In order to address sustainable policies, communities need indicators that can tell them something about the larger social and ecological system, and the relationships between the two. This study attempts to apply an existing indicator of welfare and sustainability, the Genuine Progress Index (GPI), to the regional level in Sweden. The county of Östergötland is used as a case study and the thesis is written in collaboration with Regionförbundet Östsam. The aim of the study is to develop an application of GPI methodology to circumstances of data availability at the regional Swedish level. Developed methodology used to calculate a GPI of Östergötland in 2000-2006. GPI results are put in a context of the Regional Development Program of Östergötland in order to address policy implications regarding the very meaning of growth, development, welfare and sustainability. The results indicate that GPI has increased during the time period. However, a comparison with the Gross Regional Product (GRP) shows that GRP has increased faster than GPI. It is concluded that this could be a possible trend of decreased economic efficiency, where more economic output is not sustainable in the sense that an equivalent amount of welfare is not produced. This effect is mainly an effect of increasing income inequality. It is also suggested that the Regional Development Program should distinguish growth and development as different concepts. In doing so, more sustainable policies can be adopted in the future.

Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.

Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.

Research Doctorate - Doctor of Philosophy (PhD) / Membrane fusion events are a fundamental aspect of cellular biology and underpin important processes such as organ formation and fertilisation. Within the latter, proteins that are expressed on the egg surface which are responsible for mediating sperm recognition, binding and fusion to the egg, are yet to be fully determined. Evidence does however suggest that egg surface glycophosphatidylinositol (GPI)-anchored proteins play a role in sperm binding, whilst another class of proteins, known as tetraspanins, appear to be important in downstream events of membrane fusion. Of the tetraspanins, CD9 and CD81 have been identified as fulfilling roles in membrane fusion; identifications are however yet to obtained for the important GPI-anchored protein(s). This research aimed to identify and characterise egg surface proteins implicated in sperm-egg interaction, and embodied attempts to both identify the important GPI-anchored protein(s) as well as expand upon tetraspanin studies through investigations into mice lacking the tetraspanin CD151. Throughout this research, it was hypothesised that membrane fusion events of fertilisation parallelled those of enveloped virus – host cell fusion, for which rearrangement of surface protein thiols is essential. In vitro binding and fusion experiments were utilised as functional bioassays in the investigation of factors affecting sperm-egg interaction, such as tetraspanin deletion and the xenobiotic modification of cell surface thiols, while mass spectrometry (MS)-based proteomics and bioinformatics-based analyses were employed to compile oocyte protein databases and to identify candidate proteins responsible for mediating sperm-egg interaction, such as GPI-anchored proteins. It was determined that exposing oocytes to compounds with a capacity to alkylate cell surface thiols strongly inhibited sperm-egg binding. Additionally, while CD151 deletion had no effect on sperm-egg binding, the downstream events of membrane fusion were significantly impaired. Ovaries from CD151 null mice also exhibited abnormal phenotypes. In addition, a total of 11 identifications were obtained in the search for the GPI-anchored proteins expressed within eggs, however only 6 of these were deemed to be potential mediators of sperm-egg interaction. In conclusion, the experiments outlined herein demonstrate a novel inhibitory effect for specific xenobiotics on sperm-egg interaction, and correlate the inhibitory action of these compounds with their capacity to reduce cell surface thiol labelling. A novel role for CD151 in the mediation of sperm-egg fusion was also discovered, while at the same time the important GPI-anchored protein(s) implicated in sperm-egg binding may be among 6 identified potential candidates. Together the findings reiterate the consensus that oocytes possess a cell surface protein complex responsible for mediating sperm binding and fusion as separate events, and in light of the demonstrated importance of surface thiols, that events of sperm-egg membrane fusion parallel those of enveloped virus – host cell fusion.

