No description available.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The multiplication of cells proceeds through consecutive phases of growth and division (G1, S, G2 and M phases), in a process known as the cell cycle. The transition between these phases is regulated by so-called checkpoints, which are important to ensure proper functioning of the cell cycle. For instance, mutations leading to faulty regulation of the G1/S transition point are seen as one of the main causes of cancer. Traditionally, models for biological systems that show rich dynamic behavior, such as the cell cycle, are studied using dynamical systems analysis. However, using this analysis method one cannot quantify the extent of control of an individual process in the system. To understand system properties at the process level, one needs to employ methods such as metabolic control analysis (MCA). MCA was, however, developed for steady-state systems, and is thus limited to the analysis of such systems, unless the necessary extensions would be made to the framework. The central question of this thesis focuses on quantifying the control in mathematical models of the G1/S transition by the individual cell cycle processes. Since MCA was never applied to the cell cycle, several new methods needed to be added to the framework. The most important extension made it possible to follow and quantify, during a single cell cycle, the control properties of the individual system processes. Subsequently, these newly developed methods were used to determine the control by the individual processes of an important checkpoint in mammalian cells, the restriction point. The positioning of the restriction point in the cell cycle was distributed over numerous system processes, but the following processes carried most of the control: reactions involved in the interplay between retinoblastoma protein (Rb) and E2F transcription factor, reactions responsible for the synthesis of Delayed Response Genes and Cyclin D/Cdk4 in response to growth signals, the E2F dependent Cyclin E/Cdk2 synthesis reaction, as well as the reactions involved in p27 formation. In addition it was shown that these reactions exhibited their control on the restriction point via the Cyclin E/Cdk2/p27 complex. Any perturbation of the system leading to a change in the restriction point could be explained via its e ect on the Cyclin E/Cdk2/p27 complex, showing a causal relation between restriction point positioning and the concentration of the Cyclin E/Cdk2/p27 complex. Finally, we applied the new methods, with a modular approach, to compare a number of cell cycle models for Saccharomyces cerevisiae (budding yeast) and mammalian cells with respect to the existence of a mass checkpoint. Such a checkpoint ensures that cells would have a critical mass at the G1/S transition point. Indeed, in budding yeast, a correction mechanism was observed in the G1 phase, which stabilizes the size of cells at the G1/S transition point, irrespective of changes in the specific growth rate. This in contrast to the mammalian cell cycle models in which no such mass checkpoint could be observed in the G1 phase. In this thesis it is shown that by casting specific questions on the regulation and control of cell cycle transition points in the here extended framework of MCA, it is possible to derive consensus answers for subsets of mathematical models. / AFRIKAANSE OPSOMMING: Die selsiklus bestaan uit agtereenvolgende groei- en delingsiklusse wat tot selvermeerdering lei. Die siklus word gekenmerk deur onderskeie fases (G1, S, G2 en M) wat deur sogenaamde beheerpunte gereguleer word. Hierdie beheerpunte verseker dat selvermeerdering nie ongekontroleerd kan plaasvind nie en mutasies wat lei tot foutiewe regulering van die G1/S transisiepunt word as een van die hoofoorsake van kanker beskou. Die hoofdoel van hierdie studie was om die beheer wat selsiklusprosesse op die G1/S transisie uitoefen met behulp van wiskundige modelle te kwantifiseer. Omdat biologiese sisteme soos die selsiklus ryk dinamiese gedrag vertoon, word hulle tradisioneeldeur middel van dinamiese sisteemanalise bestudeer. Die analisemetode beskik egter nie oor die vermoë om die hoeveelheid beheer wat afsonderlike sisteemprosesse op 0n sisteemeienskap uitoefen te kwantifiseer nie. Om sisteemeienskappe op prosesvlak te verstaan moet metodes soos metaboliese kontrole analise (MKA) ingespan word. MKA was egter ontwikkel om sisteme in 0n bestendige toestand te analiseer en aangesien MKA nog nooit vantevore vir selsiklus analises gebruik was nie, moes nuwe MKA tegnieke gedurende die studie ontwikkel word. Die belangrikste van die metodes maak dit moontlik om beheer (soos uitgeoefen deur die onderskeie sisteemprosesse) oor 0n enkele selsiklus na te volg en te kwantifiseer. Die nuut-ontwikkelde metodes was vervolgens gebruik om te bepaal hoe een so 0n beheerpunt in soogdierselle - die restriksiepunt - deur die onderskeie sisteemprosesse beheer word. Die studie het aangedui dat die posisie van die restriksiepunt tydens die selsiklus deur ’n verskeidenheid sisteemprosesse beheer word. Die bevinding was dat vier prosesse beduidend meer beheer op die posisie van die restriksiepunt uitoefen: Reaksies wat betrekking het op die wisselwerking tussen retinoblastoma proteïen (Rb) en E2F transkripsiefaktor; reaksies verantwoordelik vir die sintese van vertraagde responsgene en Siklien D/Cdk4 in respons tot groeiseine; die E2F afhanklike Siklien E/Cdk2 sintesereaksie; sowel as die reaksies betrokke in p27 vorming. Daar was ook aangetoon dat hierdie reaksies hul beheer op die posisie van die restriksiepunt deur die Siklien E/Cdk2/p27 kompleks uitoefen, siende enige sisteemversteuringe (wat tot veranderinge in die restriksiepuntposisie aanleiding gee) deur veranderinge in die kompleks verklaar kon word - 0n observasie wat aandui dat daar 0n kousale verhouding is tussen die posisie van die restriksiepunt en die Siklien E/Cdk2/p27 kompleks. Die nuut-ontwikkelde metodes was verder gebruik om 0n verskeidenheid selsiklusmodelle van Saccharomyces cerevisiae (bakkersgis) en soogdierselle met 0n modulêre aanpak te vergelyk om te bepaal of daar 0n massa beheerpunt in beide soogdier- en bakkersgisselle bestaan. Daar word gepostuleer dat hierdie beheerpunt verseker dat selle 0n kritiese massa by die G1/S transisiepunt bereik. Die resultate van die studie dui daarop dat bakkersgis, anders as soogdierselle, oor so 0n korreksiemeganisme beskik. Die meganisme stabiliseer die grootte van selle in die G1 fase ondanks veranderinge in die groeitempo van die selle, sodat massa homeostaties by die G1/S transisiepunt gehandhaaf word. Die studie het getoon dat moeilike vrae met betrekking tot die selsiklus beantwoord kan word deur van wiskundige modelle gebruik te maak en die probleme in die nuut-ontwikkelde metaboliese kontrole analise raamwerk te giet.

A key effect of human papillomavirus (HPV) infection is to disrupt the normal cell cycle in order to subvert the cellular DNA replication machinery. Morphologically, condylomata induced by high and low risk HPV types cannot be distinguished and many studies have shown that the pattern of viral gene expression is similar in condylomata caused by both high risk and low risk HPV types. Detailed morphological study of cell cycle protein expression has not previously been performed on condylomata infected with low risk HPV types. The findings presented suggest that the mechanisms employed by low risk HPV6 or HPV11 to subvert cellular functions in condylomata acuminata are similar to those employed by high risk HPVs, with the exception of cyclin D1 and p53 protein over-expression. The differences in p53 expression and cyclin D1 expression seen between high and low risk HPV infection, reflect the known differences between high and low risk types and are in agreement with the known differences between high risk and low risk E6 and E7 proteins. PHK transduction studies demonstrated HPV E6 and E7 induce changes in cell cycle protein expression and that there are differences in cell cycle abrogation between HPV6 and HPV16. Disruption of the p53-MDM2 interaction can lead to activation of the p53 pathway. HPV infected lesions almost always contain wild-type p53. The binding of HPV E6 to p53, and its subsequent targeting for degradation, prevents activation of the p53 pathway in HPV infected cells. Cells over expressing HPV genes were treated with Nutlin-3, a MDM2-small molecule antagonist. The findings presented suggest treatment with Nutlin-3 induces cell cycle arrest in cells expressing HPV16 E7 and HPV6 E6 and HPV6 E7. This suggests a potential role for Nutlin-3 in the treatment of HPV infected cells.

