This study was conducted to assess the regulation of glycolysis by 6phosphofructokinase (PFK) during the postmortem metabolism of beef muscle. In the first experiment, M. sternocephalicus pars mandibularis samples were excised from six randomly-selected steers. Two samples were obtained from each steer immediately postmortem; one sample was quickly immersed in liquid nitrogen and the other was stored at 4oC for 4 d. Glycogen concentrations decreased 45% from d 0 to d 4, and 39.6 ?mol/g of glycogen was still present in the tissue at d 4. Concentrations of free glucose increased (P < 0.001) from 0.84 ?mol/g at d 0 to 6.54 ?mol/g at d 4. Fructose-6-phosphate (F6P) and glucose-6-phosphate (G6P) increased (P < 0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33 ?mol/g) and was elevated to 45.9 ?mol/g by d 4. Glycolytic potential was 34.4 ?mol/g higher (P < 0.05) when measured at d 0 than at d 4. The greatest activity of PFK was measured in fresh muscle extracts, between pH 7.4-7.8; by reducing the pH to 7.0, PFK activity was depressed by nearly 50% at 1 mM F6P. In a second experiment, M. longissimus lumborum samples were excised at the 13th thoracic rib location from six randomly-selected steers. Samples were obtained at intervals ranging from 40 min to 24 h postmortem. Glycogen concentrations decreased 45% between 40 and 100 min, and tended (P &#8804; 0.10) to decrease between 100 min and 24 h (from 47 to 32 ?mol/g). Concentrations of free glucose increased (P &#8804; 0.009) from 1.0 ?mol/g at 40 min to 5.0 ?mol/g at 24 h. Concentrations of F6P and G6P increased dramatically after 100 min (muscle pH &#8804; 6.5), whereas glycogen depletion appeared to halt by 100 min. Lactate began accumulating almost immediately and tripled in concentration by 24 h. The elevation of G6P and F6P, coupled with the pH sensitivity of PFK, indicate that the postmortem decline in pH ultimately inactivates PFK prior to glycogen depletion.

La reacción catalizada por la 6-fosfofructo-1-quinasa (PFK-1) es el paso limitante de la glucólisis. Existen 3 genes que codifican para las tres isoenzimas descritas de la PFK-1: la muscular, la hepática y plaquetaria. La pérdida de la isoenzima muscular de la PFK-1 es la causante de la glucogenosis de tipo VII o enfermedad de Tarui. Ésta se caracteriza por la ausencia de actividad PFK-1 en el músculo esquelético y un déficit parcial en el eritrocito. Los síntomas más comunes son miopatía con intolerancia al ejercicio y anemia hemolítica compensada. Debido a su baja incidencia y a la ausencia de un modelo en ratón, actualmente son poco conocidas las adaptaciones fisiológicas y bioquímicas, así como la interacción entre los diferentes tejidos afectados por el déficit de PFK-1M. En este trabajo se ha generado un modelo murino de glucogenosis de tipo VII mediante técnicas de recombinación homóloga en células ES. Este animal reproducía la sintomatología característica de esta enfermedad asociada con una elevada mortalidad. Así, el músculo esquelético de estos animales presentaba un bloqueo glucolítico que provocaba el acúmulo de hexosas monofosfato y glucógeno. Este bloqueo glucolítico estaba acompañado por una severa intolerancia al ejercicio, caracterizada por una depleción del ATP muscular. Asimismo, el estudio de la musculatura implicada en la respiración reveló una severa afectación morfológica y bioquímica asociada a una posible alteración funcional de este proceso. Del mismo modo, la musculatura cardíaca también estaba afectada por la carencia de PFK-1M. Paralelamente, se estudió si este animal reproducía las alteraciones eritrocitarias descritas en los pacientes. Así, los ratones deficientes en PFK-1M presentaban una hemólisis crónica caracterizada por un elevado número de reticulocitos en sangre. Se observó también una esplenomegalia asociada a un incremento del número de precursores hematopoyéticos en este órgano. El análisis bioquímico del eritrocito evidenció un bloqueo glucolítico con una marcada reducción del 2,3DPG. Esta disminución provocaba una respuesta a hipoxia en la musculatura esquelética caracterizada por la expresión de HIF-1?. Como consecuencia de estas alteraciones musculares y eritrocitarias la musculatura esquelética de los animales Pfkm-/- no podía compensar la carencia glucolítica provocando una situación de estrés energético crónico que se reflejaba en una pérdida de la funcionalidad muscular. / The reaction catalyzed by 6-phosphofructo-1-kinase (PFK-1) is the rate limiting step of the glycolysis. There are 3 genes that codify for the described muscle, hepatic and platelet isoenzymes. The loss of the muscle isoenzyme is the cause of type VII glycogenosis or Tarui's disease. It is characterized by the absence of PFK-1 activity in the skeletal muscle and a partial deficiency in the erythrocyte. The most common symptoms are myopathy with exercise intolerance and compensated haemolytic anaemia. Due to the low incidence and the absence of a mouse model, at the present little is described about the biochemical and physiological adaptations, as well as the interaction among the different organs and tissues affected by the deficit of PFK-1M. In this work, a murine model of type VII glycogenosis has been generated using homologous recombination in ES cells. These animals reproduced the characteristic pathology of this disease associated with a high mortality. Thus, the skeletal muscle of these animals presented a block in the glycolytic pathway that caused the increase of monophosphate hexoses and glycogen. This blockade was paralleled by a severe intolerance to exercise, characterized by depletion of muscle ATP. Likewise, the study of the breathing musculature revealed a severe morphological and biochemical alteration associated to a possible functional alteration of this process. In the same way, the heart musculature was also affected by the lack of PFK-1M. In parallel, it was studied if this animal reproduced the erythrocyte alterations described in patients. PFK-1M deficient mice showed chronic haemolysis characterized by a high count of blood reticulocytes. In addition, these mice showed esplenomegaly associated with an increment in the number of haematopoietic precursors in this organ. The biochemical analysis of the erythrocytes showed also a glycolitic block with a marked reduction of 2,3DPG levels. This decrease caused a hypoxic response in the skeletal muscle characterized by the expression of HIF-1?. As a result of these muscle and erythrocyte alterations, the skeletal muscle of Pfkm-/- mice could not compensate the lack glycolysis causing chronic energy stress witch was reflected in a loss of the muscular functionality.

Els resultats que es presenten en aquesta memòria aporten informació que ens permet donar un pas més en el coneixement de la regulació transcripcional del gen Pfkfb4 i conseqüentment de la regulació de la via glucolítica en el testicle. En primer lloc, hem identificat el moment d'aparició dels gens Pfkfb3 i Pfkfb4 durant el desenvolupament del testicle de rata, tant en períodes prenatals com en períodes postnatals. Així mateix, vam aïllar fraccions cel·lulars del testicle de rata (Sertoli, Germinals i una fracció somàtica formada per una barreja de cèl·lules peritubulars i de Leydig), trobant que PFKFB4 només s'expressava en aquella fracció germinal, mentre que PFKFB3 ho feia tant a la fracció germinal com a les fraccions somàtiques (Sertoli, Leydig y Peritubulars). Pel que fa a l'expressió d'ambdues proteïnes en els espermatozoides, es va comprovar que PFKFB3 s'expressava en els espermatozoides més immadurs mentre que PFKFB4 ho feia en els madurs i que es localitzava, de forma principal, a la zona del cap de l'espermatozoide i, de forma secundària, al flagel. En segon lloc, hem identificat diferents seqüències consens en el promotor del gen Pfkfb4 on podrien unir-se factors de transcripció per regular el gen. També hem identificat el seu inici de transcripció. Entre les seqüències consens d'unió per factors de transcripció es destaquen els elements de resposta de HIF-1 (HRE), els elements de resposta de Sp-1 i els elements de resposta a c-Myc (E-box). Hem observat que el gen Pfkfb4 respon, incrementant la seva transcripció, a la hipòxia i que aquesta resposta seria depenent de la presència del factor HIF1-alfa actiu a la cèl·lula. A més, la presència de Sp-1 activa la transcripció basal del gen i en situacions d'hipòxia augmentaria la resposta a aquesta de Pfkfb4. Per la seva banda, c-Myc reduiria la resposta transcripcional de Pfkfb4 tant en normòxia com en hipòxia.En tercer lloc, hem estudiat la regulació hormonal del gen Pfkfb4, trobant que la testosterona per se no produeix cap efecte ni en el gen Pfkfb4 ni en la proteïna. Si no que la regulació hormonal ve regulada a través de les cèl·lules de Sertoli, les quals sota l'estimulació hormonal de la testosterona produeixen un factor soluble que seria el responsable de controlar l'expressió de Pfkfb4 i Pfkfb3 en les cèl·lules germinals. Aquests resultats obtinguts sobre l'expressió i regulació de dos isoenzims de la proteïna PFK-2 en el testicle, assenyalarien a la proteïna PFK-2 com a un dels punts claus en el control de la via glucolítica durant l'espermatogènesi dels mamífers.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-20T15:50:30Z No. of bitstreams: 1 Cristiane Maia Alves.pdf: 1063313 bytes, checksum: a48ab6ac753935673b07a64282e7821b (MD5) / Made available in DSpace on 2013-09-20T15:50:30Z (GMT). No. of bitstreams: 1 Cristiane Maia Alves.pdf: 1063313 bytes, checksum: a48ab6ac753935673b07a64282e7821b (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / O vírus Influenza é o agente infeccioso que mais afeta o trato respiratório dos seres humanos, resultando em altos índices de morbidade e mortalidade. Este vírus pertence à família Orthomyxovirida, apresenta RNA fita simples, octa-segmentado, de polaridade negativa e possui um envelope lipoproteico no qual estão inseridas as glicoproteínas hemaglutinina e neuraminidase. Já foi demonstrado que a infecção por Influenza gera alterações no metabolismo celular, porém, não foi descrito, até o momento, qual seria o mecanismo de ação relacionado. Em nosso trabalho, decidimos analisar mais a fundo essas alterações metabólicas relacionadas à infecção por Influenza A. Para tal, resolvemos investigar os efeitos da inibição da via glicolítica e do silenciamento da enzima chave de regulação da via, a enzima PFK-1, sobre a replicação viral. Inicialmente, utilizamos células MDCK infectadas com MOI de 5,0 e verificamos o efeito da inibição de umas das etapas finais desta via metabólica sobre a replicação do vírus. Observamos que, em 24 horas pós-infecção, a inibição da glicólise provocou uma redução da replicação do vírus Influenza, porém, em 48 horas pós–infecção, a inibição desta via promoveu um aumento da replicação viral. Verificamos, também, que o silenciamento das isoformas L, M e P da enzima PFK-1, em 24 horas pós-infecção, promoveu uma significativa diminuição do título viral. Diante disto, decidimos investigar quais as etapas do ciclo replicativo estariam sendo afetadas pelo silenciamento desta enzima e observamos que, tanto a adsorção como a penetração de material genético no núcleo celular foram afetadas pelo silenciamento da PFK-1, principalmente pela inibição da isoforma P. Esses resultados sugerem que a enzima PFK-1 tem um importante papel na infecção pelo vírus Influenza podendo, inclusive, ser um possível alvo terapêutico. / The Influenza virus is the major infectious agent that affects the respiratory tract of humans, resulting in high morbidity and mortality. This virus belongs to Orthomyxovirida family and presents single-stranded, octa-segmented, negative polarity RNA and has a lipoprotein envelope in which are inserted the hemagglutinin and neuraminidase glycoproteins. It has been shown that Influenza infection causes changes in cellular metabolism, however, not been described up to now, which would be related mechanism of action. In our work, we decided to further analyze these metabolic changes related to infection with Influenza A. To this end, we decided to investigate the effects of inhibition of glycolytic pathway and the silencing of key regulatory enzyme of this pathway, the enzyme PFK-1, on viral replication. Initially, we used MDCK cells infected with m.o,.i.5.0 to check the effect of inhibition of some final stages of this pathway on virus replication. We observed that, in 24 hours postinfection, the inhibition of glycolysis resulted in a reduction of the replication of Influenza viruses, but at 48 hours post-infection, the inhibition of this pathway leads to an increase in viral replication. We also verified that silencing of L, M and P isoforms of PFK-1, at 24 hours post-infection, caused a significant decrease in viral titer. So, we decided to investigate which steps of the replicative cycle were being affected by the silencing of this enzyme and found that both the adsorption and penetration of genetic material in the cell nucleus were affected by the silencing of PFK-1, mainly by inhibiting P isoform. These results suggest that the enzyme PFK-1 plays an important role in the Influenza virus infection and may it also be a potential therapeutic target.

The trypanosomatid parasites T. brucei, T. cruzi and Leishmania spp. are responsible for the ‘neglected diseases’ Human African Trypanosomiasis, Chagas disease and Leishmaniasis respectively. In their human infective form in the bloodstream all three trypanosomatid parasites rely heavily on glycolysis for ATP production. Phosphofructokinase (PFK) catalyses the third step of the glycolytic pathway in all organisms using aerobic respiration. It facilitates the phospho transfer from ATP to fructose 6-phosphate (F6P) to make the products fructose 1,6- bisphosphate (F16BP) and ADP. RNAi knockout of T. brucei PFK has shown the enzyme is essential for survival of the bloodstream form parasites. Trypanosomatid PFKs have a unique set of structural and regulatory differences compared to the mammalian host enzyme. These differences, coupled with the availability of trypanosomatid PFK crystal structures present an opportunity for the structure-based design of specific inhibitors against the enzyme. Here we present an enzymatic characterisation of recombinant PFKs from T. brucei, T. cruzi and Leishmania infantum trypanosomatids, their regulation by the allosteric activator AMP, and their inhibition by drug-like inhibitor compounds. Inhibitor compounds (‘CTCB compounds’) were designed against T. brucei PFK with the aim of developing novel treatments against Human African Trypanosomiasis (HAT). We describe the testing, ranking and biophysical characterisation of these compounds as part of a Wellcome Trust Seeding Drug Discovery program. We found that CTCB inhibitor compounds bound to an allosteric pocket unique to trypanosomatid PFKs. We show that the compounds are specific; neither competing with the natural substrates ATP or F6P nor inhibiting the human PFK enzyme. We describe the development and testing of highly potent and specific low molecular weight PFK inhibitors that translate to both killing of cultured T. b. brucei parasites and a cure of stage I HAT in mice models. We describe the tight, 1:1 binding of these compounds with trypanosomatid PFKs, and the thermodynamic characteristics of binding through various biophysical assays. We also show the unprecedented characterisation of the reverse PFK reaction by trypanosomatid and human forms of the enzymes. We found that PFK can also carry out the reverse enzymatic reaction, under physiologically relevant concentrations of ADP and F16BP to produce F6P and ATP. We show that the reverse reaction is also subject to allosteric regulation by AMP, and can be inhibited by the CTCB compounds with a similar potency to the forward reaction. Finally, we describe the mechanism of allosteric activation by AMP and inhibition by the drug-like compounds against trypanosomatid PFKs.

Bacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose-6-phosphate (F6P), binds along the other dimer-dimer interface. The different functional forms BsPFK can take when in the presence of F6P and PEP can be described by the following diproportionation equilibrium: XE + EA <--> XEA + E where XE is the enzyme bound to PEP, EA is the enzyme bound to F6P, E represents the apo enzyme, and XEA is the ternary complex formed when both substrate and inhibitor are bound. Currently in the Protein Data Bank (PDB) there are two relevant forms of wild-type BsPFK, the EA form and the X'E form, which represents the enzyme bound to the PEP analog, phosphoglycolate (PGA). When comparing the EA and the X'E structures, a 7° rotation about the substrate-binding interface is observed and is termed the quaternary shift. The current study uses methyl TROSY NMR to examine the different liganded states of BsPFK, and for the first time structural data for the XEA species is shown. In addition, crystallography was used to obtain the first apo structure of BsPFK. To distinguish between changes associated with the quaternary shift and those associated with the intra-subunit tertiary changes, the variant D12A BsPFK was studied using kinetics, crystallography, and NMR. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the X'E form of wild-type. Kinetic studies of D12A BsPFK, when compared to wild-type, show a 50-fold diminished F6P binding affinity, 100-fold enhanced binding affinity, and a similar coupling constant. A conserved hydrogen bond between D12 and T156 takes place across the substrate binding interface in the EA form of BsPFK. The variant T156A BsPFK shows similar binding, coupling, and structural characteristics to D12A BsPFK. PEP still inhibits these variants of BsPFK despite the fact that the enzymes are in the quaternary shifted position prior to PEP binding. Therefore the quaternary shift of BsPFK primarily perturbs ligand binding but does not directly contribute to heterotropic allosteric inhibition.

Glioblastome sind die aggressivsten und häufigsten Hirntumore beim Menschen und entziehen sich weiterhin einem kurativen Therapieansatz. Wie die meisten malignen Tumore zeigen Glioblastome den sogenannten Warburg Effekt, eine gesteigerte aerobe Glykolyse. Ein Schlüsselenzym der Glykolyse ist die 6-Phosphofrukto-1-kinase (PFK-1), deren stärkster allosterischer Aktivator Fruktose-2,6-bisphosphat (F2,6BP) ist. Die zelluläre Konzentration von F2,6BP wird von dem bifunktionalen Enzym 6-Phosphofrukto-2-kinase/Fruktose-2,6-bisphosphatase (PFK-2) reguliert. Im Menschen existieren vier PFK-2-Isoenzyme (PFKFB1-4), die gewebespezifisch exprimiert werden. Die PFKFB3 hat das höchste Kinase/Bisphosphatase- Aktivitätsverhältnis von 710:1 innerhalb der PFK-2-Familie und wird in Tumorzelllinien und verschiedenen malignen Tumoren überexprimiert. In humanem Hirngewebe wurden sechs alternative Spleißvarianten der PFKFB3-mRNA (UBI2K1–6) beschrieben, welche sich in der C-terminalen Region unterscheiden. Neuere Untersuchungen im Verlauf dieser Arbeit ergaben, dass es auch Spleißvarianten gibt, die in der N-terminalen Region variieren, sodass insgesamt 10 Spleißvarianten der PFKFB3 bekannt sind. Die spezifischen Funktionen im Zellstoffwechsel wurden bisher nur für die Spleißvariante UBI2K5 untersucht, die der anderen Spleißvarianten sind weitestgehend unbekannt. Das Ziel der vorliegenden Arbeit war, die Bedeutung der PFKFB3 Spleißvariante UBI2K4 in Glioblastomen für den Stoffwechsel und das Wachstum dieser Tumore am Modell der humanen U87-Glioblastomzelllinie aufzuklären und die These, dass die UBI2K4 eine proliferationshemmende Funktion hat, zu überprüfen. Im ersten Teil dieser Arbeit wurde mittels stabiler Transfektion von HEK-293-Zelllen mit einem pTER-Vektor und gleichzeitiger transienter Transfektion bei U87- und HEK-293-Zellen mit synthetischer siRNA ein Knockdown der UBI2K4 mRNA und des Proteins erzielt. Es stellte sich heraus, dass der UBI2K4 Knockdown in beiden Zelllinien zu einer reduzierten Viabilität und Zellproliferation führte. Die Verdopplungszeiten waren prolongiert und auch das dreidimensionale Wachstum in Soft-Agar-Kulturen war reduziert. Bei HEK-293-Zellen wurde der UBI2K4 Knockdown durch einen signifikanten Anstieg der UBI2K5 mRNA Expression kompensiert. Die UBI2K6 Expression blieb unverändert. Bei U87-Zellen, deren native UBI2K4 mRNA Expression sehr gering ist, wurde eine UBI2K5 mRNA Reduktion bei gleichzeitiger Hemmung der UBI2K4 Expression festgestellt, während die UBI2K6 mRNA leicht zunahm. Weiterhin konnte im Western Blot gezeigt werden, dass in HEK-293-Zellen neben der Spleißvariante UBI2K4, auch als Variante 4 bezeichnet, auch die neu entdeckte Variante 5 exprimiert wird. In U87-Zellen konnte nur die Expression der Variante 4 nachgewiesen werden. In einem Antikörper-Mikroarray wurde gezeigt, dass die UBI2K4 die Expression insbesondere von Proteinen mit immunmodulatorischen Eigenschaften, apoptose-induzierenden Proteinen und Proteinen der Zellkommunikation und des Metabolittransports beeinflusst. Im zweiten Teil dieser Arbeit wurde das native Protein der UBI2K4 in HEK-293- und U87-Zellen überexprimiert. Die Zellen mit einer gesteigerten UBI2K4 Proteinkonzentration konnten schneller proliferieren und zeigten eine gesteigerte Zellviabilität. DIese Ergebnisse korrelieren mit den bereits beschriebenen Ergebnissen des UBI2K4 Knockdowns im ersten Teil dieser Arbeit. Die vermutete inverse Korrelation der UBI2K4 Konzentration und der Proliferationsrate konnte hier nicht bestätigt werden. Vielmehr scheint die UBI2K4 so wie die Spleißvariante UBI2K5 der PFKFB3 ebenfalls in höheren Konzentrationen proliferationsfördernd zu wirken. Dies scheint überaus wahrscheinlich, da die im Antikörper-Mikroarray beeinflussten Proteine auch darauf hindeuten.:1 EINFÜHRUNG UND AUFGABENSTELLUNG 8 1.1 GLIOBLASTOME 8 1.2 DIE GLYKOLYTISCHE AKTIVITÄT IN TUMORZELLEN – DER WARBURG-EFFEKT 12 1.2.1 REGULATION DER GLYKOLYSE IN TUMORZELLEN 16 1.2.2 DIE 6-PHOSPHOFRUKTO-2-KINASE/FRUKTOSE-2,6-BISPHOSPHATASE ISOENZYME 18 1.3 AUFGABENSTELLUNG 26 2 MATERIAL UND METHODEN 27 2.1 LABORAUSSTATTUNG 27 2.2 CHEMIKALIEN UND REAGENZIEN 27 2.3 VERBRAUCHSMATERIALIEN 29 2.4 STANDARDS 29 2.5 ENZYME 29 2.6 ANTIKÖRPER 29 2.7 REAGENZIENSYSTEME 30 2.8 BAKTERIENSTÄMME 30 2.9 ZELLLINIEN 31 2.10 PLASMIDE 31 2.11 OLIGONUKLEOTIDE 31 2.12 RIBONUKLEINSÄUREN 34 2.13 ZELLBIOLOGISCHE METHODEN 34 2.13.1 KULTIVIERUNG UND PASSAGIEREN VON ZELLEN 34 2.13.2 BESTIMMUNG DER ZELLZAHL 35 2.13.3 KONSERVIERUNG VON ZELLEN 35 2.13.4 TRANSFEKTION UND SELEKTION 35 2.13.5 PROLIFERATIONSTESTS 38 2.14 MOLEKULARBIOLOGISCHE ARBEITEN 40 2.14.1 ARBEITEN MIT BAKTERIENKULTUREN 40 2.14.2 PRÄPARATION VON NUKLEINSÄUREN 42 2.14.3 KONZENTRATIONSBESTIMMUNG VON NUKLEINSÄUREN 43 2.14.4 AGAROSEGEL-ELEKTROPHORESE 43 2.14.5 POLYMERASE-KETTENREAKTION (PCR) 44 2.14.6 REVERSE TRANSKRIPTION 45 2.14.7 QUANTITATIVE REAL-TIME PCR 45 2.14.8 DNA-SEQUENZIERUNG 47 2.14.9 GEN-SILENCING DURCH SIRNA 48 2.14.10 ÜBEREXPRESSION DER SPLEIßVARIANTE UBI2K4 51 2.14.11 MYCOPLASMENTEST 52 2.15 PROTEINCHEMISCHE METHODEN 53 2.15.1 ZELLLYSE UND BESTIMMUNG DER PROTEINKONZENTRATION 53 2.15.2 SDS-PAGE 53 2.15.3 WESTERNBLOT-ANALYSE 54 2.16 ANTIKÖRPER MIKROARRAY 55 2.17 STATISTISCHE ANALYSEN 55 2.18 SOFTWARE 56 3 ERGEBNISSE 57 3.1 AUSWAHL DER ZELLLINIE 57 3.2 KNOCKDOWN DER UBI2K4 DURCH STABILE UND TRANSIENTE TRANSFEKTION 59 3.2.1 KLONIERUNG DER PTER-EXPRESSIONSPLASMIDE FÜR DIE STABILE TRANSFEKTION 59 3.2.2 STABILE TRANSFEKTION MITTELS PTER-PLASMIDEN 62 3.3 NACHWEIS DES KNOCKDOWN DER UBI2K4 AUF DIE MRNA EXPRESSION 64 3.3.1 KNOCKDOWN DER UBI2K4 IN STABIL TRANSFIZIERTEN HEK-293-ZELLEN 64 3.3.2 KNOCKDOWN DER UBI2K4 IN STABIL TRANSFIZIERTEN HEK-293-ZELLEN MIT ZUSÄTZLICHER TRANSIENTER TRANSFEKTION MITTELS SIRNA 65 3.3.3 KNOCKDOWN DER UBI2K4 IN TRANSIENT TRANSFIZIERTEN U87-ZELLEN 67 3.4 EXPERIMENTE MIT ZELLEN BEI KNOCKDOWN DER UBI2K4 70 3.4.1 EINFLUSS DES KNOCKDOWN DER UBI2K4 AUF DAS WACHSTUM VON HEK-293-ZELLEN 70 3.4.2 EINFLUSS DES KNOCKDOWN DER UBI2K4 AUF DIE KOLONIEBILDUNG VON HEK-293-ZELLEN IN SOFT-AGAR-KULTUREN 71 3.5 KOLORIMETRISCHE TESTS BEI KNOCKDOWN DER UBI2K4 73 3.5.1 EINFLUSS DES UBI2K4 KNOCKDOWN AUF DIE VIABILITÄT UND DIE PROLIFERATION VON HEK-293-ZELLEN 73 3.5.2 EINFLUSS DES UBI2K4 KNOCKDOWN AUF DIE VIABILITÄT UND DIE PROLIFERATION VON U87-ZELLEN 74 3.6 ERGEBNISSE DES ANTIKÖRPER MIKROARRAY 75 3.7 ÜBEREXPRESSION DER UBI2K4 DURCH STABILE / TRANSIENTE TRANSFEKTION 78 3.7.1 KLONIERUNG DER PCDNA3.1-EXPRESSIONSPLASMIDE 78 3.8 NACHWEIS DER ÜBEREXPRESSION DER UBI2K4 AUF DIE MRNA EXPRESSION 79 3.9 KOLORIMETRISCHE TESTS BEI ÜBEREXPRESSION DER UBI2K4 81 3.9.1 EINFLUSS DER UBI2K4 ÜBEREXPRESSION AUF DIE VIABILITÄT UND DIE PROLIFERATION VON HEK-293-ZELLEN 81 3.9.2 EINFLUSS DER UBI2K4 ÜBEREXPRESSION AUF DIE VIABILITÄT UND DIE PROLIFERATION VON U87-ZELLEN 82 4 DISKUSSION 85 4.1 AUSWAHL DER ZELLLINIEN 85 4.2 GENERIERUNG STABILER ZELLLINIEN MIT UBI2K4-, UBI2K5- UND UBI2K6-KNOCKDOWN 87 4.2.1 KNOCKDOWN DER PFKFB3 SPLEIßVARIANTEN DURCH STABILE TRANSFEKTION IN HEK-293- UND U87-ZELLEN 89 4.3 KNOCKDOWN DER UBI2K4 IN HEK-293- UND U87-ZELLEN MITTELS TRANSIENTER TRANSFEKTION 90 4.4 ÜBEREXPRESSION DER UBI2K4 IN HEK-293 UND U87-ZELLEN 95 4.5 MIKROARRAY 97 4.5.1 PROTEINE MIT EINER SIGNIFIKANTEN KONZENTRATIONSERHÖHUNG 98 4.5.2 PROTEINE MIT EINER SIGNIFIKANTEN KONZENTRATIONSERNIEDRIGUNG 100 5 ZUSAMMENFASSUNG DER ARBEIT 104 6 LITERATURVERZEICHNIS 107 7 ANLAGEN 129 7.1 ABBILDUNGSVERZEICHNIS 129 7.2 TABELLENVERZEICHNIS 131 8 ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT 132 9 LEBENSLAUF 133 10 DANKSAGUNG 134