<p>The neuropeptide Y (NPY) family of structurally related peptides includes NPY, peptide YY (PYY) and pancreatic polypeptide (PP). They bind to G-protein coupled receptors named Y receptors, and include in mammals Y1, Y2, Y4, Y5, Y6 and in non-mammalian vertebrates also Y7, Yb and Yc. Subtypes Y1 and Y5 stimulate appetite, while Y2 and Y4 have the opposite effect in mammals. The studies described here concern human Y1 and Y4, chicken Y6 and Y7, and zebrafish Y2. </p><p>Site-directed mutagenesis of human Y1 identified sites important for binding of NPY and PYY as well as Y1 antagonists. The results clarify contradictory findings previously reported by others and identify new sites of interaction. A three-dimensional structural model of the Y1 receptor based upon the high-resolution structure of bovine rhodopsin was generated that increases our understanding of ligand-receptor interactions and hopefully will facilitate the design of novel subtype-selective agonists and antagonists. </p><p>Two naturally occurring variants of human Y4 with substitutions R240C and V276M have been found in a sample of obese children. Functional studies in vitro showed that the cellular response to PP was greatly decreased for R240C and may provide a causative link to juvenile obesity. </p><p>The genes for chicken Y6 and Y7 were found to be located ~1 megabase apart on chromosome 13 syntenic to the human Y6 pseudogene. Y6 mRNA has widespread expression whereas Y7 mRNA was found only in adrenal gland. Truncated fragments of PYY had lower affinity to chicken Y7 and zebrafish Y2 than to Y2 from mammals and chicken. The results suggest that Y2 in mammals acquired the ability to bind truncated PYY, i. e. PYY_3-36, fairly recently, which has implications for its role in appetite inhibition.</p><p>These differences between receptor subtypes in sequences and pharmacological properties will be useful to further elucidate the structure and activation of Y receptors by site-directed mutagenesis. </p>

The neuropeptide Y (NPY) family of structurally related peptides includes NPY, peptide YY (PYY) and pancreatic polypeptide (PP). They bind to G-protein coupled receptors named Y receptors, and include in mammals Y1, Y2, Y4, Y5, Y6 and in non-mammalian vertebrates also Y7, Yb and Yc. Subtypes Y1 and Y5 stimulate appetite, while Y2 and Y4 have the opposite effect in mammals. The studies described here concern human Y1 and Y4, chicken Y6 and Y7, and zebrafish Y2. Site-directed mutagenesis of human Y1 identified sites important for binding of NPY and PYY as well as Y1 antagonists. The results clarify contradictory findings previously reported by others and identify new sites of interaction. A three-dimensional structural model of the Y1 receptor based upon the high-resolution structure of bovine rhodopsin was generated that increases our understanding of ligand-receptor interactions and hopefully will facilitate the design of novel subtype-selective agonists and antagonists. Two naturally occurring variants of human Y4 with substitutions R240C and V276M have been found in a sample of obese children. Functional studies in vitro showed that the cellular response to PP was greatly decreased for R240C and may provide a causative link to juvenile obesity. The genes for chicken Y6 and Y7 were found to be located ~1 megabase apart on chromosome 13 syntenic to the human Y6 pseudogene. Y6 mRNA has widespread expression whereas Y7 mRNA was found only in adrenal gland. Truncated fragments of PYY had lower affinity to chicken Y7 and zebrafish Y2 than to Y2 from mammals and chicken. The results suggest that Y2 in mammals acquired the ability to bind truncated PYY, i. e. PYY3-36, fairly recently, which has implications for its role in appetite inhibition. These differences between receptor subtypes in sequences and pharmacological properties will be useful to further elucidate the structure and activation of Y receptors by site-directed mutagenesis.

<p>The superfamily of G protein-coupled receptors (GPCRs) is one of the largest protein families of mammalian genomes and can be divided into five main families; <i>Glutamate</i>, <i>Rhodopsin</i>, <i>Adhesion</i>, <i>Frizzled</i>, and <i>Secretin</i>. GPCRs participate in most major physiological functions, contributing to the fact that they are important targets in drug discovery. In paper I we mined the human and mouse genomes for new <i>Adhesion</i> GPCR genes. We found two new human genes (GPR133 and GPR144) and 17 mouse <i>Adhesion</i> genes, bringing the number up to 33 human and 31 mouse genes. In paper II we describe 53 new splice variants for human <i>Adhesion</i> receptors supported by expressed sequence tags (EST) data. 29 of these variants seem to code for functional proteins, several of which lack one or more functional domains in the N-termini. Lack of certain domains is likely to affect ligand binding or interaction with other proteins. Paper III describes the <i>Glutamate</i> GPCR in human, mouse, <i>Fugu</i>, and zebrafish. We gathered a total of 22 human, 79 mouse, 30 <i>Fugu</i>, and 32 zebrafish sequences and grouped these into eight clans using phylogenetic methods. The report provides an overview of the expansion or deletions among the different branches of the <i>Glutamate</i> receptor family. Paper IV focuses on the trace amine (TA) clan of <i>Rhodopsin</i> GPCRs. We identified 18 new rodent genes, 57 zebrafish genes, and eight <i>Fugu</i> genes belonging to the clan. Chromosomal mapping together with phylogenetic relationships suggests that the family arose through several mechanisms involving tetraploidisation, block duplications, and local duplication events. Paper V provides a comprehensive dataset of the GPCR superfamily of human and mouse containing 495 mouse and 400 human non-olfactory GPCRs. Phylogenetic analyses showed that 329 of the receptors are found in one-to-one orthologous pairs, whereas other receptors may have originated from species-specific expansions.</p>

The superfamily of G protein-coupled receptors (GPCRs) is one of the largest protein families of mammalian genomes and can be divided into five main families; Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin. GPCRs participate in most major physiological functions, contributing to the fact that they are important targets in drug discovery. In paper I we mined the human and mouse genomes for new Adhesion GPCR genes. We found two new human genes (GPR133 and GPR144) and 17 mouse Adhesion genes, bringing the number up to 33 human and 31 mouse genes. In paper II we describe 53 new splice variants for human Adhesion receptors supported by expressed sequence tags (EST) data. 29 of these variants seem to code for functional proteins, several of which lack one or more functional domains in the N-termini. Lack of certain domains is likely to affect ligand binding or interaction with other proteins. Paper III describes the Glutamate GPCR in human, mouse, Fugu, and zebrafish. We gathered a total of 22 human, 79 mouse, 30 Fugu, and 32 zebrafish sequences and grouped these into eight clans using phylogenetic methods. The report provides an overview of the expansion or deletions among the different branches of the Glutamate receptor family. Paper IV focuses on the trace amine (TA) clan of Rhodopsin GPCRs. We identified 18 new rodent genes, 57 zebrafish genes, and eight Fugu genes belonging to the clan. Chromosomal mapping together with phylogenetic relationships suggests that the family arose through several mechanisms involving tetraploidisation, block duplications, and local duplication events. Paper V provides a comprehensive dataset of the GPCR superfamily of human and mouse containing 495 mouse and 400 human non-olfactory GPCRs. Phylogenetic analyses showed that 329 of the receptors are found in one-to-one orthologous pairs, whereas other receptors may have originated from species-specific expansions.

<p>Eukaryotic gene transcription plays a regulatory role in mammalian developmental processes. It has been shown that transcriptional control is an important mechanism for specification of neurotransmitter phenotypes. In the mammalian central nervous system, the transcription factor AP-2 family is one of the critical regulatory factors for neural gene expression and neuronal development. It has been shown that several genes in the monoaminergic systems have AP-2 binding sites in regulatory regions, suggesting a regulatory role of AP-2 also in the adult brain. Brainstem monoamines are implicated in the expression of personality traits and imbalances in these systems may give rise to psychiatric disorders. </p><p>The gene encoding AP-2β includes a polymorphic region consisting of a tetranucleotide repeat of [CAAA]_4-5 in intron 2. Studies on AP-2β genotype in relation to personality and platelet MAO activity, a trait-dependant marker for personality, are presented in this thesis. Furthermore, correlations between brainstem levels of AP-2α and AP-2β and monoamine turnover in projection areas in rat forebrain are reported. These results strengthen the notion that the AP-2 family is important regulators of the monoaminergic systems in the adult brain. Furthermore, two studies are presented in this thesis with analyses indicating a role for AP-2 in the molecular mechanism of antidepressant drugs.</p><p>Altogether, this thesis presents data supporting our notion that the transcription factor AP-2 family is involved in the regulation of the monoaminergic systems both pre- and postnatally, and, therefore, might be involved in the pathophysiology of neuropsychiatric disorders.</p>

<p>Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO; EC.1.4.3.6) has been reported to be high in disorders such as diabetes mellitus, chronic congestive heart failure and liver cirrhosis. Little is known of how the activity is regulated and, consequently, the cause for these findings is not well understood. Due to the early occurrence of increased enzyme activity in diabetes, in conjunction with the production of highly cytotoxic substances in SSAO-catalysed reactions, it has been speculated that there could be a causal relationship between high SSAO activity and vascular damage. Aminoacetone and methylamine are the best currently known endogenous substrates for human SSAO and the resulting aldehyde-products are methylglyoxal and formaldehyde, respectively. Both of these aldehydes have been shown to be implicated in the formation of advanced glycation end products (AGEs).</p><p>This thesis is based on studies exploring the regulation of SSAO activity and its possible involvement in the development of vascular damage. The results further strengthen the connection between high SSAO activity and the occurrence of vascular damage, since type 2 diabetic patients with retinopathy were found to have higher plasma activities of SSAO and lower urinary concentrations of methylamine than patients with uncomplicated diabetes. From studies on mice, it was also found that an SSAO inhibitor potently reduces the incorporation of methylamine-metabolite in the tissues. By quantifying SSAO-gene expression in alloxan-induced diabetes, increased transcription could be ruled out as a cause for the increased enzyme activity, thereby opening up for the possibility that the activity is regulated post-translationally. In fact, increased enzyme activity in adipose tissue was accompanied by decreased mRNA-levels, suggesting that the gene expression could be negatively controlled by the enzyme activity.</p>

<p>Oligopeptides can be used to carry cytotoxic agents in cancer chemotherapy, using tumour-associated proteins as the molecular target for selectivity. During the seventies and eighties Peptichemio, a cocktail of six alkylating oligopeptides carrying <i>m</i>-L-sarcolysin, was investigated in a wide variety of human malignancies. Positive clinical results were suggested to result from rapid and effective DNA-crosslinking following uptake in neoplastic cells, but also from antimetabolic properties of the drug. Although <i>m</i>-L-sarcolysin never reached widespread clinical use, the well established <i>para</i>-isomer melphalan still, after nearly fifty years, has a place in cancer chemotherapy.</p><p>The present study was undertaken to synthesise the melphalan containing analogue of the tripeptide P2 (L-prolyl-<i>m</i>-L-sarcolysyl-<i>p</i>-L-fluorophenylalanine ethyl ester, the main contributor to Peptichemio’s activity) and similar compounds, preferably dipeptides. The new compounds compared favourably with melphalan, <i>m</i>-L-sarcolysin and P2, considering their potency in vitro. Structure activity relationship analysis showed that the activity of melphalan dipeptides depended on the amino acid composition, sequence and end group modifications, but only to a minor degree on lipophilicity. Results suggested that the dipeptides, to exert their full cytotoxic activity, had to interact with specific biomolecules such as dipeptide transporters or peptidases. Although no active transport could be demonstrated the influence of peptide hydrolysis was obvious, thereby suggesting a rationale for increased activity as well as potential tumour selectivity in comparison with melphalan.</p><p>Preliminary in vivo studies in mice supported the results, despite equal alkylating capacity the dipeptide J1 (melphalanyl-<i>p</i>-L-fluorophenylalanine ethyl ester) and the tripeptide J3 (L-prolyl-melphalanyl-<i>p</i>-L-fluorophenylalanine ethyl ester) were more active than was melphalan on human tumour cells implanted in test animals although all drugs produced expected side effects, notably leukopenia, of similar magnitude.</p><p>In conclusion, oligopeptide derivatives of melphalan seem to provide improved cytotoxic activity and therapeutic index. Further development of such oligopeptides for clinical use seems worthwhile.</p>

<p>The antitumoural cyanoguanidine CHS 828 has shown promising activity in a number of preclinical and clinical studies. However, the mechanisms underlying the cell death induced by CHS 828 has not been clarified. This thesis describes in vitro studies of the cellular pharmacology of CHS 828.</p><p>CHS 828 induced cell death with necrosis like features in the lymphoma cell line U-937 GTB. Addition of 3-aminobenzamide, an inhibitor of ADP-ribosylation, resulted in a decreased sensitivity to CHS 828 and a shift in the mode of cell death towards apoptosis. </p><p>Mouse fibroblasts lacking the enzyme PARP-1 were more sensitive to CHS 828 compared to normal fibroblasts. CHS 828 was able to induce p53 in normal fibroblasts but this effect does not seem to be necessary to induce cell death.</p><p>Characterization of two CHS 828 resistant cell lines indicated that they were selectively resistant to cyanoguanidines. Known mechanisms of anticancer drug resistance did not seem to account for the cyanoguanidine resistance. One possible resistance mediating protein, which was upregulated in the resistant cells, was epidermal fatty acid binding protein.</p><p>A novel high content screening assay was also developed. The assay was shown to be suitable both for screening of potential novel antitumoural substances as well for mechanistic studies. In the assay, CHS 828 induced caspase-3 activity and reduction in mitochondrial membrane potential, both signs of apoptosis, in U-937 GTB cells. However, nuclei in exposed cells did not show nuclear fragmentation, one of the hallmarks of apoptosis.</p><p>CHS 828 was also shown to indirectly inhibit the proteasome activity in U-937 GTB cells. </p><p>In conclusion, the results presented provide new insights into the metabolic and molecular events involved in cell death induced by CHS 828.</p>

<p>The CNS monoaminergic systems are considered as the head engine regulating neuropsychiatric functions and personality. Transcription factor AP-2 is known to be essential for the development of the brainstem including the monoaminergic nuclei, and has the ability to regulate many genes in the monoaminergic systems. The ability of transcription factors to regulate specific gene expression, has lately made them hot candidates as drug targets. In this thesis, results indicating a role of AP-2 in the molecular effects of the antidepressant drugs citalopram and phenelzine, are presented. </p><p>A polymorphism in the second intron of the gene encoding AP-2ß has previously been associated with anxiety-related personality traits as estimated by the Karolinska Scales of Personality (KSP). In this thesis, results confirming this association, gained by using a larger material and several different personality scales, are presented. Furthermore, data is presented showing an association between the activity of platelet monoamine oxidase, a trait-dependent marker for personality, and the genotype of the AP-2ß intron 2 polymorphism. </p><p>The functional importance of the AP-2ß intron 2 polymorphism has not yet been elucidated. Included in this thesis are results showing that the AP-2ß intron 2 polymorphism is not in linkage disequilibrium with the only other described polymorphism in the AP-2ß gene, i.e. in the AP-2ß promoter (-67 G/A). Introns have in several studies been shown to include binding sites for regulatory proteins, and thus, to be important in transcriptional regulation. Results are presented demonstrating that one human brain nuclear protein binds only to the long variant of the AP-2ß intron 2 polymorphism. If this protein is involved in the regulation of the AP-2ß gene, it would affect the expression levels of the AP-2ß protein. </p><p>In general, this thesis further establishes the role of transcription factor AP-2 as a regulatory factor of importance for personality and monoaminergic functions.</p>

<p>Semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6) belongs to a family of copper-containing amine oxidases. SSAO exists as a membrane bound protein in endothelial-, smooth muscle-, and adipose cells as well as soluble in plasma. SSAO catalyses oxidative deamination of primary monoamines, which results in the production of corresponding aldehydes, hydrogen peroxide and ammonia. These compounds are very reactive and potentially cytotoxic, and are able to induce vascular damage if produced in high levels. Patients with diabetes mellitus, and with diabetic complications in particular, have a higher SSAO activity in plasma compared to healthy controls. It has therefore been speculated that high SSAO activity is involved in the development of vascular complications associated with diabetes. The aim of this thesis is to investigate the importance of SSAO in the development of disorders of a vascular origin. We have studied the transcriptional regulation of the SSAO gene, by inducing diabetes in NMRI and in transgenic mice, overexpressing the human form of SSAO in smooth muscle cells. We found that the increase in SSAO activity in diabetes is accompanied by reduced mRNA levels of the endogenous mouse gene, suggesting a negative feedback on the transcription of the SSAO gene. In addition, the transgenic mice exhibited an abnormal phenotype in the elastic tissue of aorta and renal artery. These mice have a lower mean artery pressure and an elevated pulse pressure. These results indicate that high SSAO activity in smooth muscle cells is associated with a change in the morphology of large arteries. This is likely contributing to the development of vascular complications in diabetes.</p>