéciation de U au niveau cellulaire. Les résultats démontrent une bio-association significative de U et du Ra avec les diatomées et soulignent le rôle important des groupes carboxyliques et phosphates dans l'interaction U-diatomées. Les deux mécanismes d’adsorption et d’incorporation ont pu être mis en évidence pour U et Ra, la répartition entre les fractions adsorbées et incorporées dépendant de la phase de croissance des diatomées et du temps de contact entre les micro-algues est les radioéléments. Ce travail met également en évidence la contribution significative des bactéries symbiotiquement associées aux diatomées aux interactions globales
Diatoms are ubiquitous unicellular microalgae that are commonly used as bioindicators to evaluate the impact of contaminations on the ecological health of aquatic ecosystems. In recent decades, diatoms have received increasing attention as a decontamination material for removing heavy metals from contaminated effluents or for in situ bioremediation purposes. However, the interactions between diatoms and radioelements, e.g., uranium (U) and radium (Ra), remain relatively unclear and poorly documented. Therefore, this work aims to study, at both the macroscopic and molecular level, the interaction of U and Ra with a xenic Achnanthidium saprophilum diatom culture in which a naturally occurring bacterial community is symbiotically associated to diatoms. Batch-type U and Ra bio-association experiments are performed to evaluate the adsorbed and incorporated fractions of U/Ra in the diatom culture. Besides, various microscopic and spectroscopic techniques are applied to investigate the localization and speciation of U at the cellular level. Results demonstrate the significant bioassociation of U and Ra with the diatoms and highlight the important role of carboxylic and phosphate groups in the U-diatoms interaction. Both adsorption and incorporation are observed for U and Ra in the diatom culture, with their distribution depending on the diatom growth phase and on the contact time. This work contributes to a better understanding of the interaction of U and Ra with the xenic diatom culture and also highlights the contribution of the bacteria symbiotically associated with the diatoms to the overall interactions

Les quinolones peuvent etre utilisees dans le traitement des infections par des bacteries a gram negatif ou positif, y compris les mycobacteries. Afin de determiner les mecanismes d'action de ces drogues et les mecanismes de resistance des bacteries, l'accumulation intracellulaire de la norfloxacine a ete determinee chez diverses especes bacteriennes. Dans un premier temps, l'accumulation a ete determinee chez escherichia coli et staphylococcus aureus, a l'aide de la radiometrie et la fluorimetrie. Le maximum d'accumulation est atteint rapidement avec 250 nanogramme par milligramme de poids sec pour les deux souches. Il a egalement ete verifie que le transport de la norfloxacine est passif. Dans un second temps, l'accumulation de la norfloxacine a ete etudiee chez diverses souches de mycobacteries. Les methodes ont d'abord ete appliquees a mycobacterium smegmatis, grace a un protocole permettant d'obtenir des suspensions homogenes. Apres une importante modification des techniques, notamment en fluorimetrie, l'accumulation intracellulaire de la norfloxacine est maximale apres 5 minutes d'exposition avec 45 nanogramme par milligramme de poids sec. De plus, une adsorption de la norfloxacine a la surface cellulaire a ete revelee et doit etre deduite des valeurs d'accumulation mesuree. Par ailleurs, le transport de la quinolone est passif. Les methodes d'etudes ainsi modifiees ont alors ete appliquees a d'autres souches de mycobacteries sensibles ou resistantes a la quinolone. L'adsorption a la surface cellulaire est importante et le maximum d'accumulation est atteint assez tardivement: 100 nanogramme pour les souches de mycobacterium chelonae en 10 minutes d'exposition et 40 ou 60 nanogramme pour les souches de mycobacterium avium, respectivement sensible ou resistante a la norfloxacine, en 20 minutes d'exposition. D'autre part un phenomene de resistance naturelle aux quinolones, par un systeme d'efflux actif, semble avoir ete mis en evidence chez les souches de m. Avium

