Thèse

M Moreau

Patrick Univeristé De Bordeaux

Président M Gachet

Yannicl

Paul Sabatier

M Mcfarlane

M El Ghouzzi

Robert Debré

Examinateur M Hooks

Proteomic studies of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe Membres du Jury

Études protéomiques des mutants hid1Δ et hid3Δ chez Schizosaccharmyces pombe

RÉCAPITULATIF EN FRANÇAIS

Schizosaccharomyces pombe est devenu un important système modèle pour étudier les processus physiologiques, biochimiques et génétiques chez l'homme. Ces travaux appartiennent à un projet sur la façon dont la fonction altérée de l'appareil de Golgi contribue à des maladies comme le cancer ou cause des anormalités génétiques. La protéine HID-1 de C. elegans et des humains est une protéine de l'appareil de Golgi qui appartient à la superfamille de protéines DYMECLIN. Les animaux ont à la fois un gène HID1 et un gène DYM. Chez les humains, une expression réduite de HID1 est impliquée dans la prolifération de tumeurs. La perte de DYM chez les humains mène à une déformation squelettique. S. pombe a trois gènes orthologues HID-1, mais pas de DYM. Par contraste, de nombreux eucaryotes unicellulaires et pluricellulaires n'ont qu'un gène DYM. Les mutants de S. pombe sans Hid1 et Hid3 étaient sensibles au stress oxydatif et la croissance de hid3Δ a été stoppée dans des milieux de culture minimum standard. L'insensibilité de hid3Δ au brefeldin A, mais sa sensibilité au golgicide, a démontrent que Hid3 fonctionne dans le transport antérograde à travers l'appareil de Golgi. Afin d'explorer des rapports indiquant que le renouvellement des protéines pourrait être modifié dans hid3Δ, j'ai entrepris une étude de protéomique a la quantification label-free. La régulation positive de la voie de signalisation MAPK de tension a démontré que les cellules étaient dans un état de tension dans des conditions normales de croissance. De plus, des composants de protéine dans plusieurs voies de signalisation étaient modifiés, pouvant affecter une large gamme de processus cellulaires.

MOTS CLÉS: Formation Dauer, Proteomique, Signalisation de stress

II

ENGLISH SUMMARY

Schizosaccharomyces pombe has become an important model system to study physiological, biochemical and genetic processes in humans. This work is part of a continuing project to study how altered Golgi function contributes to diseases, such as cancer, or causes of genetic abnormalities. The HID-1 protein of C. elegans and humans are peripheral membrane proteins of the Golgi apparatus and are part of the DYMECLIN superfamily of proteins. Animals have both a HID1 and a DYM gene. In C. elegans, HID-1 maintains normal cellular growth and in humans reduced expression of HID1 has been implicated in tumour proliferation. Loss of DYM in humans leads to skeletal deformation and potentially mental retardation. S. pombe has three HID-1 orthologues, but no DYM. In contrast, many unicellular and multicellular eukaryotes have only DYM. S. pombe mutants lacking Hid1 and Hid3 were sensitive to oxidative stress and growth of hid3Δ was stopped in standard minimal media. Insensitivity of hid3Δ to brefeldin A but sensitivity to golgicide A demonstrated that Hid3 operates in anterograde protein transport through the Golgi. In order to investigate reports that protein turnover might be altered in hid3Δ, I undertook a proteomics study using labelfree protein quantification. Up-regulation of the MAPK stress signalling pathway demonstrated that cells were under a state of stress under standard growth conditions.

In addition, protein components of Ras signalling, microtubule dynamics and chromatin remodelling were altered potentially affecting a wide variety of processes from cell cycle regulation to metabolism. Quantitative comparison of primary sequences between members of the HID and DYM superfamily.

KEY WORDS: Dauer Formation; Proteomics; Stress Signaling

III

Table 3.3. GO ontology references to potential function of Hid proteins from S. pombe. The effect brefeldin A (BFA) on mutant growth.

IX

Récapitulatif du projet en Français Introduction

Les protéines destinées à être sécrétées ou transférées vers un compartiment particulier sont fabriquées dans le réticulum endoplasmique (RE), puis elles doivent être traitées pour les rendre entièrement fonctionnelles. C'est une des principales fonctions de l'appareil de Golgi.

L'appareil de Golgi est divisé en compartiments appelés citernes. Les protéines issues du RE pénètrent dans l'appareil de Golgi par la face-cis et ressortent par la face-trans. Il en résulte que toutes les protéines doivent passer dans la région dite de citerne, qui contient les enzymes divers, appelés enzymes résidents, pour faire les modifications comme la glycosylation, la sulfatation ou la phosphorylisation.

L'Ubiquitine (Ub) est une petite protéine (76 aminoacides), compacte et superpliée, qui est fortement conservée dans les organismes eucaryotes. Il existe trois différences d'acides aminés entre la levure et l'homme [START_REF] Shaid | Ubiquitination and selective autophagy[END_REF]. Les cellules utilisent Ub comme une étiquette pour modifier le contenu des protéines en fonction des conditions cellulaires pendant la dégradation des protéines. La voie Ub est aussi une modification post-translationnelle des protéines qui est importante et étendue, mais ne se limite pas à la dégradation. L'ubiquitylation guide la fonction protéique dans la réparation de l'ADN, le transfert protéique, la modification de la structure protéique, la modulation de l'activité des protéines cibles, la croissance cellulaire et la transduction de signal [START_REF] Horák | The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast[END_REF]. L'ubiquitination est un processus important dans le système de la membrane endosomale qui comprend le réticulum endoplasmique et l'appareil de Golgi. L'ubiquitylation est importante et permet aux cellules de vérifier que seules les protéines fonctionnelles sont modifiées et finalement sécrétées. La plupart des protéines qui sont ubiquitinées dans une cellule sont des protéines nouvellement synthétisées qui sortent juste de ribosomes [START_REF] Kim | Systematic and quantitative assessment of the ubiquitin-modified proteome[END_REF] et il est estimé que l'ubiquitylation pourrait servir de système de contrôle de la qualité pour les protéines nouvellement synthétisées [START_REF] Chhangani | E3 ubiquitin ligases in protein quality control mechanism[END_REF].

Cela constitue aussi une partie d'un processus important de signalisation pour permettre à l'appareil de Golgi de communiquer avec le noyau. Par exemple, le contrôle des niveaux de cholestérol dans les cellules humaines passe par la voie de la protéine se liant à l'élément récepteur du stérol (SREBP). Autant chez l'homme que S. pombe, le clivage de l'extrémité Nterminale de SREBP dans la membrane de l'appareil de Golgi est initié par son ubiquitylation [START_REF] Stewart | Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex[END_REF]. Au cours des dernières années, un grand nombre de maladies a été lié XV au mauvais fonctionnement de l'appareil de Golgi, à l'inclusion de maladies cutanées, de troubles neurologiques, de dysplasies squelettiques et de troubles des tissus conjonctifs [START_REF] Bexiga | Human Diseases Associated with Form and Function of the Golgi Complex[END_REF].

Le gène de la protéine 1 de formation de larves dauer induite par des températures élevées (hid-1) de C. élégans a été trouvé dans un dépistage à la recherche de mutants du nématode, qui a adopté la formation de larves Dauer dans de bonnes conditions de croissance, mais avec une température légèrement plus élevée [START_REF] Ailion | Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans[END_REF]. HID-1 est une protéine de la membrane de l'appareil de Golgi qui semblerait être impliquée dans l'excrétion de neuropeptides dans C. elegans. Il a été montré que le gène orthologue S. pombe de HID-1 localise le déubiquitinase Ubp5 (gène orthologue S. pombe de USP7 chez l'homme) dans l'appareil de Golgi. La translation fonctionnelle chez l'homme est qu'un mauvais fonctionnement de la protéine HID1 ou son absence affectera la place dans la sous-cellule de l'enzyme déubiquitylatant USP7.

Objectifs du projet et organisation de la thèse

Le but de l'introduction fournie dans le chapitre 1 était de présenter l'idée que la modification de la fonction de l'appareil de Golgi a des effets au delà du transport vésiculaire.

Ceci est intimement lié au fonctionnement général des cellules à travers un certain nombre de systèmes de signalisation. L'ubiquitinylation était au centre de cette introduction parce que la manipulation des processus Ub est une des fonctions proposées de la protéine Ftp105/Hid3 de S. pombe. Toutefois, il est probable que la protéine Ftp105/Hid3 ait d'autres fonctions dans l'appareil de Golgi ou qu'elle puisse affecter d'autres processus quand elle est mutante. La La phylogénie et les comparaisons de séquences ont confirmé la présence de trois gènes orthologues HID1 dans S. pombe et présentaient une séquence de diversification (figure 3.3, tableau 3.2). J'ai proposé que Ftp105 soit appelé Hid3 à l'avenir. S. pombe représente un bon modèle pour étudier la fonction de Hid, parce que cela élimine les complications potentielles de la présence de protéines DYM, mais il est possible qu'il y ait une fonction redondante au sein des SpHid.

Des informations de bases de données disponibles au public indiquent que Hid1 et Hid2 se trouvent dans le cytosol/noyau avec une fonction inconnue, tandis que Hid3 est une protéine de l'appareil de Golgi (mais aussi peut-être cytosolique) et responsable de la position des protéines dans l'appareil de Golgi (tableau 3.3). Une analyse structurelle des données de séquence suggèrent que SpHid1 est plus étroitement liée aux HID animaux, mais il semble que SpHid3 a conservé les propriétés structurelles qui lui permettent de se fixer à l'appareil de Golgi Chapitre 4 : Effet des traitements de la tension sur la croissance de mutants hidΔ Des données déjà publiées suggéraient que hid1 + et hid2 + peuvent être impliquées XVII dans les réactions à la tension, parce qu'elles sont induites en réaction à une tension (figure 4.1). Il a été rapporté que le mutant hid3 est sensible à certains traitements, comme des doses de sel élevées et certains produits pharmaceutiques (www.pombase.org). Les mutants produits dans l'étude d' [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF] ont donc été testés pour déterminer leurs réactions de croissance à des tensions/traitements chimiques divers (tableaux 4.1, 4.2). Aucun des mutants simples hid1Δ, hid2Δ et hid3Δ n'était sensible à une gamme variée de tensions, sauf au stress oxydatif, qui affectait hid1Δ et hid3Δ en ralentissant fortement la croissance des cultures (figure 4.2). Il est intéressant de noter que la croissance de hid3Δ a été sérieusement affectée quand elle était cultivée dans des milieux de culture minimum (figure 4.3). On n'en connaît pas la cause, mais je spécule que hid3Δ manque d'un nutriment essentiel. Pour finir, en utilisant les inhibiteurs de l'appareil de Golgi brefeldin A (figure 4.4) et golgicide A (figure 4.5), j'ai pu déterminer que dans S. pombe, Hid3 fonctionne dans le transport antérograde des protéines (figure 4.6).

Chapitre 5 : Caractérisation métabolomique et protéomique des mutants hid1Δ et hid3Δ

de S. pombe

Ce chapitre décrit les propriétés métabolomiques et protéomiques des mutants. La métabolomique sert à juger dans quelle mesure la physiologie et la biochimie fondamentales des cellules se modifient quand une mutation génétique est introduite. Par ailleurs, les évolutions métaboliques peuvent fréquemment expliquer pourquoi la croissance cellulaire est modifiée. Les échantillons utilisés dans l'étude étaient exactement les mêmes que ceux utilisés dans l'étude transcriptomique (tableau 5.1). Un nombre limité de métabolites cellulaires a été quantitativement mesuré avec 1 H-NMR. Ces mesures étaient particulièrement remarquables en raison de l'augmentation de la quantité de glycérol et de tréhalose présents dans hid3Δ. Ces composés sont synthétisés en réaction à la tension, mais ils sont aussi produits en adaptant les cellules à un état quiescent. Une analyse en composantes principales des données du métabolite a montré que l'état métabolique de hid1Δ et de hid3Δ étaient différents de la variation contingente négative (VCN) de contrôle (figure 5.1).

La conduite d'une étude protéomique visait un but directement lié à l'implication potentielle de Hid3 dans les processus utilisant l'ubiquitine qui impliqueraient la dégradation des protéines. Il est clair que le champ de la protéomique est large et qu'il peut y avoir plusieurs façons dont l'affectation de l'appareil de Golgi peut modifier les niveaux protéiques, mais la première étape était de déterminer les protéines qui évoluaient et de combien. Des protéines totales ont été extraites des mêmes échantillons utilisés pour l'étude métabolomique. Par la XVIII suite, une électrophorèse SDS-PAGE a montré que les génotypes ne présentaient pas de grandes différences au niveau de l'abondance des protéines, puisque les schémas présentés par les bandes et les quantités de protéines apparaissaient uniformes (Figure 5.2). Les échantillons de protéine ont été envoyés à l'unité de protéomique de CGFB où 10 μg de la protéine totale de chaque échantillon a été purifiée par gel et digérée dans le gel avec de la trypsine (figure 5.3). Les protéines ont été quantifiées par une analyse du mélange résultant de peptides au moyen de la spectrométrie de masse (tableau 5.3). L'analyse en composantes principales (figure 5.4) et la quantification de protéines individuelles (Figure 5.5) a montré que la composition de la protéine hid3Δ était fortement différente de celle d'autres génotypes.

L'analyse ontologique des protéines en évolution a montré pour hid3Δ des niveaux élevés de protéines de réaction à la tension et des niveaux réduits de protéines biosynthétiques (figure 5.6), indiquant des cellules dans des conditions chroniques de tension. L'inspection de protéines individuelles qui ont été modifiées en abondance a révélé la régulation positive dans hid3Δ de points clés de la voie de signalisation MAPK de réaction à la tension (Tableau 5.5).

D'autres protéines intéressantes faisant l'objet d'une augmentation étaient les protéines de remodelage de la chromatine Obr1 et l'histone H2B qui affecteraient l'expression génique et la GTPase Ras1, qui est une protéine clé de voie de signalisation dont la fonction dépend de sa place au sein de la cellule. Ces différences montrent que l'élimination de Hid3 engendre un éventail large d'effets sur la fonction cellulaire.

Chapitre 6 : Discussion générale et travaux futurs

Ce chapitre présentait les conclusions obtenues par chaque partie de l'étude. Les principales conclusions du chapitre 3 étaient la confirmation de plusieurs gènes orthologues HID1 dans S. pombe et l'absence de DYM. En conséquent, S. pombe est un modèle qui convient dans l'étude de la fonction de Hid. Du fait de leur absence de HID, d'autres organismes comme les plantes plus grosses pourraient être utilisés pour étudier la fonction de DYM. L'étude phylogénétique n'a pas abouti à la fondation de l'origine de la superfamille de DYM, mais il est clair que ces protéines sont apparues avec la diversification majeure des organismes L'incapacité de hid3Δ à se développer dans des milieux de culture minimum suggère qu'il lui manque un nutriment essentiel qui peut être acquis par des souches non affectées. Ceci va dans le même sens que l'induction de l'expression des gènes encodant les protéines de transport [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. Le chapitre 5 a confirmé autant pour le métabolisme que les niveaux de protéines que les cellules de hid3Δ subissent une forte tension même dans des conditions normales de croissance. Les évolutions des niveaux de métabolite et de protéine sont cohérentes avec l'idée que la croissance réduite des cellules de hid3Δ peuvent être dans un état de quiescence partielle et les évolutions des niveaux de protéine présentent le mécanisme qui explique les modifications observées dans l'expression génique (figure 6.1). Toutefois la gamme large de protéines dont les niveaux ont été modifiés montre que de nombreux processus différents seront affectés dans les souches de hid3Δ. Enfin, je discute de plusieurs voies possibles de recherche allant de l'utilisation des HID et DYM humains et de C. elegans pour inverser les phénotypes hid3Δ jusqu'à la caractérisation biochimique et structurelle des associations de Hid3 avec la membrane de l'appareil de Golgi. Il faudra déterminer l'emplacement dans les sous-cellules des protéines de signalisation, comme Ras1 ainsi que les mécanismes qui servent à modifier les protéines soit par l'augmentation de l'expression des gènes, soit par la modification de la dégradation.

XX The image was taken from [START_REF] Aguiar | Investigation of protein secretion and secretion stress in Ashbya gossypii[END_REF].

Chapter 1

General Introduction

Function of the Golgi apparatus in the cell

There are two main types of cells, prokaryotic and eukaryotic, which form the basis for cellular life. They carry the basic things that all cells require, such as a cell membrane, DNA, cytoplasm, and ribosomes [START_REF] Alberts | Molecular Biology of the Cell[END_REF]. Eukaryotic cells differ from prokaryotic cells in that they can be unicellular like prokaryotes but have a diverse range of cell shapes and sizes and can combine to form multicellular organisms. On the cellular level there are fundamental differences between eukaryotic and prokaryotic cells, such as a nucleus, and organelles, such as mitochondria or plastids, that can generate energy. Eukaryotic cells also have a well-defined and intricate endomembrane system that transports proteins to different parts of the cells and allows cell to cell communication in multicellular eukaryotes. The group is interested in a part of this endomembrane system known as the Golgi apparatus, which is responsible for the processing and packaging for sorting proteins and lipids to other parts of the cell, such as the lysosome in animal cells or the vacuole in plants and fungi, or to the outside of the cell [START_REF] Glick | Organization of the Golgi apparatus[END_REF]. The Golgi is an organelle that consists of various numbers of membrane stacks that various in number and shape depending on the organism. The Golgi apparatus is comprised of four main parts, cis-Golgi, medial-, trans-Golgi and trans-Golgi network (TGN). The terms 'cis'

and 'trans' refer to the different faces of a Golgi complex. Vesicles arrive at the cis-face from the Endoplasmic Reticulum (ER) and they leave from the trans-face to travel to the plasma membrane or an interior compartment (Figure . 1.1) [START_REF] Nakano | Passage through the Golgi[END_REF].

Proteins are found everywhere in the cell as they are the 'workers' of the cell providing structure and catalysing reactions. Protein function is so closely related that when a protein does not function properly it can have bad effects on a cell leading to different types of congenital diseases, such as muscular dystrophy, Alzheimer's disease and cystic fibrosis. Most importantly for my study is the group of proteins that can be directed to specific places in the cell, for instance the plasma membrane, the cytoplasm, the nucleus, the endosomes/vacuole, and the cellular machinery that accomplishes this, namely the Golgi apparatus. The diseases mentioned above relate to disrupted protein trafficking by Golgi apparatus [START_REF] Ungar | Golgi linked protein glycosylation and associated diseases[END_REF].

Proteins destined for secretion or for transfer to a particular compartment are made in the ER and then they need to be processed to make them fully functional. That is one of the 1 main functions of the Golgi apparatus. The Golgi is divided into compartments called cisternae.

Proteins enter the Golgi from the ER by cis-face and they exit from trans-face side.

As a result, all proteins have to pass a region called cisterna which contains the various enzymes, called resident enzymes, for doing protein modifications, such as glycosylation, sulfation, or phosphorylation. There are two theories about how proteins move through the Golgi, the Cisternal Maturation Model (CMM) theory and the Vesicular Model (VM) theory. In the CMM, the Golgi cisternae themselves mature and convert to the next type and in doing so carry their cargo from cis-face to trans-face. The VM model states that proteins are passed to fixed cisternae through vesiculation [START_REF] Connerly | How do proteins move through the Golgi apparatus[END_REF]. Using GFP-labelled proteins in budding yeast, two independent laboratories provided evidence for the CMM model to show that resident proteins move through the stacks [START_REF] Losev | Golgi maturation visualized in living yeast[END_REF][START_REF] Matsuura-Tokita | Live imaging of yeast Golgi cisternal maturation[END_REF], so this remains the preferred theory. However, both types of movement are likely to occur, because proteins can be recycled back through the Golgi, which is likely mediated by vesicle transfer [START_REF] Emr | Journeys through the Golgi-taking stock in a new era[END_REF].

Golgi in S. pombe

There are a number of different Golgi structures that have been identified in various organisms, but the typical structure as cis-, medial-, and trans-cisternae plus the trans-Golgi network (TGN). These compartments have been found to differ from each other in properties, like cytochemical-staining, composition of resident enzymes, and ability to bud COPI-or clathrin-coated vesicles [START_REF] Farquhar | The Golgi apparatus (complex)-(1954-1981)-from artifact to center stage[END_REF][START_REF] Kleene | The molecular and cell biology of glycosyltransferases[END_REF][START_REF] Nilsson | Sorting out glycosylation enzymes in the Golgi apparatus[END_REF][START_REF] Rabouille | Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides[END_REF][START_REF] Staehelin | Nanoscale architecture of endoplasmic reticulum export sites and of Golgi membranes as determined by electron tomography[END_REF]. Resident Golgi proteins can move rapidly within and between compartments. For example, according to the cisternal maturation model mentioned above, glycosylation enzymes that operate in assembly line fashion to process secretory cargoes must be re-transported by to the original site of function if not degraded [START_REF] Glick | Models for Golgi Traffic: A Critical Assessment[END_REF][START_REF] Nilsson | Sorting out glycosylation enzymes in the Golgi apparatus[END_REF][START_REF] Rabouille | Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides[END_REF]. A study by [START_REF] Glick | Models for Golgi Traffic: A Critical Assessment[END_REF] tried to investigate various models known to be linked to Golgi traffic, such as vesicular transport between compartments and cisternal maturation, to provide a unifying observation for Golgi function. They concluded that no single model can easily explain all of the observations from diverse organisms. A study by [START_REF] Klute | Evolution and Diversity of the Golgi[END_REF] compared the origin and history of the Golgi by comparing proteins involved in Golgi morphology as well as those involved in trafficking, by choosing proteins that act as carriers to and from the Golgi at both the cis-and trans-faces. They concluded that the basic elements of vesicle trafficking and Golgi structural appear conserved across eukaryotes. cisterna and dispersed to the cytoplasm [START_REF] Suda | The yeast Golgi apparatus[END_REF].

A B

Many of the processes of the Golgi complex of higher eukaryotes like mammals, such as carbohydrate modification and proteolytic processing, have been found in to occur in the yeast Golgi [START_REF] Seemann | Matrix proteins can generate the higher order architecture of the Golgi apparatus[END_REF].There would not be expected to be identical functions of the Golgi between higher eukaryotes and the yeast, because the Golgi in higher eukaryotes is necessary for processing and secreting many of the factors that are necessary growth, proliferation and cell communication within a multicellular context. The internal cell structure of mammalian cells is also different to that of yeast, where mammalian cells have an endosomal system for degrading cellular components, where yeasts have a vacuole system. The structure of the Golgi apparatus among yeast is also different where yeasts like S. pombe [START_REF] Chappell | A galactosyltransferase from the fission yeast Schizosaccharomyces pombe[END_REF] and P. pastoris [START_REF] Preuss | Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy[END_REF][START_REF] Rossanese | A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae[END_REF] have a stacked lamellae structure like higher eukaryotes, but S. cerevisiae has a more dispersed type of Golgi structure lacking visible cisternae (Figure 1.2). However, Mammalian-like Golgi reassembly stacking proteins (GRASPs) have been discovered in the three yeast species noted as factors involved in the formation of Golgi stacks [START_REF] Vinke | The multiple facets of the Golgi reassembly stacking proteins[END_REF]. Moreover, in yeast and unlike the mammalian cells, the Golgi down during the cell division to partition during cytokinesis [START_REF] Preuss | Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy[END_REF][START_REF] Rossanese | A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae[END_REF].

However, even in a dispersed state, the Golgi must be functional in S. pombe in order to form the septum. More investigations are needed to identify the similarities and differences in the nature of the Golgi complex, because the Golgi is an ancient and ubiquitous feature of eukaryotic cells. In addition, the use of various yeast species will help us to understand the importance of Golgi stack formation.

In addition to the morphology of the Golgi, there are functional differences among the yeasts as revealed in differences in protein components. For example, Rho3 has distinct roles in S. pombe and S. cerevisiae. In S. cerevisiae it appears to be involved in influencing cell growth by regulating polarized secretion through the actin cytoskeleton by interacting with Exo70 and Myo2 [START_REF] Robinson | Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70[END_REF]. In S. pombe, Rho3 plays an important role in Golgi/endosome trafficking by complexing with Apm1 and other subunits of the AP-1 complex. Another main feature differing S. pombe from S. cerevisiae and other eukaryotes is that it that has only one Ras protein. There are two Ras proteins in S cerevisiae and four in mammals. This S. pombe

Ras displays differing localisation [START_REF] Fukui | Role of a ras homolog in the life cycle of Schizosaccharomyces pombe[END_REF][START_REF] Nielsen | The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription[END_REF], which is known to influence the pathways it controls, such as the byr2/byr1/spk1 mitogen-activated protein kinase cascade. It also activates the the Rho GTPase Cdc42 protein [START_REF] García | Role of Rho GTPases and Rho-GEFs in the regulation of cell shape and integrity in fission yeast[END_REF] pathway to regulate cell polarity by modulating cytoskeletal elements and maintaining elongated cell morphology and protein trafficking. The differential location of Ras raises important questions as to how do signalling proteins selectively regulate particular pathways. Mammalian and S. pombe

Ras proteins have significant sequence homology that makes S. pombe an ideal organism to study aspects of Ras signalling, as it contains only a single Ras protein.

The role of ubiquitylation

Ubiquitin (Ub) is a small (76 amino acids), compact and super-folded protein which is highly conserved in eukaryotic organisms. Between yeast and humans there are only three amino acid differences [START_REF] Shaid | Ubiquitination and selective autophagy[END_REF]. Cells use Ub as a tag to change according to cellular conditions. The cell must respond to changing conditions by producing the proteins it requires at the moment. Accordingly, it is beneficial to the cell to degrade or recycle those proteins that are not required. Ubiquitylation has evolved to be a principal mechanism where proteins are tagged by direct binding with Ub to tag them for degradation by the proteasome [START_REF] Hershko | The ubiquitin system[END_REF]. The Ub/proteasome pathway also is important to keep the cells healthy and functioning in presence of translation errors and genetic mutations that damage proteins [START_REF] Tu | The Ubiquitin Proteasome Pathway (UPP) in the regulation of cell cycle control and DNA damage repair and its implication in tumorigenesis[END_REF]. Misfolded proteins can be rescued by repair proteins called chaperones, but if not, the protein will be directed to degradation by the Ub/proteasome pathway. The Ub pathway is an important and widespread post-translational modification of proteins, but not only for degradation. Ubiquitylation guides protein function in DNA repair, protein translocation, altering protein structure, modulating activity of the target proteins, cell growth and signal transduction [START_REF] Horák | The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast[END_REF].

There are also different kinds of Ub-like proteins (UBLs), such as SUMO (small Ub-like modifier), which have some similarities in structure with Ub. In humans, there are 10 genes encoding UBLs called NEDD1 -NEDD10. Like Ub, NEDD UBLs are conserved among eukaryotes. They also have the ability to attach the target proteins to modify their function or stability, and have an important role in cell cycle control and embryogenesis [START_REF] Brown | Ubiquitylation, neddylation and the DNA damage response[END_REF]. The process of NEDD-UBL ligation is known as neddylation and it shows similarity to ubiquitylation in that NEDDs must be processed before attachment to protein substrates.

Ub ligases and deubiquitinating enzymes

Ub is added covalently to a target lysine residue on a protein target either as a single protein, as a chain of Ub proteins (poly-Ub) and/or on multiple sites on the same protein (multi-Ub). Poly-Ub is the process for tagging proteins for degradation, whereas mono-Ub generally affect protein localization and multi-Ub can affect protein function [START_REF] Wooley | The new landscape of protein ubiquitination[END_REF]. The process of ubiquitylation is a three-step process performed by what are called Ub ligases. E1 ligase is the first step and activates Ub for attachment to Ub E2 ligase, which is also called ubiquitin conjugating enzyme. Thirdly, Ub is transferred from E2 onto the C-terminus of E3, which then attaches Ub on to specific lysine residues of target proteins by ac C-terminal glycine.

E3 Ub ligases are the most abundant of the three types, because they must recognise distinct molecular targets. There are more than 600 putative E3 ligases in the human genome (Li et al., 2008a). There are two main families of E3 ligase, the HECT family and the RING E3 ligases.

The HECT family of E3 ligases transfer Ub to a target protein. The RING E3 ligases probably serve as recognition helpers for E2 to transfer Ub directly to the target [START_REF] Weissman | Themes and variations on ubiquitylation[END_REF].

If the classical function of Ub is to tag proteins for degradation, it means the function of deubiquitinating enzymes (DUBs) is likely to be protein stability. Thus, based on the on the type of Ub chain that is attached to the substrate, deubiquitinating enzymes can start to react with target proteins. There are three general roles for DUBs. First, DUBs are important for Ub maturation once synthesised, since they can be synthesized in poly Ub chains or attached to smaller peptides. Secondly, DUBs can reverse the ubiquitylation action that targets proteins for degradation and in the process maintain Ub homeostasis through Ub recycling. Thirdly, DUBs can alter the type of Ub modification of a target enzyme [START_REF] Komander | Breaking the chains: structure and function of the deubiquitinases[END_REF]. The human genome contains about 90 DUBs, but with only about 80 considered to be active [START_REF] Clague | Cellular functions of the DUBs[END_REF]. These proteins have been classed into 5 families depending on the motif architecture, UCH, OTU, MJD JAMM, and USPs. The USPs comprise the largest family DUBs with 63 members, and this family is considered to be responsible for cleaving Ub to prevent protein degradation by the proteasome. USPs have an important role to counteract the Ub ligases. In S.

pombe there are 20 DUBs, of which Ubp5 is of interest to us due to its interaction with Hid3 (Kouranti et al., 2010a).

Ubiquitylation processes in the endomembrane system

ER-associate degradation of proteins

It is interesting that most of the proteins that are ubiquitinated in a cell are newly synthesized proteins just coming off of ribosomes [START_REF] Kim | Systematic and quantitative assessment of the ubiquitin-modified proteome[END_REF]. It is thought that ubiquitylation may serve as a quality control check system for newly synthesized proteins [START_REF] Chhangani | E3 ubiquitin ligases in protein quality control mechanism[END_REF]. Within the ER there is also a quality control system that makes sure that only properly-folded, functionally-active membrane proteins are transported onto the Golgi.

It is thought that glycosylation of proteins and insertion in to the membrane makes them much more resistant to deactivation and degradation so a screening system is required to remove unfunctional proteins. This is called the ERAD system [START_REF] Kornitzer | Modes of regulation of ubiquitin-mediated protein degradation[END_REF] and associated with the Unfolded Protein Response. In humans, unproperly folded proteins appear to be recognized by molecular chaperones, such as the heat shock proteins (HSPs) and marked by phosphorylation by three particular membrane associated kinases PERK, IRE1, and ATF6. This slows down protein translation at the ER and permits targeting of unfolded proteins by an unknown E3 Ub ligase [START_REF] Buchberger | Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms[END_REF]. Ubiquitylation is the signal that the protein is to be removed from the ER membrane and sent to the proteasome for degradation. In order to help decide between which proteins are to sent for degradation and those that are kept is discrimination made by the E3 ligase, but fine-tuned by the ER resident DUBs (Zhang et al., 2013b). Except for certain components, for example the chaperones and E3 ligases used, the ERAD system is conserved in the yeasts and the study of these systems has provided much insight into the animal systems [START_REF] Buchberger | Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms[END_REF].

The SREBP signalling processes of the ER and Golgi.

The control of cholesterol levels in human cells is mediated by the Sterol Receptor Element Binding Protein Pathway (SREBP). When cholesterol levels are low, SREBP is released from the endomembrane system to activate gene involved in cholesterol biosynthesis, of which there are about 30 in humans (Espenshade and Hughes, 2007). In humans, there are two SREBP genes that encode three proteins. Each protein controls the expression of genes to make a particular class of fatty acid or cholesterol. Under conditions of high cholesterol, SREPBs are held in the ER, but under conditions of low fatty acid/cholesterol, the SREBPs are released to migrate to the Golgi. In the Golgi, the two Site proteases Site-1 and Site-2 cleave SREBP releasing the N-terminal portion to enter into the nucleus and activate the transcription of target genes [START_REF] Todd | Sterol regulatory element binding protein is a principal regulator of anaerobic gene expression in fission yeast[END_REF]. The SREBP system is conserved in S. pombe, where it regulates the synthesis of the sterol ergosterol [START_REF] Hughes | SREBP pathway responds to sterols and functions as an oxygen sensor in fission yeast[END_REF]. Interestingly, it is also the main system for low oxygen sensing and the regulation of hypoxia induced gene expression [START_REF] Hughes | SREBP pathway responds to sterols and functions as an oxygen sensor in fission yeast[END_REF]. In both humans and S. pombe, the cleavage of the N-terminus of SREBP (Sre1 in S. pombe) is initiated by their ubiquitylation by Golgi-resident E3 ubiquitin ligases [START_REF] Stewart | Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex[END_REF].

Human diseases associated with Golgi malfunction

The basic function of the Golgi complex is to locate and transport of proteins within and out of the cell. It receives proteins and lipids through the cis-face, they are modified as they traverse the Golgi and they exit through the trans-face. The failure for this process to work correctly has been associated with a number of human diseases. Alzheimer's disease is a brain disorder due to the accumulation of Beta-amyloid plaques in brain cells. It has been observed

that disruption of Golgi structure comes before the accumulation of protein plaques [START_REF] Joshi | Aβ-induced Golgi fragmentation in Alzheimer's disease enhances Aβ production[END_REF]. They report that Aβ signals Golgi fragmentation, which in turn increases APP trafficking leading to accelerated Aβ production in a feed-forward loop. The amyloid-beta (Aβ) also raises the levels of cell cycle kinase, Cdk5, which phosphorylates GM130 leading to Golgi dispersal [START_REF] Sun | Novel genetic tools reveal Cdk5's major role in Golgi fragmentation in Alzheimer's disease[END_REF]. Therefore, they concluded that there are two ways to reverse or prevent the harmful effects of beta-amyloid secretion in AD patients, first by inhibiting or a mutated Cdk5 or degrading increasing APP protein degradation through the ubiquitin-proteosome pathway. The disruption of Golgi structure has also been implicated in other neurological disorders, such as

Parkinson's and Creutzfeldt-Jakob diseases [START_REF] Gonatas | Fragmentation of the Golgi apparatus in neurodegenerative diseases and cell death[END_REF].

The previous example demonstrated how signalling processes can disrupt Golgi function. There are many examples where altered function of a Golgi protein itself results in a severe disease [START_REF] Bexiga | Human Diseases Associated with Form and Function of the Golgi Complex[END_REF]. This would be expected for a process deciding the localisation of so many numbers of proteins. In addition to that, there are many ways by which the Golgi can be affected. For example, a defect in retrograde protein transport may have as severe effects as a disruption to anterograde transport. Over the past few years, many diseases have been linked to the malfunction of the Golgi complex, including skin diseases, neurological disorders, skeletal dysplasia and connective tissue disorders. I will briefly mention some of them

here. An example of altered protein trafficking for a skin disorder is Dyschromatosis universalis hereditaria, which commonly occurs in Japan (Zhang et al., 2013a). A defect in the gene ABCB6 gene leads to accumulation of its translated protein in the Golgi causing malfunction. An example of neurological disease comes from a defect in the myelin gene, PLP1. A mutation in PLP1 causes a rare disease known as Pelizaeus-Merzbacher syndrome. This disease results from an accumulation of the misfolded PLP1 protein remaining in the ER resulting in a defect in retrograde transport of proteins from the Golgi to the ER, contributing to Golgi fragmentation [START_REF] Inoue | PLP1-related inherited dysmyelinating disorders: Pelizaeus-Merzbacher disease and spastic paraplegia type 2[END_REF]. Another Neurological disease proximal spinal muscular atrophy is caused by low expression of the SMN1 gene leading to accumulation of SMN granules in the late stage of Golgi network and stopping vesicle transport [START_REF] Ting | The spinal muscular atrophy disease protein SMN is linked to the Golgi network[END_REF]. Also, North Sea myoclonus neurological disease is where a mutant cannot localise the protein to the cis-Golgi. This has been linked to the X-linked mental retardation 1 (MRX1) and is caused by mutation in the IQSEC2 gene located on chromosome Xp11.2 [START_REF] Boissé Lomax | North Sea" progressive myoclonus epilepsy: phenotype of subjects with GOSR2 mutation[END_REF][START_REF] Corbett | A mutation in the Golgi Qb-SNARE gene GOSR2 causes progressive myoclonus epilepsy with early ataxia[END_REF].

Many malfunctioning proteins found to cause diseases have been classified under connective tissue disorders. In these cases, disrupted traffic through the Golgi does not all proper delivery of connective tissue proteins to the extracellular matrix. Examples of these are RIN2 syndrome, MACS syndrome, Gerodermia osteodysplastica, Menkes disease and Cutis laxa [START_REF] Bexiga | Human Diseases Associated with Form and Function of the Golgi Complex[END_REF]. One final example for a possible link of skeletal diseases to Golgi function is through the uncharacterized transmembrane protein TMEM165 [START_REF] Foulquier | TMEM165 deficiency causes a congenital disorder of glycosylation[END_REF]. The wild-type protein is in the Golgi complex and to the endosomal-lysosomal system. In human cells lacking this protein, trafficked protein are spread to different locations around the cell. It appears that TMEM165 has important role in Golgi glycosylation and Golgi morphological structure.

Skeletal dysplasia

There are two causes of the skeletal dysplasia Dyggve-Melchior-Clausen syndrome (DMC) that I will discuss. The first is caused by mutations that alter or eliminate the function of the RAB33B and the second is the protein DYMECLIN (DYM). DMC syndrome is characterized by short stature due to skeletal dysplasia deformities accompanied by microcephaly and intellectual delay [START_REF] Dyggve | Morquio-Ullrich's Disease: An Inborn Error of Metabolism?[END_REF]. DMC is very similar to another genetic syndrome called Smith-

McCort dysplasia (SMC). Smith-McCort dysplasia is distinguished from DMC by the lack of mental retardation, but the skeletal deformities are similar [START_REF] Khalifa | Dyggve-Melchior-Clausen syndrome: novel splice mutation with atlanto-axial subluxation[END_REF]. Both DMC and SMC are autosomal recessive disorders in that in the disease occurs when a child inherits mutant alleles from each parent and so is homozygous for mutations at that locus.

Heterozygous individuals do not appear to be affected by the disease.

parent and so is homozygous for mutations at that locus. Heterozygous individuals do not appear to be affected by the disease.

This syndrome has been reported in many countries around the world and early diagnosis is important. Preventing the disease by counselling parents to avoid passing the disease on to children is the only current means of preventing the disease, since it is a standard recessive genetic disease [START_REF] Bayrak | Dyggve-Melchior-Clausen syndrome without mental retardation (Smith-McCort dysplasia)[END_REF][START_REF] Khalifa | Dyggve-Melchior-Clausen syndrome: novel splice mutation with atlanto-axial subluxation[END_REF]. Early treatment is important to alleviate some of the aggravation of the disease, such as using surgery to join vertebrae called spinal fusion or other surgical techniques to correct obvious skeletal abnormalities.

Nowadays, pre-implantation genetic diagnosis can be used for those parents who appear to be at risk to pass the syndrome to their children. Although it is rare syndrome, it appears that the number of cases continues to increase. In 2002 there were only 58 cases known and this increased to 90 cases in 2004 [START_REF] Kandziora | Atlantoaxial instability in Dyggve-Melchior-Clausen syndrome. Case report and review of the literature[END_REF] with 15 cases attributed to nine unrelated families in Egypt with both males and females being affected by a defective DYM gene [START_REF] Santos | Portuguese case of Smith-McCort syndrome caused by a new mutation in the Dymeclin (FLJ20071) gene[END_REF].

Malfunctioning RAB33B as a cause of SmithMcCort dysplasia

RAB33B was the second locus found where mutation led to the DMC syndrome [START_REF] Alshammari | Mutation in RAB33B, which encodes a regulator of retrograde Golgi transport, defines a second Dyggve-Melchior-Clausen locus[END_REF]. RAB33B resides on chromosome 4 at position 4q31.1 and homozygosity of mutation leading to complete malfunction of the protein is the cause of DMC.

There are around 70 different RAB proteins in the human genome and they have been found in a variety of organelles [START_REF] Bem | Loss-of-function mutations in RAB18 cause Warburg micro syndrome[END_REF][START_REF] Liegel | Loss-of-function mutations in TBC1D20 cause cataracts and male infertility in blind sterile mice and Warburg micro syndrome in humans[END_REF]. RAB proteins are small intracellular GTPase involved in cellular protein trafficking. Seven of them are involved in Golgi structural maintenance, Rab1, Rab2, Rab6, Rab18, Rab33B, and Rab43. These RAB proteins likely serve to recruit other factors to Golgi membrane. Like other GTPases, such as the Ras and Rho GTPases, these cycle between a GTP-bound and a GDP-bound form. In the GTP bound form they are attached to the Golgi membrane to carry out their recruitment function. Rab

GTPases have low GTPase activity, but are activated by guanine nucleotide exchange factors (GEFs) [START_REF] Martínez-Alonso | Morpho-functional architecture of the Golgi complex of neuroendocrine cells[END_REF], which is likely their mechanism of regulation. The Rab proteins are believed to be the link between Golgi function and the regulation of protein transport [START_REF] Liu | Are Rab proteins the link between Golgi organization and membrane trafficking? Cell[END_REF] and the identification of loss of RAB33B leading to DMC is strongly supports this. RAB33B is specifically thought to be involved in retrograde protein transport through the Golgi, which also shows that anterograde and retrograde movement are closely linked [START_REF] Dupuis | Dymeclin deficiency causes postnatal microcephaly, hypomyelination and reticulum-to-Golgi trafficking defects in mice and humans[END_REF]. Several other human diseases are caused by RAB mutations including cancer. For example RAB18, RAB1GAP and RAB2GAP have been implicated Warburg Micro syndrome [START_REF] Bem | Loss-of-function mutations in RAB18 cause Warburg micro syndrome[END_REF][START_REF] Liegel | Loss-of-function mutations in TBC1D20 cause cataracts and male infertility in blind sterile mice and Warburg micro syndrome in humans[END_REF] and VPS53 in progressive cerebello-cerebral atrophy type 2 [START_REF] Feinstein | VPS53 mutations cause progressive cerebello-cerebral atrophy type 2 (PCCA2)[END_REF].

Lack of DYMECLIN function resulting in DMC syndrome.

The Dymeclin (DYM) gene responsible for DMC maps to chromosome position 18q12-21.1 [START_REF] El Ghouzzi | Mutations in a novel gene Dymeclin (FLJ20071) are responsible for Dyggve-Melchior-Clausen syndrome[END_REF]. The name DYMECLIN comes from the combination of the researchers from Denmark that first systematically reported the disease Dyggve, Melchior and Clausen [START_REF] Dyggve | Morquio-Ullrich's Disease: An Inborn Error of Metabolism?[END_REF]. This gene encodes a peripheral membrane protein located in the Golgi [START_REF] Dimitrov | The gene responsible for Dyggve-Melchior-Clausen syndrome encodes a novel peripheral membrane protein dynamically associated with the Golgi apparatus[END_REF] that is involved in protein trafficking [START_REF] Osipovich | Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic[END_REF]. The DYM protein appears to be able to shuttle back and forth between the cytosol and Golgi, thus it is likely tethered to the Golgi by fatty acid modification of the protein [START_REF] Denais | Dymeclin, the gene underlying Dyggve-Melchior-Clausen syndrome, encodes a protein integral to extracellular matrix and golgi organization and is associated with protein secretion pathways critical in bone development[END_REF][START_REF] Dimitrov | The gene responsible for Dyggve-Melchior-Clausen syndrome encodes a novel peripheral membrane protein dynamically associated with the Golgi apparatus[END_REF]. The cases of DMC that have been shown to involve DYMECLIN result from mutations in the gene that eliminate the protein [START_REF] Khalifa | Dyggve-Melchior-Clausen syndrome: novel splice mutation with atlanto-axial subluxation[END_REF]. In cells of DYM patients there were ultrastructural analomolies to organelles within cells, but the Golgi apparatus appeared normal [START_REF] El Ghouzzi | Mutations in a novel gene Dymeclin (FLJ20071) are responsible for Dyggve-Melchior-Clausen syndrome[END_REF].

Cancer as a genetic disease

The human body contains a huge number of cells and every cell, with the notable exceptions of red blood cells and platelets, contain a nucleus, where the majority of them contain 23 pairs of chromosomes, one of each inherited from the mother and father. As mentioned above for the cases of heritable genetic diseases, a pre-disposition to cancer can also be passed from parents to children. Every cell in our body has about 20,000 to 25,000 genes, of which there have been 20203 annotated proteins (www.expasy.org). This is likely to be a large underestimate of the true number of functional genes if non-coding RNAs are considered [START_REF] Griffiths-Jones | Annotating noncoding RNA genes[END_REF]. In any cell type, around half of these are activate to make a certain cell type and of this half, two different cell types differ in protein-coding genes expressed by about 2000. Thus each cell probably is different by about 2000 proteins.

Cancer is a disease when the gene expression program in cells gets out of whack.

Cancer can start from the gene expression mis-programming of one cell and the replication of this cell could lead to abnormal growth of cells in a group called a tumour. Once started, it can spread to other parts, called metastasis. Thus, the healthy tissues will be affected by these cells with over time either by the tumour growing and changing the local tissue environment or spreading to new tissues [START_REF] Bertram | The molecular biology of cancer[END_REF]. There are about 15 million new cases of cancer each year [START_REF] Ferlay | Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[END_REF]. Approximately, there are 200 types of cancers, and based on that it is classified as heterogeneous disease which may causes by different kind of factors. The most common types of cancers are breast, lung, colorectal and prostate [START_REF] Ferlay | Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[END_REF]. Many studies indicate that cancer caused by both environmental and genetic (heredity) factors. Many genetic mutations do not affect cells and cells have the ability to repair the majority of them. Not all mutations have the same effective as some prevent proteins from being made, and others alter the function of the protein made, or a mutation causes more protein made than required.

Genetic mutations can occur by different ways, such as point mutations, deletions, and insertions. Point mutations are the most common, where one base is changed to another. The effect of a mutation is also dependent on the type of cell in which the mutation occurs, for example, reproductive cells going through meiosis or somatic cells undergoing mitosis [START_REF] Wilkins | The Evolution of Meiosis From Mitosis[END_REF]. Mutations occurring in germ cells called gametes (sperm or eggs) are called hereditary or germline mutations, which is the cause of about 5 to 10 % of all cancers.

However, specific mutations within germline cells are not necessarily sufficient to initiate cancer formation. Cancer does not appear at once, and more than one mutation is needed to start tumorigenesis [START_REF] Kushi | American Cancer Society Guidelines on Nutrition and Physical Activity for Cancer Prevention: Reducing the Risk of Cancer With Healthy Food Choices and Physical Activity[END_REF]. Nevertheless, there are certain genes in which inherited mutations will cause a predisposition to cancer, if an opportunistic mutation occurs later. These types of genes are known as proto-oncogenes, such as RAS that when activated uncontrollably causes increases in hormonal signalling responses, the tumour suppressor genes p53 and pTEN, genes for DNA repair and many others [START_REF] Lee | Oncogenes and tumor suppressor genes[END_REF]. Mutations in the protein p53 have been implicated in more than 50% of all cancers [START_REF] Hollstein | Database of p53 gene somatic mutations in human tumors and cell lines[END_REF] (Bertram, 2000). Other factors, like hormones, UV/isotopic radiation, viruses, bacteria and chemical substances are commonly encountered as they are found in air, water and food. An example of how environment can determine the incidences of cancer in a population is the migration of Asian people from the far-east to America. The far east diet and little industrial/agricultural exposure led people to be classified as low-risk of prostate and breast cancer, but incidences have increased enormously in Asian populations since their immigration to America [START_REF] Kushi | American Cancer Society Guidelines on Nutrition and Physical Activity for Cancer Prevention: Reducing the Risk of Cancer With Healthy Food Choices and Physical Activity[END_REF]. A major cause of cancer is the damage to DNA resulting from exposure to an environmental factor. The damage to DNA is a major cause of the mutations discussed above. Therefore, cells have developed very complex mechanisms in order to repair DNA. For example, if a double-stranded DNA break occurs there are a number of signalling pathways that are started to induce the DNA damage response. In humans, the ataxia telangiectasia mutated (ATM) kinase is induced to phosphorylate the checkpoint kinases CHK1 and CHK2 and p53. This stop in the cell cycle allows time for the DNA repair process to be initiated. If the checkpoint is stopped for too long, then apoptosis is initiated [START_REF] Shiloh | ATM and related protein kinases: safeguarding genome integrity[END_REF]. The DNA-PK pathway is also induced to initiate non-homologous endjoining repair pathway, but it can also phosphorylate p53 to help induce its activity. It is interesting that activation of the DNA-PK pathway appears to have big effects on Golgi function.

DNA-PK directly phosphorylates the Golgi protein GOL3PH leading to Golgi breakup (Farber-Katz et al., 2014). This GOL3PH-mediated Golgi dispersal appears to be essential for cells to survive DNA damage. However, over-expression of active, phosphorylated GOL3PH can prevent cellular apoptosis by DNA damage, therefore, it provides a good example how cell activation of a protein can be oncogenic.

Ubiquitin and cancer

Ubiquitylation of proteins is one of the earliest responses of the cell to DNA damage [START_REF] Brinkmann | Regulation of the DNA damage response by ubiquitin conjugation[END_REF]. PAR polymerase 1 at double-stranded DNA breaks attracts the E3 ligase CHFR that the ubiquitylates the protein leading to its dissociation and allowing DNA repair to begin. It has also been reported that ubiquitylation of histone H2AX is important, because it then attracts the ATM kinase to initiate the check in the cell cycle. As shown above, ATM kinase phosphorylates p53 in order to help activate it. The activity of p53 is kept in check by ubiquitylation of p53 by the E3 ligase MDM2, which targets p53 for degradation by the proteasome [START_REF] Momand | The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation[END_REF]. Another E3 ligase COP1 destabilises p53 by ubiquitylation of p53 and targeting for degradation [START_REF] Dornan | The ubiquitin ligase COP1 is a critical negative regulator of p53[END_REF]. A major part of p53 stabilisation by ATM is the phosphorylation of both MDM2 and COP1, which leads to their inactivation. There are many other E3 ligases that appear to regulate p53, such as Cul4B, E6-AP and Cul4A-DDB1, as well as E4 ligases that catalyse the polyubiquitylation of proteins [START_REF] Brinkmann | Regulation of the DNA damage response by ubiquitin conjugation[END_REF]. It follows that p53 is activated by deubiquitylation and this is discussed in more detail below regarding possible functions of HID1. pTEN is also a TSG that is regulated by the ubiquitylation/deubiquitylation cycle and this is also discussed below. Therefore, it is not surprising that mutations in genes involved ubiquitin-related process had been implicated in the initiation of cancer.

Environmental stress signalling

Stresses can be defined as external factors or conditions that affect a biological system.

There are various types of environmental stresses, but are categorised into two classes, biotic and abiotic stresses. Multicellular organisms, such as animals and plants face both. [START_REF] Kim | Pathological roles of MAPK signaling pathways in human diseases[END_REF], such as the initiation of MAPK signalling pathways in response to ER stress [START_REF] Darling | The role of MAPK signalling pathways in the response to endoplasmic reticulum stress[END_REF][START_REF] Kim | Pathological roles of MAPK signaling pathways in human diseases[END_REF].

In S. pombe, the homologous pathway to the human ERK1 and ERK2 pathways is the Pmk1 MAPK pathway that is also known as the cellular integrity MAPK pathway [START_REF] Garcia | The Rho1p exchange factor Rgf1p signals upstream from the Pmk1 mitogen-activated protein kinase pathway in fission yeast[END_REF]). This appears to be an important pathway for responding to extracellular stress signals.

There is also the Spm1 MAPK pathway that appears to link environmental signals to cell morphology [START_REF] Zaitsevskaya-Carter | Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe[END_REF]). An important MAPK signalling pathway that is induced by a number of stresses is known as the Sty1-mediated MAPK pathway. Sty1 is the homolog of the human p38 MAPK and the Hog1 MAPK of S. cerevisiae. It is phosphorylated by the MAPKK Wis1 where it translocates to the nucleus to activate the transcription factor Atf1 to induce transcriptional responses [START_REF] Nguyen | Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast[END_REF].

With S. pombe having similar stress signalling processes to humans there has been a lot of interest in knowing how gene and protein expression changes in response to stress. Using microarrays, the transcriptional responses of the fission yeast Schizosaccharomyce pombe were analysed in cells exposed to five different stresses, hydrogen peroxide, heat, the heavy metal cadmium, the osmotic stress compound sorbitol, and the DNA damaging chemical methylmethane sulfonate [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF]. They identified two different types of genes changing in response to stress, a core set of genes that they called the core environmental stress response (CESR) genes and sets of genes changing for each specific stress, call the stress-specific environmental stress response (SESR) genes. The CESR genes are mainly dependent on Sty1 and Atf1 and the SESR genes have specific induction mechanism. From these early studies, global 'omics' studies have expanded to look at post-transcriptional control [START_REF] Mata | Post-transcriptional control of gene expression: a genome-wide perspective[END_REF], changes in both the transcriptome and proteome [START_REF] Schmidt | Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe[END_REF] and in the metabolome [START_REF] Pluskal | Metabolic profiling of the fission yeast S. pombe: quantification of compounds under different temperatures and genetic perturbation[END_REF]. A study to look at gene expression and protein changes in the transition from proliferation to quiescence in S. pombe showed that gene expression goes way down and the proteome changes too. However, instead of across-theboard changes, like for the mRNAs, protein levels are selectively changed and are controlled by cell size [START_REF] Marguerat | Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells[END_REF]. In this study, they also determined that most mRNAs are present in 1 to 10 copies per cell, but proteins are present from hundreds to thousands of copies. 1.6. The HID1 protein

Human HID1

A potential paralogue of DYM in humans is HID1. The two proteins have been grouped

into the same clan Golgi_traff (pfam CL0456) based on sequence and functional homology. The HID1 gene lies on chromosome 17 in a region that has been well characterised as a site where loss of heterozygosity contributes to the development of cancer [START_REF] Risk | Envoplakin, a possible candidate gene for focal NEPPK/esophageal cancer (TOC): the integration of genetic and physical maps of the TOC region on 17q25[END_REF]. The wellstudied and highly publicised tumour suppressor gene BRCA1 lies in region 17q21 [START_REF] King | The race" to clone BRCA1[END_REF].

HID1 lies nearby in the TOC region 17q25 that appeared to be associated with the onset of cancer also [START_REF] Fukino | Frequent allelic loss at the TOC locus on 17q25.1 in primary breast cancers[END_REF]. The gene that was thought to be responsible was eventually cloned and called DMC1 for Down-regulated in Multiple Cancers [START_REF] Harada | Identification of DMC1 , a novel gene in the TOC region on 17q25 . 1 that shows loss of expression in multiple human cancers[END_REF]. They classified DMC1 as a Type II Tumour Suppressor Gene, because it appeared to be reduced in expression or the transcript missing entirely in a large proportion of cancers that they studied.

The gene is on Chromosome 17 between positions 72,946,839 to 72,968,900 bp making the gene just over 22 kb long. It has 19 exons and 18 introns. It encodes a protein of 788 amino acids [START_REF] Maglott | Entrez Gene: gene-centered information at NCBI[END_REF]. General screening experiments have given an idea as to where the protein is located [START_REF] Pilot-Storck | Interactome mapping of the phosphatidylinositol 3-kinase-mammalian target of rapamycin pathway identifies deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor[END_REF][START_REF] Prasad | Human Protein Reference Database--2009 update[END_REF]. It has been found in many different tissues and organs, heart, in skeletal muscle, colon, spleen, kidney, liver and lung. It was at high levels in the brain and placenta suggesting a particular importance of this protein in these tissues. DMC1 has two synonyms by which they are both commonly referred to in databases, c17orf28 and HID1. The HID1 term is more commonly used now due to its homology with the HID-1 protein of C. elegans.

C.elegans HID-1

C. elegans has become an important model system in molecular research due to its simplicity in handling and it has similarities to human physiology. About 35% of human genes have homologues in C. elegans, and C. elegans has similar physiological behaviours, such as development of Alzheimers disease, diabetes and even depression [START_REF] Kaletta | Finding function in novel targets: C. elegans as a model organism[END_REF]. C. elegans has a short life cycle, going through an embryonic stage, four larval stages (L1-L4) and adulthood. This multicellular organism goes through its 4 larval stages under good environmental conditions, such as suitable temperature, if there is enough food and in the absence of a pheromone indicating population density. Since scientists discovered that calorie restriction and mutation affecting feeding behaviour could lead to an increased lifespan, it has become an increasingly important model organism. Furthermore, C. elegans can adopt an alternative growth form called the Dauer larval stage [START_REF] Mcgrath | Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes[END_REF], which is a reduced growth stage when environmental conditions become unsuitable or population density becomes to great (Figure 1.3). Normal growth can restart if population is diluted or food returned in the case of starvation-related Dauer formation [START_REF] Fielenbach | C. elegans dauer formation and the molecular basis of plasticity[END_REF].

The hid-1 gene of C. elegans was found in a screen looking for mutants of the nematode which adopted the Dauer formation under good growth conditions, but with the temperature slightly raised [START_REF] Ailion | Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans[END_REF]. hid-1, which stands for high-termperature induced Dauer formation, is one gene of many that have been discovered controlling dauer larva formation.

These genes can be classified into two types: Dauer formation defective (Daf-d) where larva cannot form Dauers under inducing conditions, and Dauer formation consistitutive (Daf-c) where

Dauers forms spontaneously under condition where it would grow normally. The gene hid-1 is classed as Daf-c. The Daf-c genes have become interesting due to their contribution to a pheromone sensing and signalling pathway analogous to Transformation Growth Factor Beta in humans, which plays a vital role in growth of breast cancers [START_REF] Patterson | TGF beta-related pathways. Roles in Caenorhabditis elegans development[END_REF]. The protein DAF-7 is a ligand for the receptors DAF-1 and DAF-4 to activate the transcription factors DAF-8 and DAF-14 to up-regulate genes for Dauer formation. The DAF-D proteins appear to form a system that is the antagonist of this pathway [START_REF] Patterson | TGF beta-related pathways. Roles in Caenorhabditis elegans development[END_REF]. Many other signal transduction pathways play vital role regulating dauer formation, because this stage can form under other poor growth conditions, such as high temperature or nutrient limitation. A good example is the gene daf-11, which is a (Daf-c) gene that encodes an quanylyl cyclase that appears to activate cGMP-gated channels involved in neuron development [START_REF] Coburn | A cyclic nucleotide-gated channel inhibits sensory axon outgrowth in larval and adult Caenorhabditis elegans: a distinct pathway for maintenance of sensory axon structure[END_REF]. cGMP-gated channels have a role in thermo sensation and dauer larval development [START_REF] Birnby | A transmembrane guanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate a common set of chemosensory behaviors in caenorhabditis elegans[END_REF]. A particular relevant example to my study is the function of the DAF-16

protein in insulin and oxidative stress signalling [START_REF] Dillin | Timing requirements for insulin/IGF-1 signaling in C. elegans[END_REF]. DAF-16 is the orthologue of the human FOXO4 transcription factor involved in insulin signalling [START_REF] Nakae | The FoxO transcription factors and metabolic regulation[END_REF]. In C.

elegans, insulin binds to its receptor activating the AKT kinase which phosphorylates DAF-16, thereby activating it. In humans, it is thought that deregulation of the AKT pathway via FOXO4 activation may play a key part in the development of diabetes. Interestingly, the daf-16 mutation, which is a Daf-d type, directly suppresses the hid-1 temperature induced Dauer formation phenotype [START_REF] Ailion | Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans[END_REF]. This suggested some type of antagonistic interaction between Hid-1 and DAF-16, and this could translate into a similar interaction between HID-1 and FOXO4 in humans. A potential mechanism for this has been developed through studies of both C. elegans and S. pombe.

Most of what is known about HID1 function comes from studies on C. elegans. HID-1 has been shown to be a peripheral membrane protein associated with the medial-and trans-Golgi apparatus (Wang, L. 2011), but it has also been found in dense core vesicles (DCV) and at the plasma membrane in the synaptic region of neurons [START_REF] Mesa | HID-1, a new component of the peptidergic signaling pathway[END_REF]. HID1 is highly expressed in neurons compared to other tissues in the nematode. However, like DYM, HID1

was found to dynamically associate with the Golgi membrane and its attachment is mediated by myristoylation [START_REF] Mesa | HID-1, a new component of the peptidergic signaling pathway[END_REF]. Two groups independently came to the conclusion that HID1 acts to sort neuropeptides in neuronal development and communication [START_REF] Mesa | HID-1, a new component of the peptidergic signaling pathway[END_REF][START_REF] Yu | HID-1 is a novel player in the regulation of neuropeptide sorting[END_REF]. The study of [START_REF] Yu | HID-1 is a novel player in the regulation of neuropeptide sorting[END_REF] using C. elegans investigated more closely HID-1 function in the DCVs. DCVs are formed in the trans-Golgi network (TGN), where different types of vesicles are generated. Many steps have to be carried out to convert immature DCVs to mature DCVs to be localised at the plasma membrane in order to release their proteins, peptide neurotransmitters, growth factors, amines and hormones. However, in the hid-1 mutant the DVCs produced never appeared to develop to maturity. While the luminal peptide cargoes of DCVs were reduced, the DCV membrane cargoes were increased. The researchers found that the reduction in peptide cargoes occurred because of lysosomal degradation. Therefore, it appears that HID-1 play a vital role in regulating DCV traffic by preventing the mis-sorting of peptide cargo from the plasma membrane to the lysosome.

HID1 in S. pombe

The functional orthologue of HID1 in S. pombe appears to be the protein Ftp105. I say functional orthologue because S. pombe actually has three HID1 orthologues. SPAP27G11.12 and SPBP19A11.07c are listed in PomBase as human HID1 orthologs, but they have not as of yet been published. However, a global study of protein localisation has reported that they might be located in the cytoplasm or the nucleus [START_REF] Matsuyama | ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe[END_REF]. The relationship between the three paralogs I describe in more detail in Chapter 3. Ftp105 hasn't been the subject of a particular study, but some of its function has come to light from studies investigating the function of the DUB family members in S. pombe. S. pombe is a good model for studying certain protein families like the DUBs because they have a limited number compared to humans, 20 compared to 96 (Kouranti et al., 2010a). Through tagging with GFP, it was observed that the Dub Ubp5 localises to the Golgi membrane S. pombe and this is due to interaction with Ftp105. Ftp105

was also shown to localise to the Golgi through GFP tagging.

In the Ftp105 mutant, Ubp5-GFP fluorescence becomes diffuse due to redirection to the cytosol.

In addition, through an interaction study using immunoprecipated Ftp105 through TAP-tagging, a physical interaction between Ftp105 and Ubp5 was confirmed [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. It was speculated that the function of Ftp105 may be to sequester Ubp5 to the Golgi to regulate its function. A subsequent study investigating the phosphorylation status of the S. pombe Dubs indicated that phosphorylation of Ubp5 was necessary to promote the association with Ftp105.

By this association, Ftp105 may play a role in ubiquitination processes in S. pombe and by analogy HID1 in C. elegans and humans.

Ubp5 is interesting, because it is the S. pombe orthologue of the human protein USP7/HAUSP. FOXO4 transcriptional activity is rapidly increased in response to oxidative stress by ubiquitylation [START_REF] Van Der Horst | FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP[END_REF]. Its ubiquitylation is the signal for the association of FOXO4 with USP7 in order to decrease transcriptional activity. Interestingly, lower expression of FOXO4 appears to promote gastric cancer cell line growth and increase tumour metastasis [START_REF] Su | The transcription factor FOXO4 is down-regulated and inhibits tumor proliferation and metastasis in gastric cancer[END_REF]. USP7 also plays an important role in cancer development or its prevention through two well-known tumour suppressors, p53 and pTEN. PTEN stands for Phosphatase and tensin homolog and mutation in the gene inactivating the protein have been implicated in the development of cancer. In cells that have lost pTEN phosphatase activity, the phosphoinositol-3-Kase (PI3K) signalling pathway is up-regulated leading to cancer progression. This is one of the most common ways that cancer forms [START_REF] Shaw | Ras, PI(3)K and mTOR signalling controls tumour cell growth[END_REF]. In addition, the nuclear location of pTEN is critical to the inhibition of PI3K pathway activity, which is controlled by ubiquitin. Ubiquitylation of pTEN causes it to move from the nucleus to the cytosol and so inhibiting its function as a tumour suppressor [START_REF] Song | The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network[END_REF].

Therefore, more USP7 in the cytosol/nucleus would maintain PI3K inactivation and tumour progression. The protein p53 is a strong tumour suppressor through activation of apoptotic signalling pathways. Ubiquitylation by USP7 can influence p53 in a few ways, including deubiquitination of the complex that keeps it associated with DNA to for transcriptional activation [START_REF] Zhao | Negative regulation of p53 functions by Daxx and the involvement of MDM2[END_REF], and it can directly deubiquitylate p53 in order to keep it active [START_REF] Li | Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization[END_REF]. Therefore, in contrast to the pTEN system, deubiquitylation would lead to more tumour suppressor action.

There are other examples of potential Golgi function could lead to cancer when not properly functioning, such as through the Ras GTPases. Ras is linked to mTOR signalling, which involves pTEN, through the mitogen-activated protein kinase pathway. Mutations that activate Ras or the sustained excitation of Ras signalling pathways can promote tumour growth [START_REF] Shaw | Ras, PI(3)K and mTOR signalling controls tumour cell growth[END_REF]. Not only are there Ras GTPase present in the Golgi, but Ras localisation within the cell governs their function, which is dependent in many cases on Golgimediated sorting [START_REF] Chang | Spatial segregation of Ras signaling: new evidence from fission yeast[END_REF][START_REF] Onken | Compartmentalized signaling of Ras in fission yeast[END_REF]. Furthemore, Ras activity can be affected by ubiquitinylation [START_REF] Jura | Differential modification of Ras proteins by ubiquitination[END_REF]. amino acids, which is larger than Ftp105 at 872 nucleotides long. ECM30 mutants show reduced fitness compared to wild-type strains [START_REF] Giaever | Functional profiling of the Saccharomyces cerevisiae genome[END_REF] and it has abnormal bud morphology [START_REF] Watanabe | Comprehensive and quantitative analysis of yeast deletion mutants defective in apical and isotropic bud growth[END_REF]. It has abnormal vacuolar morphology in the presence of 0.4M NaCl [START_REF] Michaillat | Identification of genes affecting vacuole membrane fragmentation in Saccharomyces cerevisiae[END_REF]. It is sensitive to a number of different stress treatments, such as hydroxyurea and heavy metals. These phenotypes can certainly be associated with disrupted protein sorting by the Golgi apparatus. A study by [START_REF] Costanzo | Charting the genetic interaction map of a cell[END_REF] to map the genetic interactions for the budding yeast, S. cerevisiae found that the proteins ECM30, UBP15, and PAR32 have a similar interaction to those genes involved in the GAP1 sorting pathway. The gene GAP1 encodes an amino acid permease present in the plasma membrane and so it is sorted by the Golgi trafficking pathway. Its levels are regulated by degradation in the vacuole, and like many plasma membrane proteins, this endocytosis and vacuolar degradation is started by ubiquitination of the protein [START_REF] Soetens | Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1[END_REF]. They validated the interactions for ECM30 by deleting each gene, ecm30∆, ubp15∆, and par32∆ mutants which cause mislocalization and less activity of the Gap1 permease phenotypes [START_REF] Costanzo | The genetic landscape of a cell[END_REF].

S.pombe as a model system

The yeasts are eukaryotic organisms that belong to the kingdom of fungi. This kingdom ranges between 1.5 to 5 million species comprising filamentous fungi, mushrooms, lichens, unicellular yeasts and both animal and plant pathogens. S.pombe is a member of the Eucarya classified under a phylum of Ascomycota that contains well-known species, such as Saccharomyces cerevisiae (baker's yeast), Aspergillus nidulans, and Neurospora crassa. It is the largest phylum within the fungal kingdom with around 65,000 species. Fungi share recent ancestor with animals making them more related to animals than to plants. A phylogenetic clade named Opisthokonta refers to the flagella that are believed to be the common ancestor of animals, fungi and protists [START_REF] Adl | The new higher level classification of eukaryotes with emphasis on the taxonomy of protists[END_REF][START_REF] Koonin | The origin and early evolution of eukaryotes in the light of phylogenomics[END_REF]. The taxonomy of S.pombe according to NCBI is the following lineage: Eukaryota/ Fungi/ Ascomycota/ Archiascomycetes/ Schizosaccharomycetales/ Schizosaccharomycetaceae/ Schizosaccharomyces. It evolved from S. cerevisiae about 1000 million years ago.

S.pombe is a unicellular fungal model organism containing three chromosomes with a small genome consisting of 13.8 million base pairs and about 4,824 genes. In terms of gene numbers it is the smallest eukaryote known [START_REF] Wood | The genome sequence of Schizosaccharomyces pombe[END_REF]. It is a cylindrical cell with a diameter of 3-4 μm and a length between 6 and 14 μm. Starvation can cause the cells to shorten making them look a lot like S. cerevisiae. The normal cell state of pombe is haploid, but it can be a stable diploid, and it has a doubling time of between 2 and 4 hours. Under conditions of stress zygotes are formed. S. pombe has many advantages as a model organism, such as fast growth on a variety of well-defined media [START_REF] Yanagida | The model unicellular eukaryote, Schizosaccharomyces pombe[END_REF]. It has very few duplicated genes, making it a good model for functional genetics. Laboratory strains are believed to be predominantly isogenic, avoiding many of the problems encountered with studies in S.

cerevisiae. But like S. cerevisiae, it is easily transformable in order to make mutants or heterologously express genes with many genetic tools, such as selective marker genes and transformation cassettes. Many groups of genes are found conserved in S. pombe and humans, but missing in other model organisms. Most importantly, it shares many features with higher eukaryotic cells, such as molecular, genetic and biochemical similarities, which can help us to study homologous genes functions and processes conserved in other organisms, such as cell cycle control. Another important advantage of studying S.pombe is that its cellular signaling pathways are similar to those of humans [START_REF] Wood | The genome sequence of Schizosaccharomyces pombe[END_REF]. It has been used mainly to study cell cycle control, mitosis, meiosis, chromosome dynamics, epigenetics, DNA repair and mRNA processing. Nowadays, S. pombe is playing a big role in post-genomics studies, because mutants can be used for RNA sequencing and proteomics approaches.

The pombe cell cycle

The main aim of the cell cycle is to transfer genetic information from a parent to two daughter cells by going through four stages: M (mitosis), G 1 (first gap phase), S (DNA synthesis)

and G 2 (second gap phase) in order to create two daughter cells [START_REF] Sherr | Principles of tumor suppression[END_REF]. The cell cycle progression is regulated by two important families of proteins the cyclin proteins and catalytic cyclin-dependent kinases (CDKs). In all eukaryotes, yeast to human, the cell division is controlled by the cyclin/CDK complex. There are different CDKs in humans, while in S. pombe yeast there is only the one CDK Cdc2 that interacts with many cyclins depending on the stage of the cell cycle [START_REF] Malumbres | Cell cycle, CDKs and cancer: a changing paradigm[END_REF]. The interaction of Cdc2 with other cyclins is a check to ensure the expression of genes necessary to carry the next phase [START_REF] Novak | Mathematical model of the fission yeast cell cycle with checkpoint controls at the G1/S, G2/M and metaphase/anaphase transitions[END_REF].

Cdc2 and interacting partners, such as cyclin Cdc13, are regulated through a negative-positive interaction through phosphorylation by Wee1 kinase, and dephosphorylation by Cdc25

phosphatase. A study of gene expression throughout the cell cycle showed that there are groups of gene expressed during each of the phases and that each phase appears to be regulated by an important TF or TF complex [START_REF] Rustici | Periodic gene expression program of the fission yeast cell cycle[END_REF]. Cell cycle control is not only genetic and must respond to the environment to make sure conditions are correct for cell division. The cell cycle and cell division can be stopped for example by nutrient limitation. Stress can also regulate the cell cycle. The MAPK stress signalling pathway through Wis1 and Sty1 are important for initiating the entry into Mitosis [START_REF] Parr | The default state of the cell: quiescence or proliferation?[END_REF][START_REF] Shiozaki | Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast[END_REF]. However, both Wis1 and Cdc2 are necessary for cells to enter and keep a quiescent cell state [START_REF] Sajiki | Genetic control of cellular quiescence in S. pombe[END_REF]. Finishing the cell cycle once started and stopping the cell cycle involve a complex interaction of genetic components [START_REF] Novak | Mathematical model of the fission yeast cell cycle with checkpoint controls at the G1/S, G2/M and metaphase/anaphase transitions[END_REF] and S. pombe represents an important model to understand the regulation of the cell cycle and identifying how cells grow and differentiate, such as cancer cells [START_REF] Malumbres | Cell cycle, CDKs and cancer: a changing paradigm[END_REF].

Proteomics

Proteomics is the study of the proteome, which has been said to be the next step from transcriptomics and refers to the large-scale analysis of proteins [START_REF] Pandey | Proteomics to study genes and genomes[END_REF]. The proteome can be defined as a complete set of proteins expressed by a cell [START_REF] Wilkins | Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it[END_REF]. Proteomics initially started with protein extracts being separated on 2-dimensional gels in order to separate the mass of proteins within a cell [START_REF] Rogowska-Wrzesinska | 2D gels still have a niche in proteomics[END_REF]. 2-D gels can be used to quantify relative amounts of proteins in cell samples through the use of protein labeling, such as DIGE, which labels proteins with fluorescent dyes, much like microarrays. Nowadays, gel-based proteomics have given way to mass-spectrometry (MS) based proteomics, particularly for being able to quantify proteins for sample comparison [START_REF] Schulze | Quantitation in mass-spectrometry-based proteomics[END_REF][START_REF] Washburn | Driving biochemical discovery with quantitative proteomics[END_REF]. The main difference between gel proteomics and MS proteomics is that MS recreates proteins from peptides, where quantification of proteins on 2-D gels comes from the entire protein and identification comes from the peptides. MS has played an important role in the development of protein quantification. Because it is fast and sensitive, it has become become the method of choice for protein identification and quantitation [START_REF] Patterson | Proteomics: the first decade and beyond[END_REF].

There are both protein-labelled techniques and label-free techniques for quantifying proteins. Labeling techniques can be done by comparing proteins from cells grown on a heavy isotope of an element found in proteins to cells grown on the corresponding light element, such as 15 N/ 14 N [START_REF] Kussmann | Profiling techniques in nutrition and health research[END_REF]. The peptides of trypsin-digested protein extracts can be tagged with a compound detectable in the mass spec and quantified that way, called isobaric tagging like the iTRAQ technology [START_REF] Gygi | Using mass spectrometry for quantitative proteomics[END_REF]. The technique becoming more and more common is a label-free technique that counts the number of times a peptide from a digested protein extract is counted in the mass spectrometer [START_REF] Kussmann | Profiling techniques in nutrition and health research[END_REF]. The general workflow in a label-free experiment is to isolate the proteins from the tissue and this can be done in ways to get only the soluble proteins, using aqueous extraction techniques or all proteins using non-aqueous solvents. The proteins can either be purified on acrylamide gels and digested with trypsin or directly digested in a tube to give the peptide mixture. The peptide mixture is then injected onto the mass spec using some type of separation device, such as liquid chromatography. A number of software programs have been developed to measure masses, count the peptide hits on the MS and convert this data into protein amounts [START_REF] Langella | Management and dissemination of MS proteomic data with PROTICdb: example of a quantitative comparison between methods of protein extraction[END_REF][START_REF] Valot | MassChroQ: a versatile tool for mass spectrometry quantification[END_REF].

There have been some proteomic studies conducted on S. pombe and the number is likely to grow in the future. As stated above, a proteomic approach was used to estimate that proteins range in amount from hundreds to thousand copies in a cell [START_REF] Carpy | Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast)[END_REF][START_REF] Marguerat | Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells[END_REF], where mRNA levels were 10-100 times less [START_REF] Marguerat | Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells[END_REF]. A study of the proteome of the sty1Δ mutant in response to oxidative stress confirmed the complexity of Sty1-mediated signalling and demonstrated that there is little correspondence between proteins levels and gene expression in response to stress. In contrast, a comparative study of the S. pombe proteome and transcriptome showed quite a bit of correlation between transcript and protein levels, especially for signalling and metabolic proteins [START_REF] Schmidt | Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe[END_REF]. What makes proteomics a particularly interesting tool is that it is designed to investigate changes in protein levels. Therefore, it is particularly well suited for studying processes where proteins are likely to change, such as those involving the ubiquitin-proteosome protein degradation pathway [START_REF] Chen | Quantitative proteomics to decipher ubiquitin signaling[END_REF]. Proteomics was used to identify proteins modified by ubiquitin [START_REF] Kim | Systematic and quantitative assessment of the ubiquitin-modified proteome[END_REF]. Label-free proteomics was been used to investigate the proteins that are differentially degraded in human cancer cells lacking a specific E3 ligase in order to identify its targets [START_REF] Burande | A label-free quantitative proteomics strategy to identify E3 ubiquitin ligase substrates targeted to proteasome degradation[END_REF]. However, it is clear that proteomics is a global analysis tool that can only generate hypotheses that need specific experimentation in order to verify them. Chapter 2 presents the methodology relating to the experimentation described in this document.

Chapter 3 describes the work to computationally analyse the HIDs and their relationships to their closest members the DYMs. Animals have both HID and DYM and their localization and action, such as dynamic association with the Golgi, appear to be very similar. The phylogenetic work was conducted to determine if there is any evidence that the two proteins have overlapping functions. It is evident that eukaryotes can have either DYM or HID or both, but animals have both. Fungi, except for rare exceptions, only have HID and plants only have DYM.

The study confirmed the presence of three HID1 orthologues in S. pombe and presented a sequence of diversification. I suggest that Ftp105 be called Hid3 in the future. Lastly, publically available data was mined in order to find potential associations of HID with other proteins that might reveal aspects of function.

Chapter 4 describes the testing of the hid mutants under different types of stresses.

Previously published data suggested that hid1+ and hid2+ may be involved in stress responses, because they are induced in response to stress [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF]. The mutants analysed were produce in the study of [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. All mutants hid1Δ, hid2Δ and hid3Δ were insensitive to a wide variety of stresses, except oxidative stress that affected both of hid1Δ and hid3Δ by greatly slowing growth of cultures. Interestingly, the growth of hid3Δ was severely affected when grown on minimal media. Why this was the case is unknown, but I speculate that hid3Δ is lacking an essential nutrient. Lastly, by use of the Golgi inhibitors brefeldin A and golgicide A, I was able to determine that Hid3 in S. pombe functions in anterograde protein transport (Figure 1.4).

Chapter 5 describes the metabolomics and proteomic analysis of the mutants.

Metabolomics is used to judge how greatly the fundamental physiology and biochemistry of cells change when a genetic mutation is introduced. Also, metabolic changes can frequently explain why cellular growth is modified. The purpose for conducting a proteomic study directly relates to the potential involvement of Hid3 in ubiquitin-mediated processes that would involve protein degradation. It is clear that proteomics is broad-scope and there can be a number of ways that affecting Golgi may change protein levels, but the first step was to determine which proteins change and by how much. Both the proteomics and the metabolomics were quite revealing in that they clearly show hid3Δ cells responding as though they were stressed with up-regulation of both stress signalling proteins and oxidative stress response proteins and down-regulation of protein synthesis genes.

Chapter 6 is the general discussion that gives the biological context of the results in terms of stress responses and metabolite and protein changes. I discuss how the results may support the idea that slow growth may be a cause of cells in a partial state of quiescence. I also present some detailed mechanisms through the protein data showing how disrupted Golgi function can alter chromatin structure and affect gene expression.

Chapter 2

Materials and Methods

Phylogenetic and computational analyses

Ortholgous and paralogous protein sequences of the HIDs and DYMs were identified by DELTA-BLAST analysis of the non-redundant protein database at NCBI. The full-length sequences matching the cut-off criteria were obtained from NCBI. All alignment and phylogenetic steps were conducted using MEGA6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. MUSCLE (Edgar, 2004) was the primary alignment algorithm used.

Growth analyses of hid mutants under stress conditions

The creation of gene replacement strains from the haploid wild-type strain (h -ade6-m26 ura4-D18 leu1-32) was done according to Bähler et al. (1998) and the detailed procedure and molecular characterisation of the mutants is given in [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. All strains were grown for ~12 h in 5 ml YES at 30 o C with shaking at 200 rpm and diluted to a cell count of 5 x 10 5 cells/ml into 5 ml of fresh media containing a the stress-inducing chemical agent. The chemical agents used, the vendor, the working and stock concentrations used and the solvent in which the agent was dissolved are given in

Determination of cell number

Three different methods were employed to determine the cell number within cultures for the growth analyses. Rough estimates of cell number and relative growth rates were obtained through spectrophotometric analysis of cultures taken at a wavelength of 600 nm. Precise numbers of cells were obtained by counting under the microscope using a Thoma haemocytometer (Life Technologies, catalogue number W30157544). Routine counting of cells for the growth analyses was done using a Nexcelom Cellometer® Mini automated cell counter and 20 μl chamber counting slides (Ozyme Inc.). For each growth experiment, the overnight culture was counted and diluted accordingly and the dilution culture recounted to obtain the starting cell number.

Genotyping of strains of S. pombe

Since many different mutants strains were used in the analysis of growth properties, strains were genotyped by PCR routinely to verify their authenticity.

Isolation of the DNA from S.pombe deletions mutants

The isolation of DNA was as follows. All strains were grown in liquid cultures of 5 ml of YES to late log-phase, and 3 ml of cells were harvested by consecutive centrifugation in a microcentrifuge spun at 6,000 rpm for 5 mins. The supernatant was removed and the pellet was washed in 1 ml of sterile distilled H 2 O containing 0.1% (w/v) Sodium azide. DNA was isolated from cell using the Wizard TM Genomic DNA isolation kit according to manufacturer's instructions (Promega). In brief, the solution was centrifuged at 6,000 rpm for 2 min to pellet the cells and the supernatant was removed. The cells were resuspended thoroughly in 300μl of a buffer containing 50mM Na 2 HPO 4 , 11.5g/l citric acid, 40mM EDTA, 1 M sorbitol, and 0.1 mg/ml Zymolyase ® 20T (Amsbio), and the tubes incubated at 37°C for 30 min to digest the cell wall.

After cooling the tubes to room temperature (~ 2 min), the cells were again pelleted by centrifugation in microfuge at 13,000rpm for 2 min, and the supernatant discarded. Next, 300μl

of Nuclei Lysis Solution was added to the tubes, the pellets were resuspended by gentle mixing and 100μl of Protein Precipitation Solution was added. The tubes were vortexed vigorously for 20 seconds and the tubes incubated on ice for 5 min. After centrifugation for 2 min and the supernatant containing the DNA was transferred to a clean 1.5ml microcentrifuge tube containing 300μl of isopropanol at ambient temperature and gently mixed by inverting the tubes until the DNA precipitate became visible. The DNA was collected by centrifugation, the supernatant was removed, and the pellet was washed with 300μl of room temperature 70%

ethanol by inverting the tubes several times. The DNA pellet was recollected by centrifugation for 2 min, the ethanol was discarded, and the pellet was allowed to dry by leaving the tube inverted on clean absorbent paper for at least 10 min. The DNA was dissolved in 50μl of DNA Rehydration Solution to which was added 1.5μl of a 1 mg/ml RNase Solution and the tubes incubated at 37°C for 15 minutes. Finally, the DNA was left to rehydrate overnight at 4°C.

Polymerase chain reaction

The primer pairs used to genotype mutant strains were 1) external to the site of insertion,

2) two sets of insert/genomic junction primers and 3) a set of internal primers for each of the marker and Hid genes. All primers were designed using Primer3 (v 0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/) and the corresponding SpHid gene sequences as templates.

All PCR primers were obtained from eurofins Genomics (http://www.eurofinsgenomics.eu).

All primers were tested by PCR on appropriate DNA or cDNA templates. PCR reactions were prepared in a volume of 10 μl consisting of 1x GoTaq Flexi Buffer, 3 mM MgCl 2 , 0.2 mM dNTP, 0.05 µM of forward and reverse primers, 0.25 Units of GoTaq DNA Polymerase, 10 ng of DNA. PCR was performed on an MJ Research thermocycler with a sequence of steps comprised of an initial denaturation step of one cycle of 1 min at 94 °C, 34 cycles of 15 sec at 94 o C, 15 sec at 55 o C, and 15 sec at 72°C, followed by an extension period of 72ºC for 5 min.

PCR products were observed by agarose (1 % w/v) gel electrophoresis using SafeView® (NBS Biologicals) as detection system.

Proteomic analyses

The strategy for combining the various mutant genotypes to yield 3 biological replicates for wild-type, negative control and hid1Δ and hid3Δ strains is given in Chapter 5.

Growth of cells

Starter cultures of 5 ml of cells for wild-type, negative control and mutant strains were grown ~ 12 hours in 5 ml volumes of YES at 30 o C with shaking at 200 rpm. The starter cultures were used to inoculate 50 ml volumes of YES to a cell count of 5 x 10 5 cells/ml, which were again incubated at 30 o C until each culture had reached a final cell count of 5 x 10 7 cells/ml. The timing of incubation was adjusted according to growth rate in order for each culture to reach this concentration of cells. Cells were harvested by centrifugation for 5 min at 6,000 rpm at 4 o C and immediately frozen in liquid N 2 . Frozen cell samples were combined at the step of homogenization to yield three independent biological replicates each containing a mixture of two mutant or control genotypes. Three independent cultures were combined to give each biological replicate of wild-type.

Extraction of total cellular protein

Total protein was extracted from a quantity of 100 mg of frozen, powdered cells (FreezerMill 6750) as specified in the Fission Yeast Handbook. The powdered cells were added to 2 ml BeadBug™ prefilled tubes (Sigma-Aldrich, Z763748), which had been cooled in liquid N 2 . A volume of 500 μl of HB buffer composed of 25 mM MOPS pH 7.2, 60 mM ßglycerophosphate, 15 mM p-nitrophenylphosphate, 15 mM MgC12, 15 mM EGTA, 1 mM DTT, 0.1 mM sodium vanadate, 1% Triton X-100, 1 mM PMSF, 1 x Yeast Protein inhibitor cocktail (Sigma-Aldrich P8215) and 1 x mammalian protease inhibitor cocktail (Sigma-Aldrich, P8340)

was added to the tube. Just as the mixture was beginning to thaw, the cells were broken open by shaking the tubes for 2 x 30 secs at a Frequency (1/s) of 30 in a TissueLyser II (Qiagen). The tubes were cooled for 30 s on ice between each shaking. The tubes were centrifuged at 13000 rpm at 4°C for 5 mins and the supernatant transferred to clean tubes for storage at -20°C.

Protein quantification and visualization

The amount of total protein in extracts was determined using the Pierce BCA™ Protein Assay Kit according to manufacturer's instructions (Perbio) and verified using the method of Bradford with the reagent purchased from Sigma-Aldrich. The BSA included within the BCA kit was used routinely to produce standard curves for quantification for both methods. The volumes of solutions for both methods were adjusted proportionally to those specified in the instructions to permit coloration reactions to be done directly in 1.5 ml microfuge tubes. Visualization of total protein extracts was done by discontinuous 1D-SDS-PAGE using 5% and 10% w/v bisacrylamide for stacking and separating gels, respectively [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. The pHs of the stacking and separating gels were 6.8 and 8.8, respectively. The running buffer was 1 x TRISglycine-SDS prepared according to [START_REF] Sambrook | Molecular cloning: A laboratory manual+ Cold Spring Harbor[END_REF]. Laemmli Sample Loading Buffer was purchased from Bio-Rad (cat # 1610747) to which 2-mercaptoethanol was added prior to preparation of samples. PageRuler™ unstained and pre-stained markers were purchased from Thermo Scientific Ltd. All PAGE was conducted using a MiniProtean™ II unit (Bio-Rad).

Proteins were visualised in-gel using QC colloidal commassie according to manufacturer's instructions (Bio-Rad).

Label-free proteomic analyses

Ten μg of total protein from each sample was loaded onto a 10% SDS-PAGE and allowed to migrate until all protein had just entered into the gel. Proteins were visualized by staining with colloidal blue (Biorad) and from each lane, a gel slice containing the protein was excised. Proteins were digested in gel using trypsin and the peptides eluted from the gel. The samples were acidified and injected into a Q-Exactive TM quadripole Orbitrap mass spectrometer (Thermo Scientific) and the data collected over a liquid chromatography run period of 120 min.

The resulting mass spectra were interrogated with Proteome Discoverer TM v 1.4 (Thermo Scientific) using the S. pombe proteome Discoverer™ v 1.4 software as template for identifying peptide masses. Proteins changing significantly among mutant and control genotypes were determined by ANOVA (threshold, p < 0.05) and only proteins with a minimum of two peptides were quantified for differential amounts. PCA was conducted in R using the work package FactoMiner. For the GO analysis, I used all proteins identified by MS data (2107 proteins) and then I used UniProt batch retrieval to convert protein names to transcript names. That resulted in total of 2309 genes. The changed genes files were constructed by taking those proteins that were 1.5-times up or down-regulated in each genotype hid1 and hid3 as compared to the negative control as explained above. Data for both genotypes were submitted together to GoMiner web server (http://discover.nci.nih.gov/gominer/GoCommandWebInterface.jsp). The settings used for the analysis were data course set to Pombase.org with the organism being S.

pombe, a bootstrap analysis with 100 randomisations of the data and the gene class size was set to range from 5-500. The analyses for the each catergory Biological Process, Cellular

Component and Molecular Function were done individually.

Immunological analyses of Hid proteins

Production of Antibodies

Antibodies were commercially produced in rabbit using synthetically generated peptides by Perbio (Thermo Scientific). The peptide sequences were designed using translation sequences of each Hid gene taken from Pombase taking into account the diversity of sequences to minimize cross-reactivity. The peptide sequences are given in Chapter 5. The immunization protocol was done over a 90 day period entailing a primary injection and 2 booster injections with Freund's adjuvant. Sera were provided from pre-immune bleeds and after first and second boosters. Two rabbits were immunized with each peptide and the rabbit with the best titer after the first booster was selected for terminal bleed. Two subsequent booster immunizations were used to increase the titer of anti-SpHid1 and anti-SpHid2 antibodies. A portion of the antibodies from the terminal bleed were purified and the purified antibodies used for testing recognition of the various Hid proteins.

Immunoblotting

Protein and peptide were loaded directly onto PVDF membrane using a slot-blot device. The visual loading of protein was done using using 0.01% Ponseau S. Immunoblotting was conducted using the WesternDot™ 625 Western Blot Kit (Invitrogen, Life Technologies). The purified anti-Hid antibodies were used for immunoblotting at a dilution of 1/500 in the appropriate incubation buffer provided with the kit. Most incubations were of first antibody were allowed to proceed for at least 12 hours. Incubations with secondary anti-rabbit antibodies were allowed to proceed for at least 2 hours. After development according to the manufacturer's instructions, the images of blots were taken using a Bio-Rad EZ-imager™.

Metabolite profiling

Metabolites were extracted from approximately 30 mg of freeze-dried S. pombe cells grown as described in Section 2.1.2.1. Metabolites were extracted according to [START_REF] Weckwerth | Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks[END_REF]) and quantified by 1 H-NMR as described in (A et al., 2004) for singlet, D for doublet, M for multiplet, and its frequency in ppm) was calculated on the assumption that the measured resonance corresponded to one proton and using an arbitrary molecular weight of 100 Da.

General Data Analysis.

Principal Component Analysis and other statistical analyses were performed in R using various work packages. Specific aspects of data analysis and how they were performed are described in the appropriate chapters.

Chapter 3

Computation analyses of the DYMECLIN protein superfamily and the HID-1 orthologues from S. pombe

INTRODUCTION

Computing has become an essential aspect of biological research. The production of large data sets has driven an interest to understand the interaction of biological components [START_REF] Wooley | The new landscape of protein ubiquitination[END_REF]. The start of any research project should begin with a detailed investigation about what is currently known on a topic, not only through literature searches, but extracting relevant information from the large number of databases. In addition, the advancement of computing has enabled investigations into aspects of biology not previously possible. From sequences of DNA we can now predict protein sequences and patterns that permit in-depth comparisons of protein function. Through phylogenetic analyses we can observe how protein function varies among organisms and also allows us to estimate the evolutionary origin of particular genes and proteins. This chapter presents important background taken from public sources that has been used as a guide for my experimentation and that of the group in general.

As presented in the general introduction of Chapter 1, the term HID, used to describe the gene and protein, was introduced in a report describing the isolation of constitutive dauer phenotype in mutants of C. elegans [START_REF] Ailion | Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans[END_REF]. The human ortholog was, until just recently, called either DMC1 for Down-Regulated in Multiple Cancers 1 [START_REF] Harada | Identification of DMC1 , a novel gene in the TOC region on 17q25 . 1 that shows loss of expression in multiple human cancers[END_REF] or C17orf28 by the nomenclature for uncharacterized genes from the human genome sequencing and annotation project. Therefore, the function of HID1 in humans is not well characterized, probably because it does not have as severe a phenotype as DYM when mutated. It appears to be more critical in the stress responses of invertebrates through the formation of Dauer larvae. It is interesting to speculate that DYM is not a lethal mutation in humans, because it could be partially complemented by HID1.

The aim of this chapter is to present currently available information resident within database to show that S.pombe is a suitable model system for investigating HID function. The computational aspect starts with a phylogenetic analysis of the relationship of the HIDs and DYMs as they are both part of the DYMECLIN superfamily. The purpose of the phylogenetic study is not to determine the accuracy of current ideas of taxonomic relationships, but simply to investigate if HID and DYM share a common evolutionary path. This may help to show if either one or both are necessary for fundamental cell function. Therefore, we find it appropriate to base our sequence sampling strategy on the taxonomic relationships presented in NCBI that have tried to include various models and not proposed a single evolutionary model. A more indepth comparison of protein properties is presented to determine common structural features among the S. pombe and animal HIDs to try to determine why Ftp105 appears to be the functional homologue of the animal HIDs. Lastly, an investigation into potential interactions of the S. pombe Hids was conducted to develop more their biological functions to guide further work.

RESULTS

Global phylogenetic analysis of HIDs and DYMs

It is interesting that pfam describes the HID motif as conserved from fungi to animals and that for the DYM as conserved from plants to animals. This would imply that 1) the proteins are only of eukaryotic origin and 2) that one or more of the major classes of eukaryotic organisms do not possess either a HID or a DYM, or both. Although a comprehensive phylogenetic and functional analysis of the HIDs represents a major work on its own, it was possible to use protein sequences present in NCBI in order to address the questions asked above. Also, it was possible to determine if the two protein families have similar evolutionary paths.

Selection of sequences from eukaryotic taxa for phylogenetic analysis

For the global phylogenetic analysis of the HIDs, we queried the Genbank nonredundant protein sequences using the HIDs and DYMs from Homo sapiens, the HID-1 orthologue from S. pombe and DYM from Arabidopsis thaliana. DELTA-BLAST with default parameters was used except that the number of sequences retrieved was set at 5000 and the significance cut-off was set at 0.01. We selected those sequences that had a significance score below the cut-off, but also for which the sequence coverage was greater than or equal to 40%.

We chose this coverage cut-off value, because we would be likely to pull-out sequences from both families using a single query sequence. For example, DELTA-BLAST with AqHID or HsHID retrieves two proteins from each of the two choanoflagellates, monosiga brevicolis and Salpingoeca rosetta, and a 40% coverage cut-off separated them into their respective families.

We also found that a 40% cut-off eliminated the vast majority of partial sequences. As described below, we identified a core sequence for the DYMECLIN superfamily that is around 200 aa, which can comprise up to 30% of the predicted full-length protein. Identification of a true core (1981,1998).

Evolutionary Genome classification HID DYM Sequence Alveolata (First Rank) Acavomonas N N N Apicomplexa Y Y Y Chromerida N N Y Ciliophora Y N Y* Colpodellidae N N N Colponemidia N N N Dinophyceae N N Y* Ellobiopsidae N N N Perkinsea Y N Y Veromonas N N N Unclassified N N N Amoebozoa (Super-group) Acromoeba N N N Archamoebae N N Y* Discosea Y Y Y Gracilipodida nd nd N Multicilia nd nd N Mycetozoa Y Y Y Phalansterium N N N Stereomyxa N N N Telaepolella N N N Tubulinea N N Y* Unclassified N N N Apusozoa (Phylum) N N Y* Breviatea N N N Centroheliozoa N N N Cryptophyta (First Rank) Cryptomonadales N N Y* Pyrenomondales Y Y Y Urgorri N N N Unclassified N N N Euglenozoa (First Rank) Diplonemida N N N Euglenida N N Y* Kinetoplastida Y Y Y Symbiontida N N N Fornicata (First Rank) N N Y* Glaucocystophyceae (First Rank) N N Y* Haptophyceae (First Rank) Coccolithales N N N Coccosphaerales N N N Isochrysidales Y Y Y Pavlovales N N Y* Phaeocystales N N Y* Prymnesiales Y Y N Reticulosphaerales N N N Syracosphaerales N N N Zygodiscales N N N Unclassified N N N Heterolobosea (First Rank) Acrasida N N Y* Creneidae N N N Pharyngomonas N N N Psalteriomonadidae N N N Schizopyrenida Y Y Y Tulamoebidae N N N Unclassified N N N Jakobida (First Rank) N N Y* Katablepharidophyta (Class) N N N Malawimonadidae (First rank) N N Y* Opisthokonta (Super-group) Choanoflagellida Y Y Y Fungi Y Y Y Metazoa Y Y Y Nucleariidae N N Y* Incertae sedia Y Y Y Oxymonadida (Second rank) N N N Parabasalia (First rank) Cristamonadida N N N Honigbergiellida N N N Hypermastigia N N N Hypotrichomonadida N N N Kofoidia N N N Spirotrichonymphida N N N Trichomonadida N Y Y Trichonymphida N N N Tritichomonadida N N N Unclassified N N N Incertae sedis N N N Rhizaria (Super-group) Acantharea N N N Cercozoa N N Y* Foraminifera Y N Y Gromiidae N N N Haplosporidia N N N Mikrocytiidae N N N Polycystinea N N N Sticholonchida N N N Unclassified N N N Rhodophyta (First rank) N N Y* Stramenopiles (First rank) Actinophryidae N N N Bacillariophyta Y Y Y Bicosoecida N N Y* Blastocystis N N Y* Bolidophyceae N N N Chrysomerophyceae N N N Chrysophyceae N N Y* Developayella nd nd N Dictyochophyceae N N N Eustigmatophyceae Y N Y Hyphochytriomycetes N N Y* Labyrinthulomycetes N N Y* Oikomonadaceae nd nd N Oomycetes Y Y Y Pelagophyceae Y N Y Phaeothamniophyceae N N N Pinguiophyceae N N N Placididea nd nd N PX clade Y N Y Raphidophyceae N N Y* Slopalinida N N Y* Synchromophyceae N N N Synurophyceae N N Y* Unclassified N N N Viridiplantae (Kingdom) Chlorophyta Y Y Y Streptophyta N Y Y Unclassified N N N Unclassified N N Y* nd No data retrievable from Genbank
structure for a targeted phylogenetic analysis based on domains was difficult at this time, because the length of this core sequence was highly variable and ranged between 70 and 400 AAs. However, a more in-depth investigation of this core structure may help to determine the minimal sequence comprising a HID or DYM motif and to unravel the evolutionary background of the DYMs.

The search was conducted using the 2 nd -rank scientific names of NCBI Taxonomy in order to determine if any particular taxon possessed either a HID or a DYM or multiple copies of either. For the search of each taxon, sequences were selected according to the criteria of similarity mentioned above, and a 2000-iteration bootstrap of the Neighbor-joining algorithm was used 1) to pare-down the number of sequences to be included in the overall phylogenetic analysis, 2) to enable segregation of sequences into HIDs and DYMs, and 3) to reveal those species with multiple HIDs and/or DYMs. In general, the sequences selected were from species for which there was a genome sequence and from species with multiple HIDs or DYMs. Details on the choice of sequences selected are given for each scientific taxon searched. A list of the taxa searched and the presence or absence of HID or DYM homologues is given in Table 3.1.

For the Alveolata, the branch Apicocomplexa, which includes the genera plasmodium and toxoplasma, produced the most hits. Interestingly, the resulting phylogenetic tree showed a larger and a small subtree. The smaller subtree of 6 sequences grouped with the HIDs (primarily) and DYMs, whereas the larger group was further removed. The smaller group was comprised of the Toxoplasma, whereas the larger group contained species of Plasmodium and Cryptosporidium from the Ciliophora. The sequence from Toxoplasma gondii GT1 (gi|523573546|) was selected for the global phylogenetic analysis, since it was more similar to the query DYMs and HIDs. The more distantly related sequences from the species of the subphyla Ciliophora were not included in the global analysis. Two sequences from the Ciliophora were selected for the global analysis, one from Oxytricha trifallax (gi|403337977|) and one from Paramecium tetraurelia strain d4-2 (gi|145523772|). One sequence from Perkinsus marinus ATCC 50983 (gi|294952762|) represented the Perkinsea. Other subphyla within the Alveolata did not contain HID or DYM homologues.

For the Amoebozoa, sequences similar to DYM and HIDs were found only in the subphyla Mycetozoa and Discosea. The two branches were analysed independently to identify sequences for the global phylogenetic analysis analysis. From the Discosea, there were three sequences from Acanthamoeba castellanii str. Neff, one (gi|470387783) that groups with the DYMs, another (gi|470454537) that groups with the HIDs and another (gi|470466316) that is more removed from the HIDs, but pulls up only HIDs with DELTA-BLAST. Each of the three sequences was included in the global phylogenetic analysis. For the Mycetozoa, the three proteins from Dictostelium Fasciculatum were also chosen, because they exhibit the same type of grouping as those from A. castellanii. One (gi|470244090|) was removed from both the DYMs and HIDs, whereas the two others, gi|470241913| and gi|470246996| grouped with the DYMs and HIDs, respectively.The search of the phylum Cryptophyta, yielded only two proteins, both of which were revealed with each of the four query sequences, and these come from Guillardia theta. This is the only member of the Cryptophyceae for which a genome sequence currently exists (http://genome.jgi.doe.gov/; [START_REF] Curtis | Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs[END_REF]. Proteins gi|551677380| and gi|551670302| align with the HIDs and DYMs, respectively, thus these two sequences were included within the global phylogenetic analysis.

Searching of the Euglenozoa yielded numerous sequences similar to both DYM and HID of the sponge proteins. However, these sequences were only within the Order Trypanosomatida. Compilation of DELTA-BLAST significant hits using each of query proteins yielded a set of 42 sequences that were used for subsequent phylogenetic analysis. Species within five represented genera of the Trypanosomatida each contained two proteins, one each that clustered with the HIDs and DYMs. The one exception was Trypsanosoma vivax, which contained a third hypothetical protein similar to both DYMs and HIDS, and pulled up these sequences on a reciprocal BLAST query. Therefore, the three sequences from T. vivax (gi|340055718|, gi|340056098|, gi|340055981|) accessions were used for the global phylogenetic analysis. Reciprocal DELTA-BLAST with the remote sequence pulls up sequences of approximately 77 amino acids, which correspond to HID isoforms from fish species of the Order Cyprinodontiformes.

For the Haptophyceae, two sequences are pulled up from the genome sequence of Emiliana huxleyi, but only one of which appears to be full-length, gi|551625400|. MSA to the four query sequences shows the full-length protein to more closely resemble the DYMs than HIDs, with coverage and significance score highest with AtDYM. Only the putative full-length sequence was included within the global phylogenetic analysis as the partial sequence more closely resembled the paralogue than any of the potential orthologues.

For the Heterolobosea, the same two proteins from Naegleria gruberi were pulled up with each of the four query sequences. The protein gi|290972455| clusters with the HIDs and gi|291000556| aligns with the DYMs.

The Opisthokonta have both DYMs and HIDs in the metazoa and fungi, but which were restricted to specific orders. The taxa Incertae sedis of the Opisthokonts has the genus Capsaspora, also with both a DYM and a HID and others of unknown function. BLAST analysis of the fungi revealed two apparent paralogues of ftp105, which we term SpHid2 and SpHid3 according to divergence and we discuss these in more detail below.

Search of the Parabasalia pulls up only one protein from Trichomonas vaginalis G3 (gi|123457024|), which has greatest similarity to the DYMs.

Search of Rhizaria pulls up a protein from Reticulomyxa filose, which is significant, but only over a very short sequence of 26% query cover of the AtDYM with 37% identity. It has an identifiable DYM domain signature of 72 aa from aa 204 to 276. It is a marine protist and reciprocal blast pulls up HID homologues from primarily aquatic creatures through this domain.

However, this domain also appears in terrestrial animals. DELTA-BLAST also recognizes another domain in subjects which corresponds to a short sequence > 70 aa starting at aa 420. This second domain is not recognized as a Dymeclin signature. Due to its short-sequence coverage, it was not included in the global phylogenetic analysis.

In order to determine which subfamily was present in the Viridiplantae, the Streptophyta and the Chlorophyta were searched independently using the each of the four query proteins.

Each query pulled up the approximately the same number of significant hits, indicating that the search parameters were stringent enough to identify members of the other family. The union of the sequences retrieved were then aligned and subjected to phylogenetic analysis using Neighbor Joining and 2000 bootstrap replicates. The resulting phylogenetic tree showed that all Streptophyta proteins grouped with the DYMs. The proteins from Oryza sativa, Arabidopsis thaliana, and the lycophyte (Lycopsida) Selaginella moellendorffii were used in the global phylogenetic analysis. Inspection of the resulting Chlorophyta tree indicates that the sequences from the Trebouxiophyceae more closely resemble AtDYM whereas those from the Prasinophytes are more variable. The sequence from Bathyococcus prasinos (gi|612400476| and one from Micromonas pusilla (gi|003056438| are clearly related to the DYMs, but the sequences from the two Ostreococcus species were further removed from even AqDYM, and one sequences from Micromonas (gi|002504004| grouped with the HIDs. Therefore, the sequences selected for the global phylogenetic analysis were Coccomyxa subellipsoidea C-169 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. The alignment was not manually modified prior to tree construction. The values represents the proportion of trees whereby removal of a sequence does not affect the overall topology of the tree. The lower the value the weaker is the support for the pairing. The scale bar represents the proportion of substitutions per site.

Global phylogenetic analysis

For the global phylogenetic, the set of protein sequences contained representative HIDs and DYMs from the BLAST searches of the individual taxa and the resulting phylogenetic tree construction. The set of proteins used contained 32 HIDs and 29 DYMs. Protein sequences from a variety of Metazoan species were used in order to determine if there was a consistent pattern of divergence between proteins within this particular taxon. This was only possible with the Metazoans noting that HIDs and DYMs were missing from the Viridiplantae and the Fungi, respectively. Phylogenetic trees were produced in Mega6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF] using both the Neighbour-Joining or Maximum-likelihood algorithms with at least 1000 bootstrap replicates.

Both boot-strap and the boot-strap consensus trees from the Neighbour-Joining analysis is shown in Figure 3.2.

The overall structure of the trees shows two distinct families of proteins with the provisional designations correctly chosen. Starting from the top of the trees and the Metazoa, it was apparent that there is a continual divergence from the human HID-1 through the Metazoa with the C. owczarzaki and the choanoflagellate M. brevicolis and S. rosetta HIDs being the next most similar. Following on in divergence were HIDs from the other First-rank taxa, including the only HID from the viridiplantae, that from the Chlorophyta species Micromonas sp. RCC299.

The fungal HIDs grouped together, except for ECM30 of S. cerevisiae, but were further diverged from the Metazoa than species from the other Supergroups. The most divergent HIDs included the ECM30 from S. cerevisiae, which paired with that from the Alveolata P. tetraurelia, and a second HID from the Amoeba A. Castellanii and a second HID from the Euglenozoan T. vivax.

The grouping of the Metazoa DYMs was similar to that for the HIDs with the species C. owczarzaki also part of the clade. Within this apparent clade were also the DYMs found within the Mucorales (fungal) species P. parasitica and A. idahoensis. Although not well supported by the bootstrap analysis, the adjoining clade contained four sequences, each from different firstrank taxa the Heterolobosea, Cryptophyta, Parabasalia and Amoebozoa. The next clade contained the DYMs from the Stramenopiles and Haptophyceae species. The Viridiplantae (Chlorophyta and Streptophyta) DYMs formed a separate clade one step further removed, whereas the furthest removed sequences were from the Amoeba A. castellanii, the Euglenozoa T. vivax, and lastly the sequences from the two choanoflagellate species.

The family of HID proteins contained species with apparent paralogues. Those most relevant to this work are the two paralogues of the S. pombe ftp105 and these will be discussed in more detail below. Not all paralogues grouped together for a species like they did in S. pombe and P. parasitica. The amoeba A castellanii has two apparent HIDs. One HID paired with that

Table 3.2. Quantitative comparison of primary sequences between members of the HID and DYM superfamily.

The values for sequence identity and similarity were calculated using the Needle algorithm for pairwise sequence alignment from the EMBOSS suite of sequence analysis tools using the default settings (www.ebi.ac.uk; [START_REF] Mcwilliam | Analysis Tool Web Services from the EMBL-EBI[END_REF]. likelihood tree differed from that of the Neighbour-joining one. It is expected that for the more diverse sequences where there is low bootstrap support for associations, that there would be differences among the various trees constructed.

A search for a core motif that could be used to facilitate alignment of sequences for the phylogenetic analysis, and which could be used to search early branching eukaryotic and bacterial species for potential ancestral proteins, has not been successful. This is because neither a definite HID nor a DYM protein family signature was clear. For the vast majority of sequences, the entire protein was designated as a HID or DYM motif. In pfam, DYM and HID1

Functional characterisation of the S. pombe HIDs

Comparison of primary sequences to eukaryotic orthologues

The BLAST analysis of the Opisthokonta Super-group revealed that S. pombe contains three putative HID1 homologues. This was confirmation of the information already present in PomBase (www.pombase.org). The question then arose as if they were paralogues or if they were orthologous to other proteins. The large phylogenetic analysis suggests that they are paralogous due to similarity in sequence. At what point the paralogues came into existence could be addressed through an in-depth phylogenetic study of the Fungi, but such a study falls outside the scope of the chapter. Nevertheless, it is apparent that not all Fungi have 3 HIDs.

The yeast S. cerevisiae appears to have one HID homologue ECM30 (Figure 3.2) and its sequence has greatly diverged from those of S. pombe. In order to determine the S. pombe HID that appears to be the direct orthologue of HID-1 from C. elegans and to establish the proper nomenclature for the S. pombe HIDs a multiple sequence alignment was conducted with the proteins HsHID-1, CeHID1, Ftp105, and the two Ftp105 paralogues, called hid1 and hid2 (Figure 3.3). The names of the two ftp105 orthologues were taken from PomBase. The pairwise identities and similarities are given in Table 3.2. SpHid1 and Ftp105 had nearly the same level of identity and similarity to CeHID1 and HsHID1 with just over 20% and 40%, respectively.

SpHid2 was slightly more divergent with less than 20% identity and around 35% similarity.

SpHid2 was almost as divergent from the other two S. pombe proteins as from those from humans and C. elegans. The pairwise comparisons with the DYMs from humans (HsDYM), C. elegans (CeDYM) and Arabidopsis (AtDYM) were also included to give a better idea of diversification than was obtainable from the trees shown in Figure 3.2. Interestingly, the degree of identity and similarity between the plant and animal DYMs was equal to or greater than those between the pombe and animals HIDs (Table 3.2).

Comparative structural properties of the HIDs from S. pombe

A search of the Gene Ontology Database AmiGO 2 revealed aspects of putative functions and/or roles for each of the S. pombe Hids (Table 3.3). From a genome-wide screen of protein localization, there is evidence that Hid1 and Hid2 can localize both to the cytosol and nucleus [START_REF] Matsuyama | ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe[END_REF], but no molecular or biological function has been given to these proteins. The same study put Hid3 into both the golgi and cytosol. There is some information about the molecular and biological functions of Hid3 that came from a study on the on the deubiquitinating family of enzymes in S. pombe [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. This study showed that this enzyme is involved in the transport of Ubp5 from the cytosol into the Golgi apparatus. Therefore, it has the molecular function of protein binding and the biological function of protein transport and localization.

Proteins are organised into structural units called domains that may have distinct functions [START_REF] Alberts | Molecular Biology of the Cell[END_REF]. Most eukaryotic proteins contain multiple domains that have a vital role in protein-protein interactions and interaction networks. Hid3 localization to the Golgi membrane and its involvement in the transport of Ubp5 from the cytosol into the Golgi would require it to have domains or features giving it protein and membrane interaction properties. Also, it is expected that the protein features of Hid1 and Hid2 will have diversified sufficiently from Hid3 in order to be able to detect particular differences among the proteins. These differences were tested by comparing domain features among HIDs from various organisms.

The ProDom database (prodom.prabi.fr, [START_REF] Servant | ProDom: automated clustering of homologous domains[END_REF] contains 56 different arrangements of domains covering 94 proteins. Included within this set is HsHID1, CeHID1, SpHid1, SpHid2 and ftp105. The structure of these proteins is mainly determined by the number and placement of the putative transmembrane helices (Figure 3.4). Prodom predicted seven transmembrane helices as has been previously reported [START_REF] Wang | HID-1 is a peripheral membrane protein primarily associated with the medial-and trans-Golgi apparatus[END_REF]. CeHID1 retained 6 of the corresponding transmembrane helices. The closest domain architecture of the S. pombe sequences to either of the animals was that for Hid1. In all three S. pombe Hids, the C-terminal helices have been replaced by long cytosolic domains. These C-terminal domains have sufficiently diversified that they are not considered to be similar according to ProDom.

Prediction of genetic and physical interacting partners

STRING database v8 [START_REF] Jensen | STRING 8--a global view on proteins and their functional interactions in 630 organisms[END_REF] was the starting point for investigating potential interacting partners for the 3 Hids proteins (Figure 3.5). This database has had frequent updates and so has been continually searched for relevant information [START_REF] Szklarczyk | STRING v10: protein-protein interaction networks, integrated over the tree of life[END_REF]. Other potential interactions, both physical and genetic have been explored through PomBase [START_REF] Wood | PomBase: a comprehensive online resource for fission yeast[END_REF]. STRING offered a convenient and logical way of searching for potential interactions by pulling together information from experiments to text-based associations. In order to develop working hypothesis for Hid proteins as strong as possible, associations based only on either co-expression or direct experimental evidence was considered. For Hid1 and Hid2, only interactions based on co-expression have been considered, since there is no current experimental evidence for any physical interaction. Gene STRING showed the known physical interaction of Hid3 with Ubp5, which is a deubiquitinase that is located in the Golgi through the action of Hid3 (Figure 3.5C). The other gene was bun107 that encodes a WD repeat protein that is involved in the positive regulation of ubiquitin protease activity and vesicle secretion. The human orthologue of Bun107 is WDR48, which is necessary for activation of deubiquitinating enzymes. The human HID1 was also queried (Figure 3.5D). USP7 is the orthologue of Ubp5 and so was shown to be associated based on the experimental evidence obtained from pombe. CSDE1 is a cold-shock domaincontaining protein for which evidence suggests is an RNA-binding protein [START_REF] Grosset | A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex[END_REF]. INPPL1 is a phosphoinositol phosphatase that negatively regulates the phosphoinositol signaling pathway [START_REF] Pilot-Storck | Interactome mapping of the phosphatidylinositol 3-kinase-mammalian target of rapamycin pathway identifies deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor[END_REF], but the evidence for this association is indirect. The association of HID1 with RAD51 is unlikely and has been pulled up through an overlap in nomenclature with DMC1 involved in DNA repair. A STRING search with the C. elegans hid-1 protein also pulls up interactions based on experimental evidence with a few proteins involved in ubiquitination processes, such as the E3 ubiquitin ligase wwp-1 and the deubiquitinase math-33 (data not shown).

The disruption of proper localisation of Ubp5 in ftp105Δ indicates that Ftp105 is capable of physical interaction with it [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. It is not known if Ftp105 can physically interact with other proteins, like those mentioned above. The other type of interaction is genetic, where alteration in expression of the protein will affect the expression or activity of other proteins necessary for normal cell function. This genetic interaction can also be extended to the idea that the expression of two genes contributes to a phenotype and that such interaction can be investigated by determining the effects on growth of double mutant genotypes [START_REF] Schuldiner | Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile[END_REF]. The basis of these types of genetic interaction studies is to identify potentially physically interacting partners [START_REF] Kelley | Systematic interpretation of genetic interactions using protein networks[END_REF]. Two major studies have presented genetic interaction maps that have been summarized in Pombase [START_REF] Roguev | Conservation and rewiring of functional modules revealed by an epistasis map in fission yeast[END_REF][START_REF] Ryan | Hierarchical modularity and the evolution of genetic interactomes across species[END_REF]. There are six interactions for hid1 only one of which is a positive genetic interaction with SPAC1952.06c, which is a predicted member of the spliceosomal complex [START_REF] Ryan | Hierarchical modularity and the evolution of genetic interactomes across species[END_REF]. Ftp105 was included in the original epistasis map construction due to its potential involvement in chromosome structure [START_REF] Roguev | Conservation and rewiring of functional modules revealed by an epistasis map in fission yeast[END_REF]. Of the 101 genetic interactions identified only 27 were positive. The only conclusion that can be made is that because the vast majorities are negative, a mutation in hid3 is more likely to have a detrimental than a positive effect on growth.

DISCUSSION

The purpose of this bioinformatic study was to determine at the start if the S. pombe

HIDs represented good target to study as models for the function of HID1 in humans. This could also relate to understanding DYM function as well, because HID and DYM proteins show common structural features at the level of protein sequence. The first objective was to determine if HIDs had a similar evolutionary background to the DYMs that might indicate similar function. It is clear that both HIDs and DYMs came into existence with the appearance of eukaryotic organisms. The early appearance of eukaryotes was a time of a great genetic explosion when gene families specific to eukaryotes arose [START_REF] Koonin | The origin and early evolution of eukaryotes in the light of phylogenomics[END_REF]. The phylogenetic comparison of HID and DYMs across the range of eukaryotic taxa revealed that the two proteins have quite different evolutionary paths. It is apparent that the lineage of neither protein conforms to the current idea of eukaryotic supergroups [START_REF] Baldauf | The deep roots of eukaryotes[END_REF] and which corresponds to poor support for this idea in recent molecular evolution studies [START_REF] Yoon | Broadly sampled multigene trees of eukaryotes[END_REF]. The major findings from the phylogenetic study were 1) the confirmation that fungi have only HID and that fungi have multiple paralogues of HID, 2) that the Viridiplantae have only DYM, 3) some taxa have neither, and 4) evidence for horizontal gene transfer within the DYM superfamily.

Having DYM or HID is not a requirement for a cell being eukaryotic as shown by a vast number of organisms not having DYM or HID. Although, we have not identified a multicellular eukaryote with the exception of species of linked algae, that do not have one or the other. Thus, it is interesting to speculate that having either is necessary for cellular communication that makes multicellularism possible, but it also suggests there may be functional overlap. The major differences that have been noted between the lineages of the two proteins, such as the HID and DYM present within the choanoflagellates, the presence of DYM within the fungal family Mucorales, and the apparently different evolutionary rates of the HIDs and DYMs within fungi and plants, respectively, directly suggest that horizontal gene transfer took place at some point within their evolutionary histories, which is known to happen between eukaryotes [START_REF] Keeling | Horizontal gene transfer in eukaryotic evolution[END_REF]. Species of Mucorales are parasitic and they can infect animals, plants or each other. For example, species of absidia can infect animals or plants and species of parasitica can infect and pass or retain genes from the absidia [START_REF] Hoffmann | The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies[END_REF][START_REF] Kellner | Transfer of genetic information from the mycoparasite Parasitella parasitica to its host Absidia glauca[END_REF].

Because the DYMs are restricted to the mucorales, it is interesting to speculate that they may have obtained them from another eukaryote, in this case from a non-fungal Opisthokont. Far reaching speculation is that they subsequently transferred them to the Viridiplantae, noting that the plant DYMs are more evolutionarily conserved to animals than the HIDs are within fungi.

The phylogeny is an ongoing process that will require more detailed analysis of sequence properties. There are a number of sequences that fell below the criteria for selection within the global phylogenetic analysis that could provide more information as to early common features between the HIDs and DYMs. Also, it would be useful to try to root the trees using a more divergent sequence, such as c10orf76, which is the most similar protein pulled up by the BLAST searches.

The phylogenetic analysis confirmed S. pombe to have 3 HIDs. They most likely evolutionary model is that there was one HID ancestoral gene in fungi and that this diverged to produce the ancestor of hid2 and the ancestor of hid1 and ftp105, with the latter two having duplicated more recently. Using conventional nomenclature Ftp105 will now be called Hid3 in subsequent chapters. Although the predicted protein topology of Hid one appears more similar to that of the HID-1 of C. elegans, it appears that Hid3 retained the interaction properties that allow it to associate with the Golgi membrane [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. HIDs from both vertebrates and invertebrates appear to associate with the Golgi membrane in a dynamic manner through N-terminal myristoylation [START_REF] Mesa | HID-1, a new component of the peptidergic signaling pathway[END_REF][START_REF] Wang | HID-1 is a peripheral membrane protein primarily associated with the medial-and trans-Golgi apparatus[END_REF] so protein topology has little to do with membrane interaction. After the duplication of hid1 and hid3, Hid1 was retained and its functional diversification to a cytosolic location provided some advantage to fungal growth. The known localisation of Hid3 is consistent with a dynamic shuttling between the cytosol and Golgi membrane with its interaction helped by anchoring through a fatty acid. N-myristoylation occurs at Gly2 once the terminal methionine is removed. All S. pombe Hids start with M-G so it remains unclear as to why Hid3 alone is a Golgi protein.

S.pombe represents a good model to study HID function in that it can be studied without the potential complications of DYM being present. However, in order for it to have implications for medical research, it would have to still have some of the same interactions that are observed for human HID1. The STRING database search was useful in highlighting some aspects of similar function. Although the associations derived from one organism are used to suggest interactions for another, the network interactions confirm that homologues of interacting partners exist and that function is conserved. There is evidence that the animal HID1 physically interacts with CSDE1, which is an RNA-binding protein involved in post-transcriptional regulation of gene expression [START_REF] Mihailovich | Eukaryotic cold shock domain proteins: highly versatile regulators of gene expression[END_REF] and cell morphology and dynamics [START_REF] Kobayashi | The control of precerebellar neuron migration by RNA-binding protein Csde1[END_REF]. The closest S. pombe homologue of CSDE1 appears to be Prp22, an ATP-dependent RNA helicase. Importantly, the link between HID function and ubiquitin-related processes appears to be conserved between fungi and animals through the direct interaction of Hid3 with Ubp5. For Humans, USP7 is the functional homologue of S. pombe Ubp5, thus the association of HID1 with USP7 in human cells could be investigated. I have proposed a simple model by which altering levels of Hid3 or its complete elimination by mutation would have effects on protein levels and indirectly on gene expression and metabolism (Figure 3.6). This model shows that Hid function needs to be studied at the level of both gene and protein expression and for its influence on metabolism.

Chapter 4

Effect of stress treatments on the growth of hidΔ mutants

INTRODUCTION

The HID1 protein of C. elegans has found to be part of the switching mechanism located in the Golgi [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF], HID1 targeting to the Golgi in C. elegans is sensitive to BFA [START_REF] Wang | HID-1 is a peripheral membrane protein primarily associated with the medial-and trans-Golgi apparatus[END_REF], and the Golgi structure appeared to be disrupted in S. pombe lacking Hid3.

In addition, the growth of the hid3Δ mutant of S. pombe is compromised and so it was essential to check if the poor growth phenotype was exacerbated by external stresses.

HID-1 of C. elegans has been associated with Dense Core Vesicles and can be transported to pre-synaptic regions of the plasma membrane in neurons. It is responsible for the correct localization of the of GTPase RAB-27 and has been implicated in neuropeptide transport [START_REF] Mesa | HID-1, a new component of the peptidergic signaling pathway[END_REF][START_REF] Wang | HID-1 is a peripheral membrane protein primarily associated with the medial-and trans-Golgi apparatus[END_REF]. HID-1 has also been associated with the medial-and trans-Golgi of C. elegans where it appears to be attached to the membrane through a myristoyl anchor [START_REF] Mesa | HID-1, a new component of the peptidergic signaling pathway[END_REF][START_REF] Wang | HID-1 is a peripheral membrane protein primarily associated with the medial-and trans-Golgi apparatus[END_REF]. Thus, the evidence obtained with the C. elegans HID-1 suggests that when present in the Golgi it is primarily involved in anterograde transport of peptides or proteins. With the recycling of components from the plasma membrane to the Golgi and from the Golgi to the ER, it is very difficult to determine if a Golgi protein is involved specifically in anterograde or retrograde transport [START_REF] Sáenz | Golgicide A reveals essential roles for GBF1 in Golgi assembly and function[END_REF].

RESULTS

An indication that a protein is involved in a the cellular mechanism to respond to and to cope with external stresses is that the gene encoding it changes expression by a stress treatment. This can either be up-regulation if the protein is needed for a new function or to increase a process or down-regulation if a particular process must be de-repressed. This is different from processes that may be down-regulated because they are non-essential or even detrimental to the tolerance of a cell to stress, although this down-regulation may be tightly controlled and important for cell survival. Up-regulated or activated PKases and TFs and downregulated or inactivated PPases are examples of a system of opposite type of control [START_REF] Shiozaki | Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast[END_REF][START_REF] Zhou | MAP kinase kinase kinase (MAPKKK)-dependent and -independent activation of Sty1 stress MAPK in fission yeast[END_REF]. The study of global gene expression in response to stress conducted by [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF] presented a good resource for a first look into the stress response gene expression of each of the hids. They studied 5 different types of stresses through separate treatments: oxidative (H 2 O 2 ), heavy metal (Cadmium), abiotic (Heat); described by [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF]. The effect on growth was determined by cell count of cells incubated in YES liquid culture over period of 12 hours for the following treatments: mitomycin C (MTC), camptothecin (CPT), hydroxyurea (HU) and methyl methane sulfonate (MMS). UV light treatment was done on colonies on petri dishes containing standard YES/agar media and monitored for at least 3 days following exposure .The letters N and Y denote 'no' difference or an 'apparent' difference, respectively, compared to negative controls VCN or VCU. Each genotype was tested at least twice with each stress treatment.

Stress treatment

Strain UV light MTC CPT HU MMS rad3∆ nd nd Y Y Y hid1∆::natMX6 iso1 N N N N N hid1∆::natMX6 iso3 N N N N N hid3∆::natMX6 iso1 N N N N N hid3∆::natMX6 iso2 N N N N N hid2∆::ura4 + N N N N N
osmotic (sorbitol) and DNA damage (MMS). The data showing the expression of each of the three hids is shown in Figure 4.1. The gene hid1 was up-regulated within the first 15 min in response to oxidative, heavy metal and heat stress. It was not up-regulated in response to osmotic stress and there was a delay in its up-regulation in response to DNA damage. The gene hid2 was also quickly up-regulated in response to oxidative, heavy metal and heat stresses, but remained unchanged in response to osmotic stress and DNA damage. The expression of hid3 was unchanged, if not decreased, in response to any of the stresses. It should be noted that no hid showed a dramatic increase in expression, but even a 2-fold increase observed for hid1 and hid2 suggested that these proteins play an active role in response to stress.

Effects of stress treatments on growth of cells lacking Hids

A role in cellular stress response was examined by testing the growth mutant genotypes under different stress treatments. Pombase lists no stresses for which hid1 and hid2 mutants have been tested, however, the hid3 mutant was reported to be sensitive to salt and heavy metal stress [START_REF] Kennedy | A genome-wide screen of genes involved in cadmium tolerance in Schizosaccharomyces pombe[END_REF]. Heat was also tested, because HID-1 of C. elegans had been identified in the process of mutant was identified the mutant of the yeast orthologue of the Hids, ECM30, is also sensitive to a number of different treatments and stresses, such as HU, but appears to be temperature insensitive (www.yeastgenome.org). Therefore, the mutants were tested using NaCl and a variety of other stresses to see if they behave differently to wild type and VC genotypes. In certain case control strains were used in order to check that the stress condition was working. For the action of the microtubule-destabilising compound thiabendazole (TBZ), the spindle assembly checkpoint kinase mutant bub1Δ was used [START_REF] Bernard | Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis[END_REF][START_REF] Kadura | The A78V mutation in the Mad3-like domain of Schizosaccharomyces pombe Bub1p perturbs nuclear accumulation and kinetochore targeting of Bub1p, Bub3p, and Mad3p and spindle assembly checkpoint function[END_REF]. To check the action of the various DNA damaging agents, such as hydroxyurea (HU), Camptothecin (CPT) and Methyl Methanesulfonate (MMS), the check-point defective mutant strain rad3Δ [START_REF] Bentley | The Schizosaccharomyces pombe rad3 checkpoint gene[END_REF] was used due to its hypersensitivity to these agents.

The stress treatments were divided into two categories, general stresses (Table 4.1) and DNA damaging agents (Table 4.2). For those stress that I tested, each genotype was tried at least twice to check for any substantial difference from either WT or VC strains. The finding have been summarised in the tables, since the only stress for which there was any noticeable difference was for hid1Δ and hid3Δ under oxidative stress. At an H 2 O 2 concentration of 0.7 mM, the generation times for both hid1Δ::natMX6 iso1 and hid3Δ::natMX6 iso1 were increased 3-4 fold from about 3.5 hours to A volume of the starter culture was added to 5 ml of YES medium containing 0.7 mM H 2 O 2 to give a starting cell concentration of 5x10 5 cells/ml. An aliquot was taken to count the initial culture and the rest of the culture was incubated for 12 hours under the conditions above. The generation time was calculated using the cell concentrations at 0 and 12 hours. Each strain was tested at least 3 times. Two-tailed, Student's t-tests for unequal variance were used to determine statistical significance between genotypes. The mutant rad3 was statistically different from WT at P < 0.01. The genotypes hid1∆::natMX6 iso1, hid3∆::natMX6 iso1, hid3∆::natMX6 iso2, hid3∆::kanMX6 hid2∆:: natMX6 iso1 and hid3∆::kanMX6 hid2∆:: natMX6 iso2 were statistically different from both WT and VCN at P < 0.01. The other mutant genotypes were not significantly different from WT or the corresponding VC at P = 0.05. more than 12 hours (Figure 4.2). The hid3Δ::natMX6 iso2 mutant proliferated slooly under these conditions that the calculated generation time could not be considered accurate as shown by the large variation in values. The hid2Δ::ura4 + mutant was not sensitive to H 2 O 2 , and interestingly, when the hid1 deletion was incorporated into the hid2Δ genetic background, the double mutant was not H 2 O 2 sensitive like the hid1Δ single mutants. In contrast, the hid3Δ hid2Δ double mutants remained H 2 O 2 sensitive.

Another stress that I had attempted to use was iron limitation. Initially, iron limitation was tested using the compound Ferrozine, which is a potent iron chelator, and when in the medium prevents iron from entering cells. Ferrozine was used at a concentration of 50 μM in both YES and EMM+S medium. There was no effect of Ferrozine on growth of mutant genotypes using YES, but it came unexpectedly that hid3Δ genotypes would not grow in liquid minimal-media cultures with or without Ferrozine. This growth defect displayed by hid3Δ in minimal media was compared with wild type (wt), Vector Control (VC) and other mutant genotypes using standard drop tests on agar plates containing only EMM with added supplements (Figure 4.3). On YES/agar media, all genotypes grew almost equally well with even the few cells plated at the lowest concentration producing colonies. In contrast, every genotype experiences some difficulty growing on minimal media alone and the hid3Δ mutants grew particularly poorly with cell growth only visible at the highest concentration of cells spotted.

hid3Δ mutants are resistant to Brefeldin A and sensitive to Golgicide A

Brefeldin A (BFA) and Golgicide A (GCA) are standard compounds used to disrupt Golgi function and are used, respectively, to discriminate between anterograde and retrograde protein transport through the Golgi. Since Hid3 is a Golgi membrane protein, the combination of the two compounds was used to determine if Hid3 worked preferentially in either direction. Preliminary tests with at various concentrations of BFA showed that concentrations of 25 μg/ml or more were lethal to cells and increased estimated generation times to 17 h or more for all genotypes.

Therefore, a range of BFA concentrations from 0 μg/ml to 15 μg/ml were used in order to try to observe differences in growth among genotyopes. There was no significant effect of BFA on WT generation time at 5 μg/ml, but the average generation time increased slightly. There was a statistically significant increase in generation time for wild type at 15 μg/ml (Figure 4.4A).

Although not significant, the VCN also showed this trend of increasing generation time with increasing concentration of BFA to 15 μg/ml. I observed the increase in generation time of hid3Δ mutants under standard growth conditions that had already been reported (Ashehri 2015). In contrast to WT and VCN, growth of the hid3Δ mutants was not affected by the lower concentration of BFA.

In order to increase confidence that the differences in growth were, a different statistical test was used. The Dunnett's test compared differences in means in relation to one particular sample, i.e. WT at each of the concentrations of BFA (Figure 4.4B). Dunnett's test bases its statistical calculation on t-tests between sample means, but does it by taking into account a common variance among samples and provide confidence limits for non-overlap of means. The interpretation of a Dunnett's test is that two sample means are considered to be non-statistically different if the arm in any direction crosses the zero-difference point. Dunnett's test showed that VCN is very similar to wild type over all concentrations, but that the means of the generation times between each of the hid3Δ mutants and wild type become similar only at the concentration of 15 μg/ml. This showed that wild type and VCN generation times are increasing, but that hid3Δ generation times are, at least, changing less dramatically than those of WT and VCN. From Figure 4.4A, it appeared that generation times for hid3Δ were not changing with BFA. The hid1Δ mutants were tested and they were no different to WT and VCN.

Like for BFA, when used at too high of a concentration (< 100 μg/ml) GCA completely abolished growth of all genotypes. A concentration of 50 μg/ml was determined to give substantial differences between the hid3Δ mutants and other genotypes (Figure 4.5). At this concentration, the generation times for wild type, VCN and hid1Δ increased, but by less than 2fold. In contrast, the generation times of the hid3Δ mutants increased on average by more than 2-fold. As mentioned previously, it was very difficult to get an accurate determination of generation time due to very little differences in cell numbers between 0 and 12 hours. Based on Student's t-tests, the effect of GCA was significant for wild type, VCN and hid3Δ. A Tukey's test of significantly different means, which is a conservative test taking into account a common variance among sample means, gave only the means of hid3Δ +/-GCA as being significantly different within genotype.

DISCUSSION

I initiated a series of test for responses to stress treatments based on the evidence that two genes orthologues to the HID1 genes of human and C. elegans appeared to be induced in response to various stresses [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF]. The variety of mutant and control genotypes constructed in the lab provided a good opportunity to determine if any of the Hids play a role in helping S. pombe cope with stress. Unfortunately, the delay in obtaining multiple alleles of the hid2Δ did not allow me to exhaustively test that mutant genotype or the double mutants lacking Hid2. The treatments tested on hid1Δ and hid3Δ covered a wide variety of abiotic and mutagenic stresses. In general, the lack of Hid1 does not appear to be detrimental to cell growth in response to abiotic and mutagenic stresses, but it does seem to be necessary for normal growth in the presence of the high oxidative stress presented by incubation directly with hydrogen peroxide. Reactive oxygen species, like hydrogen peroxide, can act as a DNA mutagen as well as induce or reduce factors that may increase cell growth and proliferation that are hallmarks of cancer progression [START_REF] Klaunig | Oxidative stress and oxidative damage in carcinogenesis[END_REF]. The observation that hid1Δ is not sensitive to other DNA damaging agents suggests that Hid1 would most likely be involved in cellular homeostasis of general processes, such as metabolism or nutrient uptake. With very little data on the hid2Δ mutant, it is not possible to comment on why the sensitivity of hid1Δ to H 2 O 2 treatment is reversed by removing Hid2.

The hid3Δ mutant is also insensitive to abiotic stresses and to DNA damaging treatments, but is sensitive to H 2 O 2 treatment. The simplest explanation for hid3Δ sensitivity to oxidative stress is that the cells are already compromised in core metabolic or transport processes, because the Golgi apparatus is already disrupted. It may also be that stress signalling itself is dramatically altered if the Golgi itself is not functioning properly as suggested by ultrastructural studies of hid3Δ mutants [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. Stress signalling components located in the plasma membrane are dependent on anterograde transport through the Golgi, but also recycle back to the Golgi, such as the GTPase Ras1 of S. pombe [START_REF] Chang | Spatial segregation of Ras signaling: new evidence from fission yeast[END_REF] It was unexpected the hid3Δ mutants would not grow in EMM+S with glucose as the sole carbon source. It had been demonstrated that strains lacking this gene could be obtained using replacement with ura4 + , which requires selection in minimal media without uracil [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. However, colonies of hid3Δ::ura4 + were quite small. The type of EMM used did make a difference on growth. All genotypes grew so poorly in EMM produced by the US company Sunrise Science Products Ltd that it wasn't possible to compare growth differences. Only the formulation produced by the UK company Foremedium gave the result shown in Figure 4.3. The effect wasn't due to potentially inhibitory properties of high glucose, since substitution with the other metabololisable sugars galactose, raffinose and fructose did not reverse the phenotype.

The poor growth phenotype could be gradually alleviated by titrating in increasing amounts of YES, which would suggest that something within the EMM is missing to support hid3Δ growth.

Although I have not investigated this greatly, it does not appear that amino acids or nitrogen are lacking as addition of particular amino acids, such as glutamine, also did not rescue the phenotype. It could be that hid3Δ is experiencing a quiescent state but with an extended Figure 4.6. Hid3 involvement in protein transport through the Golgi Apparatus. COPII complex directs protein traffic from ER Golgi. COPI complex mediates retrograde transport from Golgi to ER. Proteins (coloured circles) pass through the hierarchal series of Golgi apparatus divisions where they are can be modified and transported to the plasma membrane or secreted. The forward progress of protein movement from ER through the Golgi for transport is called anterograde protein trafficking, which is mediated by the interaction of Gea1 with Big1. The return of protein components through the Golgi or from the Golgi to the ER is known as retrograde protein trafficking and is mediated by the interaction of Gea1 with Arf1. Anterograde and retrograde protein movement can be prevented by inhibition of Gea1 interaction with Big1 or Arf1 using BFA or GCA, respectively. I propose that Hid3 is responsible for helping to maintain the structure of the medial-and trans-Golgi in order to allow complete anterograde protein movement to occur. Ubp5 is also shown because it has been shown to be recruited to the Golgi by Hid3.

lifespan. It would be interesting to investigate the fate of individual cells in minimal media to see if they were dying or in a state of quiescence. One important result of the EMM effect is that a phenotype is now available in order to test if the CeHID and HsHID, and even the CeDYM and HsDYM, can complement the phenotype. If so, this would greatly support using S. pombe as a good model system for detailed mechanistic studies of these proteins as suggested in Chapter 3.

The complementary use of BFA and GCA has provided strong evidence that Hid3 works in the direction of anterograde protein transport. In mammalian cells, the action of both BFA and GCA is to inhibit GBF1, the guanine nucleotide exchange factor (GEF) that activates the ADPribosylation factor as part of the small GTPase signalling component of the Golgi apparatus.

GBF1 is interesting in that it can either interact with BIG1/BIG2 to promote anterograde protein transport, or interact with the small GTPase Arf1 in order to promote retrograde transport [START_REF] Bonifacino | The Mechanisms of Vesicle Budding and Fusion[END_REF]. BFA and GCA inhibit the action of GBF1, but in the anterograde and retrograde directions, respectively [START_REF] Sáenz | Golgicide A reveals essential roles for GBF1 in Golgi assembly and function[END_REF].

The observation that hid3Δ is unaffected in response to BFA at low concentrations whereas WT is not suggests that the mutation mimics the effect of BFA. The opposite effect of GCA to stop hid3Δ makes sense in that GCA combined with the lack of Hid3 would completely disrupt protein trafficking through the Golgi in both directions. Figure 4.6 presents a conceptual model of how Hid3 placement within the medial-and trans-Golgi network acts as an anchor to stablise Golgi structure to allow the movement of proteins through the Golgi network. With S pombe possessing orthologues of all the major factors, it is a very good model for studying Golgi protein trafficking [START_REF] Eckler | Haploinsufficiency of the Sec7 guanine nucleotide exchange factor gea1 impairs septation in fission yeast[END_REF]. It is interesting to speculate that Hid3 may be responsible for recruiting one or more of these factors to the Golgi as it does for Ubp5.

Chapter 5

Metabolomic and proteomic characterization of the hid1 and hid3 mutants of S. pombe

INTRODUCTION

Proteins are the major mediators of the cellular phenotype and function. Therefore, there has been intense interest to understand how protein levels change in response to cell perturbations. Unlike genetic mutation, which affects one gene, the consequences of stress or genetic mutation can result in changes to hundreds of the proteins in a cell. This is a hallmark of cancer, whereby a one or a few genetic mutations that start cell proliferation can have widespread effects on protein levels and ultimately cell metabolism [START_REF] Dang | Links between metabolism and cancer[END_REF][START_REF] Jones | Tumor suppressors and cell metabolism: a recipe for cancer growth[END_REF]. Likewise, a cell's complement of proteins will change due to environmental factors, drugs, stress, growth conditions and extracellular signals. Like for gene expression, efforts continue to focus on how proteins in a tissue or organism change on a global level, where the function of a protein can be evaluated in the context of the other proteins that are present.

The term used to describe studies where as many proteins in a cell, tissue or organism are detected and quantified is called proteomics [START_REF] Washburn | Driving biochemical discovery with quantitative proteomics[END_REF]. Similar to proteomics, metabolomics is the study of the cellular or tissue metabolites. It is a powerful tool to judge the metabolic state of a cell, such as its energy demands and nutrient requirements [START_REF] Griffin | Metabolic profiles to define the genome : can we hear the phenotypes ?[END_REF].

In general, gene expression is the easiest to do and the entire predicted transcriptome of a cell can be tested using microarrays or RNAseq [START_REF] Perdacher | Next-Generation Sequencing and its Applications in RNA-Seq[END_REF]. Proteomics suffers the lack of the broad detection of proteins likely to be present in a cell. However, extraction techniques using organic solvents like phenol or ether can do a good job to extract most cellular proteins, both soluble and membrane bound. Proteins are detected and quantified using 2-D gel electrophoresis or one of the quantitative mass spectrometry techniques, like ITRAQ, SILAC or label-free [START_REF] Washburn | Driving biochemical discovery with quantitative proteomics[END_REF]. Label-free is becoming increasingly popular for proteomics. It has advantages over techniques involving labeling of proteins or peptides for mass spec detection, such as ITRAQ and SILAC/SILAN. It is less complex experimentally, less costly, and it has no limits to the number of samples compared like 2-D gel electrophoresis [START_REF] Lassout | Assessment of the influence of the patient's inflammatory state on the accuracy of a haptoglobin selected reaction monitoring assay[END_REF][START_REF] Strathmann | Current and Future Applications of Mass Spectrometry to the Clinical Laboratory[END_REF].

Getting an overall picture of metabolism through metabolomics is probably the most difficult, because metabolites have many different chemical and physical properties so that one extraction technique or detection and measurement technique is insufficient to observe all classes of metabolites. In order to judge the energetic and nutritional state of the cell, usually the alcohol soluble metabolites, such as sugars, amino acids, organic acids and nucleotides. These can be observed and quantified using either proton nuclear magnetic resonance (NMR) or mass spectrometry [START_REF] Kell | Metabolomics and systems biology: making sense of the soup[END_REF].

Where there have been countless proteomic and metabolomic studies on human tissues, budding yeast and plants, relatively few have been done on pombe. This is particular so for metabolomics. Nevertheless, there have been both proteomic and metabolomic studies related to my project. Changes to the proteome of S. pombe have been investigated in normal and quiescent cells [START_REF] Marguerat | Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells[END_REF] with attempts to measure absolute levels of transcripts and proteins. They determined that protein molecules, even for signalling proteins, can exist in hundreds to thousands of copies with transcript levels being in the ones to tens. A combination of transcriptomic and proteomics was used to investigate gene expression regulation by non-coding RNAs [START_REF] Leong | A global non-coding RNA system modulates fission yeast protein levels in response to stress[END_REF]. A parallel study using proteomics and proton NMR metabolomics was use to investigate metabolome and proteome changes in cells with altered stress signalling pathways under oxidative stress [START_REF] Weeks | A parallel proteomic and metabolomic analysis of the hydrogen peroxide-and Sty1p-dependent stress response in Schizosaccharomyces pombe[END_REF]. I undertook a combination metabolomic and proteomic study in order to obtain a better understanding of molecular changes resulting from the elimination of the Hid1 and Hid3 proteins through gene deletion. Proteomics was considered to be appropriate from the evidence suggesting that the deubiquitinase Ubp5 is mislocalised to the cytosol from the Golgi in the hid3 mutant [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. As stated in Chapter 1, relocalisation of a deubiquitinase from one compartment to another would likely have major effects on protein turnover. For the metabolomics we used proton NMR to quantify detectable ethanol soluble metabolites and a label-free LC-MS/MS approach to quantify proteins. The genotypes compared were WT, VCN, hid1Δ and hid3Δ. The metabolomics and proteomics revealed that hid3Δ cells appear to be stressed although they were grown under standard conditions. In hid1Δ, metabolic proteins were altered, mainly involving membrane-related processes. This suggests that Hid1 may be involved in maintaining metabolic homeostasis. Cell cultures were growth to a density of 5x10

7 cell/ml. The cells were collected by centrifugation, immediately frozen in liquid N 2 , and ground to fine powder using a cryomill. The genotypes were combined at this step, where equal masses of powder were combined for the two genotypes specified.

Sample

Combined Genotypes

WTa

RESULTS

Table 5.2. Metabolite absolute values as determined by 1 H-NMR. Metabolites were extracted from approximately 30 mg of dried powdered cells. Metabolites were identified and quantified according to Moing et al (2004).

Metabolite

WT hid1∆ hid3∆ VCN Isoleucine 3.8 ± 0.5 3.3 ± 0.1 3.7 ± 0.5 3.6 ± 0.3 UnkM5.56 1.7 ± 0.1 1.4 ± 0.1 b 2.9 ± 0.1 b 1.7 ± 0.0 Phenylalanine 1.6 ± 0.2 1.9 ± 0.

1 b 1.3 ± 0.1 b 1.7 ± 0.1 Tyrosine 1.3 ± 0.2 1.5 ± 0.2 1.4 ± 0.2 1.4 ± 0.1
a putative assignment b significantly different (P < 0.05) from VCN based on an unpaired, two-tailed, Student's t-test assuming equal variance.

Strategy for sample preparation for metabolite and protein profiling

Cells were grown in 50 ml cultures starting with an initial density of 5x10 5 cells/ml. Cells were grown for 16 to 18 hours until concentrations reached 5x10 7 cells/ml at which point they were harvested. This cell density was chosen as it would likely reveal the biochemical differences among strains [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]; see Chapter 4). Cell growth in 50 ml volumes ensured that there was sufficient material to conduct all aspects of the post-genomic study.

Potential biological variation within mutant and control strains was accounted for combining two independent mutation events for each genotype (Table 5.1). This scheme gave 12 samples from which polar metabolites and total proteins were extracted for 1 H-NMR and proteomic profiling, respectively.

Metabolic consequences of removing Hid1 and Hid3

The polar metabolites were extracted from dry powdered cell material using a deuterated aqueous buffer according to (A et al., 2004). It must be noted that the cells were not washed prior in order to freezing in order to minimise changes to the proteome and transcriptome, which were the priorities of the study. However, carry-over of metabolites from the YES media would probably serve to mask differences among the various genotypes, thus any identified differences would be real. The output from the analysis was 26 metabolites quantified absolutely on a mol/gm dry weight basis (Table 5.2). It is apparent that the NMR could detect the added supplements, such as adenine (adenosine), uracil, (UDP-like), lysine, leucine and histidine (likely peak at S2.12) and glucose. These metabolites were high in quantity and they varied little among genotypes. The two most abundant metabolites were glycerol and betaine, which are two osmoprotectants and these were modified in each of the mutants compared to VCN. Each mutant genotype had about 10 metabolites that were significantly different to VCN, but differences were not very great.

Since there was little difference in individual metabolites, Principal component analysis (PCA) was conducted according to sample in order to see if any general metabolic differences among genotypes were visible (Figure 5.1). The plot of percent variance versus principal component showed that 82% of variance was represented the first 3 PCs (Figure 5.1A).

Therefore, these three PCs were used for pairwise plots to reveal potential differences among genotypes (Figure 5.1B). The plot of PC3 vs PC2 shows the clustering of hid1, hid3 and VCN showing that the first 3 PCs were sufficient to describe the variation between mutant and VCN samples. In the plot of PC2 vs PC1 there was no difference between wild type and VCN samples across PC1, which represented > 40 PoV. Both mutant genotypes were separated from VCN across PC1 showing that the metabolite profiles of the mutants were different to the controls, and they were split in opposite directions. All three modified genotypes were separated from wild type across PC2 and in the same direction suggesting that the similar metabolic changes were responsible. This difference in modified genotypes compared to wild type was not apparent in the PC3 vs PC1 with VCN clustering with WT. Therefore, the variance in PC2 was something common among the modified genotypes compared to WT with the most likely explanation being that expression of the marker gene did affect their metabolism. The loadings plot of the metabolites in the dimensions PC2 vs PC1 gave some indication of the metabolites responsible for the separation (Figure 5.1C). The metabolites separating hid1 were the added compounds adenine and uracil, the aromatic amino acids phenylalanine and the amino acids glutamate and betaine. Of these, none were significantly changed from VCN indicating that they likely served to distinguish the VCN from wild type. The separation of hid3 was characterised by the organic acids, citrate and acetate, the glycerol and trehalose and the amino acids choline and its acetate-modified form Ac-choline. Of these metabolites, only trehalose and citrate were significantly different to VCN indicating that the others were also defining the difference between VCN and WT. In addition, the VCN genotypes were characterized by the hydrophobic amino acids alanine and valine and the basic amino acid lysine contributed to the separation of VCN from wild type. Wild type was characterised by the other hydrophobic amino acids leucine and isoleucine, the organic acids succinate and formate and the sugar glucose.

Proteomic study on the effects of loss of Hid proteins

Total proteins for the proteomic analysis were extracted using an aqueous buffer with 1% triton detergent. It was expected that this protocol would give mainly soluble proteins and those proteins peripherally associated with membranes. After quantification of total protein, a pre-visualisation of the protein extracts was done by 1-D SDS-PAGE, which did not reveal any major difference in banding pattern among the genotypes (Figure 5.2). The gels also indicated that sample quality and total protein quantity were not greatly different among the samples, so that the proteomic analysis would be done on samples of similar properties. This would make down-stream preparation of samples and the data analysis more straight-forward. For example, it is easier for automated software, such as Progenesis IQ, to align spectra and normalise intensity values for various spectra with peaks of similar intensity. A total 10 μg of protein was used for label-free proteomic analysis with prior gel purification and in-gel trypsin digest (Figure 5.3). The peptide mixture from the trypic digest was injected directly into a Q-Exactive™ mass spectrometer (Thermo Scientific) and proteins identified from peptides using Proteome Discoverer™ v 1.4 software. The information output from the mass spectrometer, in terms of the number of peptides quantified (average = 10783), the number of different proteins detected (average = 1836) and the number of proteins characterized by more than one unique peptide (average = 1296), indicated that the protein preparation and treatment of protein samples were quite uniform among samples (Table 5.3). On average, 1836 proteins were identified per sample, which represent approximately 36% of the predicted proteome, not including dubious and transposon sequences (www.pombase.org). This number of proteins was similar to the 1896 proteins detected in a study of nutrient stress responses to the S. cerevisiae proteome [START_REF] Gutteridge | Nutrient control of eukaryote cell growth: a systems biology study in yeast[END_REF] and the 2147 proteins detected in a study of the changing proteome in response to oxidative stress in S. pombe [START_REF] Lackner | Regulation of transcriptome, translation, and proteome in response to environmental stress in fission yeast[END_REF].

Only those proteins identified by two or more unique peptides were selected by the Proteome Discoverer software for subsequent quantification using Progenesis LC-MS v 4.0.

Proteins were quantified as the sum of the peptide intensities in order to give greater weight to most abundant peptides. The peak intensities output for each sample was normalised by a scalar factor to allow comparison directly between samples. The result was a list of 2058 proteins with normalised intensity values that was used for PCA of genotype differences (Figure 5.4). The variance in the protein data was spread much more evenly over the PCs with PC1 only covering 26 % of the variance. The first four PCs covered just over 70 % of the variance describing the data set (Figure 5.4A). However, it was clear that 4 PCs were sufficient to show differences among the genotypes as all samples clustered around the zero axis of the 4 th PC (Figure 5.4B). In the plot of PC2 vs PC1, the hid3Δ samples were removed from the other genotypes across PC1 and hid1Δ clustered with VCN. This revealed that the the hid3Δ mutation had a greater effect on altering the proteome than the hid1Δ mutation. Similar to the metabolite PCA, the protein PCA also showed a separation in the second dimension where all modified genotypes were separated from WT. Again, I conclude that the presence of the marker protein has affected the overall proteome sufficiently for it to become apparent in the PCA.

Using the normalised values, Progenesis calculated the statistically relevant differences in each protein between the WT, mutants and VCN. The number of proteins UR and DR by more than 2-fold are shown in Figure 5.5. Compared to VCN, hid1Δ had 30 and 42 proteins UR and DR, respectively, whereas hid3 had 79 proteins UR and 18 proteins DR. For the The overrepresented classes were determined by GoMiner for significantly changing proteins compared to the set of all proteins used for the normalization set. The UniProt identifiers for all proteins were first converted to the unique S. pombe gene systematic Ids before being run through GoMiner. UR and DR stand for more and less abundant, respectively, than compared to VCN. The GO terms were reduced using REVIGO (revigo.irb.hr, [START_REF] Supek | REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms[END_REF] and the numbers in parentheses show the number of GO terms combined for the GO term shown. The degree of colour shows the relative degree of statistical significance.

purposes of simplicity WT is also shown compared to VCN (Figure 5.5). Therefore, in order to get an idea on the changes to the proteome due to marker gene expression, it is necessary to reverse the UR and DR proteins of which there were 28 proteins UR and 38 proteins DR compared to WT. The greatest UR protein at 8.3-fold was the pdp3-interacting factor ptf1, which may be involved in chromatin remodelling by affecting the phosphorylation state of Pdp3. This suggests that expression of the marker may affect gene expression, and some minor gene expression differences have been reported in strains expressing the Nat R gene [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF].

A GO analysis was conducted on the differentially expressed proteins changing by more than 2-fold, however, the numbers of proteins changing were not numerous enough to give statistically relevant enrichment of gene classes, except for a few for hid3Δ. Therefore, the GO analysis was repeated but with a set of proteins changing by more than 1.5-fold. For hid1∆ there were 134 proteins > 1.5-fold increased and 144 proteins > 1.5-fold decreased compared to VCN. In hid3Δ, there were 291 and 347 proteins 1.5-fold UR and DR. Taking the cut-off at 1.5 revealed that approximately the same number of proteins were UR and DR in hid3Δ. The only cellular component enriched for either mutant was the beta-subunit complex of the proteosome core-complex. The set of DR proteins for hid3Δ were enriched in ribosome and mitochondrial proteins as might be expected for cells under stress conditions. The classes of proteins enriched in the DR set for hid1∆ were much more variable, but contained elements mainly involved membrane process associated with the mitochondria. For the biological processes, the UR proteins for hid1∆ and hid3∆ were divided clearly between metabolic and stress-response processes, respectively. Classes of proteins related to carbohydrate catabolism were also enriched in UR set of proteins for hid3∆. The four classes of that were DR in hid1∆ for the biological processes weren't sufficiently coherent to provide an explanation for their enrichment, however, the classes of DR proteins for hid3∆ were clearly related to biosynthetic processes that would be reduced in cells under stress, with amino acid and nitrogen metabolism being prominent. In the category Molecular Function the classes of enriched proteins for hid3∆ both UR and DR groups could be related to stress responses. The UR proteins were highly represented by oxidoreductases, whereas the DR classes were represented by ribosome, primary metabolic proteins and also transport proteins. The enriched classes of DR proteins for hid1∆ appeared to reinforce altered membrane function as initially revealed by the Cellular Component GOs.

Preliminary analyses of immune-detection of Hid proteins in S. pombe

Targeted antibodies to each of the Hid proteins were produced by Beere Antibodies, a subsidiary of Thermo Scientific. First, custom peptides corresponding to the extreme C-terminal sequences of each of the S. pombe Hids were synthesized chemically. The sequences used were KREGPGIFEGTDVKIFDSF for anti-Hid1, RQPTILQSRSLGEFQGRLFS for anti-Hid2 and DLLYLQLPSSVNHDSSLRNK for anti-Hid3. Antibodies were produced in rabbits using the Pierce™ 90-day protocol, which starts with an initial injection of 250 μg of peptide antigen in Freund's adjuvant and subsequent booster injections over the 90 day period with 100 μg of antigen. Antibodies from the first bleed were affinity purified using a column with peptide attached. The antibodies were delivered in purified form and as a crude serum along with a sample of the peptide for control blotting.

In order to test the antibody, I used a rapid slot-blotting technique whereby a sample of whole extract and dissolved peptide were deposited onto a PVDF membrane using a membrane holder and vacuum pump. The advantage of this system is that is allowed for rapid testing of the antibody binding in the short time available to work with it. However, it has the disadvantage that it is not possible to distinguish between true signal and background signal, because the proteins were not separated by gel. The slot-blot hybridization is shown in Figure 5.7. For all three antibodies, a signal was detected for crude extract as well as for the purified protein. The signal for Hid1 and Hid2 appeared to be equal to or slightly less than the peptide control. The signal for Hid3 was well above that for the peptide control, thus it appeared that Hid3 is much more abundant than the other two Hids. This indication must be taken with caution, because the relative antibody binding efficiencies and antibody specificities remain to be determined. These must be determined using serial dilutions of peptide and standard Western blots, respectively.

One piece of information that supports this finding is that Hid3 was the only Hid paralogue that was detected in the proteomic study being present in one WT and one VCN sample.

DISCUSSION

This chapter presents the metabolomic and proteomic analyses of the hid1Δ and hid3Δ mutants. It was not necessarily expected that the metabolite analysis would give any indication of substantial differences in metabolism among genotypes. The priority was the analysis of the proteome and transcriptome. Therefore, in order to minimize changes to the cells, they were immediately frozen after centrifugation without prior washing with water. Even though they were not washed, rapid freezing without water washed would ensure that metabolic changes were minimized. Although on a dry weight basis, the nutrients coming from the yeast extract may not be detectable by the NMR, but it is possible that the supplements would be detected as adenine-like and UDP-like substances. Nevertheless, the metabolite quantification provided some interesting results. The changes in metabolites we observe were not as dramatic as observed in the null mutant lacking Sty1 or in WT cells treated with H 2 O 2 [START_REF] Weeks | A parallel proteomic and metabolomic analysis of the hydrogen peroxide-and Sty1p-dependent stress response in Schizosaccharomyces pombe[END_REF], but the directions of metabolite changes corresponded with those of H 2 O 2 stress in WT with more glucose, glycerol, acetate, alanine, and lower formate, phenylalanine succinate, valine and leucine. Where the changes didn't match, it was in most cases that we did not observe a change in that particular metabolite, such as for lysine, choline, NADH (nicotinamide) and tyrosine. This would be expected if the stress from the lack of Hid3 was not very strong. From the stress tests, we observe that H 2 O 2 has a dramatic negative effect on the growth of hid3Δ, thus it would be interesting to check metabolite levels in the mutant under limited H 2 O 2 stress.

The study of [START_REF] Weeks | A parallel proteomic and metabolomic analysis of the hydrogen peroxide-and Sty1p-dependent stress response in Schizosaccharomyces pombe[END_REF] had metabolite assignments in addition to ours, such as for orotic acid, trimethylamine-N-oxide (TMAO), phosphocholine, glycerophosphocholine, and inosine, but we had detected trehalose and Ac-choline, which have also been detected by mass spectrometry [START_REF] Chaleckis | Unexpected similarities between the Schizosaccharomyces and human blood metabolomes, and novel human metabolites[END_REF]. Some of their novel compounds may be our unknowns or their novel compounds, inosine (adn-like) and UTP + UDP (UDP-like), may be those that we cannot exclude as being from the supplements, like adenine and uracil.

Glycerol, betaine and choline are likely osmoprotective metabolites and their increase in synthesis was hypothesized to represent a cross-over between oxidative and osmotic stress [START_REF] Weeks | A parallel proteomic and metabolomic analysis of the hydrogen peroxide-and Sty1p-dependent stress response in Schizosaccharomyces pombe[END_REF]. Since we observed a substantial increase in glycerol this suggests that hid3Δ cells are under stress. It is difficult to narrow down a particular explanation, since choline was only slightly increased and betaine decreased. Possible explanations for my observations are that the cellular stress caused by the loss of Hid3 is 1) not as severe as direct application of H 2 O 2 as mentioned above, 2) only specific stress responses are initiated, or 3) that the chronic stress over generations is somehow attenuated by certain metabolic changes. The latter possibility is supported by the suggestion that for fungi betaine does not serve as an osmoprotectant, but serves as an intermediate in the catabolism of choline [START_REF] Lambou | Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus[END_REF]. Therefore, cells may be undergoing stress conditions without the consequence of synthesizing betaine. These possibilities can be explored by using cell synchronization techniques along with the application of stresses in the mutant genotypes.

In Chapter 3, I had mentioned that potential physical interactors of Hid3 were the ATPdependent RNA helicase Prp22, and the predicted inositol polyphosphate phosphatase SPAC9G1.10c. In the proteomic analysis, I did not see any change in Prp22 protein, but the ATP-dependent helicase Has1 was significantly UR. This caused me to look a bit at the other ATP-dependent RNA helicases at both the protein and mRNA level. The gene has1+ was significantly UR by 1.7-fold as well as the ATP-depdendent RNA helicase dbp2+. Dbp2 protein was also UR suggesting that along with Has1, it is transcriptionally controlled. Two other ATPdependent RNA helicases had UR mRNA levels, prh1+ and prp43+. No ATP-dependent RNA helicases were significantly DR at either the protein or RNA level. This result is in direct contrast with the result of [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF], who showed that all of the ATP-dependent RNA helicases mentioned above are DR in response to all stresses tested. This suggests that the stress response exhibited by removing Hid3 function is fundamentally different to a typical environmentally imposed stress. It would be interesting to see how these genes change in response to oxidative stress in the hid3Δ. The putative inositol polyphosphate phosphatase SPAC9G1.10c remained unchanged on both the protein and mRNA level and so its association with Hid3 remains unclear.

The study of [START_REF] Weeks | A parallel proteomic and metabolomic analysis of the hydrogen peroxide-and Sty1p-dependent stress response in Schizosaccharomyces pombe[END_REF]) also investigated the changing proteome in response to oxidative stress. All but one of the proteins that they observed as being dependent on Sty1 in H 2 O 2 induction I also observed as being significantly increased in hid3Δ, such as Gst2, SPBC215.11c, Obr1, catalase (1.2-fold induced), and thioredoxin reductase (1.5-fold). This result in itself indicates that hid3Δ cells are stressed. This was also the conclusion drawn from transcriptional profiling of hid3Δ cells [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. In addition, I did not see induced any of the proteins that are de-repressed in the unstressed sty1Δ mutant. I did observe the induction of Gpd1, which is induced in response to osmotic stress [START_REF] Ohmiya | Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth[END_REF] and which may be responsible for the production of glycerol observed in the metabolite profiling. [START_REF] Morigasaki | Glycolytic enzyme GAPDH promotes peroxide stress signaling through multistep phosphorelay to a MAPK cascade[END_REF]. Tdh1 is very abundant compared to the other complex members (10-fold over Gpd3 and 1500-fold over Mcs4 in our study based on raw counts) so that a small reduction of 1.2-fold would unlikely compromise its contribution to the complex. The induction of Wis1 may be sufficient to activate Sty1. Wis1 is necessary for the transition of proliferating to quiescent cell types [START_REF] Sajiki | Genetic control of cellular quiescence in S. pombe[END_REF]. I observed some of the hallmarks of the quiescence transition, albeit not on such a dramatic level. These include the decrease and increase, respectively, in Tdh1 and Gpd3. However, mRNA levels of both genes increase in hid3Δ, whereas they decrease in the transition from growth state to quiescent state [START_REF] Marguerat | Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells[END_REF]. I also observed the induction of the GTPase Ypt5, which is required for cell entry into quiescence like Wis1, and the proteins for a number of genes necessary for maintaining quiescence, such as Cdc2, Nat10, Apl2, and Cts1. Is it possible that the slow growth phenotype is simply a result of elevated Cdc2, which could be phosphorylated and slow cell cycle progression? However, the kinases Chk1 and Cds1 that phosphorylate Cdc2 [START_REF] Alao | Rad3 and Sty1 function in Schizosaccharomyces pombe: an integrated response to DNA damage and environmental stress?[END_REF]) remained unchanged at both the protein and mRNA level. It is also interesting to speculate that the decreased growth phenotype (Chapter3, Alshehri 2015) is indicative of a mild quiescent state brought on by stress. The main question arising is how is the lack of Hid3 being sensed as a stress.

because of sequence similarities, HID1 had been assigned to the DYMECLIN superfamily, which is conserved from plants to humans. Although these mentions come from the literature, to my knowledge, there has not been a detailed comparison of DYM and HID1

Structure or evolutionary origins. HID and DYM are both present in animals so knowing their evolutionary origins and structural characteristics may give information on functional overlap. It wasn't possible to uncover a protein that was ancestral to both DYMs and HIDs. They are both common only to eukaryotes and are present in the eukaryotes prior to the division of the seven superphyla [START_REF] Adl | The new higher level classification of eukaryotes with emphasis on the taxonomy of protists[END_REF]. However, it was interesting to find that whereas animals have both DYM and HID, plants have only DYM and fungi have only HID. Having DYM or HID is not a requirement for a cell being eukaryotic as shown by a vast number of organisms not having DYM or HID. Although, we have not identified a multicellular eukaryote with the exception of species of linked algae, that do not have one or the other. Thus, it is interesting to speculate that having either is necessary for cellular communication that makes multicellularism possible, but it also suggests there may be functional overlap. It is interesting to speculate that DYM is not a lethal mutation in humans, because it could be partially complemented by HID1.

DYM represents a possible complication for understanding the complete function of HID1, but it is also interesting to understand how both work within the same context of Golgi function.

The major differences that have been noted between the lineages of the two proteins, such as the HID and DYM present within the choanoflagellates, the presence of DYM within the fungal family Mucorales, and the apparently different evolutionary rates of the HIDs and DYMs within fungi and plants, respectively, directly suggest that horizontal gene transfer took place at some point within their evolutionary histories, which is known to happen between eukaryotes [START_REF] Keeling | Horizontal gene transfer in eukaryotic evolution[END_REF]. Species of Mucorales are parasitic and they can infect animals, plants or each other. For example, species of absidia can infect animals or plants and species of parasitica can infect and pass or retain genes from the absidia [START_REF] Hoffmann | The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies[END_REF][START_REF] Kellner | Transfer of genetic information from the mycoparasite Parasitella parasitica to its host Absidia glauca[END_REF]. Because the DYMs are restricted to the mucorales, it is interesting to speculate that they may have obtained them from another eukaryote, in this case from a non-fungal Opisthokont. Far reaching speculation is that they subsequently transferred them to the Viridiplantae, noting that the plant DYMs are more evolutionarily conserved to animals than the HIDs are within fungi.

The annotation of the S.pombe genome revealed that there are three orthologues of HID1. Two of the orthologues had been named as human HID1 orthologue 1 (SPAP27G11.12) and HID1 orthologue 2 (SPBP19A11.07c). Reference to the human orthologue HID1 has only come about within the last year and these had been referred to as orthologues of human DMC1, the term given by [START_REF] Harada | Identification of DMC1 , a novel gene in the TOC region on 17q25 . 1 that shows loss of expression in multiple human cancers[END_REF] for the Down-regulated in Multiple Cancers 1 locus. The phylogenetic analysis indicates that in the ascomycetes the ancestral HID gene duplicated to become HID2 and the ancestor of HID1/HID3, which then duplicated to become HID1 and HID3.

The presence of three paralogues may be unusual, but interesting from the point of redundancy of function. Since Hid1 and Hid3 are the most similar they were the basis of the systems biology study of the group. Why are there three orthologues of HID1 in pombe? One hypothesis is that they split to retain the corresponding (as of yet unknown) functions of HID1 in humans and C.

elegans. There is evidence that HID1 dynamically associates with the Golgi membrane [START_REF] Wang | HID-1 is a peripheral membrane protein primarily associated with the medial-and trans-Golgi apparatus[END_REF]. This implies that HID1 has a function when associated with the Golgi membrane and another when released into the cytosol. Is it possible that Hid3 retains the function of Golgiassociated HID1 and Hid1 of that when present in the cytosol and is it possible that the function of Hid3 has been modified to become an integral protein for maintaining Golgi structure in S.

pombe [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF].

II Chapter 4 conclusions

From the work conducted on C. elegans, the conclusions were made that HID-1 functions in the transport of neuropeptides in neuronal cells, the type of cells where it is most highly expressed. This is based on the observation that HID-1 is present at the synaptic junctions of the plasma membrane as well as being associated with exocytotic vesicles [START_REF] Mesa | HID-1, a new component of the peptidergic signaling pathway[END_REF][START_REF] Yu | HID-1 is a novel player in the regulation of neuropeptide sorting[END_REF]. Although it cannot be entirely excluded that HID1 works in a retrograde manner to recycle proteins or lipids from the plasma membrane to the endosome or Golgi, it most likely has an anterograde transport role. So far for pombe, Hid3 has only been associated with the Golgi apparatus and so its role in anterograde and/or retrograde protein transport remained unknown. In general, it is difficult to differentiate between anterograde and retrograde transport processes because they are highly interdependent, but it is possible by using certain inhibitors of Golgi function. By using the inhibitors BFA and GCA, I showed that growth of hid3Δ was unaffected by low concentrations of BFA, but that of WT and VC genotypes was. At high concentrations of BFA, growth of all genotypes was dramatically slowed. This result was consistent with the broad inhibitory nature of BFA to act on the Golgi and disrupt its structure. Compared to WT, VC and hid1Δ genotypes, hid3Δ was hypersensitive to the compound GCA, which is known to inhibit retrograde protein transport from endosomes to the Golgi [START_REF] Sáenz | Golgicide A reveals essential roles for GBF1 in Golgi assembly and function[END_REF]. The broad conclusion that can be made is that in the hid3Δ mutant GCA disrupts those processes that are not affected by the lack of Hid3 and so completely abolishing Golgi function. I would like to speculate that Hid3 functions primarily to maintain anterograde protein transport in S. pombe. This is consistent with its subcellular location to the medial-and trans-Golgi as reported for C. elegans [START_REF] Wang | HID-1 is a peripheral membrane protein primarily associated with the medial-and trans-Golgi apparatus[END_REF].

The genome wide gene expression data provided by [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF] gave the suggestion that the Hid proteins may be involved in the stress responses and so should be test for their responses to stress. The genes hid1 + and hid2 + were both induced in response to multiple stresses, but hid3+ was not. Hid3 + expression was essentially unchanged suggesting a primary role in maintaining Golgi function even under stress conditions. Over the variety of stresses tested, both abiotic and DNA damaging, hid1Δ and hid3Δ genotypes were relatively unaffected, except with oxidative stress through treatment with hydrogen peroxide where both were affected. In contrast, it appeared that hid2Δ was not. There is an explanation for this similar result with hid1Δ and hid3Δ. Hid1 may be involved directly in the cellular response to oxidative stress as there are a number of important proteins DR in the mutant (see Chapter 5 below). It is likely that Hid3 minus cells are already compromised in their growth and so an added stress could further reduce it. Maybe hid3Δ cells are already sensitised for oxidative stress responses through the constitutive induction of signalling pathways and so cannot further respond to the stress. The hid3Δ mutant has been reported to be moderately sensitive to cadmium and high KCl treatment [START_REF] Kennedy | A genome-wide screen of genes involved in cadmium tolerance in Schizosaccharomyces pombe[END_REF]. Although I did not test cadmium sensitivity I did not observe sensitivity to high NaCl. It should be noted that the already reduced growth of hid3Δ was a complicating factor in trying to determine sensitivity to stress, because all evaluations had to be done on a relative basis on how mutant growth was affected proportionately to that of WT or VC genotypes. It is possible that some stress treatments may have had effects that were missed.

As mentioned in Chapter 4, it was unexpected that hid3Δ would grow very poor in Edinburgh Minimal Medium. This was observed in liquid culture monitored by cell counts as well as by spot size on agar plates using standard drop tests. It is not clear why, but growth was affected depending on the type of EMM used. All genotypes grew poorly in the EMM from Sunrise Science Products and better with the Foremedium EMM. However, it is clear that even Foremedium EMM does not support complete growth even of WT cells so there is some type of nutrient limitation. Since the growth of hid3Δ was so much worse than the other genotypes, I can conclude that the nutrient limitation effect is much more profound in hid3Δ. In addition, a nutrient limitation effect was supported by experiments where standard yeast extract media was titrated in increasing amount into EMM with the effect to improve the growth of all genotypes, but improving hid3Δ growth required higher proportions of yeast extract. The conclusion of the poor growth phenotype associated with nutrient limitation is consistent with the gene expression results showing induction of gene expression for a variety of plasma membrane nutrient transporters [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. What has been a big subject of recent research is the switch between quiescent and proliferative cell growth. The poor growth of hid3Δ on EMM is consistent with nutrient limitation inducing a quiescent growth state. Naturally, it is essential to determine which element is preventing growth of hid3Δ strains.

III Chapter 5 conclusions

Chapter 5 presented the results from the metabolomic and proteomic study of the hid mutants. These two technologies were used to try to address some of the current hypotheses about Hid function. The metabolomics would show if any large changes to metabolism occurred in the hid mutants, and the proteomics would provide some mechanistic explanation for the slow growth observed for hid3Δ. It has been proposed that hid3Δ cells may be in a sort of quiescent growth state similar to hid-1 mutants of C. elegans [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. A number of genes and metabolic conditions have been described for S. pombe cells to enter and maintain a quiescent growth state [START_REF] Gray | Sleeping beauty": quiescence in Saccharomyces cerevisiae[END_REF][START_REF] Sajiki | Genetic control of cellular quiescence in S. pombe[END_REF]. It would be expected that these properties would be present in hid3Δ cells if they were in a quiescent or partially quiescent state.

The metabolomics experiments quantified 27 metabolites in WT, control and hid1Δ and hid3Δ genotypes. Certainly, the most surprising was the difference in glucose levels between the WT and strains expressing the antibiotic marker. Why this should be the case is not clear.

The data showed two interesting differences in metabolite content between hid3Δ mutants and VC strains, which were an increase in glycerol and trehalose. An increase in these metabolites, at least in budding yeast, is seen as necessary for cells to maintain the quiescent state.

Trehalose is reported to protect quiescent cells from reactive oxygen species [START_REF] Benaroudj | Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals[END_REF] and glycerol is likely to prevent cell desiccation under long term survival. Although the changes were not dramatic, the hid3Δ mutants showed some metabolic changes consistent with a quiescent state.

The label-free proteomics turned out to be an efficient and relatively inexpensive way to determine protein changes associated with the hid deletions. I was able to observe the fate of nearly 2000 proteins present in S. pombe cells. The most striking result from the proteomics analysis of the hid mutants was the pleiotropic nature of the protein changes in hid3Δ. The lists provided in Tables 5.4 and 5.5 show that 72 and 92 proteins were significantly DE in hid1Δ and hid3Δ, respectively, at a level of 2-fold or greater. This presented somewhat of a narrow picture of the entire landscape of proteins changes seeing that totals of 467 and 712 proteins were significantly DE in hid1Δ and hid3Δ, respectively. For each mutant, the complete sets of DE proteins were used in the GO analysis in order to establish the functional classes of proteins that changed in relation to the VC genotype. The proteins changing gave interesting insights into the processes that were changing in both mutants. For hid1Δ, organelle-related membrane processes were DR and cytosolic metabolic processes were UR. This suggests a fundamental metabolic shift controlled by loss of Hid1. This did not have any effect on growth rate under standard conditions, but it compromised the mutant's ability to cope with oxidative stress.

Mitochondria are highly sensitive to increased reactive oxygen species that disrupt membrane integrity and respiration, and the lack of mitochondrial function is well known to lead to apoptosis [START_REF] Circu | Reactive oxygen species, cellular redox systems, and apoptosis[END_REF]). An interesting idea is that Hid1 in S. pombe contributes to maintaining interorganelle communication of metabolic processes and that this may be the function of HID1 dissociation from the Golgi in animals cells.

It has been proposed that Hid3 may sequester Ubp5 to the golgi to prevent it from entering the nucleus to deubiquitinate certain transcription factors. This system would be analgous to the MDM2-DAXX-USP7 complex that appears to regulate the nuclear localisation of both pTEN and p53 and subsequently affects induction of apoptotic pathways. In humans, USP7 deubiquitinates pTEN leading to its translocation to the cytosol and preventing activation of apoptotic pathways [START_REF] Song | The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network[END_REF]. When DNA damage occurs DAXX dissociates from MDM2 to disrupt the complex causing MDM2 to degrade through autoubiquitination, which is prevented by USP7 in non-stress cells. Degradation of MDM2 prevents p53 ubiquitination and enhances it lifespan allowing it to induce cell apoptotic pathways [START_REF] Zhao | Negative regulation of p53 functions by Daxx and the involvement of MDM2[END_REF]. USP7 directly deubiquitinates p53 allowing activation of apoptotic pathways [START_REF] Li | Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization[END_REF]. According to these two models, HID1 reduction would lead to increased availability of USP7 and either promote or prevent cell proliferation depending on the cancer type. In fact, it is quite rare to have both pTEN and p53 mutations in the same cancer and so they serve as backup systems [START_REF] Li | PTEN has tumor-promoting properties in the setting of gain-of-function p53 mutations[END_REF]. These types of models, although unclear, demonstrate that disruption of Golgi function could affect gene expression.

My data provides a few interesting examples of how disrupting Golgi function may affect chromatin structure and gene expression. One example is Midasin, a nuclear protein that facilitates the biogenesis of ribosomes by aiding their export by removing biogenesis factors [START_REF] Bassler | The AAA-ATPase Rea1 drives removal of biogenesis factors during multiple stages of 60S ribosome assembly[END_REF]. Midasin has significant sequence similarity to human DAXX. Midasin protein was DR by more than 2-fold in hid3Δ and so could be part of the explanation for the down-regulation of ribosome function in the mutants. Down-regulation of ribosome function and protein synthesis are symptoms of both cells under chronic stress and those entering quiescence. Two interesting proteins that were UR in hid3Δ are Htb1 and Obr1/Uhp1.

Obr1/Uhp1 has a histone fold similar to Histone H2A and it binds Histone H2B of which Htb1 is a subunit. The current model of Uhp1 function is in chromatin silencing at mating locus mat2P through dynamic chromatin interaction regulated by ubiquitination through the E1 ligase Rhp6 [START_REF] Naresh | Identification of Uhp1, a ubiquitinated histone-like protein, as a target/mediator of Rhp6 in mating-type silencing in fission yeast[END_REF]. Rhp6 was not affected in our study at the level of protein or gene expression. Obr1/Uhp1 is proposed to help maintain a semi-stable structure of chromatin that will then become stable upon Uhp1 ubiquitination and, subsequent, targeting for degradation.

The presence of Ubp5 in the cytosol/nucleus may be responsible for maintaining elevated levels of Obr1/Uhp1 by deubiquitinating it. The result would be maintaining an active euchromatin state at the mating switch locus through its interaction with H2B as opposed to H2A. Therefore, a combination of an increase in Obr1/Uhp1 and H2B along with deubiquitination would maintain the mating-type locus in an non-silent state. Induction of Interestingly, activation of pheromone sensing and signalling pathways leading to unsilencing of mating type switching are mediated by Ras signalling when Ras is located at the plasma membrane [START_REF] Onken | Compartmentalized signaling of Ras in fission yeast[END_REF][START_REF] Tamanoi | Ras signaling in yeast[END_REF]. I observed more than a three-fold increase in Ras1 in hid3Δ mutants.

These examples are speculation as to the effects that Ubp5 may have when Hid3 is deleted. It must be considered that ubiquitination/deubiquitination is a process that occurs in many different cellular compartments including the Golgi apparatus. Ras signalling itself is tuned by ubiquitination/deubiquitination processes [START_REF] Jura | Differential modification of Ras proteins by ubiquitination[END_REF]. Within the ER, ubiquitination serves as a signal to export improperly folded proteins to the cytosol for degradation by the proteosome. In contrast, it is believed that deubiquitination serves as a check that properly folded/processed proteins are not targeted for degradation (Zhang et al., 2013a). A very good example is the SREBP signalling processes in lipid metabolism. SREBP is an ER resident protein that is shuttled to the Golgi where it is activated by ubiquitination to subsequently be cleaved for release into the cytosol and transport to the nucleus [START_REF] Stewart | Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex[END_REF].

There are a number of additional noticeable and interesting features to the set of UR proteins in hid3Δ. Discussed quite extensively was the induction of the MAPK stress-response signaling pathway and oxidative stress-response proteins, such as the complement of shortchain dehydrogenases, NADPH dehydrogenases and glutathione metabolism proteins. One point that needs to be added here is that the induction of many of these proteins, such as the short-chain dehydrogenase Osr1 and Wis1 appear to be under transcriptional control [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF] and so demonstrating that hid3Δ is under chronic stress and is trying to adapt or cope with it. One way to cope with chronic stress is either through cell death, which is not really an option for single cell organisms, or to adopt a quiescent state of growth. The metabolomic and protein profiling are consistent with the hypothesis that cells are in a quiescent state [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF]. One interesting question is if adoption of a quiescent state is compatible with activation of mating-type switching. Indications are that is it. Nitrogen starvation induces mating-type switching [START_REF] Sun | Global analysis of fission yeast mating genes reveals new autophagy factors[END_REF] as well as quiescence [START_REF] Sideri | Parallel profiling of fission yeast deletion mutants for proliferation and for lifespan during long-term quiescence[END_REF]. In addition, the induction of autophagy, which may be important in the nutrition of quiescent cells is depend on mating type [START_REF] Sun | Global analysis of fission yeast mating genes reveals new autophagy factors[END_REF]. A major contrast of the proteomic results from those of the transcriptics is the lack of UR of transporter proteins, but the genes are UR. Some transporter proteins mainly involving phosphate transport were shown to be DR. This result supplies part of the mechanism to explain the changes in gene expression. The lack of transport capacity leads to starvation of an element (unknown at this time) leading to increased expression of the genes to make more protein. Even if protein is being made under a reduced biosynthetic capacity, it is unlikely that they are reaching the plasma membrane, because of disrupted Golgi function, and so are being degraded.

IV Future work

The phylogeny is an ongoing process that will require more detailed analysis of sequence properties. There are a number of sequences that fell below the criteria for selection within the global phylogenetic analysis that could provide more information as to early common features between the HIDs and DYMs. Also, it would be useful to try to root the trees using a more divergent sequence, such as c10orf76, which is the most similar protein pulled up by the BLAST searches. In an experimental approach is will be necessary to express the human and C. elegans HIDs and DYMs in hid3Δ in order to see if any complement the poor growth phenotype on minimal media. It has recently been reported that hid3Δ strains are hypersensitive to the drug bortezomib [START_REF] Takeda | Identification of genes affecting the toxicity of anti-cancer drug bortezomib by genome-wide screening in S. pombe[END_REF]. The sensitivity to this drug is another way to test for functional complementation with the animal proteins.

It will certainly be necessary to investigate the reason for both hid1Δ and hid3Δ to be sensitive to oxidative stress. The complementation of the oxidative stress sensitive phenotype in the hid1Δ hid2Δ double mutant suggests that it may be possible to look for genetic suppressors in order to identify the genes responsible. Likewise, it will be necessary to determine why EMM has such a negative effect on hid3Δ growth. It may be possible to test if the re-addition of any particular component(s) to the minimal media may be able to stimulate hid3Δ growth.

From a biochemical aspect it would be interesting to determine the orientation of Hid3 in Golgi membranes and why it is important for maintaining Golgi Structure. There are tools that have been established for plants that can be adapted for use in S. pombe [START_REF] Søgaard | GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues[END_REF] and the use of the antibodies will be important for these studies. The antibodies may also be useful for identifying proteins that interact with Hid3 as has been done for Upb5 with a TAPtagged Hid3 [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. The purpose of showing the antibody results, although very preliminary, is that I am developing some of the tools necessary to investigate protein interactions with Hid3, which I had discussed quite extensively in Chapter 3. In addition, they can be used to investigate the subcellular localization of the Hids, through in immuno hybridization for example.

Besides mentioning Ras1, I have not discussed the dramatic changes to GTPase signaling proteins that I observed. This includes the large number of actin cortical patch assembly proteins that were seen to be UR. First, it will be necessary to determine if their subcellular location, particularly that of Ras1, changes in hid3Δ.

A major question is what is regulating protein changes in hid3Δ? For cell stress mRNA and protein levels are generally correlated [START_REF] Lackner | Regulation of transcriptome, translation, and proteome in response to environmental stress in fission yeast[END_REF] and this is what we see for the limited metabolic genes and proteins related to stress. There are three possible mechanisms to explain the change in protein levels. One way is through altering transcription to produce more or less mRNA for translation. This can be addressed by looking at levels of directly transcribed mRNA that are integrated into polysomes called Ribosome profiling [START_REF] Ingolia | Ribosome profiling: new views of translation, from single codons to genome scale[END_REF]. mRNA from polysomes can be quantified using RNAseq and this can be associated with the protein composition [START_REF] Fuchs | Proteomic analysis of ribosomes: translational control of mRNA populations by glycogen synthase GYS1[END_REF]. The second way is through mislocalisation of Ubp5 leading to altered ubiquitination in the cytosol and nucleus. Protein turnover under ubiquitination/deubiquitination control involving Ubp5 can be determined by use of the ubp5 mutant. Finally, proteins that are mislocalised due to disruption of the Golgi complex could be determined by using a brefeldin A to disrupt the Golgi complex. For example, Ras1 is a very good example of a protein that does not show transcriptional regulation of its levels in hid3∆.

The gene expression analysis conducted by [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF] showed that ras1+ transcript levels do not change in the mutant compared to VCs, WT or hid1Δ genotypes. Disruption of the Golgi apparatus may remove Ras1 from one of a number of Golgi located D3 ubiquitin ligases, therefore, preventing its ubiquitination and subsequent turnover. Alternatively, disruption of the Golgi may bring Ras1 in more frequent contact in order to be ubiquitinated. This would entail a combined study to identify proteins changing in common and distinctly. This could be expanded to include both oxidative stress signalling and nutrient signalling.

Figure 3

 3 Figure 3.1.Presence of HID (A) or DYM (B) orthologues in the higher phylogenetic groupings.

Figure

  Figure 3.2.Phylogenetic groupings of the HIDs and DYMs.

Figure 3

 3 Figure 3.3. Multiple sequence alignment comparing S. pombe Hids with HID1 from C. elegans and H.sapiens.

Figure

  Figure 3.4.Comparison of HID domain structures.

Figure 3

 3 Figure 3.5.STRING output for pombe and human HID interactions.

Figure

  Figure 3.6.Model of Hid3 function.

Figure 4

 4 Figure 4.1.Relative expression of hid genes under stress.

Figure 4. 2

 2 Figure 4.2 Effects of oxidative stress on mutant growth.

Figure

  Figure 4.3.Differential growth of mutants on minimal medium.

Figure 4

 4 Figure 4.5.The effect of Golgicide A (GA) on mutant growth.

Figure

  Figure 4.6.Hid3 involvement in protein transport through the Golgi Apparatus.

Figure 5

 5 Figure 5.1.Global comparison of metabolite data among mutant and WT genotypes.

Figure 5

 5 Figure 5.2.Denaturing PAGE of protein extracts used for proteome analyses.

Figure 5

 5 Figure 5.3.Denaturing PAGE preparation of samples for trypsin digest.

Figure 5

 5 Figure 5.4.PCA of normalised values of quantified proteins.

Figure 5

 5 Figure 5.5.The numbers of proteins significantly altered in amount.

Figure 5

 5 Figure 5.6.Ontology analysis of proteins with significantly altered amounts.

Figure

  Figure 5.7.Immuno-detection of Hid proteins.

  question demeure aussi concernant le rôle des deux gènes paralogues Ftp105/Hid3. Les travaux du groupe, dont ce projet de thèse fait partie, utilisent des technologies postgénomiques de transcriptomique, protéomique et métabolomique pour avoir une meilleure idée de ce que font ces protéines, pour étudier les réactions des mutants à la tension sans remplacer les gènes hid, puis pour déterminer les conséquences sur le métabolome et le protéome des mutants. Les données de ces travaux sont présentées dans trois parties principales (chapitres 3 à 5) pour finir par une discussion générale sur le projet et une proposition d'orientations futures des travaux (chapitre 6). Les résultats sont résumés pour chacune de ces parties. Le chapitre 2 détaille la méthodologie et n'a donc pas été inclus. XVI Chapitre 3: Analyses de calcul de la superfamille de protéines DYMECLIN et les gènes orthologues HID-1 issus de S. pombe Le chapitre 3 décrit les travaux de calcul pour analyser les HID et leurs relations avec leur membre le plus proche, le DYMECLIN (DYM). Les travaux phylogénétiques ont été conduits pour déterminer s'il pouvait être prouvé que des fonctions des deux protéines se chevauchaient. La présence ou l'absence de HID et DYM a été vérifiée dans les supergroupes et les taxons de premier rang de NCBI (tableau 3.1). Les animaux présentaient des protéines HID et DYM, leur lieu et leur action, comme l'association dynamique avec l'appareil de Golgi, semblaient être très similaires. Il est évident que les eucaryotes peuvent avoir des protéines DYM et/ou HID tandis que les animaux ont les deux. Les champignons, sauf quelques exceptions rares, n'ont que des protéines HID et les plantes n'ont que des DYM (figure 3.1, 3.2).

(

  figure 3.4). Enfin, des données disponibles au public ont été examinées pour découvrir des associations possibles de chacun des Hid avec d'autres protéines qui pourraient révéler des aspects de fonction (figure 3.5). Les Hid n'ont présenté qu'un nombre très faible d'associations, mais il a été démontré que Hid3 était associé à Ubp5 comme des rapports l'ont indiqué. De cette association, un modèle de base a été créé pour montrer comment la modification des niveaux de protéine Hid3 peut affecter les niveaux d'autres protéines à travers Ubp5 et au final le métabolisme et l'expression génique (figure 3.6).

Figure 1 . 1 .

 11 Figure 1.1. Trafficking through the endomembrane system in yeast. Proteins synthesized in the rough endoplasmic reticulum (ER) are passed to the Golgi apparatus by COPII protein complex coated vesicles in endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC).As the proteins and lipids traverse the Golgi, they are processed and/or modified and subsequently sorted to various compartments within the cell, such as the vacuole of plasma membrane, or secreted outside of the cell. The image was taken from[START_REF] Aguiar | Investigation of protein secretion and secretion stress in Ashbya gossypii[END_REF].

Figure ( 1

 1 Figure (1.2). Diagram of the Golgi apparatus and ER exit sites in yeast species. (A) Ordered Golgi stacks locate close to the ERES (light blue) in P. pastoris similar to S. pombe. The cis cisterna (red) of Golgi stack faces ERES. (B) in S. cerevisiae, Golgi cisternae are it matures into a medial and a trans

Figure 1 . 3 .

 13 Figure 1.3. The alternative growth stages of C. elegans. C. elegans starts in a small larval stage.Under good growth conditions it grows into an adult stage and can reproduce (Low population density). Under conditions of high population density where food might become limiting a pheromone is emitted which causes it to form a quiescent growth form called a Dauer larva. The image was reproduced from[START_REF] Mcgrath | Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes[END_REF].

  HID1 ortholog in S. cerevisiae, ECM30 By sequence, the most similar protein to HID1 in S. cerevisiae is called ECM30. It is still referred to in the Saccharomyces Genome database (yeastgenome.org) as a putative protein of unknown function. The gene is located on Chromosome XII between nucleotide positions 1007421 and 1011245 and the CDS is 3825 nucleotides long. It encodes a protein of 1274

1. 9 .

 9 Aims of the project and organisation of the thesis. The purpose of the Introduction of Chapter 1 was to introduce the idea that modification of Golgi function has effects beyond vesicular transport. It is intimately tied to the overall functioning of the cells through a number of signalling systems. Ubiquitinylation was the focus of this introduction, because manipulation of Ub processes is one of the proposed functions of the Ftp105/Hid3 protein of S. pombe. However, it is likely that Ftp105/Hid3 has other function in the Golgi or can affect other processes when mutated. There is also the question of what are the two Ftp105/Hid3 paralogues doing. The group work, which this thesis project is part of, is using post-genomic technologies of transcriptomics, proteomics, and metabolomics to give more clues as to what these proteins are doing., to investigate the stress responses of mutants without the hid genes replaced, and then to determine the consequences on the metabolome and proteome of the mutants.

Figure 1 . 4 .

 14 Figure 1.4. Schematic diagram of the possible role of S. pombe Ftp105/Hid3-mediated protein trafficking from the Golgi.

Figure 3 . 2 .

 32 Figure 3.2. Phylogenetic groupings of the HIDs and DYMs. (A) Neighbour-joining tree based on 2000 bootstrap replicates. (B) The bootstrap consensus tree of that shown in A. The trees were constructed in Mega6 using an alignment produced from MUSCLE[START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. The alignment was not

Figure 3 . 3 .

 33 Figure 3.3. Multiple sequence alignment comparing S. pombe Hids with HID1 from C. elegans and H. sapiens. The alignment was generated by MUSCLE in Mega6. Gaps, indicated by dashes, were incorporated to maximize the alignment, and positions of aligned residues are listed on the right side of the alignment. An asterisk indicates identical residues among the aligned sequences, a colon indicates conservation between amino acids with similar properties, and a period indicates conservation between amino acids of weakly similar properties.

  .org/clan/Golgi_traff) that contains 1159 domains representing 20 domain structures.Of the 754 DYM sequences, 664 have the DYM motif covering the entire sequence. Of the 405 HID1 sequences, 274 have the HID1 motif covering the entire sequence.

Figure 3 . 4 .

 34 Figure 3.4. Comparison of HID domain structures. The determination and alignment of domains was done using the ProDom website. Searches for particular entries were done using the Uniprot designations: HsHID1, uniProt Q8IV36; CeHID1, uniProt Q7Z135; SpHid1, uniProt Q9P7M6; SpHid2, uniProt Q9HDV3; ftp105 uniProt O13776. The red rectangles represent transmembrane helix domains, the blue rectangles represent cytosol-related domains, and black lines represent undefined sequences.

Figure 3 . 5 .

 35 Figure 3.5. STRING output for S.pombe and human HID interactions. The interactions were pulled from the STRING database by search term using either the gene-specific identifier SPAP27G11.12 for SpHid1 (A) and SPBPG19A11.11c for SpHid2 (B) or the gene name in the case of S. pombe ftp105 (C) and the human HID1 (D). The coloured circles (nodes) represent specific proteins or genes and the connecting lines (edges) represent a potential interaction. Only interactions representing direct experimental evidence (dark blue edges) or gene co-expression (gray edges) related evidence are shown. The confidence cut-off used for the association was 0.4.

Figure 3 . 6 .

 36 Figure 3.6. Model of Hid3 function. The known role of Hid3 is to facilitate the transport of Ubp5 into the Golgi apparatus (A). Upon elimination of Hid3, Ubp5 remains in the cytosol where it may remain active in the process of protein deubiquitination. This activity would expect to alter directly (solid arrows) protein levels and indirectly (dashed arrows) gene expression and metabolism.

  hid1 has been noted to show co-expression with two uncharacterized genes SPAC6B12.07c and SPCC18.03, which appear to have ubiquitin ligase activity and zinc ion binding activity (a PomBase notation). It also co-expressed with pof11, part of the ubiquitinatin ligase complex and for which there is evidence that the encoded protein binds Skp1 (Figure3.5A). Gene hid2 is coexpressed with mug191, a potential mannosidase involved in cell wall process, two uncharacterized genes, SPAPB2B4.06 and SPBC8E411.08c, potentially associated with the mitochondrion, an uncharacterized gene SPBC1711.11 potentially involved in vesicular transport and autophagy, and xrc4, which is involved in DNA repair, the protein of which has been localised to the nucleus and cytosol, like Hid2 (Figure3.6B).

Figure 4 . 1 .

 41 Figure 4.1. Relative expression of hid genes under stress. The values at times 15 and 60 min are relative to the zero time set to the arbitrary unit of 1. The figures were adapted from those taken from the Bähler laboratory website (http://128.40.79.33/projects/stress/) as

Figure 4 . 2

 42 Figure 4.2 Effects of oxidative stress on mutant growth. Cells of each mutant strain were grown overnight in standard YES at 30 °C with shaking at 200 RPM. The cells were counted using the Cellometer™ Mini (Ozyme Ltd). A volume of the starter culture was added to 5 ml of YES medium containing 0.7 mM H 2 O 2 to give a starting cell concentration of 5x10 5 cells/ml. An aliquot was taken to count the initial culture and the rest of the culture was incubated for 12 hours under the conditions above. The generation time was calculated using the cell concentrations at 0 and 12 hours. Each strain was tested at least 3 times. Two-tailed, Student's t-tests for unequal variance were used to determine statistical significance between genotypes. The mutant rad3 was statistically different from WT at P < 0.01. The genotypes hid1∆::natMX6 iso1, hid3∆::natMX6 iso1, hid3∆::natMX6 iso2, hid3∆::kanMX6 hid2∆:: natMX6 iso1 and hid3∆::kanMX6 hid2∆:: natMX6 iso2 were statistically different from both WT and VCN at P < 0.01. The other mutant genotypes were not significantly different from WT or the corresponding VC at P = 0.05.

Figure 4 . 3 .Figure 4

 434 Figure 4.3. Differential proliferation of mutants on minimal medium. Cells of each mutant strain were grown overnight in standard YES at 30°C with shaking at 200 RPM. The cells were counted using the Cellometer™ Mini (Ozyme Ltd) and a series of 10-fold dilutions of cells were made starting from 5x10

Figure 4 . 5 .

 45 Figure 4.5. The effect of Golgicide A (GA) on mutant proliferation. (A) Comparison of generation times in the absence and presence of GA. Preparation of cell pre-cultures, treatment (but with 50 μg/ml GA in DMSO), and calculation of generation time were done as described in the legends to Figures 4.2 and 4.3. Statistical difference between means for each genotype with and without GA were determined using Student's t-tests. This was not done for VCN with n = 2. Only for the hid1∆::natMX6 iso1 genotypes was there no significant effect of GA (threshold P = 0.05, n = 3). The stars indicate genotypes with statistically significant different means with and without GA based on a Tukey's post-hoc test (n = 5 for WT and hid3Δs). (B) Dunnett's post-hoc test for significantly different means as described in the legend to Figure 4.4

Figure 5 . 1 .

 51 Figure 5.1. Global comparison of metabolite data among mutant and WT genotypes. Metabolite data was obtained by 1 H-NMR profiling. The same samples were analysed as used for the proteomic study. (A) Variance in data as a function of Principal Component. (B) Pair-wise scores plots using the first 3 PCs. PoV represents the Percentage of Variance. (C) Loadings plots describing the scores plot of PC2 vs PC1. The amino acids are represented by the appropriate 3-letter code. UDP stands for uridine diphosphate, Ac for acetate and Adn for adenine dinucleotide. Unknown compounds are represented by the number of peaks comprising the signal (S for singlet, M for multiplet) and the shift in PPM from the external standard.

Figure 5

 5 Figure 5.2. Denaturing PAGE of protein extracts used for proteome analyses. Gels of cell protein extracts from VCN and WT (A) and mutant (B) genotypes, respectively. Total protein in each extract was determine by the Bradford method using BSA as standard. A total of 25 g of protein for each sample was prepared in Laemmli loading buffer and loaded onto a 10% SDS denaturing gel. After migration, the proteins were stained in QC Colloidal Blue (Bio-rad 161-0803. The VCN samples in (A) and the hid3∆ samples in (B) have been moved from the right side of the gel to the left side of the gel for the purposes of labeling the image. The molecular masses of the markers M are given on the right. The small letters represent an individual sample as explained in Table 5.1.

Figure 5

 5 Figure 5.3. Denaturing PAGE preparation of samples for trypsin digest. Ten g of total protein were loaded on to a 8% SDS-PAGE gel and migrated until all the proteins had entered into the gel. The square boxes (~0.5 x ~0.5 cm) represent size of the gel that was cut out for doing the in-gel trypsin digest. M stands for the molecular mass markers. The small letters represent individual samples for each genotype as shown in Table 5.1.

Figure 5

 5 Figure 5.4. PCA of normalised values of quantified proteins. PCA was conducted on normalized intensities of common proteins as determined by the Progenesis software. (A) Variance in data as a function of Principal Component. (B) Pair-wise scores plots using the first 3 PCs. PoV represents the Percentage of Variance. The number of proteins used for the PCA analysis was 1911 after removal of proteins for which samples had missing data. The PCA analysis was conducted in R using the package FactoMineR.

Figure 5 . 5 .

 55 Figure 5.5. The numbers of proteins significantly altered in amount. The numbers of proteins changing were determined by comparison to the VCN genotype. The numbers represent proteins changed in amount by more or less than 2-fold. The determination of relative protein amount was done using Progenesis LC-MS v 4.0 for proteomics (nonlinear DYNAMICS). Progenesis calculated statistical significance using ANOVA with a P-value cut-off of < 0.5.

Figure 5

 5 Figure 5.6. Ontology analysis of proteins with significantly altered amounts. The overrepresented classes were determined by GoMiner for significantly changing proteins compared to the set of all proteins used for the normalization set. The UniProt identifiers for all proteins were first converted to the unique S. pombe gene systematic Ids before being run through GoMiner. UR and DR stand for more and less abundant, respectively, than compared to VCN. The GO terms were reduced using REVIGO (revigo.irb.hr,[START_REF] Supek | REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms[END_REF] and the numbers in parentheses show the number of GO terms combined for the GO term shown. The degree of colour shows the relative degree of statistical significance.

Figure 5 . 7 .

 57 Figure 5.7. Slot blot immunolabeling of SpHid proteins in protein extracts of wild-type cells. Cells were grown in standard YES as described in Materials and Methods. Antibodies were generated in Rabbits by PerBio using peptides designed to yield antibodies exhibiting minimal cross-reactivity. The positive control is the antigen peptide for each protein to test cross-reactivity. Labeling and detection was conducted using Invitrogen's WesternDot™ 625 Western Blot Kit according to manufacturers instructions.

Table of Contents Récapitulatif en Français & Mots Cléts II English Summary & Key Words III Table of Contents IV Introduction

  Diagram of the Golgi apparatus and ER exit sites in yeast species.

	Figure 1.1.	Trafficking through the endomembrane system in yeast.
	Figure 1.2
	Figure 1.3.	The alternative growth stages of C. elegans.
	Figure 1.4	Schematic diagram of the possible role of S. pombe Ftp105/Hid3-mediated protein
		trafficking from the Golgi.
	Chapter 2	Materials and Methods
	Table 2.1	Chemical Stress agents.
	Table 2.2	List of genotypes used for growth analyses
	Chapter 3	Computation

analyses of the DYMECLIN protein superfamily and the HID-1 orthologues from S. pombe.

Table 3 .

 3 1.List of evolutionary lineages containing putative DYMs or HIDs. Table3.2.

Table 4 .

 4 1.A summary of the effect of general stresses on the growth of mutant genotypes.Table 4.2.A summary of the effect of DNA damaging agents on growth of mutant genotypes.

Table 5

 5

	.2.	Metabolite absolute values as determined by	1 H-NMR.
	Table 5.3.	Summary statistics for proteomic analyses.
	Table 5.4.	List of proteins in hid1∆ with significantly altered amounts.
	Table 5.5.	Proteins significantly altered in abundance in hid3∆.

2 Charaterization of mutant growth and stress responses 2.2.1 Growth of S. pombe All

	4 %, w/v) plates at 30 o C for 3
	days for short-term storage at 4 o C. Routine growth of cells was in 5 ml liquid cultures of YES
	media at 30 o C with shaking at 200 rpm. Edinburgh Minimal Medium (EMM) and EMM with agar
	adenine, histidine and lysine were purchased from Sunrise Science Products via ELIS Solution
	(Erpent, Belgium). In order to test for growth defects under normal conditions, wild-type and
	mutant strains were inoculated into 5 ml of YES medium and incubated for 12 hours at 30 o C
	with shaking at 200 rpm. Cells were diluted into fresh YES or EMM medium at a cell number of
	5 x 10

Tree construction was done in MEGA6 by either the Neighbour Joining or Maximum-Likelihood methods. Confirmation of functional identity as either DYM or HID, that is no other conflicting identification, was done by rescreening of the NCBI, or a database for a specific organism (e.g. Wormbase, Pombase, etc.) by BLAST analysis. Closer phylogenetic relationships were investigated through syntenic relationships among organisms, such as the fungal HIDs through the YGOB genome browser, and protein structural and functional features found in ProDom, pFam, InterPro and UniProt.

Mapping of protein-protein interactions was performed using the Strings database (www.stringdb.org).

2.strains of S. pombe were stored at -80 o C in YES medium (0.5 % w/v yeast extract, 3 % w/v glucose, 225 mg/l each of adenine, leucine, uracil, histidine and lysine) containing 25 % reagent grade glycerol (VWR). Cells were grown on YES/Agar (1.5 cells/ml and incubated at 30°C with shaking at 200 rpm.

Table 2 .1 Chemical Stress agents.

 2 The following table provides the type and concentration of the chemical stress treatment and the solvent in which it was reconstituted.

	Drug	Vendor		[Working]	[Stock]	Solvent
	Hydroxyurea (H.U)	Sigma		3mM, 5mM and 10mM	0.1g	10 ml DMSO
	Methyl Methanesulfonate	Fluka		0.0025%, 005% and 0.0075%	100µl	9.9 ml H2O
	Thiabendazole (TBZ)	Sigma	10, 12.5, 15, 20, and 25 μg/ml	0.1g	10 ml DMSO
	Camptothecin	Sigma		0.5 μg/ml and 0.8 μg/ml	0.1g	10 ml DMSO
	Phleomycin	InvivoGen	1, 2.5, 5 and 10 μg/ml	0.1g	10 ml H2O
	Brefeldin a	Sigma		5, 15, 20, 25 μg/ml	10mg	1 ml DMSO
	Golgicide a	Sigma		50 μg/ml	10mg	1 ml DMSO

Table 2 .2. List of genotypes used for experimentation.

 2 All mutant genotypes include the parental, wild-type genotype as well as the modified hid locus.

	Strain	Genotype	Source
	Wild-type	ade6-m26 ura4-D18 leu1-32	McFarlane, BP90
	VCK	kanMX6	Alsherhi
	VCN1	natMX6	Alsherhi
	VCN2	natMX6	Alsherhi
	VCU1	ura4 +	Alsherhi
	hid1∆ iso1	hid1∆::natMX6	Alsherhi
	hid1∆ iso3	hid1∆::natMX6	Alsherhi
	hid2∆ iso1	hid2∆::ura4 +	Alsherhi
	hid2∆ iso2	hid2∆::kanMX6	Alsherhi
	hid2∆ iso3	hid2∆::kanMX6	Alsherhi
	hid3∆ iso1	hid3∆::natMX6	Alsherhi
	hid3∆ iso2	hid3∆::natMX6	Alsherhi
	hid3∆ iso3	hid3∆::kanMX6	Alsherhi
	hid3∆ iso4	hid3∆::kanMX6	Alsherhi
	hid3∆ iso3 hid2∆ iso1	hid3∆::kanMX6 hid2∆::natMX6	Alsherhi
	hid3∆ iso3 hid2∆ iso2	hid3∆::kanMX6 hid2∆::natMX6	Alsherhi
	hid3∆ iso4 hid1∆ iso1	hid3∆::kanMX6 hid1∆::natMX6	Alsherhi
	hid3∆ iso4 hid1∆ iso2	hid3∆::kanMX6 hid1∆::natMX6	Alsherhi
	hid2∆ iso1 hid1∆ iso1	hid2∆::ura4 + hid1∆::natMX6	Alsherhi
	hid2∆ iso1 hid1∆ iso2	hid2∆::ura4 + hid1∆::natMX6	Alsherhi

  Table 2.1. For abiotic stresses, such as heat or cold, diluted cultures were placed under appropriate conditions with shaking at 200 rpm. Cold and heat treatment consisted of growth of cells at 23 o C and 37 o C, respectively. All stress treatments lasted for 12 hours. The genotypes subjected to stress treatments are given in Table 2.2. Growth data is

generally presented as Generation Time (GE), i.e. the doubling-time of the culture, and was calculated from starting and end cell counts according to the equation GE =12/(3.3*Log(N t=12 /N t=0 ) where N t=12 was the number of cells at 12 h and N t=0 was the starting number of cells. S.pombe cells were grown on YES medium appropriate supplements. To block ER-to-Golgi transport, cells were treated with various concentrations of BFA 0, 5 and 25 μg/ml for 12 hours. Cells treated with DMSO were used as controls. A concentration of 50 μg/ml was used for GCA to test the strains.

  . Briefly, the cells were extracted with a mixture of H 2 0/ethanol/chloroform and the aqueous phase recovered and dried under vacuum. The extracts were resuspended in 200 mM phosphate buffer pH 6.0 in D 2 O and analyzed at 500.162 MHz on a Bruker spectrometer (Bruker Biospin Avance). Metabolites were quantified using the metabolite mode of AMIX software (Bruker Biospin v. 3.5.6) based on the number of protons comprising the corresponding resonance. Concentrations in the NMR tube were converted to amounts per g fresh weight using the mass of sample extracted. Citrate, formate, fumarate, glutamate, lactate and malate are expressed as μg of the acid form. The concentration of NMR unknown compounds (named according to the form of the resonance, S

Table 3 .1. List of evolutionary lineages containing putative DYMs or HIDs.

 3 The HIDS from H. sapiens and S. pombe and the DYMS from H. sapiens and A. queenslandica were used to screen NCBI Genbank for putative homologues at the phylum or subphylum level. Sequences were recovered that exhibited a DELTA-BLAST score of <0.001 and sequence coverage of at least 40%. The highest-level scientific names were taken from NCBI taxonomy and their classification as given in[START_REF] Adl | The new higher level classification of eukaryotes with emphasis on the taxonomy of protists[END_REF] or Cavalier-Smith

Table 3 .3. GO ontology references to potential function of Hid proteins from S. pombe.

 3 The gene ontology listings were taken from AmiGO 2 (amigo.geneontology.org).

	Protein	Ontology listings	GO accession	Function	Ref.
	Hid1	Nucleus	GO:0005634	cellular_component	1
		Cytosol	GO:0005829	cellular_component	1
	Hid2	Nucleus	GO:0005634	cellular_component	1
		Cytosol	GO:0005829	cellular_component	1
	Hid3	protein binding	GO:0005515	molecular_function	2
		Golgi apparatus	GO:0005794	cellular_component	1
		Cellular protein
		Localization	GO:0034613	biological_process	2
		Cytosol	GO:0005829	cellular_component	1

1

[START_REF] Matsuyama | ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe[END_REF]

2

[START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]

Table 4 .1. A summary of the effect of general stresses on the proliferation of mutant genotypes.

 4 The effect on growth was determined by cell count of cells incubated in YES liquid culture over period of 12h. Abbreviations are: TBZ, thiabendazole; BFA, brefeldin A; GA, golgicide A. The letters N and Y denote 'no' difference or an 'apparent' difference, respectively, compared to negative controls VCN and VCU expressing the marker gene. Each test was performed at least twice for each genotype, except where not determined (nd).

	Stress treatment

Table 4 .2. A summary of the effect of DNA damaging agents on proliferation of mutant genotypes.

 4

Table 5 .1. Scheme of preparation of samples for post-genomic studies.

 5

Table 5 .4. List of proteins in hid1∆ with significantly altered amounts.

 5 Those proteins are shown that were significantly changed in abundance 2-fold UR or DR in the mutant than in the WT. Proteins were identified using the SEQUEST algorithm in Proteome Discoverer™ (Thermo Scientific).

	UniProt	Protein		Ratio
	Accession	Name	hid1∆/VCN	Description
	2-fold UR
	Q9US41	Urg1		5.3	Uracil-regulated protein
	Q9US43	Urg2		3.3	Putative uracil phosphoribosyltransferase
	P78831	Ght5		2.8	High-affinity glucose transporter
	O94505	Tol1		2.7	3'(2'),5'-bisphosphate nucleotidase
	O60181	SPBC23E6.06c	2.7	3,4-dihydroxy-2-butanone 4-phosphate synthase
	Q9HEQ9	Tcg1		2.6	Single-stranded TG1-3 DNA-binding protein
	O14351	SPBC30D10.05c 2.5	Uncharacterized oxidoreductase
	Q9P7F8	Get3		2.4	ATPase
	Q09717	Sbp1		2.4	Ran-specific GTPase-activating protein
	O14293	SPAC9E9.09c		2.4	Putative aldehyde dehydrogenase-like
	Q9US39	SPAC1039.02		2.3	Uncharacterized protein
	Q9P782	SPBC1711.08		2.3	Uncharacterized protein
	Q9UTJ7	Sdh1		2.3	Probable succinate dehydrogenase,mitochondrial
	O42699	Rpl19b		2.3	60S ribosomal protein L19-B
	Q09816	SPAC16C9.02c	2.3	S-methyl-5'-thioadenosine phosphorylase
	Q9P7B4	SPAC521.03		2.3	Uncharacterized oxidoreductase
	P31406	Vma1		2.3	V-type proton ATPase catalytic subunit A
	O75000	L12		2.3	60S ribosomal protein L12
	P54874	Hcs1		2.3	Hydroxymethylglutaryl-CoA synthase
	O36032	Grx1		2.2	Glutaredoxin-1
	Q9P7D4	SPBP4H10.15		2.2	Probable aconitate hydratase 2
	O74178	Kes1		2.2	Protein kes1
	O13991	SPAC26H5.09c	2.2	Uncharacterized oxidoreductase
	Q09184	Cdb4		2.2	Curved DNA-binding protein
	P31317	Arg3		2.1	Ornithine carbamoyltransferase,mitochondrial
	O74893	Rps20		2.1	40S ribosomal protein S20
	P87144	Ths1		2.1	Threonine--tRNA ligase, cytoplasmic
	Q9P7M9	Rib1		2.1	Probable GTP cyclohydrolase-2
	Q9UT27	Pvg1		2.0	Pyruvyl transferase
	Q9C0U6	Xks1		2.0	Xylulose kinase
	O94373	Atp4		2.0	ATP synthase subunit 4, mitochondrial
	2-fold DR
	O94582	Trp3		9.1	Probable anthranilate synthase component 1
	Q9Y7U0	SPCC63.14		8.6	Uncharacterized protein
	O74440	Rpn8		8.4	26S proteasome regulatory subunit
	O94601	SPCC622.14		5.8	Uncharacterized protein
	Q9P6R6	Atp16		4.6	ATP synthase subunit delta, mitochondrial
	O42940	Pho88		4.2	Inorganic phosphate transport protein
	O74417	SPCC14G10.04	4.0	Uncharacterized protein
	Q09822	Cdc15		3.7	Cell division control protein 15
	O94258	Los1		3.2	Exportin-T
	O43024	SPBC354.10		2.9	CUE domain-containing protein
	Q9C0W7	Apl3		2.9	AP-2 complex subunit alpha
	O14192	SPAC56E4.03		2.9	Aromatic amino acid aminotransferase
	O74455	SPCC16C4.10		2.8	Probable 6-phosphogluconolactonase

Table 5 .5. Proteins significantly altered in abundance in hid3∆.

 5 Shown are those proteins more (UR) and less abundant (DR) in the mutant vs WT at a level of 2-fold or greater. Proteins were identified using the Proteome Discoverer™ (Thermo Scientific) and relative quantities were determined using the Progenesis QI™ proteome quantification software (nonlinear DYNAMICS).

	UniProt	Protein	Ratio
	Accession	Name	hid3∆/VCN	Description
	2-fold UR
	Q7Z9I3	SPCC663.08c	26.6	short chain dehydrogenase
	Q7Z9I4	SPCC663.06c	15.1	short chain dehydrogenase
	O94467	SPBC23G7.10c	14.9	Putative NADPH dehydrogenase
	O59827	Gst2	5.4	Glutathione S-transferase 2
	Q9Y876	Psh3	5.2	ER chaperone SHR3 homologue Psh3
	Q09720	SPAC31A2.04c	5.1	Probable proteasome subunit beta type-4
	P04913	Htb1	4.3	Histone H2B-alpha
	P36627	Byr3	4.1	Cellular nucleic acid-binding protein
				homolog
	O74409	SPCC1223.09	3.3	Uricase
	O74875	SPCC330.03c	3.3	Uncharacterized heme-binding protein
	P08647	Ras1	3.2	Ras-like protein 1
	O74440	Rpn8	3.2	26S proteasome regulatory subunit
	O14224	SPAC6F12.06	3.2	Rho GDP-dissociation inhibitor
	Q9Y7U0	Eis1	3.2	eisosome assembly protein eis1
	O60071	Enp1	3.1	bystin family U3 and U14 snoRNA
				associated protein (predicted)
	O42869	Spa2	3.1	cell polarity protein Spa2
	Q9Y7K0	SPBC216.03	3.0	UPF0659 protein
	O94312	Pdp2	2.9	PWWP domain-containing protein 2
	Q10367	Glo3	2.9	ADP-ribosylation factor GTPase-
				activating protein
	O94246	Gcs2	2.8	glutamate-cysteine ligase regulatory
				subunit (predicted)
	Q10172	Pan1	2.8	Actin cytoskeleton-regulatory complex
				protein
	O74135	Cki3	2.7	Casein kinase I homolog 3
	P41891	Gar2	2.7	nucleolar protein required for rRNA
				processing
	Q92357	Cfr1	2.7	Chs five related protein Cfr1
	O14295	Plr1	2.7	Pyridoxal reductase
	O43021	SPBC354.07c	2.6	Oxysterol-binding protein homolog
	O14368	Hsp16	2.6	Heat shock protein 16
	O60184	Ssn6	2.6	General transcriptional corepressor
	O13791	Slt1	2.6	Schizosaccharomyces specific protein
	Q10488	Etr1	2.5	Probable trans-2-enoyl-CoA reductase,
				mitochondrial
	O42940	Pho88	2.5	Inorganic phosphate transport protein
	P30821	Obr1	2.5	ubiquitinated histone-like protein Uhp1
	O94315	SPBC215.11c	2.5	Uncharacterized oxidoreductase
	Q09859	Mxr1	2.4	Probable peptide methionine sulfoxide
				Reductase
	O94601	SPCC622.14	2.4	GTPase activating protein (predicted)
	Q02592	Hmt1	2.4	vacuolar transmembrane transporter Hmt1
	P41820	Bfr1	2.3	brefeldin A transmembrane transporter
	Q9Y7N9	SPCC1450.12	2.3	PX domain-containing protein

cells/ml (left column). A volume of 5 ml was placed for each spot onto petri dishes containing either YES/agar or EMM plus supplements. The EMM was purchased from Formedium Ltd (Norfolk, United Kingdom). Plates were incubated for 4 days before images were taken.

Acknowledgments

This work was funded by Tabuk University in Saudi Arabia. I extend my fondest thanks to my family, who supported me during my PhD journey. My deep gratitude also goes to my supervisor, Dr. Mark Hooks, for all his guidance, support and encouragement. Many thanks are extended to all the people who supplied me with strains, especially Dr. David Pryce. I also would like to thank Dr. Marc Bonneu and Dr. Stephane Claverol of the Proteomics Platform at the Centre Génomique Fonctionelle de Bordeaux for conduction the protein quantification proteomics data. I also gratefully acknowledge the support from all my colleagues, past and present, and especially the members of the INRA Labs. X

(gi|545359709|) from the Trebouxiophyceae and all the Prasinophyte sequences, except

Ostreococcus tauri, which was highly similar to Ostreococcus lucimarinus (gi|145341226|).

The list of the taxa searched and the presence or absence of HID or DYM homologues is given in The lists of proteins that were UR and DR at 2-fold or more are shown for hid1 and hid3 are shown in Tables 5.4 and 5.5, respectively. In agreement with the GO analysis inspection of the list of proteins significantly UR in hid1 does not provide any direct indication of specific processes that were affected. The top two induced proteins were related to uracil metabolism and may reflect the requirement to add supplements to the growth media. The list of DR proteins contains the GTPase activating factor Glo3 and two proteins involved in microtubule formation at the plasma membrane, Cdc15 and Hob3. In contrast, the list of UR proteins for hid3 reveals increased stress responses and signalling processes related to stress. The top four proteins comprised of the two short-chain dehydrogenases SPCC663.08c

(26.6-fold) SPCC663.06c (15.1-fold), a putative NADH dehydrogenase (14.9-fold) and Gst2

(5.4-fold) are induced in response to oxidative [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF] and/or iron starvation stress [START_REF] Gabrielli | Cells lacking pfh1, a fission yeast homolog of mammalian frataxin protein, display constitutive activation of the iron starvation response[END_REF]. Other stress-induced proteins listed are uricase, the pyridoxal reductase Plr1 and the heat-shock proteion Hsp16, all which in induced in response to oxidative stress [START_REF] Chen | Global transcriptional responses of fission yeast to environmental stress[END_REF]. GTPase signalling proteins were well represented on the list by the Rho dissociation inhibitor SPAC6F12.06, Ypt2, and many directly related to vesicular transport, such as Ras1, Glo3, Hob3, Stm1 and Ypt5. In addition, there were some GTPase-signaling related proteins, such as gap1, that were induced in the range of 1.5 to 2-fold. Proteins involved in other signaling processes were also induced, such as the cytosolic translational activator Byr3, the transcriptional repressor Ssn6, the hsp-interacting protein Sti1, the cyclin-dependent protein kinase Cdc2, the MAP kinase kinase Wis1 and glyceraldehydes 3-dehydrogenase Gpd3. Other signaling proteins that were increased are associated with vesicular transport, such as the casein kinase Cki3, the Chs five related protein Cfr1, the calnexin homolog Cnx1, and the TF Hmo1. Members of two interesting classes of proteins were induced as well and these have to do with chromatin remodeling and microtubule/cytoskeleton organization. The chromatin structure/remodeling proteins were the histone H2B-alpha subunit Htb1, the PWWP domaincontaining protein Pdp2, and the ubiquitinated histone-like protein Uhp1 (Obr1). There were 7

proteins UR that are involved in microtubule/cytoskeleton organization, Abp1, Arc1; Dph1; Hob3; Pan1; Slt1 and Spa2. The list of DR proteins in hid3 reinforced the decrease in metabolic processes shown by the GO analysis. Of the 347 proteins DR by more than 1.5-fold in hid3Δ, 34 were ribosomal proteins or proteins involved in ribosome biogenesis. It also suggested that transport processes that could be affected.

Chapter 6

General discussion and future work

The experimentation that is presented in this work went in three phases. The first phase was to conduct a bioinformatic analysis of the HIDs in order to establish the ground-work for using S. pombe as a model for HID function in animals, particularly humans. The second phase built on this, and on preliminary growth and morphology characterisation of the mutants conducted by [START_REF] Alshehri | Characterisation of transcriptional properties of the hid1Δ and hid3Δ mutants of Schizosaccharomyces pombe[END_REF], as a functional study in order to investigate the stress responses of the hid mutants, particularly hid1 and hid3, and the role of Hid3 in the Golgi. The third phase involved a proteomic study to determine if protein levels would change according to the stress responses and various models that had been proposed in the literature to ascribe a function to Hid3. I have divided this discussion into four sections each relating to each of the Chapters of the thesis and a presentation of future areas of work. I hope that is it realised that this discussion couldn't cover all hypotheses or mechanisms that may arise from the experimentation, particularly from the proteomics.

I. Chapter 3 conclusions

From the literature, there were three major ideas regarding the potential function of HID1 in humans. The first one came in 2001 with the publication by [START_REF] Harada | Identification of DMC1 , a novel gene in the TOC region on 17q25 . 1 that shows loss of expression in multiple human cancers[END_REF]) that deletion or reduction of expression of HID1 (then c17orf28 or DMC1) contributed to cell proliferation in a number of cancer types. The second was showing that HID-1 of C. elegans is required for normal vegetative growth of the worm [START_REF] Ailion | Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans[END_REF]. The third came from S.

pombe itself showing that elimination of Hid3 (then and still Ftp105) caused mis-localisation of the deubiquitinase Ubp5 from the Golgi to the cytosol [START_REF] Kouranti | A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity[END_REF]. More recently, general screening of S. pombe mutants in relation to quiescence due to nitrogen starvation suggested the involvement of Hid3 in maintaining normal growth [START_REF] Sideri | Parallel profiling of fission yeast deletion mutants for proliferation and for lifespan during long-term quiescence[END_REF]. Seeing that there are strong similarities between S. pombe and human cell physiology regarding Golgi function, it is possible that S. pombe Hid3 would serve as a good model to address the functions mentioned above.

The computational approach was attempted to identify aspects of HID function that were not already present in the literature, including potential complications for using S. pombe as a model system. The various databases, such as NCBI, Uniprot, etc. referred to HID1 as being conserved from fungi to humans, which is quite small on the evolutionary scale. In NCBI,