Prostaglandin E2 (PGE2) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE2 mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP1, EP2, EP3 and EP4. The EP3 prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP3 receptor isoforms and their effects on gene regulation.We generated HEK 293 EBNA cell lines expressing the EP3-Ia, EP3-II, or EP3-III isoforms. After confirming the functional expression of each of these isoforms, we examined their activation of cellular signal transduction pathways.We found that each of these isoforms utilize distinct mechanisms to regulate ERK 1/2 phosphorylation and that these differences lead to unique regulation of the downstream effectors ELK-1 and AP-1. We also found MAPK dependent differences in regulation of cell proliferation. The EP3-III isoform increases cell proliferation in a MAPK dependent manner while the EP3-Ia dose dependently regulates cell proliferation via Gαi and not ERK 1/2. Activation of the EP3-II receptor had no effects on cell proliferation.To study differential gene regulation by these three EP3 receptor isoforms, we conducted microarray studies. Over 300 genes were differentially regulated by these isoforms. Quantitative real-time PCR analysis was used to validate 15 candidate genes. Five genes were chosen for further analysis of protein expression using immunoblotting, but only one of these, WT-1, was significantly increased following treatment with PGE2. WT-1, a transcription factor important for kidney and heart development, was strongly upregulated by PGE2 stimulation of the EP3-II receptor, but only weakly by the other isoforms.In conclusion, these studies show that the human EP3 prostanoid receptor isoforms are capable of distinct regulation of both signal transduction pathways and gene transcription. Elucidating the differential functions of EP3 receptor isoforms may allow for greater understanding of the diverse functions attributed to this receptor and their physiological functions.

Activation of pathways resulting in an overexpression of oncoproteins, reliant on cap-dependent translation, or mutations of key proteins in a pathway can be advantageous to cancer cells but creates heightened protein quality control pressure. Because of this, there has been an interest in targeting enzymes involved in protein synthesis and protein quality control: such as the eukaryotic initiation factor, eIF4A, a DEAD-box RNA helicase involved in translation initiation, and p97, an AAA+ chaperone involved in protein quality control. Despite some successes in discovering both eIF4A and p97 inhibitors, many of these compounds have pharmacological setbacks. The work in this dissertation defines new inhibitors of eIF4A and p97 with unique mechanisms of action. As described in chapter 2, we demonstrated that a marine-derived sesquiterpene, elatol, can modulate the ATPase activity of eIF4A. We provide further evidence that this molecule inhibits cap-dependent translation. Because there is no clear consensus on the mechanism of action for elatol, we hypothesized that the mechanism of toxicity attributed to elatol is likely through inhibition of cap-dependent translation initiation by targeting eIF4A. In chapter 3, we adapted a colorimetric assay to identify natural products that modulate the ATPase activity of p97 from which withaferin A (WFA) was identified. Because proteostasis modulation can connect each of the reported modes of action of WFA, we hypothesized that the primary mode of cytotoxic action of WFA is through inhibition of protein quality control machinery. Through medicinal chemistry efforts, we were able to improve WFA's biochemical and cellular activities as well as shifting the activity toward p97 and away from the proteasome. The work described in chapter 4 reports that dehydrocurvularin (DHC) and its chlorinated analogs are covalent modifiers of p97 and that the selectivity toward p97 can be attributed, in part, to the electronic effects of the chlorines. Taken together, this work highlights the significance of targeting protein translation and quality control, by modulation of eIF4A and p97 activity respectively, as promising anticancer therapeutics.

Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates a battery of downstream genes that contain the antioxidant response element (ARE) in their promoter regions, including intracellular redox-balancing proteins, phase II detoxifying enzymes, and transporters. These Nrf2-dependent proteins work in collaboration to protect against many diseases where oxidative stress plays an essential role in disease onset and progression. Consequently, it is imperative to understand the basic molecular mechanisms of how Nrf2 is regulated so that this pathway can be targeted for disease prevention and treatment.Nrf2 is mainly regulated at the protein level by the ubiquitin proteasome system. Under basal conditions Nrf2 is constantly ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and subsequently degraded by the 26S proteasome. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, other mechanism responsible for modulating Nrf2-ARE signal remains to be explored. This dissertation identifies three molecular mechanisms that are important in understanding how the Nrf2-Keap1 pathway is regulated: (i) In Chapter 2, KPNA6 was identified and characterized as a negative regulatory mechanism of the Nrf2 pathway, which mediates Keap1 nuclear import and represses the Nrf2-dependent antioxidant response at post-induction phase. (ii) In Chapter 3, I identified PARP-1 as a new transcription co-activator of Nrf2, which augments ARE-specific DNA binding of Nrf2 and enhances the transcription of Nrf2 target genes. This indicates a novel function of PARP-1 and reveals another layer of regulation of Nrf2. (iii) In Chapter 4, I demonstrated that XBP1 and SYVN1 are involved in regulating the Nrf2 pathway in a Keap1-independent mechanism. During ER stress, XBP1s upregulates transcription of SYVN1, which is an ubiquitin E3 ligase. SYVN1 accelerates the clearance of Nrf2 protein through promoting ubiquitination of Nrf2, and subsequent proteasomal degradation. Moreover, we observed an inverse correlation between XBP1s/SYVN1 and Nrf2 expression in the end stage alcoholic cirrhosis liver samples, implying a pathological role of ER stress-oxidative stress crosstalk. Taken together, these findings further our understanding of how the Nrf2-Keap1 pathway is regulated, providing novel targets of chemoprevention or chemotherapy.

Cumulative evidence suggests that constitutively elevated levels of proteotoxic stress represent a specific vulnerability of malignant cells that can be targeted by pharmacological modulation of the intracellular proteotoxic stress response. According to this emerging mechanism, small molecule stress modulators may induce deviations from protein homeostasis causing cytotoxicity confined to malignant cells already at a high set point of constitutive proteotoxic stress leading to functional impairment and even cell death. In contrast, normal cells with sufficient protein degradation capacity can tolerate the extra dysfunctional protein overload. My graduate research has focused on testing the feasibility of repurposing clinically used non-oncological drugs for experimental chemotherapy targeting metastatic melanoma cells. The following specific aims were pursued: (1) To identify clinically used non-oncological drugs that preferentially induce cytotoxicity in melanoma cells but not primary melanocytes through upregulation of proteotoxic and/or oxidative stress; (2) To explore the specific molecular mechanisms underlying induction of melanoma cell apoptosis by lead compounds focusing on oxidative and proteotoxic stress modulation; (3) To explore efficacy of selected lead compounds for antimelanoma intervention in a murine xenograft model. First, we demonstrate feasibility of using the FDA-approved redox-active D-cysteine-derivative D- penicillamine for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo through induction of the unfolded protein response (UPR). Second, we demonstrate that the antimicrobial oligopeptide thiostrepton displays dual activity as a selective prooxidant and proteasome inhibitor causing proteotoxic stress that preferentially targets malignant melanoma and multiple myeloma cells. Third, we demonstrate for the first time that the clinically used 4-aminoquinoline antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced melanoma cell death. Taken together, our data indicate the chemotherapeutic potential of small molecule proteotoxic stress inducers and strongly suggest feasibility of repurposing specific non-oncological drugs for proteotoxic stress-directed antimelanoma intervention.

Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In the studies presented herein, MTK1 is shown to be required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced extracellular acidification and cell migration in MCF-7 breast cancer cells. Furthermore, it was shown that HRG stimulation leads to association of MTK1 with tyrosine phosphorylated HER3 in MCF-7 and T-47D breast cancer cells. The MTK1/HER3 association was dependent on HER2 activation and was decreased by pre-treatment with the HER2 inhibitor, lapatinib. Furthermore, HER2 does not directly associate with MTK1, but phosphorylates HER3 transiently. MTK1 also has a role in the ERK1/2 MAPK signaling pathway in response to heregulin (HRG) stimulation in T-47D and MCF-7 breast cancer cells. In addition to MTK1, Shc, Grb2 and GIT1 proteins are all involved in the ERK1/2 MAPK pathway in response to growth factor stimulation. MTK1 was also shown to associate with activated ERK1/2, GIT1, Shc, Grb2 and p85 of PI3K in response to heregulin stimulation. ERK1/2 kinase activity is involved in aberrant signaling that leads breast cancer progression. GIT1 is a scaffolding protein that is linked to growth factor mediated ERK1/2 signaling in cell migration. Moreover, we also identify the actin interacting region (AIR) on MTK1 and disruption of actin cytoskeletal polymerization with cytochalasin D inhibited the interaction between HER3 and MTK1, indicating that f-actin (which is needed for cell migration) is required for the MTK1/HER3 association. Additionally, HRG stimulation leads to extracellar acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.

Mental disorders, like anxiety and depression, are a major health problem and can appear early in life. Developing new compounds for anxiety and other mental disorders is desirable and requires good animal models. Two widely used tests to evaluate fear and anxious behavior in rodents are the open field (OF) and elevated plus maze (EPM) tests. The models are simple and it has been argued that different environments give different behavior profiles and measure different kind of behavior. The multivariate concentric square fieldTM (MCSF) is a new test that gives the rodent opportunity to visit different environments and to evaluate more parameters in one test. Validation of the tests is mainly done with adult animals. The aim with this study is to study adolescent rat behavior in the OF, OF with start box, EPM and MCSF tests and to see if there is any difference in running trials in the morning or in the afternoon. A total of 48 adolescent male Wistar rats were divided into 12 rats per group. The groups were OF and MCSF, OF with start box and MCSF, EPM and MCSF, and repeated testing in MCSF. Each session was 20 minutes and there was one week between the two tests. Three rats where run in the morning and three in the afternoon. Statistical analysis did show significant difference in some parameters in the comparison between running trials in the morning or in the afternoon. Repeated testing in the MCSF yielded differences in trial two compared to trial one. No significant difference was found in the trend analysis. The results show individual differences and with larger groups results may have been more liable. In this study some differences were found between morning and afternoon groups. Lots of data have been generated and there are many opportunities to use the same data for additional analyses.

Many blood cell mechanisms in the human body are working all the time to maintain haemostasis in the blood vessels. Once a wound arises platelets are alerted via different substances to cover the wound and prevent loss of blood. Most of the times these mechanisms do stop the blood, and further heal the wound. During other circumstances the platelet-covering continues to form a thrombus, preventing the blood to flow and instead causes myocardial infarction or stroke. There are several risk factors triggering development of circulatory diseases such as obesity, lack of exercise, smoking, infection and stress. This thesis describes the interaction between the two platelet thrombin receptors PAR1 and PAR4, together with the interaction of the oral pathogen Porphyromonas gingivalis (with thrombin-like gingipains), and the cross talk with the stress hormone epinephrine and its α2A adrenergic receptor. Until now PAR1 is thought to be the most important thrombin receptor due to its high affinity for thrombin. From a phylogenetical and patophysiological point of view there must be a reason why platelets express two different thrombin receptors. Today PAR4 is considered less important, but this thesis implies that PAR4 plays an important role in platelet signaling and haemostasis. The results show that bacteria pre-stimulated platelets, followed by epinephrine gives a strong and full aggregation and calcium mobilization, in both aspirinated and non-aspirinated human platelets. The amount of bacteria does not itself, or epinephrine alone give aggregation or calcium mobilization. This mechanism is dependent on both Rgp type gingipain released from P. gingivalis, and PARs in an interaction with the α2A adrenergic receptor. Further, results reveal that PAR4 interacts and cross talks with the platelet α2A-adrenergic receptor in aspirinated platelets. Neither of the two platelet purinergic P2Y-receptors (P2Y12 and P2Y1) contribute to this action, but the purinergic P2X1 does. In aggregation studies a low dose of PAR4 activating peptide (AP), but not PAR1-AP, followed by epinephrine results in a strong aggregation and in a calcium mobilization. ATP secretion measurements did reveal that ATP was released during epinephrine stimulation, which indicate that ATP and P2X1 have a key role in this event. By blocking P2X1 both aggregation and calcium mobilization were abolished, but not by blocking P2Y12 and P2Y1. Inhibition of PI3-kinase, both epinephrine-induced calcium mobilization and aggregation were significant reduced. In non-aspirinated platelets PAR1 synergizes with the α2A adrenergic receptor and P2X1. In conclusion, this thesis suggests that PAR4 plays an intriguing and important role in platelets with inactived cyclooxygenase 1.  The results described in this thesis contribute to an increased knowledge of the platelet thrombin receptors.

Depression är den näst mest kostsamma sjukdomen för samhället efter hjärt-kärlsjukdom, främst på grund av långa sjukskrivningsperioder. Sjukdomen kan uppstå när som helst från sex månaders ålder, men prevalensen ökar med åldern. Det finns ett antal stressrelaterade faktorer som skulle kunna leda till depression, så som stor sorg, verbala eller fysiska övergrepp samt en svår barndom. Vad som orsakar sjukdomen är ännu inte helt känt, men det finns teorier att halterna av serotonin och noradrenalin är lägre hos deprimerade personer. Behandling som används är olika former av samtalsterapi, men även läkemedel så som selektiva serotoninåterupptagshämmare (SSRI). Det finns teorier som sammankopplar användandet av SSRI med självmord, framförallt hos personer ≤19 år. Syftet med detta litteraturarbete var att undersöka om SSRI preparat har någon effekt på depression hos barn och ungdomar och om de är säkra eller kan få allvarliga konsekvenser så som självmord. Sökningar i PubMed gjordes för att hitta relevanta artiklar. Fem av de åtta inkluderade studierna rapporterade olika effekter och säkerhet hos olika SSRI preparat bland barn och ungdomar, jämfört med placebo. Två andra studier undersökte förekomsten av suicidalitet till följd av läkemedlen. Den sista studien jämförde toxikologiska data från Rättsmedicinalverket med receptregistret på antidepressiva läkemedel från  Socialstyrelsen. Endast två av de fem studerade preparaten (fluoxetin och citalopram) hade en bättre effekt än placebo i hela populationen och ytterligare ett (sertralin) hade bättre effekt hos ungdomar. Det begicks inga självmord i studierna.    De studier som har granskats i detta arbete tyder på att olika SSRI preparat har olika bra effekt samt olika säkerhetsprofiler. Det sågs inget tydligt samband mellan behandlingen och självmord, men en något förhöjd risk för suicidalitet.

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Botulinumtoxin: För och emot dess användning i skönhetsbranschen Bakgrund: Botulinumtoxin (BTX) är ett kraftfullt neurotoxin som produceras av den grampositiva och anaeroba bakterien Clostridium botulinum. BTX används i medicinskt syfte vid behandling av till exempel kronisk migrän, spasticitet efter stroke, och urinläckage vid nervskada. Utöver det används BTX inom skönhetsbranschens som då injiceras i mycket låga doser under huden i muskler för att minska rynkor i olika ansiktsregioner Syfte: Användningen och lämpligheten inom skönhetsbranschen har ifrågasatts , och syftet med detta arbeta har därför varit att utvärdera och ta ställning till om BTX fortsättningsvis bör användas i kosmetiskt syfte. Metod: Litteraturstudie där vetenskapliga artiklar söktes i databasen PubMed med kombinationer av sökord, artiklar som sedan kunde användas för att besvara den aktuella frågeställningen. Resultat: Olika studier har visats att BTX är ett effektivt och säkert toxin vid skönhetsbehandling, men flera studier visade också att BTX-relaterade biverkningar uppkom efter skönhetsbehandling. Slutsats: BTX bör inte fortsätta användas för skönhetsbehandling trots positiva effekter mot rynkor i ansiktet som visades i studierna. Injektion med BTX kan bidra till att kunder i skönhetssalonger blir svårbedömda patienter vid besök hos läkare i primärvården som inte förstår att de problem patienten söker för beror på BTX-inducerade biverkningar efter skönhetsbehandling med BTX.