Research Doctorate - Doctor of Philosophy (PhD) / Membrane fusion events are a fundamental aspect of cellular biology and underpin important processes such as organ formation and fertilisation. Within the latter, proteins that are expressed on the egg surface which are responsible for mediating sperm recognition, binding and fusion to the egg, are yet to be fully determined. Evidence does however suggest that egg surface glycophosphatidylinositol (GPI)-anchored proteins play a role in sperm binding, whilst another class of proteins, known as tetraspanins, appear to be important in downstream events of membrane fusion. Of the tetraspanins, CD9 and CD81 have been identified as fulfilling roles in membrane fusion; identifications are however yet to obtained for the important GPI-anchored protein(s). This research aimed to identify and characterise egg surface proteins implicated in sperm-egg interaction, and embodied attempts to both identify the important GPI-anchored protein(s) as well as expand upon tetraspanin studies through investigations into mice lacking the tetraspanin CD151. Throughout this research, it was hypothesised that membrane fusion events of fertilisation parallelled those of enveloped virus – host cell fusion, for which rearrangement of surface protein thiols is essential. In vitro binding and fusion experiments were utilised as functional bioassays in the investigation of factors affecting sperm-egg interaction, such as tetraspanin deletion and the xenobiotic modification of cell surface thiols, while mass spectrometry (MS)-based proteomics and bioinformatics-based analyses were employed to compile oocyte protein databases and to identify candidate proteins responsible for mediating sperm-egg interaction, such as GPI-anchored proteins. It was determined that exposing oocytes to compounds with a capacity to alkylate cell surface thiols strongly inhibited sperm-egg binding. Additionally, while CD151 deletion had no effect on sperm-egg binding, the downstream events of membrane fusion were significantly impaired. Ovaries from CD151 null mice also exhibited abnormal phenotypes. In addition, a total of 11 identifications were obtained in the search for the GPI-anchored proteins expressed within eggs, however only 6 of these were deemed to be potential mediators of sperm-egg interaction. In conclusion, the experiments outlined herein demonstrate a novel inhibitory effect for specific xenobiotics on sperm-egg interaction, and correlate the inhibitory action of these compounds with their capacity to reduce cell surface thiol labelling. A novel role for CD151 in the mediation of sperm-egg fusion was also discovered, while at the same time the important GPI-anchored protein(s) implicated in sperm-egg binding may be among 6 identified potential candidates. Together the findings reiterate the consensus that oocytes possess a cell surface protein complex responsible for mediating sperm binding and fusion as separate events, and in light of the demonstrated importance of surface thiols, that events of sperm-egg membrane fusion parallel those of enveloped virus – host cell fusion.

Le trafic intracellulaire est un processus fondamental qui maintient l'homéostasie cellulaire. Les RAB GTPases sont des régulateurs clés du trafic intracellulaire. RAB6 est la RAB résidente la plus abondante du Golgi. RAB6 est un régulateur clé de l'homéostasie Golgienne. Mon projet de thèse s'est intéressé à l'étude de la fonction de RAB6 dans la sécrétion post-Golgienne. Des études précédentes ont montré que la déplétion de RAB6 inhibe l'arrivée à la membrane plasmique de différents cargos : dans des cellules HeLa, NPY et VSV-G, et TNFα dans les macrophages. Nous avons donc émis l'hypothèse que RAB6 pourrait être un régulateur général de la sécrétion post-Golgienne. A l'aide de cellules MEFs RAB6 KO, nous avons d'abord montré que la sécrétion de toutes les protéines nouvellement synthétisées est inhibée. Pour comprendre les mécanismes entraînant cet effet, nous avons étudié le rôle de RAB6 dans le transport post-Golgien de trois types différents de cargos : GPI-APs (PLAP et CD59), collagen X, une protéine soluble, et une protéine transmembranaire TNFα. Afin de synchroniser le transport de cargos, nous avons utilisé le système RUSH. Ainsi, nous avons montré que RAB6 est présent sur les vésicules post-Golgiennes contenant les 3 types de cargos et que la déplétion de RAB6 affecte leur sécrétion. Les effecteurs de RAB6 sont aussi impliqués: Myosine II dans leur fission du Golgi, KIF5B dans leur transport vers la périphérie cellulaire, ELKS dans leur arrimage à la membrane plasmique. Finalement, nous avons pu montrer que les 3 cargos sont présents dans les mêmes vésicules post-Golgiennes avec RAB6. Ces résultats montrent que RAB6 régule la sécrétion de différents cargos. / Intracellular trafficking is a fundamental process which ensures cell homeostasis. RAB GTPases are key regulators of intracellular trafficking. RAB6 is the most abundant Golgi resident RAB and is a key regulator of Golgi homeostasis. My Ph.D project focused on understanding the function of RAB6 in post-Golgi secretion.Previous reports have shown that RAB6 depletion impairs the arrival at the plasma membrane of different cargoes: in HeLa cells, NPY and VSV-G and TNFα in macrophages. We thus hypothesized that RAB6 could be a general regulator of post-Golgi transport steps. Using MEF cells from RAB6 KO mice, we first showed that the secretion of all newly synthesized proteins is affected. To decipher the mechanisms leading to this inhibition, we have then investigated the role of RAB6 in the post-Golgi transport of three different classes of proteins, GPI-anchored proteins (such as Placental Alkaline phosphatase or PLAP and CD59), collagen X, a soluble protein, and the transmembrane protein TNFα. In order to synchronize transport of newly-synthetized cargoes along the secretory pathway, we used the RUSH system. Here, we show that RAB6 is present on post Golgi vesicles containing the three types of cargo and that RAB6 depletion affects their secretion to the plasma membrane. RAB6 effectors are also implicated: Myosin II for their fission from the Golgi, KIF5B for their transport to the cell periphery, ELKS/RAB2IP2 for their docking with the plasma membrane. Finally, we could show that these three cargoes are present in the same post-Golgi transport carriers with RAB6. Altogether, these results show that RAB6 regulates the secretion of a wide number of cargo proteins.

Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling.

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