ACTH (hormônio adrenocorticotrópico) e fatores peptídicos de crescimento regulam a proliferação celular em células adrenocorticais. Resultados preliminares de ensaios biológicos, cromatografia de heparina-sefarose e \"Westem blotting\" indicaram a produção de fatores peptídicos de crescimento das famílias de PDGF e de FGF na linhagem celular adrenocortical Y-1. No entanto a relação entre o estímulo promovido por ACTH e a expressão de fatores peptídicos de crescimento e sua contribuição para o controle do ciclo celular em células adrenocorticais ainda não estão definidas. Os principais objetivos deste projeto consistiram em: 1. Detectar e caracterizar os fatores peptídicos de crescimento e seus correspondentes receptores expressos em células adrenocorticais; 2. Investigar a regulação da expressão de fatores peptídicos de crescimento sob o estímulo de ACTH; 3. Definir os mecanismos de ação para os fatores peptídicos de crescimento localmente expressos e sua contribuição na regulação do ciclo celular. Os modelos experimentais utilizados foram: a linhagem Y-1 de células tumorais de adrenocorticais de camundongo e culturas primárias de células adrenocorticais de rato. Os resultados obtidos foram: 1. Fatores peptídicos de crescimento e seus respectivos receptores, correspondentes a PDGF (A e B), FGF2 (isoformas de alto e de baixo peso molecular), IGF (1 e 2), TGFβ (1, 2 e 3), VEFG-A, TGFα e EGF, foram detectados por RT-PCR e confirmados por clonagem e sequenciamento; 2. mRNAs correspondentes às formas de \"splicing\" alternativo para FGF2, bem como as isoformas de baixo (LMW-FGF2) e de alto (HMW-FGF2) peso molecular para a proteína FGF2 foram detectados em células Y-1. Os níveis basais de LMW-FGF2 foram aumentados sob estímulo de ACTH; 3. Com relação à distribuição subcelular em células adrenocorticais, LMW-FGF2 endógeno (produzido sob estímulo de ACTH) foi encontrado predominantemente no citoplasma, enquanto LMW-FGF2 exógeno (recombinante) foi internalizado para o núcleo de maneira dose e tempo-dependente; 4. Receptores para FGF foram detectados e caracterizados em células adrenocorticais. As isoformas detectadas, correspondentes a FGFRlb, FGFR2c e FGFR3b, foram reguladas sob tratamentos de ACTH e de FGF2. A expressão de FGFRs foi silenciada utilizando-se RNAs de interferência (RNAi), permitindo investigar a contribuição da sinalização promovida por FGF e seus receptores no ciclo celular. / ACTH and locally produced peptide growth factors regulate the proliferation in adrenocortical cells. Our previous results of biological assays, heparin-sepharose chromatography and Western blot indicated that PDGF and FGF-like factors are synthesized in Y-1 adrenocortical cell line. But the interaction among the ACTH stimulation and locally produced peptide growth factors, and their contribution for the cell cycle control, remains to be investigated. The main objectives of these project were: 1. To characterize peptide growth factors and their respective receptors expressed in adrenocortical cells; 2. To investigate the regulation of the expression of peptide growth factors under ACTH stimulation; 3. To find the mechanism of action of the locally produced peptide growth factors and to determine their contribution in the cell cycle control. The experimental models utilized were the Y-1 mouse adrenocortical cell line and primary cultures from rat adrenocortex. The results found were: 1. Several peptide growth factors and their respective receptors, corresponding to PDGF (A and B), FGF2 isoforms, IGF (1 and 2), TGFβ (1 , 2 and 3), VEGF-A, TGFα and EGF were detected by RT-PCR and confirmed by cloning and sequencing; 2. Two mRNAs splicing variants, as well the low (LMW-FGF2) and the high (HMW-FGF2) molecular weigh isoforms for FGF2 were detected in adrenocortical cells. The very low basal level of the endogenous LMW-FGF2 in untreated cells was up regulated after the ACTH stimulation; 3. In regard to the intracellular distribution of FGF2 in adrenocortical cells, the endogenous (ACTH induced) LMW-FGF2 was found predominantly in he cytoplasm, but the exogenous (recombinant) LMW-FGF2 was internalized into the nucleus in a time and dose dependent manner; 4. FGFRs corresponding to FGFRlb, FGFR2c and FGFR3b isoforms were detected and characterized in adrenocortical cells. All of them were up regulated under ACTH and FGF2 treatments. The expression of FGFRs could be silenced by RNAi approach, allowing to investigate the contribution of the FGF signaling for the cell cycle control.

ACTH (hormônio adrenocorticotrópico) e fatores peptídicos de crescimento regulam a proliferação celular em células adrenocorticais. Resultados preliminares de ensaios biológicos, cromatografia de heparina-sefarose e \"Westem blotting\" indicaram a produção de fatores peptídicos de crescimento das famílias de PDGF e de FGF na linhagem celular adrenocortical Y-1. No entanto a relação entre o estímulo promovido por ACTH e a expressão de fatores peptídicos de crescimento e sua contribuição para o controle do ciclo celular em células adrenocorticais ainda não estão definidas. Os principais objetivos deste projeto consistiram em: 1. Detectar e caracterizar os fatores peptídicos de crescimento e seus correspondentes receptores expressos em células adrenocorticais; 2. Investigar a regulação da expressão de fatores peptídicos de crescimento sob o estímulo de ACTH; 3. Definir os mecanismos de ação para os fatores peptídicos de crescimento localmente expressos e sua contribuição na regulação do ciclo celular. Os modelos experimentais utilizados foram: a linhagem Y-1 de células tumorais de adrenocorticais de camundongo e culturas primárias de células adrenocorticais de rato. Os resultados obtidos foram: 1. Fatores peptídicos de crescimento e seus respectivos receptores, correspondentes a PDGF (A e B), FGF2 (isoformas de alto e de baixo peso molecular), IGF (1 e 2), TGFβ (1, 2 e 3), VEFG-A, TGFα e EGF, foram detectados por RT-PCR e confirmados por clonagem e sequenciamento; 2. mRNAs correspondentes às formas de \"splicing\" alternativo para FGF2, bem como as isoformas de baixo (LMW-FGF2) e de alto (HMW-FGF2) peso molecular para a proteína FGF2 foram detectados em células Y-1. Os níveis basais de LMW-FGF2 foram aumentados sob estímulo de ACTH; 3. Com relação à distribuição subcelular em células adrenocorticais, LMW-FGF2 endógeno (produzido sob estímulo de ACTH) foi encontrado predominantemente no citoplasma, enquanto LMW-FGF2 exógeno (recombinante) foi internalizado para o núcleo de maneira dose e tempo-dependente; 4. Receptores para FGF foram detectados e caracterizados em células adrenocorticais. As isoformas detectadas, correspondentes a FGFRlb, FGFR2c e FGFR3b, foram reguladas sob tratamentos de ACTH e de FGF2. A expressão de FGFRs foi silenciada utilizando-se RNAs de interferência (RNAi), permitindo investigar a contribuição da sinalização promovida por FGF e seus receptores no ciclo celular. / ACTH and locally produced peptide growth factors regulate the proliferation in adrenocortical cells. Our previous results of biological assays, heparin-sepharose chromatography and Western blot indicated that PDGF and FGF-like factors are synthesized in Y-1 adrenocortical cell line. But the interaction among the ACTH stimulation and locally produced peptide growth factors, and their contribution for the cell cycle control, remains to be investigated. The main objectives of these project were: 1. To characterize peptide growth factors and their respective receptors expressed in adrenocortical cells; 2. To investigate the regulation of the expression of peptide growth factors under ACTH stimulation; 3. To find the mechanism of action of the locally produced peptide growth factors and to determine their contribution in the cell cycle control. The experimental models utilized were the Y-1 mouse adrenocortical cell line and primary cultures from rat adrenocortex. The results found were: 1. Several peptide growth factors and their respective receptors, corresponding to PDGF (A and B), FGF2 isoforms, IGF (1 and 2), TGFβ (1 , 2 and 3), VEGF-A, TGFα and EGF were detected by RT-PCR and confirmed by cloning and sequencing; 2. Two mRNAs splicing variants, as well the low (LMW-FGF2) and the high (HMW-FGF2) molecular weigh isoforms for FGF2 were detected in adrenocortical cells. The very low basal level of the endogenous LMW-FGF2 in untreated cells was up regulated after the ACTH stimulation; 3. In regard to the intracellular distribution of FGF2 in adrenocortical cells, the endogenous (ACTH induced) LMW-FGF2 was found predominantly in he cytoplasm, but the exogenous (recombinant) LMW-FGF2 was internalized into the nucleus in a time and dose dependent manner; 4. FGFRs corresponding to FGFRlb, FGFR2c and FGFR3b isoforms were detected and characterized in adrenocortical cells. All of them were up regulated under ACTH and FGF2 treatments. The expression of FGFRs could be silenced by RNAi approach, allowing to investigate the contribution of the FGF signaling for the cell cycle control.

Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T15:15:29Z (GMT). No. of bitstreams: 1 Souza_EdmarciaElisa_D.pdf: 15682912 bytes, checksum: e58bfe67bbf5f3bc0979ec28db650292 (MD5) Previous issue date: 2014 / Resumo: As proteínas Neks (NIMA-related kinases) representam uma família de 11 quinases humanas nomeadas Nek1 a 11 que compartilham 40 a 45% de identidade de sequência com o regulador mitótico NIMA identificado em Aspergillus nidulans. O sinergismo entre os mecanismos que dirigem a mitose é essencial para a adequada divisão celular e sua desregulação é correlacionada ao aparecimento de cânceres humanos. As Neks são essenciais para progressão do ciclo celular e por isso têm recebido especial atenção como alvos para terapia do câncer. A Nek7 humana, por sua vez, contribui para formação do fuso mitótico e biogênese dos centrossomos. Neste trabalho, nós revelamos a Nek7 como uma quinase multifuncional. Nossos estudos demonstraram um amplo espectro de proteínas de interações com a Nek7 humana, classificadas dentro de múltiplas categorias funcionais, sobretudo, da divisão celular. Alguns novos parceiros de interação também são seus potencias substratos e, ainda, localizam com a Nek7 em estruturas essenciais para a mitose e citocinese. Nós evidenciamos ainda, que através de mecanismos distintos, os domínios N- e C-terminal de Nek6 e Nek7 podem contribuir diferencialmente para a regulação e catálise e podem proporcionar a base estrutural para a independência funcional dessas quinases na sinalização celular. Além disso, usando estudos baseados microscopia confocal e RNAi (RNA interference), nós mostramos que o interactor de Nek7, a proteína RGS2, é necessária para organização e orientação do fuso mitótico. Células em metáfase suprimidas de RGS2 apresentaram fenótipos tais como: prisão na mitose; defeitos na tensão dos cinetocóros e alinhamento dos cromossomos; desorganização do fuso mitótico; perturbação na redistribuição de proteínas do polo do fuso envolvidas em nucleação; mal-orientação do fuso mitótico; e redução de microtúbulos dos ásteres e de dinâmica. Além disso, tanto a supressão quanto a superexpressão das formas selvagem e quinase dead de Nek7 prejudicaram o recutamento de ?-tubulina para o polo do fuso. Esses achados introduz a participação de RGS2 na mitose e indicam que esta pode atuar cooperativamente com Nek7 para a precisa organização e formação do fuso mitótico. Por fim, empregando biologia de sistemas, nós mostramos um compreensivo interactoma das Neks, destacando para um possível crosstalking de todos os membros da família nos processos de regulação de centríolos e mitose; função ciliar e ciliopatias; e resposta a dano de DNA / Abstract: The Neks (NIMA-related kinases) proteins represent a human kinases family named Nek1 to 11 that share 40 to 45% of sequence identity with the established NIMA mitotic regulator, identified in Aspergillus nidulans. The synergism of the mechanisms that drive mitosis is essential for proper cell division and its dysregulation is correlated with the occurrence of human cancers. The Neks are essential for cell cycle progression and therefore have received attention as targets for cancer therapy and other diseases. Human Nek7 contributes to mitotic spindle formation and centrosome biogenesis. Herein, we reveal Nek7 as a multifunctional kinase. Our proteomic studies have demonstrated a broad spectrum of interaction proteins of human Nek7 classified into multiple functional categories, especially, cell division. Some new interaction partners are also potential Nek7 substrates and localize in key structure during mitosis and cytokinesis. We also evidenced that, through different mechanisms, the N- and C- terminal domains of Nek7 and Nek6 can differentially contribute to the regulation and catalysis and provide the basis for a functional independence of Nek6 and Nek7 in cell signaling. Furthermore, using studies based in confocal microscopy and RNAi we showed the Nek7 interactor, RGS2 protein, is required for organization and orientation of the mitotic spindle. Metaphase cells RGS2-depleted showed phenotypes such as: arrest in mitosis; defects in tension kinetochores and alignment chromosomes; disruption of the mitotic spindle; disturbance in the proteins redistribution of the spindle pole involved in nucleation and microtubule dynamics; mitotic spindle misorientation; and astral microtubules reduction. Furthermore, the suppression or both overexpression of Nek7 wild type or kinase dead impaired the recruitment of ?-tubulin to the spindle pole. These findings introduce the involvement of RGS2 in mitosis and indicate that it may act cooperatively with Nek7 for proper mitotic spindle organization and formation. Finally, employing systems biology, we showed a comprehensive Neks interactome, highlighting for a possible crosstalking of all family members in centrioles and mitosis regulation; ciliary and ciliopathies function; and response to DNA damage / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Background: Different types of cardiomyopathies are associated with variable hypertrophic response. <p>A number of growth factors are thought to play a role in pathologic cardiac remodeling. <p>Aims: We compared the modulation of the TGF-ƒÒ superfamily and IGF-1 signaling pathways and their target genes, the cell cycle regulatory proteins in tachycardia-induced dilated cardiomyopathy, a model with no detectable hypertrophy and in ischemic cardiomyopathy, a model with a marked hypertrophic reaction. <p>Methods: In the first study, endomyocardial biopsies were obtained weekly in 15 dogs, during the development of tachycardiomyopaty. Genes involved in the myostatin-TGF-ƒÒ-Activin-A/Smad signaling pathway, p21 and cyclin D were quantified and correlated to echocardiographic measures of hypertrophy. In the second study, myocardial tissue samples were obtained in 8 dogs with a healed myocardial infarction, in 8 dogs with heart failure induced by overpacing and in 7 healthy dogs. We measured gene expression of IGF-1, its receptor (IGF-1R) and cyclins A, B, D1, D2, D3 and E and correlated them to the level of hypertrophy. <p>Results: Tachycardiomyopathy was characterized by chambers dilation with no identifiable hypertrophy. Ischemic cardiomyopathy was characterized by eccentric hypertrophy. In tachycardiomyopathy, Activin-A mRNA was 4-fold higher than at baseline. Smad7 was overexpressed in severe heart failure; p21, a direct target gene of the Smad pathway was upregulated 8-fold and cyclin D1 was down-regulated. In that model, IGF-1 was overexpressed but neither IGF-1R nor any of the cyclins studied.<p> In ischemic cardiomyopathy, IGF-1, IGF-R, and cyclins B, D1, D3 and E gene expression were upregulated.<p> In tachycardiomyopathy, Activin-A and p21 were inversely correlated to the thickness of the interventricular septum. In normal dogs and in the both models of cardiomyopathy, IGF-1R was correlated to the thickness of the interventricular septum and to cyclins. <p>Conclusions: Taken together, these results agree with the notion that Activin-A, IGF and cyclins are involved in the modulation of hypertrophic response observed in cardiomyopathies. <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / The MDM2-p53 feedback loop is a well-characterized pathway. p53 is a transcription factor and regulates the transcriptional expression of genes that encode proteins responsible for cellular senescence, cell cycle arrest, apoptosis, and DNA repair. Various cellular stresses can result in p53 activation, including hypoxia, DNA damage by agents such as UV or IR, oncogenic signaling, nucleotide depletion and nucleolar stress from perturbation of ribosomal biogenesis. Under normal conditions, MDM2’s role in the pathway is to inhibit p53 function by directly binding to this protein and facilitating its ubiquitylation and 26S proteasome-mediated degradation. Under stressful cellular conditions, certain proteins interact with and rescue MDM2’s inhibition of p53. For example, upon exposure to small amounts of Actinomycin D, rRNA transcript synthesis is stalled resulting in the release of various ribosomal proteins including RPL5, RPL11 and RPL23; each of which has been shown to bind MDM2 within its central acidic domain and inhibit its ability to destabilize p53. Although the RPL5 binding region of MDM2 have been mapped in prior investigations, the MDM2-binding region(s) of RPL5 have yet to be characterized. By employing RPL5 deletion mutagenesis and in vitro GST-fusion protein-protein association assays with purified proteins, this dissertation attempts to elucidate those regions of RPL5 that may interact with MDM2. Normalizing RPL5-WT to 1.00, our study reveals that the basic N and C-terminals of RPL5 appear to bind with MDM2 while RPL5’s central region displays negligible binding to the central acidic domain of MDM2. Also, the possible meanings of these RPL5 MDM2 binding domains are discussed along with their utilization in potential future applications.

No description available.

Confidentiel / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished