To all the people that have been supporting me throughout this

journey

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Åkerberg H, Fällmar H, Sjödin P, Boukharta L, Gutiérrez-de-Terán H, Lundell I, Mohell N, Larhammar D (2010) Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions. Regulatory Peptides, 163(1-3): 120-129.

II Fällmar H, Åkerberg H, Gutiérrez-de-Terán H, Lundell I, Mohell N, Larhammar D (2011) Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes. Neuropeptides. 45(4):293-300.

III Xu B, Fällmar H, Boukharta L, Gutiérrez-de-Terán H, Lundell I, Mohell N, Åqvist J, Larhammar, D (2011) Investigation of residues in the human neuropeptide Y/peptide YY receptor Y2 involved in peptide binding based on homology modeling and docking. Manuscript.

IV Fällmar H, Sundström G, Lundell I, Mohell N, Larhammar D (2011) Neuropeptide Y/peptide YY receptor Y2 duplicate in zebrafish with unique introns displays distinct peptide binding properties. Accepted for publication in Comparative Biochemistry and Physiology

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11 The NPY-family of peptides .................................................................... 12

Structure ............................................................................................... 12 Function ............................................................................................... 12 Evolution ............................................................................................. 13

The NPY-family of receptors ................................................................... 14 G protein-coupled receptors ................................................................ 14 Evolution ............................................................................................. 16 The Y1 subfamily ................................................................................ 18 The Y2 subfamily ................................................................................ 19 The Y5 subfamily ................................................................................ 20

Appetite regulation and energy homeostasis ............................................ 22 The hypothalamus ................................................................................ 23 Regulation of food intake in fish ......................................................... 26

GPCR crystal structures and mutagenesis studies .................................... 26

Materials and methods .................................................................................. 27 Selection of positions for site-directed mutagenesis ................................ 27 Site-directed mutagenesis and receptor cloning ....................................... 28

Gateway® cloning ................................................................................ 28 QuikChange® II site-directed mutagenesis .......................................... 28

Transfection and harvesting ..................................................................... 29 Expression of receptor proteins for detection by microscopy .................. 29 Receptor binding experiments .................................................................. 29

Ligands ................................................................................................ 29 Saturation and competition assays ....................................................... 30

Statistical analyses of binding experiments .............................................. 30 Computational Y1 and Y2 receptor modeling ......................................... 30

Homology modeling ............................................................................ 30 Docking of hNPY ................................................................................ 31

BLAST searches and phylogenetic analyses ............................................ 32

Results and discussion .................................................................................. 33 Paper I and II ............................................................................................ 33 Paper III .................................................................................................... 36 Paper IV ................................................................................................... 37

Concluding remarks and future perspectives ................................................ 39 Paper I, II and III ...................................................................................... 39 Paper IV ................................................................................................... 40

Svensk sammanfattning ................................................................................ 42

Acknowledgements ....................................................................................... 44

References ..................................................................................................... 46

Abbreviations

ARC Arcuate nucleus 125I-PYY/NPY Iodinated PYY/NPY A2AR Adenosine 2A receptor Bmax Maximum binding capacity cAMP Cyclic AMP CXCR4 Chemokine receptor type 4 DMH Dorsomedial nucleus of the hypothalamus GFP Green fluorescent protein GI Gastrointestinal GPCR G protein-coupled receptor hPYY Human PYY HEK Human embryonic kidney Kd Dissociation constant Ki Inhibition constant Leu31, Pro34-hNPY Human NPY with Leu and Pro introduced at

position 31 and 34 in the peptide LHA Lateral hypothalamic area NPY, NPY3-36, NPY13-36 Neuropeptide Y, truncated NPYs NTS Nucleus of the solitary tract pNPY Porcine NPY PCR Polymerase chain reaction PP Pancreatic polypeptide PVN Paraventricular nucleus PYY, PYY3-36 Peptide YY, truncated PYY VMH Ventromedial nucleus of the hypothalamus wt Wildtype Y1, Y2,…Y7, Y8 Neuropeptide Y receptor Yn zf Zebrafish

1R/ 2R 1/2-adrenergic receptor

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Introduction

A body is a complex structure. It is made up of many different organs in close communication with each other. These organs consist of a variety of cell types which can interact with each other via direct contact, i.e. through larger cell adhesion molecules, or with the use of secreted ligands and membrane bound receptors. Ligands can be anything from small neurotransmitters to peptides and hormones or exogenous compounds depending on the target receptor. The majority of them are under the control of the brain.

In order to function properly, a body needs energy. That is achieved by food consumption, which is one of the basic prerequisites for animal survival. Appetite is rigidly regulated by a well-controlled system of interaction between several hormones, neuropeptides, neurotransmitters and metabolites. This system in humans, however, is today challenged by the increased access to food in combination with less physical activity. In a recent meta-analysis it was stated that 500 million people worldwide are considered obese (Finucane et al., 2011) and this further reinforces obesity as an epidemic. The major peripheral and central structures involved in energy intake regulation and expenditure are the gustatory system, the gastrointestinal tract, the pancreas, the liver, the muscles, the adipose tissue, the caudal brainstem, the hypothalamus and parts of the cortex and limbic system (Lenard and Berthoud, 2008).

The NPY families of peptides and receptors are involved in several central as well as peripheral functions in the body and comprise a very important system in appetite regulation. Increased knowledge about the NPY system will contribute to the understanding of metabolism and physiology and thus be useful in the development of strategies against diseases.

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The NPY-family of peptides Structure Neuropeptide Y (NPY) was first isolated from porcine brain by Tatemoto and coworkers in 1982 (Tatemoto, 1982; Tatemoto et al., 1982). NPY was found to show structural and biological similarities to peptide YY (PYY) and pancreatic polypeptide (PP). Together they constitute the NPY-family of peptides (Larhammar, 1996). They all have a protein structure shape of a “U” consisting of an extended polyproline helix and an -helix connected by a -turn, commonly referred to as the PP-fold (Glover et al., 1984; Schwartz et al., 1990). They are 36 amino acids long with an amidated carboxy terminus (Figure 1). Subsequently, it was confirmed that this 3D structure was retained in solution (Darbon et al., 1992; Keire et al., 2000; Li et al., 1992). Nevertheless, NPY has been found to adopt a more flexible structure than PYY and PP at low concentrations in solution (Bettio et al., 2002; Lerch et al., 2004). In mammals, the peptides NPY and PYY also exist in truncated forms after cleavage by the enzyme dipeptidyl peptidase IV (Mentlein et al., 1993). However, due to the third amino acid in the N-terminal of the fish PYY peptides being a proline, cleavage with this enzyme cannot happen in these species (Mentlein, 1999).

Figure 1.Schematic picture of the human NPY peptide structure showing the characteristic PP-fold. The polyproline helix is highlighted in light gray followed by the -turn in white, the -helix in black and the amidated carboxy terminus in dark grey.

Function NPY is a neurotransmitter involved in the regulation of several physiological processes including cardiovascular functions, anxiety, circadian rhythm,

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reproduction, growth, bone formation and appetite (Lee and Herzog, 2009; Morales-Medina et al., 2010; Pedrazzini et al., 2003). The first reports of a correlation between NPY and food intake were published in 1984 (Clark et al., 1984; Levine and Morley, 1984). Since then, several studies have confirmed these findings and NPY is today described as one of the most orexigenic (appetite stimulatory) endogenous neuropeptides in the brain. NPY is released in the hypothalamus prior to food intake and is known to increase food intake mainly via the Y1 and Y5 receptors (Gehlert, 1999; Lecklin et al., 2002; Lecklin et al., 2003; Zhang et al., 2011).

In contrast to NPY, both PP and PYY are hormones released postprandially (after a meal) from endocrine cells: the former in the pancreas and the latter in the small intestine, the colon as well as in the pancreas (Cox, 2007). The endogenous truncated PYY(3-36) has been found to have an inhibitory effect on appetite in humans (Batterham et al., 2003; Batterham et al., 2002). That both full length PYY and PYY(3-36) lead to delayed gastric emptying is well known from animal studies (Allen et al., 1984; Chelikani et al., 2004). More recently, these peptides have also been demonstrated to result in an increased satiety in humans (Witte et al., 2009). A positive correlation between high plasma PYY(3-36) levels and exercise has been reported (Ueda et al., 2009). PYY has also been detected in the hypothalamus of the human brain (Morimoto et al., 2008) but its function in the central nervous system has yet to be delineated. It was not until recently that the first report on the characterization of appetite regulatory effects of PYY in a non-mammalian vertebrate, i.e. in the goldfish, was published (Gonzalez and Unniappan, 2010). It was shown that intraperitoneal and intracerebroventricular injections of gold fish PYY inhibited food intake by approximately 30%.

Evolution The three members in the NPY-family of peptides show high amino acid sequence identity to each other with approximately 70% between NPY and PYY and approximately 50% between PP and the other two (Cerda-Reverter and Larhammar, 2000). NPY and PYY are generally highly conserved, particularly NPY, throughout vertebrates (Cerda-Reverter and Larhammar, 2000; Conlon, 2002; Larhammar, 1996; Sundstrom et al., 2008). For instance, the NPY amino acid sequence from rat differs at only one position compared to chicken and at only three positions compared to marbled electric ray (Blomqvist et al., 1992). The PP gene arose through a local duplication of the PYY gene in an early tetrapod and has evolved much more rapidly with only 50% identity between human and chicken (Larhammar, 1996). A whole genome doubling in the teleost fishes generated duplicates of the genes coding for both NPY and PYY, resulting in the four peptide

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genes NPYa, NPYb, PYYa and PYYb (Sundstrom et al., 2008). In the zebrafish, the NPYb gene was subsequently lost.

The NPY-family of receptors G protein-coupled receptors The receptors for NPY and its related peptides belong to the large G protein-coupled receptor (GPCR) superfamily and more specifically the rhodopsin-like, also denoted class A, clan among these (Fredriksson et al., 2003; Fredriksson and Schioth, 2005). GPCRs are characterized by seven transmembrane -helices connected by six loops of varying length (Palczewski et al., 2000), with an extracellular amino terminus and an intracellular carboxy terminus (Figure 2). Some general structural features of the rhodopsin-like GPCRs are that the transmembrane ligand-binding site in the receptors is a hydrophobic cavity with a variety of key H-bonding residues that are interacting with the ligands (Congreve et al., 2011). However, for peptide binding GPCRs, such as the NPY-family of receptors, it is believed that it is only a small part of the ligand that binds in this site and that a larger part of the interaction is happening on the extracellular part of the receptors. Moreover, the extracellular loop 2, between transmembrane region 4 and 5, is situated above this docking site. Although this loop vary in length between the receptors it is still generally believed to be involved in ligand binding, especially for peptide binding GPCRs. There is a highly conserved tryptophan residue, W6.48, in transmembrane region 6 named the “toggle switch” (Holst et al., 2010). Upon agonist binding this residue is thought to rotate and thus give rise to a conformational change in the -helices leading to an active state of the receptor. The ionic lock, i.e. a salt bridge between residue 3.50 and 6.30, has been observed in the inactive rhodopsin structure and it is supposed to hold the receptor in an inactive conformation (Hofmann et al., 2009). An ionic lock has not, however, been observed in the other inactive crystal receptor structures solved. This is explained to be the result of crystallization artifacts or that the ligands used were not fully inverse agonists and thus the structures might not represent entirely inactive receptor states (Topiol and Sabio, 2009). Finally, the last common structural feature among the GPCRs is the involvement of the intracellular loop 2 and 3 in G protein binding. The rhodopsin-like GPCRs are further classified based on their individual peptide binding profiles.

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Figure 2. Receptor model of a GPCR. hY1 based on a chimeric hCXCR4/hA2AR receptor template. The -helices are color labeled anticlockwise from -helix 1 in blue to -helix 7 in red. The fade green/red background is the cell membrane and the fade blue visualize the external and internal cellular milieu. Picture was kindly provided by Hugo Gutiérrez-de-Terán.

Upon activation the receptor undergoes a conformational change leading to activation of an intracellular G protein (Deupi and Kobilka, 2007). The G proteins can be divided into three major groups; Gi/o, Gs and Gq/ll and they are classified depending on their downstream signaling pathways. In mammals, all NPY receptor subtypes signal primarily via the Gi/o pathway, thus inhibiting adenylyl cyclase and cAMP production. Subsequent responses include mobilization of intracellular Ca2+ and modulation of Ca2+ and K+ channels (Michel et al., 1998; Mullins et al., 2002).

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Evolution One single NPY receptor gene is believed to be the ancestor of the entire vertebrate NPY receptor family. Two local duplications of this ancient gene and two subsequent rounds of whole genome doublings have resulted in a repertoire of seven receptor genes in the gnathostome ancestor, i.e. Y1, Y2, Y4, Y5, Y6, Y7 and Y8 (Figure 3) (Larhammar and Salaneck, 2004; Salaneck et al., 2008; Wraith et al., 2000). There have been reports with pharmacological data for a proposed Y3 receptor but the corresponding gene has so far not been found in any genome (Larhammar et al., 2001), thus it probably does not exist. Additional duplication events and lineage-specific losses have given rise to a varying set of receptors in the different vertebrate lineages. The mammalian lineage seems to have lost Y7 and Y8. Y6 is a pseudogene in primates, pig (Wraith et al., 2000) and guinea pig (Starback et al., 2000), and nonexistent in rat, but has been shown to be functional in both mouse and rabbit (Larhammar and Salaneck, 2004). Y5 and Y6 have been lost in the teleost fish lineage. The Y8 receptor is present in amphibians and cartilaginous fishes and this gene has duplicated to generate Y8a and Y8b in the teleost fish lineage (Salaneck et al., 2008). Thus, the only ones that seem to have retained all seven ancestral receptors are the lineage of amphibians (Sundström et al. in preparation) and the cartilaginous fishes (Larsson et al., 2009).

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Figure 3. Schematic drawing illustrating the evolution of the NPY-family of receptors. A single ancestral Y gene is believed to have given rise to a repertoire of seven receptors in the ancestral gnathostome after two local duplications and two whole genome doublings. Additional duplication events and lineage-specific losses have given rise to the varying set of receptors in the mammalian, amphibian and teleost fish lineages. Crossed-out boxes represent lost genes or genes yet to be found. Picture modified from Larhammar et al. 2004.

AncientY5

AncientY2

AncientY4/8

AncientY5

AncientY2/Y7

AncientY1/6

AncientY

AncientY2

AncientY1/Y5

AncientY5

AncientY2

AncientY1

Genomedoubling

Y4

Y5Y2 Y1

Y7 Y6

Y4

Y8

Y5Y2 Y1

Y repertoire in gnathostome ancestor

Local duplication

Local duplication

1st genome doubling

2nd genome doubling

Y8b

Y7 Y6

Y4

Y5Y2 Y1Y2-2

Y8aY8

Y7 Y6

Y5Y7 Y6

Y4

Y5Y2 Y1

Y8

Mammalian lineage Amphibian lineage Teleost fish lineage

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The NPY-receptor subtypes were primarily distinguished based upon their different pharmacological binding profiles to NPY analogs and truncated peptides. Moreover, the receptors can be grouped into three subfamilies: Y1, Y2 and Y5, based on their degree of amino acid sequence identity (Larhammar and Salaneck, 2004). The additional members in each subfamily are, in the Y1 subfamily: Y4, Y6 and the Y8, in the Y2 subfamily: Y2-2 and Y7, and finally the Y5 subfamily with a single member. The subfamilies share only 27-31% sequence identity to one another and are therefore considered the most divergent receptors known to interact with the same peptide ligands (Larhammar et al., 2001).

The Y1 subfamily Y1 This was the first NPY receptor to be cloned. Its coding sequence was determined from rat forebrain cDNA in 1990 (Eva et al., 1990; Krause et al., 1992). It was subsequently cloned in human (Herzog et al., 1993; Larhammar et al., 1992) and the human gene was localized to chromosome 4 (Herzog et al., 1993). Furthermore, the Y1 receptor is well conserved across vertebrates (Larhammar et al., 2001; Larsson et al., 2009) which suggest that it has great functional importance.

Expression of the Y1 receptor in rat has been found primarily in the cortex, hippocampus, thalamus and specific nuclei of the hypothalamus and amygdala (Parker and Herzog, 1999) as well as in vascular smooth muscle cells in humans (Abounader et al., 1999). It has been shown to be involved in food intake (Kanatani et al., 2001; Lecklin et al., 2002; Lecklin et al., 2003), anxiolysis (Wahlestedt et al., 1993) and vasoconstriction (Wahlestedt et al., 1990).

The pharmacological profile of the Y1 receptor is characterized by high affinity for NPY, PYY and Pro34-analogues of NPY, whereas truncated peptides and PP are significantly less potent at this receptor (Larhammar et al., 1992; Michel et al., 1998). The first antagonists against the Y1 receptor, BIBP3226 (Rudolf et al., 1994) and SR120819A (Serradeil-Le Gal et al., 1995), were designed to mimic the C-terminus of NPY. Since then several new antagonists have been synthesized, with higher specificity and better affinity.

Y4 When the Y4 receptor was cloned in human (Bard et al., 1995; Lundell et al., 1995; Yan et al., 1996), it was found to be the primary binding target for PP (Michel et al., 1998). Sequence comparisons revealed that it belongs to the Y1 subfamily (Larhammar and Salaneck, 2004). Whereas the other NPY receptors are expressed in the brain, the Y4 receptor is predominantly found

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in the colon, small intestine and pancreas (Lundell et al., 1995). The Y4 receptor is the mediator of PP-induced physiological functions such as inhibition of gallbladder secretion as well as pancreas and gut motility. PP is the preferred ligand to the Y4 receptor in rat, mouse and humans (Bard et al., 1995; Lundell et al., 1995) but not in chicken (Lundell et al., 2002) or amphibians (Sundström et al. in preparation) where PYY and NPY have equal affinity for the receptor. When first discovered in zebrafish this receptor was referred to as Ya (Starback et al., 1999).

Y6 A functional Y6 receptor has so far only been characterized in mouse and rabbit among mammals (Gregor et al., 1996; Matsumoto et al., 1996; Mullins et al., 2000). It corresponds to a pseudogene and non-functional receptor in primates due to a frameshift mutation (Burkhoff et al., 1998; Matsumoto et al., 1996). In chicken it also gives rise to a functional receptor (Bromee et al., 2006). Its physiological role, however, is poorly investigated. Phylogenetic analyses show that it belongs to the Y1 subfamily (Larhammar and Salaneck, 2004).

Y8 The last member in the Y1 subfamily is Y8. This receptor has so far only been found in amphibians (Sundström, in preparation), teleost fishes (Larsson et al., 2008; Ringvall et al., 1997; Salaneck et al., 2008; Starback et al., 1999) and elephant shark (Larsson et al., 2009). Thus, it has been lost in amniotes. The extra copy of Y8 that is present in teleost fishes is thought to be the only additional copy remaining after the teleost-specific third whole genome duplication event (Meyer and Van de Peer, 2005). The teleost Y8 receptors have been named Y8a and Y8b and were initially called Yc and Yb (Arvidsson et al., 1998; Larson et al., 2003; Larsson et al., 2008; Lundell et al., 1997; Ringvall et al., 1997; Salaneck et al., 2008).

The Y2 subfamily Y2 Y2 was the second NPY receptor to be cloned in humans (Gerald et al., 1995; Rose et al., 1995). By showing high affinity for PYY and NPY it was characterized as a receptor with a similar pharmacological binding profile as Y1 (Michel et al., 1998). Additionally, Y2 also displayed high affinity for truncated peptide ligands and markedly decreased affinity for Pro34-analogues of NPY and PYY (Gerald et al., 1995; Rose et al., 1995). There are several Y2-specific non-peptide antagonists of which three are commercially available: BIIE0246 (Doods et al., 1999), JNJ 5207787

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(Bonaventure et al., 2004; Jablonowski et al., 2004) and SF 11 (Brothers et al., 2010).

The genes for receptors Y2 and Y5 have been localized to the same chromosomal segment as Y1 in human (Ammar et al., 1996; Herzog et al., 1997) and pig (Wraith et al., 2000) as well as in chicken, leading to the conclusion that the three genes arose from a common ancestral NPY receptor gene (Wraith et al., 2000). Furthermore, the Y2 sequence is equally well conserved between species as Y1 (Larhammar and Salaneck, 2004).

The expression of the Y2 receptor is widespread in the human central nervous system with particularly dense regions in the hypothalamus and the hippocampus, but it has also been detected in peripheral tissues (Gehlert et al., 1996). In the brain it is primarily located presynaptically having a modulatory role in presynaptic neurotransmitter release (Stanic et al., 2011). The main physiological functions directly correlated with activation of the Y2 receptor are regulation of energy homeostasis (King et al., 2000), circadian rhythm (Huhman et al., 1996) and memory retention (Redrobe et al., 2004). It has also been found to be involved in angiogenesis (Ekstrand et al., 2003). A silent single nucleotide polymorphism in the Y2 gene in a cohort of Swedish men has been reported to be associated with obesity (Lavebratt et al., 2006). In this thesis a novel NPY receptor gene, found in the genomes of the zebrafish and medaka, is presented for the first time. It has been classified as a Y2-like receptor in Paper IV since it clusters with other Y2 receptors in the phylogenetic analysis and is thus denoted Y2-2. Due to its location on chromosome 1 in zebrafish, next to the Y2 receptor gene, it is most probably the result of a local duplication. The expression in zebrafish was found in the eye and gastrointestinal tract but its functional role has yet to be investigated.

Y7 Y7 is another member in the Y2 subfamily based upon sequence phylogeny. In contrast to Y2, the truncated NPY and PYY peptides do not bind to the receptor with similar high affinities as the intact peptides. The receptor gene has so far been identified in the genomes of chicken (Bromee et al., 2006), amphibians, teleost fishes (Fredriksson et al., 2004) and elephant shark (Larsson et al., 2009). Thus this gene seems to have been lost in mammals. The functional role of this receptor is still unknown.

The Y5 subfamily Y5 The Y5 receptor gene was first cloned from rat hypothalamus (Gerald et al., 1996). Due to its large third intracellular loop and a total length of 445

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amino acids, it is the largest of all NPY receptors. Immunohistochemistry studies in rat brain have revealed an overlap of Y1 and Y5 receptor expression in cerebral cortex, hippocampus, hypothalamus, thalamus, amygdala, and brainstem (Wolak et al., 2003). NPY in the hypothalamus is known to induce its appetite stimulating effect via the Y1 and Y5 receptors (Feletou et al., 2006). However, subtype-specific agonist stimulation of Y1 and Y5 in guinea pigs revealed that they had distinct action profiles on various feeding parameters and it was suggested that they probably modify different phases of feeding behavior (Lecklin et al., 2003). The binding profile of the Y5 receptor is characterized by equally good binding to NPY and PYY as well as the truncated versions NPY3-36 and PYY3-36 (Michel et al., 1998).

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Appetite regulation and energy homeostasis With the increasing problem of obesity worldwide, understanding the mechanisms regulating food intake and energy expenditure has become more urgent than ever. Increased fat and sugar content in the food, in combination with food being more easily accessible, and our everyday life being less active than it has ever been, are major factors contributing to this escalating problem.

Our drive to eat, however, is fundamental and throughout human evolution the search for food has probably taken up much of our active time in order to survive. Thus, a hypothesis is that the regulation of food intake has been evolutionarily selected to prioritize feeding whenever food is accessible. Consequently, our bodies are less well suited for excess availability of food as is the case in many developed nations.

There are numerous organs in the body that are involved in the regulation of food intake. The major peripheral components are the gustatory system, gastro-intestinal (GI) tract, pancreas, liver as well as muscle and adipose tissues. All of these structures are in bidirectional contact with the brain via neural connections, hormones and metabolites. In the brain, the structures involved in appetite regulation include mainly the caudal brainstem, the hypothalamus and parts of the cortex and limbic system (Lenard and Berthoud, 2008).

The gustatory system and GI tract send relevant nutritional information to the caudal brainstem via the gustatory and vagal afferents, respectively (Hameed et al., 2009; Shimura et al., 1997). The hypothalamus receives central inputs mainly from the brainstem as well as peripheral inputs via circulating hormones and metabolites (Wynne et al., 2005). The cortico-limbic system is mainly involved in the brain’s higher functions such as learning, memory, reward, emotion and decision making: factors that are all of importance in the psychological aspects of food intake (Lenard and Berthoud, 2008).

In general, signals originating in the periphery that influence energy expenditure and food intake can be divided into two categories: satiety signals phasically secreted during meals, and adiposity signals that are more tonically active (Woods and D'Alessio, 2008). The major peripheral satiety signals are cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), PYY(3-36) and PP. The majority of them signal via the vagus nerve up to the brainstem. Insulin and leptin are the two major peripheral adipose signals and there is evidence that they, in contrast to the other satiety signals, act more directly on the hypothalamus (Valassi et al., 2008).

All factors that affect appetite, including those in the central nervous system, can be further divided into orexigenic and anorexigenic factors depending on whether they induce or reduce appetite, respectively (Arora and Anubhuti, 2006; Dhillo, 2007). The major orexigenic factors are NPY, agouti-related peptide (AgRP), melanin-concentrating hormone (MCH),

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orexin and ghrelin. Some of the anorexigenic factors are alpha-melanocyte-stimulating hormone ( -MSH) from the precursor called POMC, cocaine- and amphetamine-regulated transcript (CART), leptin, insulin, corticotrophin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), CCK, GLP-1, PYY(3-36) and PP.

The hypothalamus The hypothalamus is the most important brain structure involved in food intake regulation. There are five substructures in the hypothalamus, i.e. the arcuate nucleus, the paraventricular nucleus, the ventromedial nucleus, the dorsomedial nucleus and the lateral hypothalamic area, that each has a specific role in appetite and satiety regulation. See Figure 4 for an overview.

The arcuate nucleus The arcuate nucleus (ARC) receives and sends signals regulating energy homeostasis. It is located close to the median eminence, in contact with the third ventricle, and in close proximity to the anterior pituitary, which enables the inception of peripheral peptides and proteins from both the cerebrospinal fluid and the blood. Leptin, released in the periphery in proportion to adipose tissue, and insulin, released from the pancreas in response to increased blood glucose levels, are both known to activate neurons in the ARC (Valassi et al., 2008). The two distinct groups of neurons in this structure involved in energy homeostasis are NPY/AgRP- and -MSH/CART-containing neurons (Hill et al., 2008). High leptin signaling inhibits NPY/AgRP neuron activation and stimulates -MSH/CART neuron activity whereas low levels result in the opposite situation. Ghrelin is the only orexigenic peripheral peptide known and it conveys its appetite-stimulating effect through activation of NPY/AgRP neurons in the ARC. Elevated levels of PYY3-36 after food intake have been suggested to inhibit feeding via the activation of Y2 receptors in the arcuate nucleus (Batterham et al., 2002).

The paraventricular nucleus Adjacent to the dorsal part of the third ventricle, in the anterior hypothalamus, there is a structure named paraventricular nucleus (PVN) on either side of the third ventricle. This is the main site for secretion of the anorexigenic hormones CRH and TRH (Arora and Anubhuti, 2006). Moreover, several neuronal pathways involved in energy homeostasis from neighboring structures converge here, i.e. NPY/AgRP as well as -MSH/CART projections. Y1 and Y5 receptors are expressed in the PVN where they are thought to mediate NPY’s orexigenic effect (MacNeil, 2007). The anorexigenic peptide -MSH is released here and binds to the melanocortin 4 receptor upon stimulation of -MSH/CART neurons in the ARC. (Dhillo, 2007). Interestingly, AgRP inhibits

-MSH by binding the same receptor (Neary et al., 2004).

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The ventromedial nucleus The third important structure is the ventromedial nucleus of the hypothalamus (VMH) which is mainly acting as a satiety centre. Studies in animals have shown that lesions of either VMH or PVN lead to hyperphagia (overeating) and obesity (King, 2006). NPY has been shown to, via Y1 receptors, inhibit VMH neurons expressing leptin receptors by hyperpolarizing them and thus decrease their ability to fire action potentials (Chee et al., 2010), therefore disturbing the anorexic output of these neurons.

The dorsomedial nucleus The dorsomedial nucleus of the hypothalamus (DMH) is a central hypothalamic structure and as such it is known to integrate and process information from both the ventromedial and the lateral hypothalamic areas (Bernardis and Bellinger, 1998). Neuronal NPY/AgRP and -MSH/CART projections from the ARC terminate here (Wynne et al., 2005). The DMH itself projects to the PVN where it indirectly regulates the hypothalamic-pituitary axis by changing the sensitivity of TRH neurons to thyroid hormones (Hillebrand et al., 2002). Recently, a study done by Yang et al. showed that overexpression of NPY in the DMH of lean mice lead to an increase in food intake and body weight (Yang et al., 2009). Moreover, they showed that the ablation of NPY reduced the hyperphagia and obesity in obese mice.

The lateral hypothalamic area The lateral hypothalamic area (LHA) contains glucose-sensitive neurons and is thus involved in mediating hypoglycemia (low blood glucose) and subsequent hyperphagia (Bernardis and Bellinger, 1996). This is the structure where the orexigenic neuropeptides MCH and orexin are produced. NPY/AgRP and -MSH/CART neurons from the ARC are also known to project here (Wynne et al., 2005).

Finally, there are many reciprocal connections between the hypothalamus and the brainstem, in particular with the nucleus of the solitary tract (NTS). This region has a high density of Y1 and Y5 receptors, and NPY-containing neurons have been shown to project from NTS to the PVN (Wynne et al., 2005). On the other hand, vagal afferent fibers terminate in the NTS and food intake inhibition has been found to be mediated through the activation of CCK-A receptors in the vagal nerve (Kopin et al., 1999; Lin and Miller, 1992). The peripherally released GLP-1 and PYY(3-36) are also thought to affect the hypothalamus via the NTS and the vagal afferent nerve (Arora and Anubhuti, 2006). It has been suggested that PP exerts some of its anorectic actions via the brainstem but this is yet to be confirmed (Larsen and Kristensen, 1997).

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Figure 4. Schematic picture of the main structures in the hypothalamus and the neuropeptides involved in appetite regulation and energy homeostasis. Neuropeptides written in blue are anorexigenic and those in red orexigenic. Pluses indicate a stimulatory and minuses an inhibitory effect. Thick black arrows represent neuronal NPY/AgRP and -MSH/CART pathways projecting from arcuate nucleus on left and right side, respectively. However, there is bilateral symmetry in the hypothalamus. Structures in the hypothalamus are light yellow and structures in the periphery grey. Abbreviations: cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), agouti-related peptide (AgRP), melanin-concentrating hormone (MCH), alpha-melanocyte-stimulating hormone ( -MSH), cocaine- and amphetamine-regulated transcript (CART), corticotrophin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH), lateral hypothalamic area (LHA), nucleus of the solitary tract (NTS), gastrointestinal tract (GI).

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Regulation of food intake in fish Homologs of many of the mammalian appetite-regulating peptides have in recent years been identified and characterized in teleost fishes (Volkoff et al., 2005). However, the fishes constitute a huge phylogenetic group of approximately 30,000 species with a high level of diversity regarding morphology, ecology and behavior and thus precaution should be taken when talking about appetite regulation in fish. Nevertheless, the major differences found so far between mammals and fish is that leptin is expressed and produced mainly in the liver of the fish and not fat tissue (Gorissen et al., 2009) and that MCH in fish acts as a anorexigenic and not a orexigenic neuropeptide (Matsuda et al., 2009).

GPCR crystal structures and mutagenesis studies The facts that GPCRs are embedded in the cell membrane with multiple transmembrane regions and are highly flexible have made them very difficult to purify and crystallize. The first group to succeed was Palczewski et al. who published a high resolution structure of bovine rhodopsin using X-ray crystallography (Palczewski et al., 2000). It was not until 2007 that the next three-dimensional GPCR structure was revealed, i.e. the human 2-adrenergic receptor (h 2R) (Rasmussen et al., 2007). Since then there has been a steady stream of publications every year of new receptor crystal structures: the h 2R in inactive state (Cherezov et al., 2007; Rosenbaum et al., 2007) and active state (Rasmussen et al., 2011a; Rasmussen et al., 2011b; Rosenbaum et al., 2011), the turkey 1-adrenergic receptor (t 1R) (Warne et al., 2011; Warne et al., 2008), the bovine rhodopsin without ligand (Park et al., 2008), the human adenosine 2A receptor (hA2AR) (Jaakola et al., 2008), the human chemokine receptor type 4 (hCXCR4) (Wu et al., 2010) and the human dopamine D3 receptor (hD3R) (Chien et al., 2010).

These receptor models, and in particular the rhodopsin model, have formed the basis for numerous models of receptor proteins that have not yet been crystallized. In order to create more accurate models, the computer-based calculations are often combined with mutagenesis studies (de Graaf and Rognan, 2009). By using computer programs where mutagenesis data can be taken into account when creating the receptor structures, information of the ligand-receptor complex can be revealed, regarding both amino acid interactions and the subsequent receptor conformational changes. Knowing that the majority of the drugs on the pharmaceutical market targets GPCRs (Lundstrom, 2005; Overington et al., 2006), this approach will hopefully contribute in the design of new pharmaceuticals for specific GPCRs.

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Materials and methods

Selection of positions for site-directed mutagenesis The investigated positions of the hY2 receptor in this thesis are Thr2.61, Tyr2.64; Gly2.68; Tyr3.30, Gln3.32, Thr3.40; Leu4.60; Tyr5.38, Leu6.51, Gln6.55; Val6.58, Tyr7.31 and His7.39 (Figure 5). Positions in Paper I and II were selected based on sequence alignments as well as previous mutagenesis results in hY1 whereas positions in Paper III were chosen from the receptor modeling.

The residues mutated in this thesis are named according to the system for numbering GPCRs of Ballesteros and Weinstein (Ballesteros and Weinstein, 1995). Briefly, the numbers are the coordinates of the position, where the first number denotes the transmembrane region in question and the second is the number relative the most conserved amino acid (i.e. number 50) in each transmembrane region. The first amino acid is the one present in the wild type receptor and the second the introduced new amino acid.

The NPY-family receptor sequences were downloaded from the Ensembl database (www.ensembl.org), GenBank (www.ncbi.nlm.nih.gov) and the elephant shark genome project (esharkgenome.imcb.a-star.edu.sg). Many of these sequences were determined in our laboratory. Also, yet unpublished sequences determined in our lab were included in the alignments. Multiple sequence alignments of the sequences were made using Clustal W (Thompson et al., 1994) with standard settings as implemented in Jalview 2.4 (Clamp et al., 2004).

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Figure 5. Snake view of the hY2 receptor. Numbered residues were mutated in this thesis. Grey residues show the most conserved amino acid in each helix.

Site-directed mutagenesis and receptor cloning Gateway® cloning The mutated receptor genes in Paper I and II were generated by a two-step PCR using the coding region of the wild-type (wt) hY2 receptor gene as template. The primers in the first PCR were mutant specific and the primers in the second PCR were specific for the Gateway® system. The final PCR product was transferred into an expression vector and cloned using the Gateway® system according to the manufacturer’s instruction (Invitrogen). The final expression vector carried a gene for green fluorescent protein (GFP) downstream of the insertion resulting in a final receptor protein with a C-terminal GFP-tag. The mutant receptors were control sequenced to confirm the introduced mutation.

QuikChange® II site-directed mutagenesis QuikChange® II site-directed mutagenesis kit (Stratagene) was used to generate mutants in Paper III according to manufacturer's protocol. Briefly, the mutations were introduced through one PCR with primers specifically designed for each mutation. A pcDNA-DEST47 vector (Invitrogen) inserted with the coding region of the wt hY2 receptor was used as parental template. The mutant receptors were control sequenced to confirm the introduced mutation.

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Transfection and harvesting Human embryonic kidney (HEK) 293 cells were transfected with the receptor vectors using Lipofectamine 2000 Transfection reagent (Invitrogen) according to the manufacturer’s instructions. In Paper I, II and IV, the cells were grown for 24 hours in serum free DMEM (Dulbecco’s modified Eagle’s medium) containing penicillin, streptomycin and amphotericin after transfection. This was followed by growth for another 24 hours in DMEM medium with fetal calf serum to obtain cells with transient expression. In Paper III the cells were incubated directly in this serum containing medium for 48 h after transfection. Cells were subsequently harvested, resuspended in binding buffer and aliquots were stored in -80 (Paper I and II) or -20 °C (Paper III and IV). The protein concentration of the cell batches were determined using the Bio-Rad Protein Assay (Paper I and III) or Bio-Rad DC Protein Assay (Paper II and IV) with bovine serum albumin as a standard.

Expression of receptor proteins for detection by microscopy The HEK 293 cells were seeded on coverslips coated with 0.1 mg/ml Poly-D-lysine before transfection conducted as described above. The wt hY2 or zfY2-2 transfected cells served as a positive control whereas cells transfected without any plasmids were used as a negative control. The transfected cells were washed with PBS twice and fixed with 4% paraformaldehyde for 10 min. After rinse with PBS, 100 µl of DAPI (0.5 µg/ml) was applied to the coverslips and incubated at room temperature in dark for 15 min. The coverslips were rinsed with PBS twice and dried, mounted with mounting medium, and fluorescence images were taken in an inverted confocal microscope (Zeiss LSM 510 Meta) (63xoil objective (NA=1.4)) and with the LSM software.

Receptor binding experiments Ligands The radioligand used in the receptor binding assays was 125I-pPYY with a specific activity of 4000 Ci/mmol (Amersham) in Paper I and II and 2200 Ci/mmol (Perkin Elmer) in Paper III and IV.

The peptide ligand used in the competition assays in Paper III was hPYY3-36. This ligand and additionally pNPY, pNPY13-36 and Leu31, Pro34-hNPY were used in Paper II (all purchased from Bachem). Paper IV included studies with the truncated ligands mentioned above and with hPYY

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(Bachem), zfNPY and zfPYYa (both from Eli Lilly and Company). zfPYYb in Paper IV was supplied in crude form by GL Biochem Shanghai and purified as previously described (Fredriksson et al., 2006).

The Y2-selective non-peptide antagonist used in all Papers presented herein was BIIE0246 (Boehringer Ingelheim).

Saturation and competition assays Saturation experiments were carried out with serial dilutions of radioligand and competition experiments with serial dilutions of the competing ligands. Non-specific binding was defined in the presence of 100 nM hPYY (Paper I), 1 M hPYY (Paper II and III) or 1 M zfPYYb (Paper IV).

All binding experiments were performed in a final volume of 100 l (Paper I, II and IV) or 200 l (Paper III) and incubated in room temperature for three hours. Saturation studies were performed in at least duplicates, the competition studies in triplicates and all binding studies were repeated independently at least three times. The incubation was terminated by filtration with Tris buffer through Filtermat A,GF/C filters (PerkinElmer) pre-soaked in 0.3% polyethyleneimine (Sigma-Aldrich) using a Tomtec cell harvester (Orange). The filters were dried and covered with MeltiLex A melton scintillator sheets (Wallac Oy). The radioactivity of the filters was counted using a Wallac 1450 Microbeta counter.

Statistical analyses of binding experiments The data from the binding experiments were analysed with nonlinear regression curve-fitting (GraphPad Prism software). Linear regression using Scatchard transformation was performed for each saturation experiment and the Hill coefficients were calculated for all competition experiments. The competition experiments were also tested for one- and two-site fitting in Paper I, II and IV. The two-site model was accepted if each site accounted for >20% of the receptors and if it significantly improved the curve fit (p<0.05; F test). The pKd- and pKi- values (from the formula -log K) for all receptor mutants were compared with the values for the wt receptor using one-way ANOVA followed by Dunnett’s multiple comparison test.

Computational Y1 and Y2 receptor modeling Homology modeling The sequences of hY1 (Paper I) and hY2 (Paper I, II and III) were retrieved from the UniProt database (Boeckmann et al., 2003). An alignment of the

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two sequences and the sequences of the crystallized GPCRs available at the time for each Paper were generated by the ClustalW program (Thompson et al., 1994). The GPCR modeling and simulation toolkit (http://gpcr.usc.es) was used to provide additional information about sequence identity for each of the secondary structural elements (Paper II and III).

The crystal structures of h 2AR (Cherezov et al., 2007; Rasmussen et al., 2007) (Paper I), hA2AR (Jaakola et al., 2008) (Paper I, II and III) and a chimeric hCXCR4/hA2AR receptor (Wu et al., 2010) (Paper III) were selected as templates and respective pairwise crystal structure/Y receptor alignment was used as input for homology modeling using the program Modeller v9.7 (Sali and Blundell, 1993). The exact protocol for building the models differed slightly depending on which template used, mainly due to differences in the extracellular loops and parts with low sequence identity (see Paper I, II and III for details). Briefly, an initial pool of homology models was generated and the best model out of these was then subjected to extracellular loop refinement with the LOOPMODEL routine as implemented in Modeller v9.7. Out of these loop-refined models a best final model was selected.

At each modeling step several criteria were taken into account. For consensus scoring of the models the Modeller objective function and the DOPE assessment score (Shen and Sali, 2006) were used and the stereochemical quality of the modeled receptor was validated and approved by the programs WHATCHECK (Hooft et al., 1996) and PROCHECK (Laskowski et al., 1993). Addition of hydrogens was performed with PDB2PQR software (Dolinsky et al., 2004).

Docking of hNPY In Paper III the hA2AR based hY2 receptor model was used for docking of hNPY. Initially, automated docking of the conserved C-terminal dipeptide fragment of the natural agonists with acetylated N-terminus (CH3C(O)-R35-Y36-NH2) was performed using the GOLD 4.0 software (Jones et al., 1997). The binding site was defined using a 25 Å radius sphere centered approximately halfway between Thr2.61 and Gln6.55 in the receptor model.

The full hNPY peptide was built by homology modeling starting from the crystal structure of the avian pancreatic peptide (aPP, PDB code 2BF9) by means of the Modeller package (Sali and Blundell, 1993). The best one out of 15 models was selected according to the DOPE-HR scoring function (Shen and Sali, 2006). The protein-protein docking program HADDOCK (Dominguez et al., 2003) was used to elucidate the binding mode of the hNPY peptide to the hY2 receptor. Mutagenesis data and the docking results of the dipeptide were used to bias the docking. For further details see Paper III.

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BLAST searches and phylogenetic analyses BLAST searches (Altschul et al., 1990) in Paper IV where performed in the Ensembl database release 61 with the known zebrafish Y2 sequence (XP_001343301) as query. BLAST hits covered by Ensembl protein or automatic GenScan predictions (Burge and Karlin, 1997) were selected for further analysis. Divergent or incomplete protein predictions were manually edited, following the consensus rule for splice donor and acceptor sites as well as sequence identities to other family members.

All identified members in the Y2 subfamily from zebrafish, medaka and stickleback were used together with the known full-length Y2 and Y7 amino acid sequences from zebrafish, fugu, tetraodon, human, chicken, mouse and elephant shark (for ID nr see Fig.1 in Paper IV) to construct an alignment using ClustalW 2.012 with standard settings. Phylogenetic trees were constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates in ClustalX 2.012 (Larkin et al., 2007). Phylogenetic maximum likelihood (PhyML) trees were constructed using the online execution of the PhyML 3.0 algorithm (Guindon and Gascuel, 2003) with 100 bootstrap replicas.

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Results and discussion

Paper I and II Site directed mutagenesis and binding studies were performed in both Paper I and II in order to investigate the receptor-ligand interactions of the hY2 receptor with different ligands.

The three positions, Tyr2.64, Val6.58 and Tyr7.31, in Paper I were selected based on previous results of the corresponding positions in the hY1 receptor. One hY1 study reported decreased affinity and even lost binding of 125I-NPY for receptor mutants with Ala introduced at these positions (Sautel et al., 1995). In addition, 125I-pPYY has also been shown to lose binding to these hY1 Ala mutants (Sjodin et al., 2006). The results from both of these studies suggested that these positions are important for ligand interaction in hY1. Moreover, in the first study Tyr2.64, Phe6.58 and His7.31 were proposed to form a hydrophobic binding pocket for the C-terminal end of the peptides (Sautel et al., 1995).

In Paper II the positions Gln2.68, Leu4.60 and Gln6.55 were also selected based on results from mutagenesis studies in hY1. In hY1, tested ligands showed decreased affinities as well as lost binding when the corresponding positions were mutated (Du et al., 1997; Kanno et al., 2001; Sautel et al., 1996; Walker et al., 1994). Thr3.40 in Paper II was selected solely from the NPY receptor sequence alignment.

The five mutant receptors Tyr2.64Ala, Tyr2.64Phe, Val6.58Ala, Tyr7.31Ala and Tyr7.31His were created in Paper I and the four mutants Gln2.68Asn, Thr3.40Ile, Leu4.60Ala and Gln6.55Ala were created in Paper II. All the mutant receptors were expressed in HEK 293 cells and pharmacologically characterized with the radioligand 125I-pPYY, the peptide ligands pNPY, pNPY13-36, hPYY3-36 as well as the hY2 specific non-peptide antagonist BIIE0246. In addition, the mutant receptors in Paper I were also tested with Leu31, Pro34-hNPY in the binding assays. The Kd- and Ki-values obtained from the binding studies are summarized in Table 1.

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Table 1. Summary of the Kd- (125I-pPYY) and Ki- (pNPY, pNPY13-36, hPYY3-36 and BIIE0246) values for the wt hY2 and the mutant receptors in Paper I, II and III. Ki- and Kd-values are given in nM. The number of stars indicates the statistical significance level for each mutant receptor when compared to the wt; *p<0.05, **p<0.01 and ***p<0.001.

In addition to the mutagenesis and binding studies performed in Paper I, four in silico-based receptor models were generated, two of hY1 and two of hY2. The publication of several GPCR crystal structures recently made it possible to create models based on the h 2R (Cherezov et al., 2007; Rasmussen et al., 2007) and the hA2AR (Jaakola et al., 2008). One purpose of creating these models was to re-evaluate the proposed hydrophobic pocket suggested from the rhodopsin-based model presented earlier (Sautel et al., 1995; Walker et al., 1994).

In Paper I, the affinity of the radioligand for the receptor mutant Tyr 2.64 Phe was lost. In addition, all tested ligands showed significantly decreased affinity when an Ala mutation was introduced at this position. When Tyr7.31 was substituted to His the radioligand also lost binding to the receptor. An Ala exchange however, resulted in a decreased affinity of pNPY and the

Mutants Paper 125I-pPYY pNPY pNPY13-36 hPYY3-36 BIIE0246

wt I 0.028±0.01 0.98±0.2 6.7±0.8 0.37±0.03 1.3±0.2 Tyr2.64Ala I 0.16±0.03** 5.1±0.7** 33±1** 3.3±0.2** 5.0±0.5* Tyr2.64Phe I nbVal6.58Ala I 0.040±0.01 1.5±0.5 7.3±0.6 0.57±0.1 1.0±0.2 Tyr7.31Ala I 0.053±0.01 2.5±0.08* 21±4** 2.9±0.2** 2.5±0.8 Tyr7.31His I nb

wt II 0.026±0.005 0.81±0.2 4.0±0.7 0.32±0.03 1.2±0.1 Gly2.68Asn II 0.022±0.003 1.1±0.2 7.8±4.9 0.87±0.2** 0.71±0.1Thr3.40Ile II 0.015±0.004 0.10±0.04** 1.8±0.7 0.14±0.02** 2.7±0.6*Leu4.60Ala II 0.024±0.004 1.9±0.7 7.2±0.4 0.67±0.07* 53±9**Gln6.55Ala II 0.011±0.002* 0.11±0.01** 5.4±1.5 0.03±0.01** 3.3±0.6**

wt III 0.010±0.001 0.34±0.1 2.1±0.3Thr2.61Ala III 0.037±0.006*** 200±40*** 0.53±0.1*Gln3.32Glu III 0.018±0.002 19±5*** 13±2***Gln3.32His III 0.028±0.001*** 47±7*** 0.40±0.1**His7.39Gln III 0.020±0.002 3.1±2*** 11±2**Gln3.32Hi+His7.39Gln III nbTyr3.30Leu III 0.007±0.001 0.61±0.1 0.50±0.04*Tyr3.30Ala III 0.018±0.004 2.0±0.5** 1.1±0.03Tyr5.38Leu III 0.017±0.002 3.8±1*** 0.60±0.1Tyr5.38Ala III 0.027±0.005** 13±7*** 1.4±0.4Leu6.51Ala III 0.022±0.003* 9.7±3*** 0.9±0.1Tyr3.30Leu+Tyr5.38Leu III 0.094±0.03*** 13±4*** 2.8±2Tyr5.38Leu+Leu6.51Ala III 0.038±0.002*** 29±15*** 1.0±0.7Tyr3.30Leu+Leu6.51Ala III 0.026±0.005** 4.7±0.4*** 1.4±1

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truncated ligands to this mutant. The Val6.58Ala mutant retained a binding profile similar to the wt hY2 receptor. Leu31, Pro34-hNPY had such low affinity that an accurate Ki value could not be determined. To summarize, the ligands either showed decreased or lost affinity for all but one mutant receptor in Paper I.

The results from the manual docking of an amidated Tyr36 (mimicking the C-terminal binding end of the NPY-peptide) into the hY1- and hY2-receptor models were in agreement with the results from the binding studies. The docking model suggested that the residues Tyr2.64 and Tyr7.31 could indeed be in contact with the ligand in the hY2 hA2AR based model. The hydroxyl group in Tyr2.64 could form a hydrogen bond with Tyr36 and Tyr7.31 could interact with the amide bond of the same amino acid. This was consistent with their changed binding profiles after the introduced mutations. In addition, Val6.58 was situated too far away in the model to have a hydrogen interaction with the ligand, also is in good agreement with the binding results. However, the hydrophobic pocket suggested by Sautel et al. was possible in the hY1 2R-based model, also explaining the larger affinity changes seen when these positions are mutated in hY1.

In Paper II, hPYY3-36 showed decreased affinity for Gly2.68Asn and Leu4.60Ala but increased affinity for Thr3.40Ile and Gln6.55Ala. Increased affinity of pNPY was also observed for the Thr3.40Ile and Gln6.55Ala mutants. The radioligand 125I-pPYY displayed increased affinity for the Gln6.55Ala mutant. Furthermore, BIIE0246 had decreased affinity for Thr3.40Ile, Leu4.60Ala and Gln6.55Ala.

The binding results in Paper II were subsequently compared to the hY2 hA2AR-based model. According to this model, the Thr3.40Ile mutation might lead to a stronger hydrophobic interaction with Phe6.44, a position shown in the 2R to be an important switch in receptor activation (Rasmussen et al., 2011a). This stabilization of the receptor in an active receptor confirmation could explain the enhanced affinity of pNPY and hPYY3-36 for the mutant. The mild changes in affinities of the ligands for the Gly2.68Asn mutant receptor were unexpected when compared to the modeling result. Since it is located at the top of transmembrane region 2, it was proposed to form an interaction with the first extracellular loop. Thus, this residue, although the affinity of the truncated Y2-specific peptide hPYY3-36 is decreased, does not seem to be in direct contact with the peptide. The decreased affinity of the same ligand for Leu4.60Ala is probably due to a lost hydrophobic interaction.

A comparison with the hY2 A2A-based modeling result made it hard to interpret the increased affinities of 125I-pPYY, hPYY3-36 and pNPY for the Gln6.55Ala receptor mutant. The orientation of this residue towards the binding cavity in the model suggested either a direct contact with the peptides or an intra-receptor interaction stabilizing the extracellular loop 2 conformation. The exchange of the larger amide Gln to the much smaller

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uncharged Ala was thus expected to disrupt these contacts and result in decreased affinities. Furthermore, in favor of the first modeling theory, this residue has been shown important in receptor binding in other GPCRs (Jaakola et al., 2008; Rasmussen et al., 2011a; Wieland et al., 1996). However, the latter is more in agreement with the increased affinity seen for three of the peptides tested herein since the removal of the interaction with the extracellular loop 2 could result in a more accessible binding cavity.

The results from the binding experiments with the small non-peptide antagonist BIIE0246 indicate different sites of interaction compared to the peptide ligands. The large decrease in affinity obtained by exchanging the bulky Leu for an Ala at position 4.60 strongly suggests a direct hydrophobic interaction with the antagonist. In addition, the antagonist also showed decreased affinity for the mutated residues Thr3.40 and Gln6.55. Interestingly, all three residues are oriented towards the cavity between the transmembrane regions 3,4,5 and 6 in the receptor model that has previously been suggested to be the cavity for antagonist binding in hY1 (Kanno et al., 2001; Sautel et al., 1996).

Paper III The hY2 hA2AR-based model built in Paper I was used for hNPY docking: first with only the C-terminal dipeptide and then the full length peptide. Based on these docking results, six amino acid residues were selected for mutagenesis and subsequent binding studies. 125I-pPYY was used as the radioligand and hPYY3-36 and BIIE0246 as the competing ligands. During the progress of the study, the crystal structure of the hCXCR4 was published (Wu et al., 2010). Since this was the first structure of a peptide-binding GPCR to be solved, a second hY2 receptor model was built based on the hCXCR4. This model was used for comparison with the docking results obtained from the hA2AR-based model.

The results from the docking into the hY2 hA2AR-based model revealed the residues Gln3.32 and His7.39 to be part of a hydrogen bonding network with the amidated C-terminal residue Tyr36 of the peptide. Thr2.61 was further found be part in this network by interacting with these two residues with an additional hydrogen bond. The residues Tyr3.30, Tyr5.38 and Leu6.51 were all hydrophobic residues identified to possibly interact with the side chain of Tyr36 in the docking model.

Mutation of Gln3.32 into the charged equivalent amino acid Glu affected both agonist and antagonist binding, suggesting that a negative charge at position 3.32 has an unfavorable effect on the polar contacts between the receptor and ligand. This was further supported by an even more decreased agonist affinity when His was introduced. The antagonists’ affinity, however, was increased to the Gln3.32His mutant. Replacement of His7.39

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with Gln resulted in decreased affinity of both hPYY3-36 and BIIE0246 for the mutant receptor, indicating that they are both in contact with this residue. The lost affinity of the reciprocal double mutant Gln3.32His+His7.39Gln could support the presence of a direct contact with these residues but could also be the result of the altered properties in the surrounding of each residue. However, since receptor expression was still observed, if the latter occurred it did at least not disturb the transport of the receptor to the cell membrane. The decreased affinity of the antagonist BIIE0246 for Gln3.32Glu and His7.39Gln suggests that the hydrogen network interacting with the amidated C-terminal of the agonist probably is applicable in a depicted antagonist binding model as well. 125I-pPYY and hPYY3-36 showed decreased affinity for the Thr2.61Ala mutant, probably resulting in a less stable receptor, in line with the predictions from the docking model. On the contrary, BIIE0246 showed slightly increased affinity for Thr2.61Ala. Thus, although it might be involved in the hydrogen network as suggested above, it most likely does so in a different way.

The only statistically significant change in affinity of the agonists for the mutant Tyr3.30Ala was observed with hPYY3-36. In addition, the antagonist showed a small increase in affinity for the Tyr3.30Leu mutant. These weak effects on receptor binding, however, were consistent with the new hY2 CXCR4-based model where this residue is oriented towards the membrane. Both the agonists 125I-pPYY and hPYY3-36 showed decreased affinities to Ala mutations of Leu6.51 and Tyr5.38. However, only hPYY3-36 was affected by the Ty5.38Leu mutant. Moreover, both the agonists displayed decreased affinity for the double mutants Tyr3.30Leu+Tyr5.38Leu, Tyr5.38Leu+Leu6.51Ala and Tyr3.30Leu+Leu6.51Ala. These results imply that the residues Tyr5.38 and Leu6.51 are indeed important for ligand binding presumably by interacting with the side chain of Tyr36.

Paper IV The number of NPY receptor genes in various species is known to vary. This is due to differential losses after whole genome doublings in early vertebrate evolution as well as local duplications. In this study, the known zebrafish Y2 sequence was used in a BLAST search in the Ensembl database release 61 against several fish genomes. By aligning the hits and building phylogenetic trees it was possible to identify a novel Y2-like receptor gene in the zebrafish and medaka genome. This gene was named Y2-2. The chromosomal location and intron-exon structure of the gene differed between the two species. In zebrafish Y2-2 contained two introns and was located on chromosome 1 next to Y2. In medaka Y2-2 contained three or more introns and was located on chromosome 18 whereas Y2 was localized on a short scaffold.

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To analyze the binding characteristics of the zfY2-2, the coding sequence was inserted into a vector and used for cell transfection and subsequent binding studies. Saturation binding experiments with 125I-pPYY showed one binding site with a Kd of 0.056 ± 0.005 nM. Nevertheless, four of seven ligands tested in the competition assays displayed two-site binding curves. The reason for this is not understood. This is unexpected since the agonist radioligand only bound to one receptor conformation up to the highest concentrations used, i.e. 250 pM. Thus, it should not be able to detect any additional conformations by the competing ligands. However, one possible explanation for this could be that the radioligand binds two receptor conformations with so similar affinities that they are not detected in the saturation curve. Another possibility is that the competition binding reaction did not reach equilibrium resulting in apparent biphasic curves.

If comparing the Ki and Kihigh values, all ligands except pNPY13-36 (Ki=642 ± 133 nM) bound in a nanomolar range to the receptor (0.15–2.4 nM). The variation within the affinities of the endogenous zebrafish peptides was smaller in zfY2-2 compared to zfY2 with an approximately 4- compared to 46-times difference. The Ki of the severely truncated pNPY13-36 for zfY2-2 was in good agreement with the result from rainbow trout Y2 and Y7 but was 262-fold higher than to zfY2. The human Y2 selective antagonist BIIE0246 displayed a Kihigh for zfY2-2 equal to hY2 but with a more than 56-times difference compared to zfY2, chicken Y2 and rainbow trout Y2 and Y7. Though, if comparing the Kilow of the antagonist for zfY2-2 with the zfY2 it showed less than a two-fold difference.

A functional assay measuring the inositol phosphate production upon binding of the antagonist to the zfY2-2 receptor resulted in an 83% response compared to the basal level. This indicates that BIIE0246 may act as a weak inverse agonist of the receptor. Expression of the receptor was found in the eye and gastrointestinal tract by real-time PCR. Since the distribution of PYY and NPY in various species is localized in the gut and the brain, respectively, one could assume that the zfPYYa and zfPYYb are the preferred ligands in the gastrointestinal tract and zfNPY in the eye of the fish.

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Concluding remarks and future perspectives

Paper I, II and III The focus in Paper I, II and III was to identify amino acid positions in the hY2 receptor that are important for ligand interaction. In total we investigated thirteen residues, chosen from sequence comparisons of the receptor subtypes, within and between different species. The positions were also selected based on previous results from hY1 mutagenesis studies (Sautel et al., 1995; Sjodin et al., 2006) as well as on results from receptor modelling and docking (Paper III). The results presented here, in combination with previously published mutagenesis data for both hY2 and hY1, form the basis for further computerized modelling of the NPY receptors. These models will, in combination with ligand docking simulations of both agonists and antagonists, reveal common denominators for peptide ligand interactions and might contribute to the development of new NPY receptor-specific pharmaceuticals. Hopefully, the modelling will also enhance our understanding of how various NPY receptor subtypes have become so structurally divergent and, in spite of this, are still able to interact with the same peptide ligands, i.e. NPY and PYY. The receptor modelling might also help reveal an explanation for that Y2 can bind, and be activated by, the truncated endogenous PYY3-36 peptide, whereas Y1 cannot.

The hydrophobic pocket in the hY1 receptor suggested by Sautel et al. was re-evaluated in a previous study in our laboratory (Sjodin et al., 2006). Agreement between the studies was found regarding the binding results of the three residues in question (2.64, 6.58 and 7.31) but a new receptor model presented by Sjödin et al. did not support the hydrophobic pocket hypothesis. The results from Paper I presented herein show a plausible hY1 receptor model that is in agreement with Sautel et al. However, this binding pocket is not possible in hY2 according to our receptor models based on both h 2R and hA2AR. The results in Paper I in combination with Paper II and III indicate that the ligands have fewer, or possibly different, points of interaction in the hY2 receptor compared to the hY1 receptor.

Moreover, since the agonist ligands displayed increased affinity for the mutant receptors Gln6.55Ala and Thr3.40Ile, it would be interesting to test if this also reflects an increased intracellular response. This can be done by measuring the decrease in cAMP production in the cell or, like in Paper IV, using a chimeric G protein in an inositol phosphate assay.

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An ongoing project focuses on further investigation of how the residues Gln3.32, Thr2.61 and His7.39 interact with each other and with the ligands, in the suggested hydrogen bonding network identified from the ligand docking simulations in Paper III. Ala and Leu will be introduced at positions 3.32 and 7.39 and tested in binding experiments in order to see if this abolishes a hydrogen bond or polar interaction, respectively. In the near future, more residues will be identified that will contribute to the determination of the structures of the NPY-family of receptors.

During the course of this thesis, there has been rapid progress in the field of GPCR structures with the release of several new high-resolution crystal structures. This has encouraged the modeling work performed herein and has provoked the initiation of new receptor modeling projects in our lab. Preparation of the Y1 and Y2 models for molecular dynamics simulations is in progress.

Paper IV There is a variation in the NPY receptor repertoire in various animal lineages despite their recent common origin in vertebrate evolution. This is mainly due to gene losses, such as the loss of the Y8 receptor in the mammalian lineage. Additionally, in the teleost fish lineage gene duplications have occurred. An extra Y8 gene copy has been identified (in the articles referred to as Yb and Yc) (Arvidsson et al., 1998; Larson et al., 2003; Larsson et al., 2008; Lundell et al., 1997; Ringvall et al., 1997; Salaneck et al., 2008).

In this Paper an additional NPY receptor sequence was identified in the genomes of zebrafish and medaka. The bioinformatics analyses performed clearly showed that this novel NPY receptor was a Y2-like receptor. Since this new gene is most likely the result of a local duplication of Y2, and not a genome doubling event, the gene was named Y2-2. Based on the sequence alignments it became obvious that the two Y2-2 gene sequences differed from previous Y2 genes by having introns, two in zebrafish and three in medaka, though positioned differently in the two species. This implied that the introns were introduced in each species independently. However, introns in the coding region are rare in the NPY receptor family. So far, Y1 is the only gene in tetrapods that has an intron and in fishes Y8a has received three introns in the tetraodon and fugu lineage (Larsson et al., 2008).

The binding profiles of the zfY2 and Y2-2 receptors differed the most in their interaction with the truncated NPY-analogue and the antagonist. This information will be useful in future NPY receptor-modeling and docking studies. In order to investigate the zfY2-2 receptor further, it would be relevant to follow up the binding experiments with functional studies.

Although Y2-2 expression could be seen in the gastrointestinal tract and eye in the reverse-transcriptase PCR analyses it would be interesting to

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estimate the expression levels using a quantitative method, like qPCR or in situ hybridization. The latter would also allow the identification of the specific cells expressing the gene. This knowledge could further be used to design Y2-2 gene specific morpholinos for studies of cell biology, physiology and behavior.

In particular, it would be interesting to try to investigate its involvement in appetite regulation. Maybe, since the Y2-2 appears to be the zebrafish NPY receptor with the highest affinity for the antagonist BIIE0246, this ligand could be used in animal studies to evaluate the blocking of Y2-2.

Finally, this study emphasizes the complex evolutionary history of the NPY receptors by highlighting the more diverse NPY system in the teleost fishes compared to the mammals.

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Svensk sammanfattning

Det långsiktiga målet med forskningen som den här avhandlingen är en del av är att kunna bidra med en bättre kunskap om neuropeptid Y-systemet, ett system bestående av både peptider och receptorer. Den kunskapen kan bli användbart för utveckling, och så småningom framställningen, av nya läkemedel mot en rad olika sjukdomar. Neuropeptid Y-systemet i människa är nämligen, med de tre kroppsegna peptiderna neuropeptid Y (NPY), peptid YY (PYY) och pankreatisk polypeptid (PP) och receptorerna Y1, Y2, Y4 och Y5, inblandade i en uppsjö av olika funktioner i kroppen. Några som bör nämnas är aptit, vilket har varit fokus för denna avhandling, men även hjärt-kärl-reglering, ångest, reglering av dygnsrytm, fortplantning, tillväxt och benbildning. Det bedrivs mycket forskning för att försöka identifiera selektiva och potenta substanser som påverkar NPY-systemet och störst är intresset att framställa läkemedel mot övervikt. I synnerhet söker man substanser som stimulerar Y2- och Y4-receptorerna eftersom detta leder till dämpad aptit, samt inhiberande substanser mot Y1- och Y5-receptorerna vilka är aptitstimulerande. NPY-receptorerna karaktäriseras baserat på hur bra de olika peptiderna binder till dem, dvs. vilken affinitet de har till receptorerna. Y2-receptorn skiljer sig från de andra receptorerna genom att den binder förkortade NPY- och PYY-peptider med hög affinitet.

De första tre artiklarna som inkluderas i avhandlingen (Paper I-III) är baserade på experiment utförda på människans Y2-receptor. Aminosyror på vissa positioner i receptorn byttes ut varpå bindningen av olika peptider testades mot dessa ”nya” modifierade receptorer. På så sätt gick det att identifiera positioner som verkar vara viktiga för peptidens bindning till receptorn. Denna information användes sedan löpande genom alla tre arbetena i framställningen av tredimensionella datamodeller av den mänskliga Y2-receptorn.

Positionerna som byttes ut valdes på något av de tre följande sätten: 1) De motsvarande positioner som i tidigare bindningsstudier i den mänskliga Y1-receptorn visat på intressanta resultat. 2) Strukturerna för alla kända NPY-receptorer jämfördes inom och mellan arter och positioner som skilde sig eller var bevarade mellan dem identifierades. Hypotesen var att de bevarade positionerna skulle kunna tyda på en evolutionärt viktig funktion vid, exempelvis, interaktionen med en peptid. Alternativt, om positionen bara skilde sig i Y2-receptorerna, skulle den kunna bidra till denna receptors

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förmåga att binda förkortade peptider. 3) Utifrån datamodellering av Y2-receptorn och efterföljande dockning av NPY till modellen. På så sätt kunde positioner i receptorn med troliga interaktioner till peptiden identifieras på förhand.

Totalt byttes 13 positioner ut och de modifierade receptorerna testades i bindnings experiment med tre eller flera peptider. Resultaten jämfördes med resultaten från tidigare publicerade Y1-studier. Vidare användes resultaten till att ge feedback till datamodelleringen och på så sätt optimera Y2-receptormodellen. Slutsatsen från dessa arbeten är i korthet att Y2 verkar ha färre, alternativt andra, interaktionsställen än Y1-receptorn. Denna information kommer vara användbar i framtida dockningsstudier och i förståelsen av hur de till strukturen relativt olika receptorerna fortfarande kan binda och aktiveras av samma peptider.

Det sista arbetet som ingår i denna avhandling (Paper IV) skiljer sig från de övriga tre. Med hjälp av genomen (arvsmassorna) för olika arter som finns fritt tillgängliga i databaser på nätet identifierades en ny NPY-receptor i fiskarterna zebrafisk och medaka. Strukturinformationen för den nya receptorn jämfördes med de andra NPY-receptorerna och på så sätt kunde den identifieras som en Y2-lik receptor och således blev den döpt Y2-2. Jämförelse mellan generna för Y2- och de bägge Y2-2-receptorerna visade att de senare hade en annorlunda uppbyggnad: bägge innehöll icke-kodande delar, introner, i den proteinkodande delen, två introner i zebrafisken och tre i medaka. Dock satt de på olika ställen och avspeglar således troligen separata händelser i evolutionen.

Efter att det konstaterats att Y2-2-receptorn fanns uttryckt i vävnader hos zebrafisken testades den i bindningsexperiment med flera peptider för att klarlägga dess specifika bindningsprofil. ZebrafiskensY2-2-receptor skilde sig från zebrafiskens Y2-receptor framför allt i den mycket försämrade bindningen av den förkortade NPY-peptiden och den förbättrade bindningen till den inhiberande substansen BIIE0246. Denna kunskap kan i framtiden komma att vara användbar för förståelsen av exakt hur peptiderna binder till receptorerna. Detta kan i sin tur förenkla framställningen av receptorspecifika substanser, t.ex. vid framställningen av läkemedel. Eftersom Y2-receptorn i övriga arter har visats ha en inhiberande effekt på aptit så skulle det vara intressant att göra en studie där man studerar fiskars ätbeteende efter Y2-2-specifik stimulering. Kunskapen om aptitreglering hos fisk är idag begränsad men verkar, med några få undantag, fungera liknande den hos däggdjur. Dock, med tanke på variationen mellan de många olika fiskarterna med avseende på var de lever, hur och vad de äter, fortplantar sig, med mera, så bör man förstås inte förvänta sig att aptit regleras likadant hos alla fiskar.

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Acknowledgements

My greatest gratitude goes to my supervisor Dan Larhammar who has been an excellent mentor and friend throughout these years. You have shown me the essence of how true science is conducted and how fun it can be. Ingrid Lundell and Nina Mohell, my two co-supervisors have both contributed with their vast knowledge about my experiments. You have both been indispensable.

I have had the advantage to be part of a research group consisting of great people. Helena Åkerberg was the fantastic person that introduced me to mutagenesis and cloning. Now, although I have had my doubts, in the hindsight, I think you did right doing that. ;) Christina Bergqvist, our outstanding lab technician, you are unique and I am so glad I have had the pleasure of sharing office with you all this time – with all that comes with that! Daniel Ocampo Daza, with your infinite knowledge and warm personality you have had me fallen in love with you many times. :) Bo Xu, my newest partner in crime who had been indispensable ever since you started: I wish you all the best in life. Chus (Xesús M.) Abalo, a more devoted and inspiring researcher is hard to find. Remember, all you want you can have. Do not forget to notify me when your book is published! Jasna Pruner, your devotion and drive will take you far. David Lagman, the most fish-like person I know. :) Jenny Widmark, I will miss your happy face that always puts me in a good mood.

Other, former members in the group not to forget: Görel Sundström, I will put every one of my children on the patchwork quilt you made for us! Tomas Larsson, your humor and kind personality did help on dull days here at BMC. Big thanks also to all the students that have passed through our lab over the years. Especially Robin Luu, Andrea T. Gerholm, Katarina Anderson and Magnus Grill - you were all students of mine and will therefore always be extra special to me. :)

Not to forget the people in the neighboring groups: Hanna-Linn Wargelius, you have shown me a strength and single-mindedness rarely seen. Josefin Jacobsson, when your house is done I will come visit! Maria Hägglund, positivism in person. :) Nicole Neumann, I am impressed of what you have achieved.

I have had the advantage of getting to know very competent and friendly people through our research collaborations: Lars Boukharta, Hugo

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Gutiérrez-de-Terán, Henrik Keränen and Johan Åqvist. You have provided a third dimension to my research ;) and I really appreciate it!

Birgitta Hägerbaum, Marita Berg, Ulla Jonson, Emma Andersson, Lena Karlsson, Maria Sundien and Mariana Hooli: you have all contributed with your helpfulness and knowledge in various areas where my understanding has been low – thanks for that!

To all other people here at BMC, I would like to thank you for contributing to a good and welcoming working environment!

Many hugs and kisses to my beloved parents, Anja and Håkan, for raising me with the guts to start a project like this. I guess it is in my genes. ;) My sister-in-law, Helene, your inspiration and confident boosts has been more than needed. To my sister Lisa and brother Peppe with families, I know I have had your support. My second family: Britt-Marie, Peo, Andreas, Anna and Terés – dinners with you in Nåntuna have brighten up many of my days!

To my friends outside work: Anna and Johan, you are special to me and moving closer to you feels just great. Peter and Lina, more inventive people are impossible to find! Anna and Jakob, we have many Geocaches to find together yet. :) My book club friends: Sandra, Jill, Sanna and Cathrine, our dates have widened my view on literature and I hope we will continue forever. Looking forward to do TjejVasan with you! All my board game friends: Mia, Viktor, Elin, Jesper, Sigrid, Christian, Daniel, Mattias and other members in BORK, nothing can distract my mind as well as a game of Hacienda or Puerto Rico with you. :) Mikaela, Anders, Melle and Pär, thanks for all the get-togethers we have had – more of those! Having a baby in the middle of my PhD studies was indeed a good thing. :) Tone and Jonas, soon we will have two gardens to let the children out in – we just provide them with watering cans and then we can enjoy some good wine while watching them. :) Helena and Anders, our baby boys will be best friends! Elin and Thobias, our Wednesday’s family dinners have been great – you have to come visit us in Nåntuna.

Finally, the support I have got from my husband David is immeasurable. Sometimes I feel that you have done the hard work of my thesis. You have fulfilled all the house duties when I have been working, to that I owe you. Whenever I have had a down period you have been there to lift me up. And best of all, you gave me the funniest reason ever for having a break in my PhD studies – Harry! This little son of ours has made me realize that there is more to this world than just fulfilling your own needs. You two are, and will always be, in my heart.

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