Mitochondrial dysfunction has been implicated as one of the primary causes of Parkinson's disease (PD). The proteins PINK1, a serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are mutated in many genetic cases of PD. In healthy individuals, Parkin is recruited to damaged mitochondria and leads to autophagic degradation of mitochondria in a process termed mitophagy. Following depolarization of the mitochondrial membrane, PINK1 is stabilized on the outer mitochondrial membrane, and triggers Parkin translocation from the cytosol to mitochondria. Precisely how this phenomenon is regulated is still unclear. We employed RNA interference (RNAi) technology in a 384-well format to identify novel genes that are required for Parkin recruitment to mitochondria. We identified ATPase inhibitory factor 1 (IF1) as the strongest hit required for Parkin recruitment following treatment with the protonophore CCCP. We show that IF1 is upstream of PINK1 and Parkin, and required to sense mitochondrial damage by allowing the loss of membrane potential. In cells treated with CCCP, the absence of IF1 permits the ATP synthase to run freely in reverse, consuming ATP to maintain potential across the inner mitochondrial membrane, thus blocking PINK1 and Parkin activation. Interestingly, Rho0 cells, that lack mitochondrial DNA, have downregulated endogenous expression of IF1 in order to maintain mitochondrial function. Overexpression of IF1 in Rho0 cells results in the depletion of mitochondrial membrane potential and the initiation of mitophagy. These data demonstrate a unique role for IF1 in the regulation of mitochondrial quality control that has not been explored in the etiology of PD.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-109) Also available in print.

Regensburg, Univ., Diss., 2009.

Publicación a texto completo no autorizada por el autor / El gen WRN codifica a la proteína del síndrome de Werner (WRN), una proteína multifuncional con actividad helicasa y exonucleasa. Datos previos indican que WRN está asociada a la maquinaria traduccional y relacionada con las vías metabólicas que regulan la síntesis de macromoléculas, la producción de energía y el balance de óxidoreducción (redox) implicados en la proliferación celular. La proteína ribosomal eucariota S3 (eRPS3) es un elemento importante de la subunidad ribosomal menor 40S que promueve el reconocimiento del codón de inicio y la interacción con el ácido ribonucleico mensajero (ARNm) para el desarrollo de la síntesis proteica. La asociación entre la proteína ribosomal humana S3 (hRPS3) y WRN ha sido descrita previamente mediante ensayos de co-inmunoprecipitación, inmunoprecipitación y pulldown usando extractos totales de células embrionarias de riñón. Se identifica la interacción entre WRN, presente en la fracción citoplasmática de células HeLa, y la hRPS3. Mediante el uso de la tecnología de ADN recombinante se sintetizó en E. coli la proteína hRPS3 fusionada a una cola de seis histidinas (hRPS3-6xHis). Esta proteína de fusión fue inmovilizada por cromatografía de afinidad en una resina de agarosa con iones níquel, y se usó conjuntamente con la fracción citoplasmática de células HeLa que contenía a WRN para llevar a cabo el ensayo de pull-down. El análisis por Western blot del pull-down evidenció la presencia de WRN en la resina que contenía a la proteína de fusión hRPS3-6xHis. Este resultado demuestra la interacción entre WRN presente en el citoplasma y la hRPS3 que es un elemento importante de la maquinaria de traducción. / Tesis

Cyanobacterial secondary metabolites are a rich source of bioactive compounds with potential utilization in pharmacology. The aim of this study was to evaluate the effect of the novel compound nostatin A isolated from the cyanobacterium Desmonostoc muscorum. Project was focused on the extraction, purification and characterization of the cytostatic effect caused by this novel compound and its naturally occurred structural analogues. The cytostatic activity of nostatin A and its analogs was evaluated in HeLa cell line. Experiments based on microscopy, flow cytometry and HPLC-HRMS techniques were performed in order to clarify the cytostatic effect of nostatin A in HeLa cells and its mechanism of the action.

Mitochondrial dysfunction has been implicated as one of the primary causes of Parkinson's disease (PD). The proteins PINK1, a serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are mutated in many genetic cases of PD. In healthy individuals, Parkin is recruited to damaged mitochondria and leads to autophagic degradation of mitochondria in a process termed mitophagy. Following depolarization of the mitochondrial membrane, PINK1 is stabilized on the outer mitochondrial membrane, and triggers Parkin translocation from the cytosol to mitochondria. Precisely how this phenomenon is regulated is still unclear. We employed RNA interference (RNAi) technology in a 384-well format to identify novel genes that are required for Parkin recruitment to mitochondria. We identified ATPase inhibitory factor 1 (IF1) as the strongest hit required for Parkin recruitment following treatment with the protonophore CCCP. We show that IF1 is upstream of PINK1 and Parkin, and required to sense mitochondrial damage by allowing the loss of membrane potential. In cells treated with CCCP, the absence of IF1 permits the ATP synthase to run freely in reverse, consuming ATP to maintain potential across the inner mitochondrial membrane, thus blocking PINK1 and Parkin activation. Interestingly, Rho0 cells, that lack mitochondrial DNA, have downregulated endogenous expression of IF1 in order to maintain mitochondrial function. Overexpression of IF1 in Rho0 cells results in the depletion of mitochondrial membrane potential and the initiation of mitophagy. These data demonstrate a unique role for IF1 in the regulation of mitochondrial quality control that has not been explored in the etiology of PD.

Die Krebserkrankung ist bis zum heutigen Zeitpunkt eine große Belastung in unserer Gesellschaft. Obwohl es stets Fortschritte in der Entwicklung neuer Therapiemöglichkeiten gibt, stellt die Behandlung auch in der modernen Medizin eine enorme Herausforderung dar. Darum besteht bis heute ein hoher Bedarf an neuen und weiterentwickelten Behandlungsmöglichkeiten. Um die Proliferation einer neoplastischen Zelle zu beeinflussen, stellen die Biomasse und die Energie einen grundlegenden Ansatz dar. Hier bieten sich vor allem die Aminosäuren als wesentlicher Baustein der Zellmasse und der Energieträger „Glukose“ an, wodurch sich die beiden Ansätze einer Protein- bzw. Aminosäure-Restriktion und einer Glukose-Restriktion ergeben. Ziel ist es durch eine veränderte Stoffwechsellage einen Low-Energy-Metabolismus (LEM) zu induzieren, welcher die Zelle in einen sich selbst regenerierenden, antiproliferativen Zustand versetzt. Zusätzlich sollte untersucht werden, ob sich die beiden Ansätze grundsätzlich als Therapieform gegen das Plattenepithelkarzinom (HNSCC) eignen. Zudem sollte ein Modell einer humanen Zelllinie erstellt werden, mit Hilfe dessen sich ein LEM auf metaboler Ebene charakterisieren lässt. Die Ergebnisse zeigen, dass Zellen unter konstanter Glukose-Restriktion teils sensitiver auf Todesliganden reagieren. Außerdem wirken Kalorien-Restriktions-Mimetika antiproliferativ auf HNSCC Zellen. Hinzu kommt, dass eine Methionin-Restriktion Einfluss auf die Genexpression jener Gene hat, die mit der LEM-Signalkaskade in Zusammenhang stehen. Zuletzt lieferte die massenspektrometrische Analyse von mehr als 150 Metaboliten der humanen Zelllinie HeLa ein detailliertes Bild ihres Metabolismus unter Methionin-Restriktion. Durch die Definition eines charakteristischen Fingerabdrucks nach 72 h und eines kleinen Fußabdrucks aus wenigen Metaboliten, konnte ein humanes Modellsystem etabliert werden, dass zukünftig u.a. die schnelle Analyse von Kalorien-Restriktions-Mimetika ermöglicht. / Cancer continues to be a major burden in our society to this day. Although there is always progress in the development of new treatment options, treatment remains an enormous challenge even in modern medicine. That is why there is a high demand for new and advanced treatment options to this day. To influence the proliferation of a neoplastic cell, biomass and energy represent a fundamental approach. In this context, amino acids as an essential building block of the cell mass and the energy carrier "glucose" are particularly suitable, resulting in the two approaches of protein or amino acid restriction and glucose restriction. The aim is to induce a low-energy metabolism (LEM) by changing the metabolic state, which will put the cell into a self-regenerating, anti-proliferative state. In addition, it should be investigated whether the two approaches are suitable in principle as a form of therapy against head and neck squamous cell carcinoma (HNSCC). Furthermore, to establish a model of a human cell line that can be used to characterize LEM at the metabolic level. The results show that cells under constant glucose restriction are partly more sensitive to death ligands. Moreover, caloric restriction mimetics have an antiproliferative effect on HNSCC. In addition, methionine restriction has an impact on gene expression of those genes related to the LEM signaling cascade. Most recently, mass spectrometric analysis of more than 150 metabolites from the human cell line HeLa provided a detailed picture of their metabolism under methionine restriction. By defining a characteristic fingerprint after 72 h and a small footprint consisting of a few metabolites, a human model system could be established that will allow, among other things, the rapid analysis of caloric restriction mimetics in the future.

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Inom högstadieskolan är det flera yrkesprofessioner som bör samarbeta med skolans studie- och yrkesvägledningsuppdrag för att eleverna ska få med sig rätt förutsättningar till att kunna göra väl underbyggda studie- och yrkesval. Forskning har visat att det är viktigt med tydlighet kring studie- och yrkesvägledningens organisering och mål för att få arbetet med vid vägledning att fungera. Historiskt sett har lärare haft en central roll i skolans studie- och yrkesvägledning. I tidigare läroplaner har studie- och yrkesvägledning ingått i samtliga ämnen och idag har läraren fortfarande ett ansvar över att stärka elevers självkännedom som underlag för val. Syftet med den här studien är därför att undersöka hur högstadielärare ser på den vida vägledningen och hur uppdraget studie- och yrkesvägledning som hela skolans ansvar arbetas med. Kvalitativa semistrukturerade intervjuer har genomförts med åtta yrkesverksamma högstadielärare. En tematisk analys visade att högstadielärarna har bristande kännedom om sin roll i arbetet med studie- och yrkesvägledning eftersom de inte fått någon utbildning i ämnet. Viljan och engagemanget bland informanterna parallellt med att det finns ett behov av att ge lärarna rätt förutsättningar för att kunna fullfölja det som står i läroplanen diskuteras.