Дисертації з теми "Nuclear role"

The view on how protein kinases regulate gene expression have recently expanded to include not only transcription factors but also histones, chromatin remodelers or components of the basal transcription machinery, which are directly modified on genomic loci. For the shuttling kinase DYRK1A (dual-specificity tyrosine-regulated kinase), most of its nuclear-associated functions can be explained by DYRK1A cytosolic activities, questioning a role for DYRK1A within the nuclear compartment. In the present study, through an unbiased proteomic approach the first “DYRK1A nuclear interactome” have been generated. DYRK1A interacts with several components of the basal transcriptional machinery as well as with the pre-mRNA processing machinery. Moreover, evidences uncovering a new role for DYRK1A as a transcriptional regulator of specific target genes have been generated. Genome-wide DYRK1A-chromatin analysis shows that the kinase is recruited to RNA polymerase II proximal promoters, via a highly conserved palindromic sequence, and also to RNA polymerase III-dependent promoters. Growth-dependent induction of the expression of a subset of target genes (protein coding and tRNAs) depends on DYRK1A protein levels and/or activity. In addition, downregulation of DYRK1A leads to a reduction in cell size. DYRK1A could therefore work by sitting on promoters of specific genes and act on different components of the basal transcription and/or mRNA processing machinery to modulate gene expression.
Resultados recientes han puesto de manifiesto que la regulación de la expresión génica por proteína quinasas va mas allá de su modulación de la actividad de factores de transcripción, ya que tanto histonas como remodeladores de cromatina o componentes de la maquinaria basal de transcripción puedes ser sustratos de fosforilaciones directamente en regiones genómicas reguladoras. La proteína quinasa DYRK1A (dual-specificity tyrosine-regulated kinase) está presente tanto en el núcleo como en el citoplasma de células de mamífero, si bien la mayoría de sus actividades nucleares pueden ser explicadas por fosforilaciones que ocurren en el citosol, lo que ha planteado dudas sobre si esta quinasa posee funciones específicamente nucleares. En este trabajo, se ha definido el primer "interactoma" nuclear de DYRK1A mediante una aproximación proteómica no sesgada, que ha permitido mostrar que DYRK1A interacciona con componentes de la maquinaria basal de transcripción así como con complejos implicados en el procesamiento del pre-mRNA. Los resultados también han puesto de manifiesto un nuevo papel de DYRK1A como regulador de la transcripción de un grupo específico de genes relacionados con la traducción de proteínas. Análisis a nivel genómico de la presencia de DYRK1A en cromatina muestra que la quinasa es reclutada a regiones proximales de promotores dependientes de la RNA polimerasa II, mediante una secuencia palindrómica altamente conservada, así como a genes dependientes de la RNA polimerasa III. La inducción de la expresión de un grupo de estos genes diana (tanto codificantes como tRNAs) en respuesta a factores de crecimiento depende de DYRK1A. Además, la reducción en los niveles de DYRK1A provoca una reducción en el tamaño de las células. Los resultados permiten proponer un modelo por el que DYRK1A podría regular directamente la expresión de genes diana mediante la fosforilación, en regiones reguladoras promotoras, de diferentes componentes de la maquinaria basal de la transcripción y/o de los complejos proteicos implicados en el procesamiento del mRNA.

The eukaryotic cell nucleus is characterized by a defined spatial organization of the chromatin, which relies on the physical tethering of many genomic loci to the inner surface of the nuclear envelope. This interaction is mainly mediated by lamins and lamin-associated proteins, which create a protein network at the nuclear periphery called nuclear lamina. Man1 is a member of a lamin-associated protein family known as LEM-domain proteins, which are characterized by the presence of a highly conserved domain, called LEM, that mediates the interaction with the chromatin. Data obtained with the yeast Man1 homolog Src1 underline the importance of this protein in different processes of the cell cycle, such as chromosome segregation, nuclear pores assembly, gene expression, chromatin organization and maintenance of genome stability, while in animal models, the function of Man1 has been associated to the regulation of developmental signalling pathways during embryogenesis. In this study, truncated recombinant mutants of Man1, containing the LEM domain, were shown to inhibit nuclear assembly and alter nuclear pore formation when added to Xenopus laevis cell-free extracts. Moreover, Xenopus nuclei assembled in the presence of Man1 truncated fragments were characterized by defects in chromatin organization, DNA replication and accumulation of DNA damage and, as a consequence, they failed to progress through mitosis. Furthermore, mouse embryonic stem cells (mESCs) depleted for Man1 showed evident signs of spontaneous differentiation, indicating inability in the maintenance of stem cell features. Intriguingly, preliminary analysis of Man1-knockout mESCs transcriptional profile showed an alteration of gene expression at the level of pericentromeric and telomeric regions, underlining a potential link between Man1 and genomic stability of these particular regions. In conclusion, this study illustrates the importance of Man1 in ensuring the proper chromatin organization necessary to support different cellular and DNA metabolic processes.

Ce travail de thèse s'articule autour de deux questions scientifiques importantes à propos des galaxies actives : quels sont les mécanismes transportant le gaz et quel est le role de la galaxie sur l'activité nucléaire ? Nous avons donc mené une étude observationnelle approfondie et statistique du gaz et des étoiles, pour comparer la morphologie et cinématique des galaxies actives et non-actives sur differentes échelles spatiales, en utilisant des données spectroscopiques optique et radio. Nos résultats montrent que dans les régions centrales des galaxies actives la cinématique des étoiles est régulière alors que le gaz est perturbé. Ces perturbations suggèrent un lien entre la dynamique au centre des galaxies et les mécanismes d'alimentation du noyau actif. Enfifin les données radio et optique sont combinées pour analyser la cinématique galactique dans son ensemble. Cette étude nous
permet de sonder à differentes échelles spatiales les perturbations liées à l'alimentation du noyau actif.

The differentiation of preadipocytes into adipocytes is one of the most intensely studied biological processes and has led to the understanding of a number of signaling pathways involved in cell growth and differentiation. The nuclear phosphatidyl inositol (pl) signalling pathway, centred around the enzyme phospholipase C (PLC) βI has been demonstrated to be involved in a number of cellular processes such as mitogenesis and differentiation. Recently components of another lipid signalling pathway, that involving prostaglandins, have also been shown to localise at or within the nucleus. To date, there have been no reports demonstrating the role of either of these nuclear pathways during the differentiation of adipocytes. The 3T3-L1 preadipocyte cell line was used to study the role of nuclear pI and prostaglandin lipid signalling in adipocyte differentiation. In Chapter 3 data is presented to show that two phases of nuclear PLC βl activity occur during adipocyte differentiation. The first phase occurs within minutes of differentiation being induced, is regulated by the actions of extracellular signal-related kinase (ERK) l/2 and protein kinase c (pKC) α, and is involved in the mitotic clonal expansion phase of differentiation. The second phase of activity occurs from day 2 of differentiation, is regulated independently of ERK 1/2 and pKC α and is involved in the regulation of the cell cycle factors cyclin D3 and cyclin-dependent kinase (cdk) 4. In Chapter 4 I investigate the nuclear localisation of prostaglandin synthases, how their localisation is altered by differentiation, and the role of these enzymes in adipocyte differentiation is characterized through inhibitor studies. Cytosolic phospholipase A2 (cPLA2) cyclooxygenase (COX) 1 and 2, microsomal prostaglandin E2 synthase (mPGES), prostacyclin synthase (PGIS), lipocalin prostaglandin D2 synthase (L-PGDS) are localised to the nuclear membrane and their localisation changes during differentiation. The COX 1 and2 enzymes also localise within the nucleus and this COX 1 localisation appears to be cell cycle dependent. COX 2 is functionally coupled to mPGES and PGIS and plays a role in adipocyte differentiation as inhibition of COX 2 with a specific inhibitor (NS 398) completely blocks adipocyte differentiation. COX 1 is functionally coupled with L-PGDS and appears to be involved in lipid metabolism as the inhibition of COX 1 with a specific inhinitor (SC 560) partially blocks lipogenesis. In Chapter 5, I investigate the localisation of prostaglandin receptors in the differentiation process. 3T3- L1 cells express the prostaglandin E2 (EP4) receptor, prostaglandin F2α (FP) receptor and the prostaglandin D2 (DP) receptor but these do not have a nuclear localisation. Activation of the DP receptor is involved in lipid metabolism as the DP receptor antagonist, BW A868C, partially inhibits lipogenesis. lnternalisation of prostaglandins appears to be necessary for differentiation to occur fully as treatment of differentiating 3T3-L1 preadipocytes with the anionic transport inhibitor bromocresol green (BCG) partially inhibits carbaprostacyclin (cPGI2) induced differentiation and completely inhibits that caused by cyclopentenone prostaglandins. Thus, nuclear lipid signalling pathways appear to play a role in the process of adipocyte differentiation and a proper understanding of these pathways may lead to a better understanding of fat cell development and the role it plays in obesity and its related diseases.
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Trafficking of macromolecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs) is an essential process in eukaryotic cells. For example, proteins containing a nuclear localisation sequence (NLS) are imported into the nucleus, whereas proteins containing a nuclear export sequence (NES) are exported into the cytoplasm. Many nucleoporins, the proteins from which NPCs are constructed, contain FxFG-repeats, sequences based on a hydrophobic core (of sequence PhexPheGly, where x is usually a small residue) and a hydrophilic linker. Previous work has shown that carrier molecules, including importin-β and Nuclear Transport Factor 2 (NTF2), bind to FxFG-repeats, and addition of soluble FxFG-repeats inhibits nuclear trafficking processes. This Thesis characterises the interactions between FxFG nucleoporin repeats and both NTF2 and importin-β. Binding studies on a rationally-designed NTF2 mutant localise the FxFG binding site to near Trp7. Although I show that the interaction between FxFG repeats and NTF2 is weak, I also show that the interaction is essential for NTF2 to mediate the nuclear import of Ran in permeabilised cells. An X-ray crystal structure of importin-β (residues 1-442) in complex with FxFG repeats is presented which shows, for the first time, how FxFG-repeats interact with a carrier molecule. The crystal structure is used to produce mutants of full-length importin-β which are deficient in FxFG repeat binding, and yet retain binding to RanGTP and importin-α. These mutants are used to demonstrate that an interaction with FxFG repeats is essential for importin-β to mediate nuclear protein import of a substrate containing a classical NLS. A model for how RanGTP may release importin-β from FxFG repeats is proposed.

To further investigate the role played byRrp6p in RNA metabolism, my work aimed at identifying and characterising potential Rrp6p-associated complexes. Density gradient analyses revealed no pool of free Rrp6p, but the majority of Rrp6p was apparently associated with complexes other than exosome. In addition to the exosome, Rrp6p appeared to be associated with a 10S complex and two RNase A sensitive complexes that may correspond to pre-40S and pre-60S ribosomes. Proteomic analyses identified Gbp2p and Srp1p as potential components of the 10S complex. However, genetic analyses did not reveal defects in stable RNA synthesis in gbp2 or srp1 mutant strains. Microarray analysis identified a small number of mRNAs that were upregulated in the gbp2D strain. Purification of exosome complexes identified the nuclear protein Rrp47p as a substoichiometric component. Rrp47p was shown to be required for most Rrp6p functions in stable RNA synthesis but not for Rrp6p function in RNA surveillance. To better understand this similarity, I analysed the Rrp47p-exosome and the Rrp47p-Rrp6p interactions. Around 10% of the exosome was found to be associated with both Rrp6p and Rrp47p. However Rrp47p did not appear to be part of the 10S Rrp6p complex, suggesting that the surveillance functions of Rrp6p may be performed in this complex. These results have provided new insights into the function of Rrp6p in nuclear pre-mRNA turnover and into its potential association with complexes other than exosome.

Most bulge-dominated galaxies host a supermassive black hole and yet ongoing nuclear activity is observed in only ~5% of nearby galaxies. Reignition of dormant black holes is therefore required and a key unanswered question is whether the ignition mechanism is related to the galaxy host properties, in particular the fuel transportation mechanisms. The nuclear activity in distant and luminous quasars is related to galaxy interactions and the high-luminosity active galactic nuclei (AGN) are found in giant elliptical galaxies associated with past mergers of lower mass galaxies. In contrast, the low-luminosity AGN, such as the Seyfert galaxies, are not found preferentially in interacting systems or in barred galaxies and fuelling these low-luminosity AGN remains an unsolved problem. Recent results suggest the presence of identifiable dynamical differences between Seyfert and intive galaxies in the central kpc regions. Probing the dynamics of Seyfert galaxies requires spectroscopic data and integral-field spectroscopy (IFS) is particularly well-suited to the investigation of complex structures of nearby galaxies. A comprehensive and statistically-significant study of the neutral gas, ionised gas and stellar kinematics of a well-defined distance-limited sample of Seyfert galaxies paired with control inactive galaxies with carefully matched optical properties was then initiated, using the VLA and the AURON integral field unit (IFU).

Thesis (Ph. D.)--Michigan State University. Dept. of Microbiology and Molecular Genetics, 2006.
Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.

Les facteurs de transcription de la superfamille des récepteurs nucléaires jouent de multiples rôles dans le développement et la fonction des lymphocytes T régulateurs (TREG). Les TREG sont des cellules régulatrices/suppressives qui contrôlent les réponses d’autres types cellulaires et l’homéostasie locale des tissus.Comme les TREG sont actives au sein de divers organes, tant à l’homéostasie qu’en conditions inflammatoires,ils doivent répondre à la fois aux contexte local au sein du tissus et à un environnement immunologiquement agressif tout en préservant leurs propriétés tolérogéniques au cours du temps. Ces caractéristiques apparemment antinomiques sont contrôlées par un réseau transcriptionnel complexe au sein duquel le facteur de transcription FOXP3 joue un rôle prédominant. Au cours des dernières années, de nombreuses études se sont intéressées aux TREG présent dans les tissus non lymphoïdes (NLT). Ces populations ont été étudiées aussi bien à l’homéostasie qu’en conditions inflammatoires dans diverses pathologies. Des facteurs de transcriptions spécifiques d’un tissus ou d’une fonction déterminées ont été mis en évidence et leur rôle régulateur dans le développement, l’activation, la migration et l’immunosuppression a été caractérisé. RORa est un récepteur nucléaire qui contrôle le développement cérebellaire et hépatique, le métabolisme systémique, la différenciation des lymphocytes auxiliaires TH17, des cellules lymphoïdes innées (ILC) de type 2 et 3. RORa est fortement exprimé dans les TREG des NLT, y compris dans le tissus adipeux viscéral (VAT), l’intestin et la peau. . . .Ces populations de TREG exprimant RORa ont été associées à diverses pathologies. Cependant seule une étude récente a été consacrée à leur rôle précis. L’implication de RORa dans de nombreuses fonction, sa forte expression au sein des TREG des NLT nous a poussé a étudier le rôle de ces TREG exprimant RORa dans diverses pathologies. Dans ce butit, nous avons généré des souris spécifiquement déficientes pour RORa au sein des TREG (RORaFoxp3/Foxp3 ). Nous avons émis l’hypothèse que RORa contrôle le développement ou la fonction des TREG en conditions homéostatiques et dans des pathologies inflammatoires des NLT. Aussi nous avons caractérisé le phénotype des animaux RORaFoxp3/Foxp3 et en particulier les TREG du VAT à l’homéostasie, où la réponse de type 2 est protectrice et dans un modèle d’obésité (et d’insulino-résistance) induit par l’obésité (DIO) dans laquelle nous avons mis en évidence un rôle protecteur important des TREG exprimant RORa dans ces deux conditions expérimentales. Nous également étudié la contribution de ces cellules dans un modèle d’inflammation allergique (AAI) induite par un acarien (HDM) caractérisé par une forte réponse de type 2 et montré une aggravation de la pathologie. Pour étudier le mécanisme moléculaire de l’action de RORa au sein des TREG, nous avons procédé à une analyse transcriptomique des cellules isolées dans diverses conditions expérimentales in vivo et in vitro et avons étudié le rôle de RORa dans les modifications épigénétiques au sein des TREG en caractérisation l’acétylation des histones dans le génome entier. Cette étude nous a permis de mieux appréhender comment les TREG étaient régulées par un facteur nucléaire à l’homéostasie et en conditions inflammatoires. Les récepteurs nucléaires représentent des cibles thérapeutiques intéressantes compte tenu de leur action pléiotropique et de leurs ligands de petite taille. Compte tenu de l’importance des TREG dans l’homéostasie tissulaire et dans de nombreuses pathologies, cibler de tels facteurs au sein d epopulations cellulaires spécifiques représente une stratégie prometteuse dans le case de RORa et des TREG
Transcription factors of the nuclear receptor superfamily have a vast influence on development and function ofregulatory T cell (TREG) cells. TREG cells are suppressive immune cells of adaptive immune system. Their mainfunctions are control of inflammatory response mounted by other immune cells and maintenance of localtissue homeostasis. As TREG act at various sites of the body and both in homeostatic and inflammatory state,they need to adequately respond to local tissue-specific cues as well as adapt to aggressive immuneenvironments while preserving their long-lasting tolerogenic properties. This is achieved by weaving complextranscriptional networks, converging at transcription factors with various coordination functions, the mainbeing forkhead box P3 (FOXP3). During last few years, many studies focused on TREG cells found innon-lymphoid tissue (NLT). These populations of TREG are examined in the contexts of homeostasis and manyinflammatory diseases, and tissue- or function-specific transcription factor (TF) were assigned to some ofthem as regulators of development, activation, proliferation, stability, migration and suppressive functions.Retinoic acid receptor-related orphan receptor alpha (RORa) is a nuclear receptor, which controls cerebellumdevelopment, liver and whole-body metabolism and differentiation of T-helper (TH)17, type 2 innate lymphoidcells (ILC2) and type 3 innate lymphoid cells (ILC3). RORa is highly expressed in NLT TREG, includingpopulations in visceral adipose tissue (VAT), intestine and skin, and gets more and more mentions in thearticles dedicated to TREG in NLT. These RORa-expressing populations of TREG were all shown to be involvedin various pathologies. However, RORa role in TREG was directly addressed only once in a recent study. It’sactive involvement in various processes, high expression in NLT TREG and lack of knowledge make RORa anattractive target for investigation, to deepen current view of homeostasis control by TREG and thus betterunderstand mechanisms of development of associated diseases. To attain these objectives, a mouse strain withTREG-specific RORa deficiency was generated. Our central hypothesis is that RORa controls development orfunction of TREG cells in homeostasis of NLT and potentially in inflammatory diseases. For studying a role ofRORa in NLT TREG during control of tissue homeostasis, in particular, VAT TREG, we have charachterizedphenotype of untreated RORaFoxp3/Foxp3 mice and challenged mice with a model of diet-induced obesity(DIO). In both cases we have found an important role of TREG-expressed RORa. To further investigate a roleof RORa in TREG during pathologies and it’s contribution to various types of immune response we have testedan involvement of RORa in TREG in the model of allergic pathology, namely house dust mite (HDM)-inducedallergic airway inflammation (AAI) model.To elucidate molecular mechanisms of RORa action in TREG cells, we have performed gene expression profilingof TREG cells from examined tissues and conditions in vivo, as well as in vitro. We also have studied a role ofRORa in epigenetic landscape of TREG cells in vitro by probing histone acetylation marks genome wide. As aresult of this study, we have gained a broader understanding of TREG control by nuclear receptors and TF ingeneral in homeostatic conditions and during inflammation. Nuclear receptors proved to be useful targets fortherapeutic agents thanks to their versatile functions inside the cell and to ligand-dependency. Given thecrucial importance of TREG cells in organismal homeostasis and their involvement in numerous pathologies,targeting particular cues inside these cells may be a powerful tool in new treatment strategies. Results of ourstudy might serve as a basis for development of novel pharmaceutical agents targeting RORa

The nucleus of mammalian cells has been proven to be highly organised. A recent study on interphase chromosome positioning has identified low serum induced rapid chromosome repositioning. Chromosome 10 initially localised at an intermediate position in normal proliferating human dermal fibroblasts (HDF) was found to relocate to the nuclear periphery 15 minutes after the cells have been incubated in low serum. Whereas chromosome X has remained in a peripheral position. The relocation of chromosome 10 has been shown to be dependant on both actin and myosin functions. In this project we have further investigated the possible role of nuclear myosin I in chromosome 10 repositioning. Using siRNA to block the expression of the nuclear myosin I (NMI) we were able to identify this nuclear myosin as necessary for the rapid repositioning of chromosome 10. Furthermore, using image analysis software we investigated the effect of the NMI knock down on the overall nuclear size and shape. The analysis has revealed that while the nuclear size of normal proliferating cells remained unchanged after the low serum incubation both in cells expressing the NMI and NMI depleted cells, the knock down of the NMI seems to have affected the nuclear shape when the cells were subjected to the serum incubation. On the other hand, the analysis of the chromosome territories area has revealed significant differences in the chromosome territories sizes before and after the low serum incubation, in normal proliferating HDF cells .

A high resolution NMR probe was modified with gradient coils (31 mm diameter) for the measurement of translational diffusion and for microscopic imaging, and a larger set of gradient coils (15 cm diameter) was constructed for surface coil diffusion measurements. The magnitudes of the gradients produced by these coils were determined from the linewidths and lineshapes of gradient spectra. In diffusion experiments using the pulsed gradient method of Stejskal and Tanner, induced eddy currents and slow variation of the magnetic field at the sample interfered with measurements at short echo times. For these experiments the known diffusion coefficient of water was used to determine the effective gradient in each experiment. The diffusion coefficient of acrylonitrile was measured from the decay of single, double and triple quantum echoes using a modified pulsed field gradient spin echo pulse sequence. In the second part of this thesis, three examples of living systems were studied. The first involved the application of pulsed gradient spin echo measurements to characterize the motion of water and lipid, in-vivo, in human forearm. Spin echo spectra from human forearm gave a water signal that was attributed to extracellular water because of relatively long spin-spin relaxation time (0.8 s) compared to that of intracellular water (20-30 ms). Comparison of the diffusivity of water, from experiments at two different echo times suggest that the major part of the motion of water, in-vivo, was due to directionally randomized bulk flow rather than molecular diffusion. The second application involved the chemical shift resolved mapping of the proton distribution, in one-dimension, along the anteroposterior direction, of pupae of the Douglas-fir cone moth Barbara colfaxiana. Proton distribution maps showed that the distribution of the aqueous fluid depended upon the vertical orientation, head pointing upward or downward, of the pupae. Finally, two dimensional images of mature caps of the marine alga Acetabularia mediterranea were obtained using the normal spin echo sequence as well as with T₁, T₂ and diffusion contrasting. D₂O—contrasting was obtained by briefly submerging the caps in D₂O. All of these images showed features resembling the radial structure of the caps. The resolution was estimated by comparison with microscopic views of the caps and was found to be 0.1 mm, determined as the smallest distinguishable feature in the image.
Science, Faculty of
Chemistry, Department of
Graduate

Transcriptional factor NF-κB family has been demonstrated to play an important role in tumor pathogenesis and serve as a potential target in cancer therapy. However, it is necessary to clarify the specific functions of NF-κB members, which would provide the basis for the selective blockade and reduction of therapeutic side effects resulting from unspecific inhibition of NF-κB members. We firstly explored the role of NF-κB p105/p50 in melanoma pathogenesis in vivo. We found that the expression of NF-κB p105/p50 significantly increased in dysplastic nevi, primary melanoma and metastatic melanoma compared to normal nevi (p=0.0004, χ²test). NF-κB p105/p50 nuclear staining increased with melanoma progression and strong NF-κB p105/p50 nuclear staining was inversely correlated with disease-specific 5- year survival of patients with tumor thickness >2.0 mm (p=0.014, log-rank test). Multivariate Cox regression analysis revealed that nuclear expression of NF-κB p105/p50 is an independent prognostic factor in this subgroup. Moreover, we found that upregulation of NF-κB p50 enhanced melanoma cell migration whereas siRNA knockdown inhibited cell migration.. In addition, overexpression of NF-κB p50 induced RhoA activity and Rock-mediated formation of stress fiber in melanoma cells. To further elucidate how NF-κB p50 is involved in melanoma, we analyzed the gene expression profile in melanoma cells overexpressing NF-κB p50 by cDNA microarray. We found that IL-6 mRNA was highly induced by NF-κB p50. Since IL-6 is a newly reported proangiogenic factor in cancer, we hypothesized that NF-κB p50 could promote melanoma angiogenesis by upregulating IL-6. Indeed, using the proliferation assay of human umbilical vein endothelial cells (HUVECs) we demonstrated that the conditioned medium from melanoma cells overexpressing NF-κB p50 promoted HUVEC proliferation in vitro. In vivo matrigel plug assay showed that tumor vascularity was significantly enhanced by NF-κB p50. Furthermore, silence of NF-κB p105/p50 decreased IL-6 expression and overexpression of ATF3 abrogated the effect of NF-κB p50 on the induction of IL-6. Taken together, our data indicate that NF-κB p105/p50 may be an important marker for human melanoma progression and prognosis as well as a potentially selective therapeutic target.
Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate

Proper embryonic development requires precise genetic regulation of cell growth and differentiation. Organogenesis, the origin and formation of internal organs, must be exquisitely choreographed to ensure correct temporal and spatial patterning of functional organs within the developing organism. The liver is a vital organ responsible for hundreds of essential metabolic functions, but the intricate pathways controlling organ specification, differentiation, and positioning have not been fully elucidated. Uncovering the molecular mechanisms involved in hepatogenesis will enhance our understanding of normal liver development as well as inform the design of therapeutics to combat liver disease. Nuclear receptors are evolutionarily recent signal transducers that occupy a special niche in gene regulation, acting as direct connections between a ligand and its downstream transcriptional target. Nuclear receptor signaling governs many physiological processes, however its impact on liver development is not well understood.

Patients who are diagnosed with pancreatic ductal adenocarcinoma (PDAC) face a dismal prognosis. One reason for this is the dense stroma that is a characteristic of PDAC, which may preclude drugs from accessing the tumour cells. Pancreatic stellate cells (PSCs) are the key cell responsible for desmoplasia in PDAC and it is becoming clear that they are a promising target for therapy. Over-expression of FGFs and their receptors is a feature of PDAC and correlates with poor prognosis, but whether their expression impacts on PSCs is unclear. The aim of my research was 1) to understand the role and function of nuclear FGFR1 and FGF using 2D based assays; 2) to use a physiologically relevant 3D organotypic model to study the effects of blocking nuclear FGFR1 and FGF2 in PSCs; 3) to assess whether this target could provide a novel therapeutic strategy in PDAC. At the invasive front of human pancreatic cancer, FGF2 and FGFR1 localised to the nucleus in activated PSCs but not cancer cells. Inhibiting FGFR1 and FGF2 in PSCs, using RNAi or chemical inhibition in vitro, resulted in significantly reduced cell proliferation, which was not seen in cancer cells. Cancer cells co-cultured on top of collagen/Matrigel gels together with PSCs showed marked invasion of both cancer cells and PSCs. However, FGFR inhibition blocked invasion of both PSCs and cancer cells. FGFR inhibition resulted in cytoplasmic localisation of FGFR1 and FGF2, in contrast to vehicle-treated conditions where PSCs with nuclear FGFR1 and FGF2 led cancer cells to invade the underlying extra-cellular matrix. Strikingly, abrogation of nuclear FGFR1 and FGF2 in PSCs abolished cancer cell invasion. These findings suggest a novel therapeutic approach, where preventing nuclear FGF/FGFR mediated proliferation and invasion in PSCs leads to disruption of the tumour microenvironment, preventing pancreatic cancer cell invasion. Thus, for 6 patients with PDAC which is resistant to conventional chemotherapy, targeting the stroma by blocking nuclear FGFR1 and FGF2 in PSCs identifies a novel therapeutic approach.

The lipid hydroxylase Clk-1 catalyses an essential step in the mitochondria localised ubiquinone biosynthetic pathway that is conserved throughout eukarya. Like many canonical mitochondrial proteins, murine Clk-1 is targeted and imported into mitochondria by virtue of a ~4kDa N-terminal mitochondrial targeting domain that is cleaved following translocation into the mitochondrial matrix. Clk-1 mutants in C. elegans and heterozygous Clk-1+/- mice exhibit increased longevity and delayed development rates compared to wild type individuals, with associated changes in oxidative stress signalling pathways. Previous work in the laboratory identified human Clk-1 as a potential interactor of Sin1, a component of the mammalian target of rapamycin signaling pathway, which has also been associated with modulations that affect lifespan. Here the interaction between Clk-1 and Sin1 is further characterised and Clk-1 is identified as a potential substrate of the Sin1-associated kinases cAMP dependent kinase, protein kinase C and unc-51-like kinase 1. Interestingly, a fraction of Clk-1 was observed residing in the nucleus, in addition to its mitochondrial localisation. The sequence determinants for Clk-1 nuclear localisation were found to be in the same N-terminal region required for mitochondrial localisation and a single point mutant was identified that translocated to the mitochondria but not the nucleus. Oxidative stress treatment was shown to increase the level of uncleaved Clk-1 and this form was enriched in nuclear and chromatin fractions. Clk-1 was found to be associated with over 1000 genomic loci following Clk-1 chromatin immunoprecipitation followed by promoter microarray analysis. Cells stably expressing the Clk-1 non-nuclear point mutant displayed decreased resistance to oxidative stress-induced cell death and increased levels of oxidative species following treatment with exogenous stress. In addition, c-Jun N-terminal kinase signaling was enhanced in these cells in response to tumour necrosis factor-α stimulation. Microarray analysis of these cells showed both positive and negative transcript changes compared to wild type Clk-1 expressing cells which was significant for over 2000 genes. Functional clustering analysis identified enrichment for gene groups associated with glycolytic and tricarboxylic acid cycle metabolism, Wnt signaling, and several specific differentiation and oncogenic pathways. Many of the genes identified are reported to be regulated by promoter methylation, and this was confirmed for glutathione-S-transferase P1 that had significantly decreased expression in mutant Clk-1 expressing cells. Loss of Clk-1 nuclearisation or Clk-1 activity within the nucleus could therefore be acting as part of specific differentiation pathways either during early development or following differentiation of specific cell lineages. Nuclear Clk-1’s ties to respiration and cell survival pathways, and sensitivity to oxidative stress, could also implicate it in oncogenic progression and/or the Clk-1 ageing phenotype.

Cellular senescence is a phenomenon characterised by stable and durable states of proliferative arrest induced by various stress stimuli. The abundance of senescent cells in tissues increases over a lifetime and, among other functions, they have been proposed to play a pivotal role in ageing. Hutchinson-Gilford progeria syndrome (HGPS) is a disease characterised by segmental premature ageing. It is caused by the expression of a persistently farnesylated lamin A isoform. Strikingly, HGPS patients show an increased abundance of senescent cells. Therefore the relationship between the farnesylated lamin A precursor, prelamin A, and senescence was studied in this thesis. Cellular models of ageing in human dermal fibroblasts were established. They relied on either replicative exhaustion or prelamin A accumulation. An unbiased genome-wide transcript analysis of the ageing models revealed that a significant part of the common senescence programme during cellular ageing can be replicated in young cells by accumulating prelamin A. The most prominent and overlapping expression changes were observed in pathways regulating inflammation, lipid metabolism and cholesterol homeostasis. Six genes, identified as underexpressed consistently across the studied ageing models, were tested functionally in the context of senescence regulation by prelamin A. The ability of prelamin A-accumulating cells to induce inflammation has also been demonstrated by multiplexed detection of secreted cytokines, chemokines and other factors. This confirms that a significant overlap between replicative and prelamin A-induced senescence exists not only at mRNA level changes, but is also observed at the level of secreted proteins. Finally, nuclear morphology was studied in the ageing models, with a particular interest in the formation of a nucleoplasmic reticulum. We identified NOGO/Reticulon-4 as a new protein involved in the process of NR formation, and also demonstrated that new NR channels require incorporation of newly synthesised lipid and protein components. The research presented in this thesis offers a new insight into a role of the prelamin A maturation process in senescence and ageing.

Polycystic kidney diseases (PKDs) are a group of disorders resulting in the formation of multiple renal cysts, and are characterised by renal interstitial inflammation, cell proliferation and apoptosis. The nuclear factor (NF)-κB system regulates the transcription of genes involved in inflammation, growth and apoptosis, but it is unknown whether NF-κB has a role in the pathophysiology of PKD. Hence, the aim of this thesis was to determine the endogenous expression and activity of NF-κB in PKD, and to begin to elucidate its role in this disease. Chapter One. Chapter One demonstrates that PKD is a complex disease involving several pathophysiological processes (particularly proliferation and inflammation) and multiple dysfunctional signalling pathways. The chapter discusses the current and emerging therapies and argues that new translational therapies should focus on targeting ‘master regulators’ that simultaneously control several pathways of PKD. The chapter then provides evidence supporting the case for NF-κB as one such key gene regulator that may be involved in PKD, and introduces the main hypothesis that NF-κB expression and activity are upregulated in PKD, and may mediate cyst expansion in this disease. Chapter Two. Chapter Two presents the methodology and preliminary results of two methods that were attempted but not achieved due to technical limitations. These include the 3D collagen cyst model, which aimed to provide information on the effect of NF-κB inhibition on cyst expansion, and the electrophoretic mobility shift assay (EMSA), which aimed to characterise the DNA binding activity of NF-κB proteins in renal tissue. Chapter Three. Chapter Three examined the endogenous renal expression of NF-κB proteins in Lewis Polycystic Kidney rats (LPK, a genetic orthologue of human nephronophthisis-9) compared to healthy Lewis controls from postnatal weeks 3 to 20. Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis. Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and were present until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBα protein levels were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human autosomal and recessive PKD (ADPKD and ARPKD). This study indicated that several NF-κB proteins are consistently expressed in CECs in human and experimental PKD, and may be a constitutive and early pathological feature of cystic renal diseases. Chapter Four. Chapter Four tested the hypothesis that an inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), reduces the progression of PKD in vivo. Chronic administration of PDTC to LPK rats from postnatal weeks 4 until 11 was associated with a slower increase in total kidney volume (TKV) compared to vehicle treatment. By serial magnetic resonance imaging (MRI) at weeks 5 and 10, the relative within-rat increase in TKV was 1.3-fold greater, while the increase in cyst volume was 1.4-fold greater, in LPK rats treated with vehicle compared to the higher-dose PDTC group. By week 11 in LPK rats, PDTC had attenuated kidney weight to body weight ratio by 25%, and decreased proteinuria by 66%, but did not improve renal dysfunction. PDTC did not alter histological interstitial inflammation and fibrosis, or renal cell proliferation in LPK rats. The phosphorylated form of the NF-κB p105 subunit was increased in CECs of LPK rats, but was not altered by PDTC. Moreover, PDTC did not significantly alter nuclear expression of the p50 subunit or NF-κB (p65)-DNA binding. In conclusion, PDTC reduced renal cystic enlargement and proteinuria without decreasing inflammation or NF-κB activity in LPK rats. Chapter Five. This chapter expanded upon the findings of Chapter Three by examining the mechanism by which PDTC reduces renal cyst growth, in vitro. Chapter Five tested the hypothesis that PDTC reduces the proliferation of immortalised ADPKD CECs in an NF-κB-dependent manner. Serum-induced proliferation was similar between normal human kidney cortical (HK-2) cells and ADPKD cells over 72 hours. PDTC decreased proliferation in both ADPKD and HK-2 cells, but these anti-proliferative effects were delayed in ADPKD cells compared to HK-2 cells. Basal NF-κB-dependent luciferase reporter activity was lower in ADPKD cells compared to HK-2 cells. Classical NF-κB stimulants, lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α, increased NF-κB luciferase activity in HK-2 cells, whereas in PKD cell lines, NF-κB activity was only induced by TNFα. However, neither stimulant altered proliferation in any cell line. PDTC reduced TNFα-stimulated NF-κB activity in HK-2 cells only. In conclusion, this study indicated that PDTC had anti-proliferative properties in ADPKD cells but did not consistently alter NF-κB activation, suggesting that other signalling pathways are likely to be involved in its ability to attenuate renal cyst growth in vivo. Chapter Six. Given the efficacy of the mammalian target of rapamycin complex 1 (TORC1) inhibitor, sirolimus, in slowing the rate of TKV growth in previous pre-clinical models of PKD, Chapter Six investigated whether sirolimus can also modulate NF-κB activity in PKD when administered at different stages of disease. Male LPK rats and Lewis controls were administered either vehicle or sirolimus, during early-stage (postnatal weeks 3 to 10) or late-stage disease (weeks 10 to 20). In early-stage disease, sirolimus completely prevented the increase in MRI-assessed TKV but only partially reduced the percentage cyst area (by 19%), and did not affect the decline in endogenous creatinine clearance (CrCl). Rats treated in late-stage disease only had a partial reduction in TKV, with no change in percentage cyst area and marginal improvement in CrCl. Sirolimus reduced TORC1 but did not alter the expression of markers of TORC2 activation or the expression of NF-κB-dependent genes (CCL2 and TNFα). Late but not early sirolimus treatment increased p65 NF-κB DNA binding activity in Lewis and LPK kidneys. Therefore, the time at which sirolimus was initiated, determined the drug’s efficacy in attenuating cystic growth. However, sirolimus did not alter cystic microarchitecture, significantly improve renal function, or decrease NF-κB expression and activity in LPK rats. These data suggest that additional approaches to normalise cellular dedifferentiation and inflammation are required to completely arrest the progression of PKD. Chapter Seven. Chapter Seven highlights the novel findings of this thesis, which were namely, the first-time localisation of several NF-κB proteins to the CECs in human and experimental PKD; demonstration of a decrease in TKV and cyst volume with PDTC in an animal model of PKD in vivo; and the identification of non-canonical NF-κB signalling in PKD. It puts forward the potential implications of this thesis, such as the capacity of dithiocarbamates to provide renoprotective effects in cystic renal disease. It also underlines the lack of demonstration of a functional relationship between NF-κB and cyst growth by this work, and proposes recommendations to experimentally investigate this concept in future in vitro studies (e.g. a 3D cyst model) as well as in vivo studies (particularly the use of NF-κB gene deletion in an orthologous model of ADPKD).

Breast cancer is a great source of morbidity and mortality in the developed world, being the most common cause of cancer death in Australian women and affecting roughly 1 in 10 women. The development and progression of cancers is a multi-stage process, involving numerous perturbations to normal cellular functions, especially to those genes which control cellular growth, cellular differentiation and DNA repair. Over time, these alterations combine to change normal cells into cancerous ones that typically no longer respond to normal control stimuli and grow with great rapidity. The nuclear receptors are a family of proteins which accept incoming signals from various molecules, and then alter gene expression or affect cell behavior directly. The steroid nuclear receptors receive transducer signals from hormones such as estrogen and testosterone and are intimately involved in affecting cellular growth and differentiation. Stimulation of the steroid receptors have been used as successful treatment avenues, so how these genes behave in cancer is of great interest. Accordingly, this study has examined the expression of various nuclear receptors and some related genes in a number of tissue samples derived from breast tumours and from surrounding areas, and examined how various pathological parameters affect their expression. Tissue samples were first microdissected to separate tumour tissue from the surrounding stroma and then subjected to RNA extraction procedures to allow gene expression to be quantitated. RNA was then converted into cDNA by reverse transcription and the genes of interest amplified and quantitated using semiquantitative polymerase chain reaction. Expression data was then normalized by expressing it as a ratio of the gene of interest to the 18S ribosomal gene and differences in expression analysed by analysis of variance (ANOVA) testing. The first portion of the study dealt with the progesterone and glucocorticoid receptors in tumour tissue. Progesterone is an anti-mitogenic signal for breast tissue and the stimulation of the receptor is a useful part of combined hormonal therapy for breast cancer. The glucocorticoid receptor plays a similar role to the progesterone receptor, slowing breast tissue growth and promoting apoptosis. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the glucocorticoid and progesterone receptor genes and was normalized using 18S. Analysis of the expression data by ANOVA showed that the progesterone and glucocorticoid receptors were more highly expressed in grade 3 tumour tissue (p=0.023 and p=O.00033, for the progesterone and glucocorticoid receptors, respectively). Most advanced tumours cease being responsive to growth repressing hormones, so these results indicated that the control of the expression of these receptors in tumour tissue was more complicated than might have been expected. The increase in expression observed may be the result of intact growth preventing feedback mechanisms which have malfunctioned in some manner. There are several mechanisms the tumours may be using to escape an increase in progesterone or glucocorticoid sensitivity. The mRNA detected may simply not be being translated into completed proteins or be being spliced into isoforms of the receptors that favour tissue growth. The second portion of the study dealt with the estrogen receptors alpha and beta in tumour tissue. The estrogen receptors control estrogen signaling and are highly important in breast cancer, since estrogen is the primary growth inducing hormone for breast tissue. For the estrogen receptors alpha and beta, the complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR and were normalized using 18S. ANOVA analysis found that the expression of the estrogen receptors did not change significantly in any grade of cancer (p= 0.057 and p=O.7'38, for ESRα and ESRβ, respectively) nor in tissues that are negative for the estrogen receptor alpha protein, a poor prognostic factor in breast cancer (p= 0.794 and p=O.7l6, for ESRα and ESRβ, respectively). These results indicated that the loss of estrogen receptor in advanced cancers is not controlled at the level of mRNA. The mRNA observed in this study may be being spliced into alternate and possibly inactive isoforms, or may be being degraded post transcriptionally, preventing estrogen stimulation in these tumours. This could prove an excellent area for further study, since if the mechanism that prevents or distorts ESR mRNA translation can be discovered, it would allow manipulation of one of the most important treatment avenues for breast cancer. The third portion of the study dealt with the expression of the androgen receptor in tumour tissue. Androgens are a strong anti-growth signal in breast tissue and despite the side effects that androgens have on women, they have been used to treat breast cancer with some success. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the androgen receptor gene and normalized using 18S. The results obtained for androgen receptor expression in tumour tissue showed that androgen receptor expression was significantly elevated in grade 2 and grade 3 tumour tissue, as well as in ESRα negative tumours (p= 0.014 and p=O.O25, respectively). An increase in expression in late stage tumours would seem to be unusual for an anti-mitogenic receptor, however many advanced breast tumours have been found to be receptive to androgen stimulation, even when they no longer respond to other hormones. The increased expression of AR may be a normal response to cellular over-growth, or it may be a mechanism by which the tumour prevents stimulation by other growth retarding hormones, by sequestering all available receptor co-enzymes with a receptor that is unlikely to be stimulated. The fourth portion of the study examined the expression of the estrogen alpha, estrogen beta, progesterone, glucocorticoid and androgen nuclear receptors in stromal tissue derived from the tumours studied in previous chapters. Tumours have been observed in other studies to manipulate the activities of the cells that surround them through the release of cofactors and vice versa. These cofactors include the steroid hormones, among others, and hence the study of how the tumour and stroma interacts is a valuable extension to the results obtained in the previous sections. PCR was performed for all nuclear receptors, except for the estrogen receptor alpha, in the complete tissue population of 25 samples of tissue derived from the stroma of the grade 1, grade 2 and grade 3 tumours used in the previous studies as well as the control tissues. Due to difficulties in PCR optimization for estrogen receptor alpha, only three stromal samples from each grade and four controls were able to produce results, for a total population of 13 samples. Of all the receptors tested, only the progesterone and glucocorticoid receptors displayed significant changes in expression in stromal tissue, with PgR having significantly lower expression in all stromal samples compared to control, while GR was more highly expressed in stroma derived from high grade tumours (p= 5.908x107 and p=2.761x105, for PgR and GR, respectively). UR expression was also increased in stroma derived from ESRα negative tumours (p=5.85x105). These alterations reflect the kind of stimulation a tumour is likely to apply to the surrounding stroma, using progesterone to stimulate the cells into differentiating to provide a more suitable environment, hence the loss in PgR expression. The increase in GR expression may be the result of the high level of growth stimulating factors that tumours produce, priming the local cells to be more sensitive to growth suppressors, a situation that is also mirrored in results previously obtained for the tumour tissue. The fifth part of the study concerned the expression of the nuclear receptor coactivator 1 and nuclear receptor co-activator 3 genes. These proteins are required for the activation and function of the nuclear receptors and both have been implicated in cancer development, being found to be over-expressed in several tissues and cell lines. As integral parts of the nuclear receptor pathway, their level of expression is important for determining how effective any nuclear receptors present will be when stimulated. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for both nuclear receptor co-activator genes and normalized using 18S. The result of ANOVA analysis on the NCoA data showed that NCoA3 expression remained unaltered in all grades of cancer and stroma and in both ESRα positive and negative tissue. NCoA1 however, was significantly upregulated in grade 3 tumours as compared to grade 1 tumours and also in ESRα negative tumours. This increase in expression would seem to indicate that these tissues would be more capable of acting on any received hormonal stimulation. That this increase in expression occurs in more advanced cancers could be evidence that the nuclear receptor expression observed in prior sections is resulting in NR splice variants that favour, rather than repress, growth, as advanced cancers usually do not respond normally to hormonal stimulation. The final part of the study investigated the possibility of correlations between the expression of the nuclear receptors, between the nuclear receptor co-activators and between all of the tested genes and other pathological parameters, including tumour size, metastasis, site of tumour, carcinoma in situ invasiveness, age of patient and the presence of calcification. The data generated in the prior studies was analyzed using ANOVA for categorical data and correlation analysis for numerical data. The ANOVA and correlation analysis revealed a number of interactions between these factors, which provide additional information on the relationships between the tested genes. Expression of the progesterone receptor was found to be correlated with the expression of GR, AR and NCoA1 (p= 0.022, p=O.OO3, and p=O.0i9, respectively). Likewise the expression of GR and AR were found to be correlated (p=O.O29). Additionally, AR was found to be associated with tumour size (p=O.O36) while GR was found to be associated with both tumour size and metastasis (p= 0.006 and p=7.6x106, respectively). ESRα and ESRβ expression were found to be negatively correlated (p=O.O44M), as were patient age and the amount of ductal carcinoma in situ invasion. Given the results of previous analyses, it is not surprising that PgR, GR, AR and NCoA1 expression are related, and the negative correlations between ESRα and ESRβ expression, as well as between age and ductal carcinoma in situ invasion have been documented in other studies. Hence, these results provide reinforcement for previous observations, as well as providing new information, particularly on AR and GR.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text

The nuclear lamina is a filamentous network that underlies the nuclear envelope. Lamina components include the family of LEM domain (LEM-D) proteins, named for LAP2, emerin and MAN1. Mutations in genes encoding LEM-D proteins cause tissue-restricted human disease, even though these genes are globally expressed. To understand the contributions of the LEM-D proteins to nuclear lamina function, investigations of the Drosophila LEM-D proteins was undertaken. The Drosophila genome encodes four LEM-D proteins and this thesis describes work done on the Drosophila homologues of MAN1 and emerin, Drosophila MAN1 (dMAN1) and Otefin (Ote). Chapter 2 describes the generation and phenotypic analyses of dMAN1 mutants. These mutants display a range of tissue-specific defects associated with an increase in BMP/Dpp signaling. This suggests that dMAN1 downregulates BMP/Dpp signaling at the nuclear periphery. Chapter 3 describes the identification and phenotypic analyses of ote mutants. Loss of Ote is associated with a tissue-specific defect of the female germline where ote mutant females display defects in germline stem cell (GSC) maintenance. Loss of Ote causes defects in the germline cells, the cap cells of GSC niche and an increased sensitivity to Dpp signaling in both germline and somatic cells. These findings support models suggesting that laminopathies arise from dysfunction of the homeostasis in stem cell populations. Taken together, these studies suggest that the nuclear lamina may play tissue-specific roles through regulation of signal transduction pathways. Our data also support the use of Drosophila as a system to elucidate the mechanistic basis of diseases associated with defects in the nuclear lamina.

Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed April 9, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Nuclear actin-related proteins (Arps) have 20-30% identity to conventional actin and many are found to be in chromatin remodelling complexes. There are two families of chromatin remodelling complexes, one of which carries out covalent modification on histones such as acetylation. The other is an ATPase complex, which alters the nucleosomal spacing, and nuclear Arps are found in both complexes. These complexes are believed to be required for transcriptional activation by increasing the accessibility of the transcription machinery to the target DNA. The alp5-1134 mutant was isolated from a screen for temperature-sensitive (ts) mutants with altered polarity and shows severe mitotic defects. Cloning of alp5+ revealed that Alp5 is an essential actin-related protein, most similar to budding yeast Arp4 and human BAF53. Alp5 localises to the nucleus and immunoprecipitates with Mst1, a histone acetyltransferase. These results strongly indicate the role of Alp5 in chromatin remodelling process, as its homologues. Given the interaction between Alp5 and Mst1, its function in histone acetylation was investigated both genetically and biochemically. It was found that Alp5 is required for acetylating the N-terminus tail of histone H4 lysine residues, and functionally counteracts with the histone deacetylases Clr6, Hst4 and Sir2. At the restrictive temperature, the alp5-1134 mutant shows a mitotic delay due to the activation of a spindle assembly checkpoint, which suggests a defect in the kinetochore-spindle interaction. This study also reveals that the function of Alp5 is required for the transcriptional repression at the core centromere region. Possible roles of Alp5 in mitosis are discussed.

Masters of Commerce
Highly enriched uranium (HEU) is one of the most dangerous materials in the world, because it is a key ingredient in making a nuclear bomb. If a terrorist organisation can get HEU, it would be close to making a nuclear bomb. After South Africa disarmed its nuclear weapons, it kept HEU that was extracted from the nuclear bombs. The US tried to persuade South Africa to blend down its HEU into low enriched uranium (LEU) or give it up for safekeeping. However, South Africa refused to give it up. After a breach at Pelindaba, a national key point facility where South Africa stores its HEU, the US intensified its efforts to pressure South Africa to give its HEU up. It even promised incentives to South Africa should they agree to give it up, but South Africa refused. The US used the nuclear terrorism narrative to justify its initiative to eliminate vulnerable materials in the world. However, South Africa is yet to be swayed. This is odd since South Africa's refusal can negatively affect its credentials as a nuclear nonproliferation and disarmament champion and its image as a norm entrepreneur. The objective of the study was to understand the role played by HEU in South Africa's nuclear diplomacy. It was to explore HEU as a factor in the state's nuclear diplomacy and to understand the power of having HEU in nuclear negotiations, as well as what SA intends to do with its HEU. The study is framed theoretically by drawing on foreign policy theory, namely middle-power theory, and revisionism. It juxtaposed middle power, reformist, and revisionist positions with status quo foreign policy to analyse the role of HEU in South Africa's nuclear diplomacy. As a middle power with a moral high ground, South Africa hoped that it can affect change in the nuclear regime. However, when this did not occur its foreign policy shifted to a revisionist character that is discontent with the status quo in the nuclear regime. SA is dissatisfied with the current nuclear order and wants it revised towards liberal values such as equality and nondiscrimination. It views the current nuclear order as nuclear apartheid.

In recent years, Iran’s growing obsession with achieving an independent technological capability for enriching uranium has led many regional and international states to conclude that Tehran is actually pursuing a nuclear weapons programme. This has ensured that Iran’s nuclear programme has remained at the top of the international agenda for well over a decade now. Against this backdrop, and through developing and applying an analytical framework based on the concepts of the internal security dilemma and the politics of nuclear symbolism this thesis examines the extent to which internal/transnational ethno-religious challenges have contributed to the shaping of Iran’s nuclear motives. The existing literature analysing Iran’s assertive pursuit and acquisition of a breakout nuclear capability has primarily emphasised the external challenges faced by Tehran as the main driving forces behind Iran’s nuclear ambitions. Informed by the insights of structural realism, which takes the state’s internal coherence for granted, the literature has largely overlooked the role of ethnic diversity and divisions in determining Iran’s nuclear goals. Iran, however, is a multi-ethnic state and challenges of ethnicity and ethnic diversity are central to the shaping of Tehran’s threat perception and national security strategy. The thesis argues that Iran’s non-Shiite and non-Persian major ethnic groups, empowered by their co-ethnics and Tehran’s rivals in the Middle East, have been a potent challenge to Iran’s domestic security and the Islamic Republic’s authority in the country’s ethnic regions. Based on the balance of probabilities, the thesis argues that these challenges have influenced Iran’s nuclear motivations. Drawing on original sources, the thesis demonstrates that members of the ruling elite in Tehran have pursued the politics of nuclear symbolism and have used the country’s nuclear achievements (1) to consolidate national unity; (2) to enhance the Islamic Republic’s domestic reputation for strength; and, thus, (3) to protect the security of a unified Iranian nation-state in a region where nation-states are constantly threatened by ethno-sectarian conflict, war, and rivalry.

Bone morphogenetic protein 2 (BMP2) is a secreted growth factor that is essential for proper embryonic development and proliferation. Our laboratory discovered a nuclear variant of BMP2 (nBMP2) which is produced when translation is initiated at an alternative start codon within the BMP2 gene. When translation occurs at the downstream start codon, the resulting protein lacks the ER signal peptide, thereby allowing cytoplasmic translation and nuclear localization. Our aim is to distinguish the role of this nuclear localized variant from secreted BMP2. Overexpression of nBMP2 in HEK293 and HT29 cell lines resulted in a higher percentage of cells in proliferative phases of the cell cycle. We determined that nBMP2 does not regulate cell cycle progression by inducing hyperphosphorylation of retinoblastoma protein (Rb), but it may regulate the cell cycle by interacting with ROC1. In order to examine the role of nBMP2 in vivo, we have generated a mouse model in which a mutation of the nuclear localization signal (NLS) disrupts nuclear localization of nBMP2. Aberrant crypts were more abundant in nBmp2NLStm azoxymethane (AOM) treated mice than in wild type mice. Furthermore, H&E staining of colonic tissue showed that mutant mice have increased levels of dysplasia and aberrant crypt foci. This work suggests that nBMP2 is involved in regulating cell cycle progression and proliferation, and therefore may play a role in tumorigenesis.

Andrei Gromyko, a veteran Foreign Minister of the USSR, shocked the world last winter with a particular revelation in his memoirs: the late Chairman Mao Zedong of China had a plan to lure United States troops into the heartland of China and then wipe them out with atomic weapons made v/ith Soviet help. 1 We may question the truth of Gromyko’s memoirs. The Chinese Foreign Ministry has already done so. But the story of Sovietcooperation in China's nuclear industry, both for peaceful and military purposes, cannot be denied. It is one that tells why the Soviets and Chinese became close allies in the early 50s and why they drew apart several years later. It is the contention of this writer that the Sino-Soviet dispute cannot be fully understood without giving due weight to the disagreements the Chinese and Soviets had over nuclear technology issues.