Les bases moléculaires de la maladie d'Alzheimer n'ont pas encore été clairement établies mais il semblerait que la dérégulation de l'homéostasie des métaux, et plus particulièrement celle du cuivre, soit étroitement liée à la pathogénèse de la maladie et à ses dépôts caractéristiques de peptide amyloïde (Aß). Des thérapies basées sur la modulation de l'homéostasie en cuivre ont vu le jour. Dans ce contexte, les complexes de cuivre et de bis(thiosemicarbazones) ([Cu(btsc)]) ont été proposés pour le traitement de la maladie d'Alzheimer. En effet, ces complexes sont supposés moduler la concentration en cuivre et modifier la production du peptide Aß dans les neurones. Par ailleurs, il est proposé que les complexes [Cu(btsc)] puissent être réduits dans la cellule. Cependant, à notre connaissance, la réduction intracellulaire de ces composés n'a jamais pas été démontrée. Ainsi, le but de notre étude était d'améliorer la compréhension du mécanisme d'accumulation intracellulaire des complexes de [Cu(btsc)]. Nos résultats révèlent que l'accumulation intracellulaire du cuivre dans les cellules est très élevée et que ces composés ne sont pas substrats de la P-glycoprotéine. Cette protéine est un élément clé dans la faible perméabilité de la barrière hémato-encéphalique. Par ailleurs, nous n'avons pas détecté de réduction intracellulaire des ions cuivriques. Enfin, une fois dans la cellule, les complexes subissent une agrégation, ce qui suggère fortement que l'agrégation des complexes est la force motrice responsable de leur accumulation intracellulaire. Leur localisation intracellulaire est en partie diffuse mais une quantité non négligeable se localise autour des gouttelettes lipidiques
The molecular basis of Alzheimer’s disease has not been clearly established, but disruption of brain metal ion homeostasis, particularly copper and zinc, might be closely involved in the pathogenesis of this disease and its characteristic ß-amyloid neuropathological features. The use of complexes of copper with bis(thiosemicarbazones) ([Cu(btsc)]) has been proposed for the treatment of Alzheimer’s disease. Their mode of action could involve the modulation of the concentration of copper or zinc, and it has been suggested that these compounds can modulate the production of ß-amyloid peptide at the neuron level. Furthermore, it has been reported that [Cu(btsc)] complexes can be reduced inside the cells. However, to our knowledge the intracellular reduction of these compounds has never been demonstrated. Thus, the goal of our study was to increase understanding of the mechanism of intracellular accumulation of [Cu(btsc)] complexes. Our results reveal that the intracellular concentration of copper inside the cells is very high and that these compounds are not P-glycoprotein substrates. This protein is a key element of the low permeability of the blood–brain barrier. Furthermore, no intracellular reduction of cupric ions was detected. Finally, once inside the cells, the complexes undergo aggregation, strongly suggesting that aggregation of complexes is the driving force responsible for their intracellular accumulation. Their intracellular localization is partly scattered but a significant amount is localized around lipid droplets

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

碩士
大葉大學
生物科技碩士在職學位學程
104
Trehalose is a non-reducing disaccharide which widely present in various living organisms and be used as energy storage or a carbon source. When the organism is under extreme growth environment, such as high salt, high pH, high temperature, low temperature, or abnormal osmotic pressure etc., the cells will secrete more trehalose to protect themselves. To investigate the effect of different reverse culture conditions on trehalose accumulation in the yeast cells. In this study, we plan to stimulate the secretion of trehalose in vivo using different reverse environment. Different kinds of stress factors were used as follow : pH (pH 6.0 ~ 9.0); temperature (20 ℃~40 ℃); incubation time (1 to 5 days); different nitrogen source and concentration: NH4NO3, peptone, yeast extract, NH4Cl, KNO3, NH4H2PO4, and (NH4)2SO4 (0.5 - 1.0%，w/v); various inorganic salts and concentration: MgCl2．6H2O, CaSO4, FeSO4．7H2O and MgS04．7H2O (0.5 -1%，w/v). After high-temperature extraction , the result shows that the addition of 1% KNO3, 1% MgCl2．6H2O in the medium, and incubated at 30 ℃ for 4 days, to effectively enhance 7.2 times of the trehalose accumulation in vivo. The optimized reverse fermentation conditions, for intracellular trehalose accumulation of Candida rugosa can be another new selection for further industrial applications in the future. Key Words: Candida rugosa, Extraction, Fermentation, Stress, Trehalose

Background: Tissue non-specific alkaline phosphatase (TNAP) is an enzyme which functions within the body to catalyze the hydrolysis of pyrophosphate to phosphate, and is a well-known mediator of bone mineralization. It has also been identified as a positive mediator of intracellular lipid accumulation (ICLA) in both murine and human preadipocytes as well as in the hepatocellular cell line HepG2. However, the mechanism through which TNAP functions to control ICLA is not known. Both osteoblasts and adipocytes are both of mesenchymal origin and thus may share conserved mechanisms through which TNAP functions. Within bone, TNAP converts pyrophosphate (which inhibits mineralization) to phosphate. This phosphate is essential to the mineralization process through binding to hydroxyapatite crystals, and it also activates the transcription of genes whose products function in osteoblast differentiation, including NRF2. This thesis therefore aimed to determine the role of both pyrophosphate and TNAP-generated phosphate in ICLA. In addition, it is possible that TNAP may interact with other proteins, as it is known that TNAP is able to dephosphorylate proteins such as tau. This thesis therefore aimed to determine whether TNAP binds to other proteins in the context of ICLA. Lipids are not only stored within hepatocytes and adipocytes, but are also found in cells of the adrenal cortex, and TNAP is known to be expressed within such cells. Therefore, this thesis also aimed to determine whether TNAP is involved in the accumulation of cholesterol esters within lipid droplets in the adrenal cortex. Methods: To determine the effect of high intracellular pyrophosphate levels on ICLA, 3T3-L1 cells (a preadipocyte cell line) were cultured in the presence and absence of probenecid, an inhibitor of the pyrophosphate transporter ANK, and induced to accumulate lipids. Lipid accumulation was monitored through Oil red O staining. The effect of probenecid treatment on TNAP activity and intracellular pyrophosphate levels was also analysed. To determine whether TNAP functions in ICLA by producing phosphate for gene induction, 3T3-L1 cells were stimulated to undergo ICLA in the presence and absence of the TNAP inhibitor levamisole, which in turn blocks ICLA. Levamisole treated cells were also incubated with phosphate to see if this would overcome the inhibitory effect of levamisole on ICLA. The ability of phosphate to induce gene expression of NRF2 was determined through real-time PCR. In addition, an NRF2 expressing plasmid was transfected into cells treated with the TNAP inhibitor levamisole to determine if this would also overcome the block on ICLA caused by TNAP inhibition. In silico analysis identified TRAF2 as a potential binder of TNAP. The expression of TRAF2 during ICLA was determined through real time PCR, and the effect of overexpression of TRAF2 on intracellular lipid accumulation was determined through the transfection of a TRAF2 expressing plasmid in cells induced to undergo ICLA. To determine whether TNAP modulates lipid accumulation in cells of the adrenal cortex, the Y1 murine adrenocortical cell line was cultured in the presence and absence of TNAP inhibitor levamisole, and ICLA measured by Oil Red O staining. The location of TNAP within Y1 cells was identified by histochemical staining. Results: Cells treated with probenecid showed increased pyrophosphate levels (expressed as a % of levels observed at baseline) when compared to untreated controls (155.5 ± 15.1 % vs 51.1 ± 18.9 %; p=0.001) after 24 hours of culture. Increased pyrophosphate levels resulted in ICLA within 3T3-L1 cells surpassing levels seen in untreated controls (507.4 ± 30.4 % vs 337.6 ± 16.17 %; p=0.004). This increase in pyrophosphate was coupled to an increase in TNAP activity within the initial 24 hours (291.5 ± 72.8 % vs baseline of 100%; p=0.038) compared to that seen in control experiments (103.43 ± 24.3 % vs baseline of 100%; p=0.848). Cells treated with levamisole showed minimal ICLA and when exogenous phosphate was added, lipid levels were reconstituted to levels similar to that seen in cells induced to accumulate lipids in the absence of levamisole (284.01 ± 62.52% vs 275.86 ± 35.52%; p= 0.83). In the presence of levamisole plus exogenous phosphate, NRF2 expression was upregulated within 1 hour of treatment to levels greater than that seen in the absence of phosphate but presence of levamisole (216.64 ± 19.24% vs 98.28 ± 3.79%; p=0.004). Expression of NRF2 (through transfection with an NRF2 expression plasmid) in cells deficient in TNAP activity (via levamisole treatment), and induced to accumulate lipids, was not able to completely reconstitute ICLA when compared to cells not treated with levamisole (193.72 ± 16.51 vs 326.46 ± 47.64; p = 0.019), but ICLA was still greater than that observed at baseline. In silico analysis predicted that TNAP would bind to TRAF2, yet neither band shift assays nor immune co-precipitation showed evidence of this. However, TRAF2 mRNA was down regulated within 3T3-L1 cells during adipogenesis, reaching levels of 15.27 ± 10.27% (p= 0.014) of baseline (levels prior to induction of intracellular lipid accumulation) by day 4 of lipid accumulation. Overexpression of TRAF2 during adipogenesis markedly reduced intracellular lipid accumulation (147.88 ± 11.28% vs 326.46 ± 47.64%; p=0.028 (after 8 days of culture)). In Y1 cells TNAP activity is upregulated during ICLA, reaching 233 ± 37.56% (p=0.019 vs. baseline) of baseline levels within the initial 24 hours. Inhibition of TNAP activity through levamisole treatment resulted in a decrease in ICLA when compared to cells not treated with levamisole. Histochemical analysis showed that TNAP activity was localised to the lipid droplet. Discussion and Conclusions: Within 3T3-L1 cells TNAP mediates intracellular lipid accumulation through the generation of phosphate. The phosphate is able to increase the expression of NRF2, however it is likely that NRF2 is not the only gene whose expression is regulated by TNAP-generated phosphate. It was found that TNAP and TRAF2 do not bind to each other in the context of ICLA; however TRAF2 is a negative mediator of ICLA through a TNAP-independent mechanism. Functional TNAP is necessary for the accumulation of cholesterol esters within the Y1 cell line, suggesting that TNAP is essential for lipid accumulation in cell types that store lipids in intracellular membrane-bound droplets in the form of triglycerides or cholesterol esters.
GR2018

Most (99%) mitochondrial proteins are nuclear-encoded and must be imported into mitochondria. Deficits in mitochondrial protein import (MPI) affect mitochondrial function and can cause neurodegenerative diseases. I hypothesized that MPI was regulated by iCa2+. In differentiated PC12 cells, treatment with the Ca2+ ionophore (A23187; 24h, 0.15uM) increased iCa2+, ROS generation and promoted neurite outgrowth. Western blot and flow cytometry in live cells showed that A23187 increased levels of mitochondrial proteins; mtHSP70 and mtGFP in mitochondria and autoradiography confirmed that A23187 increased the import of mtGFP. A23187 also slowed intramitochondrial mtGFP degradation. Increased MPI was not associated with mitochondrial biogenesis, but appeared partially dependent on cAMP. In rat cortical neurons, mtHSP70 also increased after A23187 treatment. These results show that, in neurons, increased iCa2+ can regulate MPI. Further, increased iCa2+ can slow intramitochondrial protein degradation. These results indicate that MPI is labile and may be altered in response to neuronal activity.

碩士
嘉南藥理科技大學
生物科技系暨研究所
92
Abstract Oxidative stress increasing in vivo is an important risk factor in the coronary heart disease development and correlated with the formation processing of atherosclerosis. It has been reported that consumption of a traditional diet rich in vegetables and fruits obviously decreases the risk of coronary heart disease. The purpose of this study is to explore the antioxidant capacity of aqueous extract of Welsh onion green leaves (WOE) and whether WOE can modulate intracellular lipid accumulation in RAW 264.7 macrophages. The results showed that WOE in 0.01 – 1.0 mg/ml can scavenge superoxide radicals, nitric oxide, ferrous ions and inhibit xanthine oxidase activity in a dose-dependent manner. And WOE in 0.5 – 2.0 mg/ml inhibit protein tyrosine residue nitration in mouse heart homogenate in vitro. On the other hand, WOE at 0.5 mg/ml can block lipopolysaccharide (LPS)-decreased scavenger receptor B1 (SR-B1) and ATP- binding cassette transporter A1 (ABCA1) expression in macrophage RAW 264.7 cells. Quercetin (20 ?嵱) and kaempferol (30 ?嵱), the major components of WOE, also show the similar inhibitory action on SR-B1 and ABCA1. Furthermore, VLDL and oxLDL decreases SR-BⅠ expression in macrophages. WOE (0.5 mg/ml), Quercetin (20 ?嵱) and kaempferol (30 ?嵱) block VLDL-decreased ABCA1 expression in macrophages. These results may be contributed by WOE’s direct or indirect actions in PPAR ? activation or inflammatory inhibition in macrophages. Finally, WOE (0.5 mg/mL), Quercetin (20 ?嵱)、Kaempferol (30 ?嵱) and 9-cis Retinoic acid (RA；40 ?嵱) also decrease oxLDL-induced intracellular lipid accumulation in macrophages. These results implied that WOE might have benefit in protect consumers from oxidative stress and cardiovascular diseases.

The inwardly-rectifying K+ current (IK1) is important for heart cell function because it sets the resting potential, influences cell excitability, and promotes repolarization of the action potential. My objective was to investigate the modulation of IK1 by extracellular K+ (K+o) and intracellular K+ (K+i). IK1 was recorded from whole-cell-configured guinea-pig ventricular myocytes that were dialyzed with Na+-free EGTA-buffered pipette-filling solution and bathed with Na+ or NMDG+ solution that contained agents that inhibit non-IK1 currents. Lowering K+o from standard 5.4 to 2 and 1 mM shifted the reversal potential (Erev) of IK1 in accord with calculated K+ equilibrium potential (EK), and altered IK1 amplitude in accord with conductance (GK1)? ?K+o. Surprisingly, myocytes bathed with 0-mM K+ solution had a small outward IK1 at holding potential (Vhold) ?85 mV. This IK1 was attributed to channel-activation by T-tubular K+ (K+T) whose concentration is sensitive to the flow of T-tubular IK1. K+T in myocytes bathed with 0-mM K+ solution was ? 3.2 mM at Vhold ?85 mV, but ? 0.3 mM following large K+T-depleting flows of inward IK1 at ?160 mV. Results consistent with interplay of IK1 and K+T were also obtained in experiments on myocytes bathed with 2-, 5.4-, and 15-mM K+ solution. Myocytes were dialyzed with pipette solutions that contained 0-140 mM K+ to investigate modulation by K+i. When IK1 at Vhold was kept small, Erev varied with pipette K+ in a near-Nernstian manner (i.e., Erev ? EK); however, when IK1 (Vhold) was large and inward, Erev was markedly negative to nominal EK. Findings in experiments that involved shifting Vhold, changing K+o, and application of Ba2+ and Cs+ suggest that the magnitude/direction of IK1 strongly affects the concentration of K+ in submembrane cytoplasm. Classical GK1-voltage parameters GK1max and V0.5 (but not slope factor) were affected by decreases in K+i: GK1max declined by ? 25% per decade decrease in K+i, and V0.5 shifted approximately as 0.5 ? EK-shift. The latter findings are discussed and compared with those of earlier studies on the dependence of inwardly-rectifying K+ conductance on K+i.

碩士
台北醫學院
醫學研究所
88
(1) Recent investigations have indicated that amphetamine, a psychostimulator, produces its central effect by activation of the NMDA (N-methyl-D-aspartate) receptor, a subtype receptor of the glutamate receptor. Our previous study using [3H]TCP receptor ligand binding to assess this receptor/channel complex had shown that amphetamine could directly inhibit the NMDA-coupled ion channel by acting at multiple sites on the receptor. In the present study, we examined this specific action of amphetamine and compare the effect of d-amphetamine、l-amphetamine、Methamphetamine on the NMDA receptor-mediated 45Ca2+ accumulation. This study confirm that amphetamine could direct inhibit the function of NMDA receptor, and the d-amphetamine、l-amphetamine、Methamphetamine have similar effect on the NMDA receptor-mediated 45Ca2+ accumulation. (2) Apoptosis (also termed programmed cell death, PCD) is a mode of cell death in which the cell plays an active role in its demise. In the present study, we investigate the characteristic of apoptosis in the primary cortical neuronal culture. We find that the cell in our rat primary cortical cell culture undergoing apoptosis by the examination of microscope. We confirm the presence of apoptosis in this culture by several biochemical methods. Including taking picture of neuronal culture under microscope, examine the apoptotic cells by TUNEL assay and immunocytochemistry, and quantify the mRNA level of bax, c-fos, c-jun by RT-PCR. Besides, we also examine the LDH response of culture cells. In addition, we also detect the effect of NMDA (N-methyl-D-aspartate) (100M), MK-801 (10M), morphine (10M), and naloxone (10M) on the apoptosis of this culture in DIV6, 12, 18 culture. We find that our rat primary cortical cell culture undergoing spontaneous apoptosis expressed as increase in the LDH level, increase in the number of TUNEL positive neuron in a two-phase manner. This apoptotic phenomenon is accompanied with increase gene expression of c-fos, and likely the expression of bax gene. Addition of NMDA to culture also induces significant increase in LDH response accompanies with apoptosis. But MK-801, morphine, or naloxone has no effect on LDH response and apoptosis. However, NMDA or MK-801 decrease c-fos on DIV 6 culture, but increase c-fos on DIV 18 culture. Morphine and naloxone didn’t have any effect on the c-fos gene expression. NMDA increase apoptotic neurons on DIV 12 and 18 culture, but it has no effect on DIV 6 culture. This study confirm that NMDA increase apoptosis in this culture, but MK-801, morphine, or naloxone didn’t have any effect on the apoptosis in this culture.

Thesis (Ph.D.)--State University of New York at Buffalo, 2005.
Title from PDF title page (viewed on Mar. 21, 2006) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Oseroff, Allan R. Includes bibliographical references.

Articular cartilage has a low propensity for self-repair, due to which 27 million people are affected by osteoarthritis every year in North America. The current repair techniques used for cartilage defects possess flaws that reduce long-term clinical success. Tissue engineering carries with it the promise of engineering hyaline-like cartilage with physical and biochemical properties, similar to that of native cartilage. This being said, the primary objective of my project was to engineer clinically relevant sized articular cartilage constructs. To achieve my objective, first, I investigated the effect of continuous culture on cartilaginous tissue growth. Constructs grown under continuous media flow significantly accumulated more collagen and glycosaminoglycan, and displayed a stratified morphology, similar to that found in native cartilage. The second goal was to further increase chondrocyte proliferation, and extracellular matrix (ECM) accumulation. To achieve this, constructs were grown in a bioreactor with media supplemented with 14 mM sodium bicarbonate (NaHCO3). Constructs cultivated in the bioreactor with NaHCO3 supplementation exhibited a significant (p<0.05) increase in ECM accumulation (a 98-fold increase in glycosaminoglycans and a 25-fold increase in collagen content), cell proliferation (a 13-fold increase), and thickness (a 28-fold increase) compared to all other conditions (static and reactor without NaHCO3 supplementation). The third goal was to engineer cartilage constructs with as little cells as possible, reducing donor site morbidity. From the results obtained, it was evident that the monolayer constructs outperformed all the other constructs (pellet, biopsy, and minced). The final goal was to understand the underlying reason for the increased proliferation. First, I investigated if there were any differences present in intracellular pH (pHi) and intracellular buffering capacity. Second, I determined the role of extracellular pH (pHe) on cell proliferation. In an effort to accurately achieve this, I, for the first time, have reported on measuring pHi of chondrocytes while still in culture (2D and 3D cultures) using a confocal microscope. This study demonstrated the importance of extracellular environments, such as pHe, extracellular buffering capacity, and the presence of carbon dioxide and bicarbonate ions for chondrocyte proliferation.
Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2012-10-30 19:19:32.026