This work involves thermal photon production from the Quark Gluon Plasma (QGP) and Hadronic Gas (HG) in heavy ion collisions. Using a 3D + 1 hydrodynamic model (MUSIC) to describe the evolution of the system after thermalization of a Au+Au collision, we compute the total production of thermal photons. We use effective models for mesonic interactions in HG and the quark gluon plasma. We compare photon production and elliptic flow from both sources and look at how the centrality and the equations of state influence the photon observables.We do this using ideal (non-dissipative) hydrodynamics, but also with viscous hydrodynamics. We examine the effects on the hydrodynamic properties (temperature, flow) and on the photon production of the inclusion of shear viscosity in the hydrodynamic equations. We then also look at another effect of viscosity, which is that the distribution functions of quarks and mesons need to be corrected. In our approach, we compute the necessary integrals for the viscous corrections and tabulate the results as a function of the temperature and energy. We then interpolate between these values. We look at how this affects photonic yield and elliptic flow. We also examine the effect of fluctuating initial conditions on the photon production.We find that viscosity does have a significant impact on the photonic yield, and an even more important effect on the elliptic flow of photons. Fluctuating initial conditions also lead to an appreciable effect on the yield. For all this, we restrict our study to photons with an energy between 0.2 and 4 GeV, which is the range where thermal photons account for a significant amount of the total yield. We show that photons make excellent probes for heavy ion collisions as they are very sensitive to many important theoretical concepts in our model of heavy ion collisions.
Cet ouvrage concerne la production de photons thermaux provenant du Plasma Quarks Gluons (PQG) et du gaz hadronique (GH) dans les collisions d'ions lourds. Nous utilisons un modèle hydrodynamique 3D + 1 (MUSIC) pour décrire le système après la thermalisation d'une collision Au+Au, avec lequel nous calculons la production totale de photons thermaux. Nous utilisons des modèles effectifs pour les interactions mésoniques dans le GH et dans le PQG. Nous comparons la production totale et l'écoulement elliptique des photons pour les deux sources et regardons comment la centralité et les équations d'état influencent les observables photoniques.Nous utilisons un modèle hydrodynamique idéal (non dissipatif), mais aussi un modèle incluant la viscosité. Nous regardons comment les propriétés hydrodynamiques (température, écoulement) sont modifiées par la viscosité de cisaillement et son effet sur la production de photons. De plus, la viscosité a aussi pour effet d'apporter une correction aux fonctions de distribution des quarks et des mesons. Dans notre approche, nous calculons numériquement les intégrales nécessaires pour les corrections visqueuses et tabulons les résultats en fonction de la température et de l'énergie du photon et interpolons entre ces valeurs. Nous examinons comment cela affecte la production totale de photons ainsi que l'écoulement elliptique. Nous regardons aussi l'effet des conditions initiales aléatoires.Nos calculs montrent que la viscosité a un effet significatif sur la production de photons, et surtout sur l'écoulement elliptique de ceux-ci. Les conditions initiales aléatoires ont aussi un effet très appréciable sur la production totale de photons. Pour tous les éléments mentionnés ci-haut, nos calculs sont restreints à des énergies du photon de 0.2 GeV à 4 GeV, qui correspond à la plage d'énergie pour laquelle les photons thermaux contribuent de façon significative à la production complète de photons (incluant tous les autres procédés pertinents). Nous montrons que les photons font d'excellentes sondes pour les collisions d'ions lourds car ils sont très sensibles aux concepts théoriques importants dont notre modèle dépend.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and composed of proteins called nucleoporins. NPCs as such control the bidirectional traffic of proteins and RNAs between the nucleus and the cytoplasm in eukaryotic cells whereas individual nucleoporins were found to be implicated in other cellular processes such as, cell division, kinetochore assembly, gene expression and cell migration. A prime example for nucleoporin functional versatility can be seen in Nup153. Nup153 is since its discovery known to be a central player in nucleocytoplasmic transport, but additionally participates directly or indirectly, for example, in gene expression and cell cycle control. In this context, it was previously shown that altered levels of Nup153 led to mitotic abnormalities, particularly in cytokinesis and in the spindle assembly checkpoint (SAC). The SAC promotes accurate chromosome separation to ensure the faithful segregation of genetic material to daughter cells. Nup153 was found to interact with the SAC protein Mad1. In the present study, we have further dissected the interaction between Nup153 and Mad1 and investigated the function of the Nup153-Mad1 complex in human cells. By using the high resolution imaging technique “in situ proximity ligation assay”, we found that Nup153 and Mad1 interact with each other exclusively in the presence of a NE, from late mitosis to prophase. By in vitro binding assays, we have confirmed the direct interaction between Nup153 and Mad1 and furthermore identified two independent Nup153-binding sites in Mad1. We have also provided some evidence that Nup153 interacts also with SUMO-modified Mad1.It was previously shown that depletion of Nup153 had no obvious effect on Mad1 and SAC activity. In the present study, we have shown by time-lapse imaging microscopy that the depletion of Mad1 led to a delayed recruitment of Nup153 at the reforming NE during anaphase in living cells, which was often accompanied by a prolongation of anaphase. Furthermore, Mad1 depletion led to alterations in the NE architecture, which were characterized by a change of the membrane curvature at the NPC-NE interface. This was followed by an expansion of the spacing between the inner and outer membranes as seen by electron microscopic and three-dimensional structured illumination investigations. This suggests an implication of Mad1 in a mechanism related to the NE reformation and stability independent of the SAC. Mad1 depletion also resulted in redistribution of the ER network and mitochondria throughout the cell as seen by fluorescence microscopy. Nup153 depletion coincided with the NE abnormalities and alteration of these organelles similar to that seen in Mad1-depleted cells. Further, by fluorescence microscopy, we have shown that Nup153 depletion, but not of Mad1, partially affected the localization of the cytoplasmic nucleoporins in human and in mouse cells and thus the NPC integrity. In conclusion, altogether, our results suggest that Nup153 is essential for NE and NPC integrity. Nup153 has likely separable roles in this context: one in post-mitotic NE reformation with Mad1 and one in interphase in NPC assembly. Nup153-Mad1 complex has a function independent of the spindle checkpoint, but important for the establishment of an intact NE architecture.
Les pores nucléaires sont des structures enchâssées dans l’enveloppe nucléaire et composées de protéines appelées les nucléoporines. Ces pores nucléaires contrôlent le trafic bidirectionnel des protéines et des ARNs entre le noyau et le cytoplasme dans les cellules eucaryotes tandis que les nucléoporines individuelles sont également impliquées dans d’autres processus cellulaires tels que la division cellulaire, l’assemblage des kinétochores, l’expression génétique et la migration cellulaire. Un exemple primordial de la versatilité fonctionnelle des nucléoporines peut être observé à travers Nup153. Depuis sa découverte, Nup153 est connue pour être un élément clé dans le transport nucléo-cytoplasmique, mais il a également été démontré qu’elle participait directement ou indirectement à l’expression génétique et au contrôle du cycle cellulaire. Dans ce contexte, nous avons montrés précédemment que des niveaux altérés de Nup153 menaient à des anomalies mitotiques, particulièrement en cytokinèse et dans le point de contrôle de l’assemblage du fuseau mitotique (SAC). Le SAC assure la ségrégation correcte du matériel génétique entre les cellules filles. Il a été montré que Nup153 interagit avec la protéine du SAC Mad1. Dans cette étude, nous avons utilisé une technique d’imagerie de haute résolution, « in situ proximity ligation assay » pour disséquer davantage l’interaction entre Nup153 et Mad1 dans les cellules humaines. Nous avons montré que ces deux protéines interagissent exclusivement au niveau de l’enveloppe nucléaire, depuis les dernières phases de la mitose jusqu’à la prophase. Par des expériences d’interaction in vitro, nous avons également identifiés sur Mad1 deux sites de liaison indépendants pour Nup153. Nous avons également fourni des indications que Nup153 interagit aussi avec une forme SUMOylée de Mad1. La déplétion de Mad1 menait à un recrutement tardif de Nup153 au niveau de l’enveloppe nucléaire en cours de reformation en anaphase dans les cellules vivantes et à des altérations de l’architecture de l’enveloppe nucléaire, caractérisées par un changement de la courbure membranaire au niveau de l’interface pore nucléaire-enveloppe nucléaire. Suite à cela, une expansion de l’espace entre les membranes nucléaires internes et externes a été observée par microscopie électronique. Ceci suggère une implication de Mad1 dans un mécanisme lié à la stabilité de l’enveloppe nucléaire indépendant du SAC. La déplétion de Mad1 résultait également en une redistribution du RE et des mitochondries à travers la cellule. La déplétion de Nup153 coïncidait avec des anomalies similaires au niveau de l’enveloppe nucléaire et des organelles. De plus, la déplétion de Nup153 affectait partiellement la localisation des nucléoporines cytoplasmiques, contrairement à la déplétion de Mad1. Ensemble, nos résultats suggèrent que Nup153 est essentielle pour l’intégrité des pores nucléaires et de l’enveloppe nucléaire. Nup153 semble avoir deux rôles, un au niveau de la formation de l’enveloppe nucléaire en fin de mitose, en complexe avec Mad1 et un autre rôle au niveau de l’assemblage des pores nucléaires. Le complexe Nup153-Mad1 a une fonction indépendante du SAC, mais importante pour l’établissement d’une enveloppe nucléaire intacte.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished