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Melanoma is a highly aggressive tumour with a poor prognosis for patients with advanced disease because it is resistant to current therapies. Therefore, the development of novel strategies for melanoma treatment is important. The characterization of the molecular mechanisms underlying melanoma proliferation, progression, and survival could help the development of novel targeted melanoma treatments. The MAPK and PI3K pathways both play important roles in melanoma progression. In the MAPK pathway, DUSP6, which acts as a phosphatase to negatively control the activation of ERK1/2, is involved in the development of human cancers. The MAPK pathway also regulates expression of the DNA repair gene ERCC1 following EGF treatment. ERCC1 is essential for nucleotide excision repair, which is one of the major systems for removal of cisplatin induced DNA lesions. The aims of this project were: 1, to investigate the molecular changes in our immortal mouse melanocyte cell lines that were needed for them to form tumours in a xenograft model; 2, to investigate whether the MAPK pathway regulates ERCC1 following cisplatin treatment and protects melanoma cells from death. Through comparison of the RAS/RAF/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways between our immortal mouse melanocyte cell lines and their tumour derivatives in our xenograft model, we identified a molecularly distinct subtype of mouse melanoma characterized by reduced ERK and AKT activity and increased expression of DUSP6. Functional analyses employing ectopic overexpression indicated that increased expression of DUSP6 enhanced anchorage independent growth ability and invasive ability in our mouse melanocytes, suggesting that increased DUSP6 expression may contribute to melanoma formation in the xenograft assay. We also demonstrated that higher expression of p-ERK suppressed invasion, but not anchorage independent growth, in our subtype of mouse melanoma by enforced expression of constitutively active MEK1 and MEK2. In addition, the role of DUSP6 in classical human melanoma was investigated in this Genetic changes in melanoma progression study. Inhibition of anchorage independent growth and invasion were observed after exogenous expression of DUSP6 in human melanoma cells. This suggested that DUSP6 played different roles in classic human melanoma than in our distinct subtype of mouse melanoma. Our study also investigated the phosphorylation level of ERK1/2 and the mRNA and protein level of ERCC1 and its partner XPF in the human melanoma cell line following cisplatin treatment. Significant increases in expression of p-ERK, ERCC1 and XPF were found in cisplatin treated cells. Moreover, a MEK inhibitor inhibited ERCC1 induction by cisplatin, but did not significantly affect XPF induction. This suggested that the MAPK pathway was involved in regulation of ERCC1 but not XPF. Furthermore, the DUSP6 level decreased after cisplatin treatment and overexpression of DUSP6 inhibited ERCC1 and XPF induction and reduced resistance to cisplatin. DUSP6 seems to play a crucial role in resistance of melanoma to cisplatin. In addition, a novel larger ERCC1 transcript was identified in human cell lines and was found to be upregulated by cisplatin. The ratio of larger ERCC1 transcript relative to the normal ERCC1 transcript increased following cisplatin treatment. The functions of this larger ERCC1 transcript in cisplatin resistance deserve further study.

Titel: Vårdpersonalens erfarenheter av att använda vårdplanen Liverpool CarePathway (LCP) i palliativ vård - en litteraturstudieBakgrund: De senaste åren har det rapporterats mycket kring LCP, mestadelsnegativt. I några länder där vårdplanen LCP använts, ändras nu vårdplaner förpalliativ vård, däribland Sverige.Syfte: Denna litteraturstudie syftar till att beskriva vårdpersonalens erfarenheter avatt använda vårdplanen LCP.Metod: Åtta artiklar med kvalitativ ansats användes till litteraturstudien. Artiklarnasresultat lästes, analyserades och kategoriserades.Resultat: I litteraturstudiens resultat framkom två kategorier med sexunderkategorier: Möjligheter i användandet av LCP och Hinder i användandet avLCP.Konklusion: Det råder delade meningar om dokumentationen inom LCP.Vårdplanen upplevs fungera som ett bra stöd i palliativ vård, speciellt förnyutexaminerad vårdpersonal. Den förbättrade kommunikationen inom vårdteametbidrar till stärkt patientsäkerhet, medan bristande utbildning inom LCP kan leda tillatt patientsäkerheten inte kan garanteras.

gC1qR was originally discovered as a C1q receptor specific to the globular head domain of C1q, the first subcomponent of the classical pathway of complement activation. During the same period, calreticulin (CRT), formerly called as cC1qR, was described as a receptor for the collagen region of C1q and collectins. Although much work has been carried out with relation to CRT-CD91 complex, the biological implications and structure-function studies of C1q-gC1qR interaction has not been further explored. With passage of time since 1994, it has become evident that gC1qR is also a multi-functional pattern recognition receptor that can recognise pathogens in addition to acting as a modulator of inflammation at the site of injury or infection. In this thesis, a recombinant form of gC1qR using a T7 promotor expression system was expressed and examined for its interaction with individual globular head modules of C1q A, B and C chains (ghA, ghB and ghC, respectively). A number of single residue substitution mutants of ghA, ghB and ghC modules were also analysed for their interaction with gC1qR in order to map complementary binding sites. Concomitant expression of gC1qR and C1q in the adherent monocytes with, and without proinflammatory stimuli was analysed by qPCR in order to establish autocrine/paracrine basis of C1q-gC1qR interaction. In addition, experiments were carried out to examine if C1q-mediated anti-lymphoproliferative effect can be altered by gC1qR. Subsequently, using the wild type and mutants of ghA, ghB and ghC modules, the interaction of DC-SIGN and SIGN-R, a newly discovered partner of C1q and gC1qR on the dendritic cell surface, was examined. Experiments are underway to understand how a trimolecular complex involving C1q, gC1qR and DC-SIGN participate during HIV-1 infection. Structure-function studies involving gC1qR and HCV core protein and HIV-1 gp41 have also been carried out to localise domains of gC1qR responsible for viral pathogenesis. The last chapter dwells on a newly discovered ability of gC1qR to upregulate bradykinin 1 receptor on the endothelial cell surface, thus its role in altering vascular permeability and the contact system. The thesis describes (1) localisation of interacting sites between C1q and gC1qR and their togetherness in co-expression under pro-inflammatory conditions and possibly suppression of immune cell proliferative response; (2) localisation of complementary binding sites between DC-SIGN, gC1qR and C1q and its possible implications in HIV-1 infection and antigen presenting cells such as dendritic cells; (3) localisation of interacting sites between gC1qR and HCV core protein as well as HIV-1 gp41 peptides with potential to propose a therapeutic peptide; and (4) ability of gC1qR to upregulate bradykinin 1 and 2 receptors on endothelial cells and its newly identified function as a modifier of inflammation.

Titel: Vårdpersonalens erfarenheter av att använda vårdplanen Liverpool CarePathway (LCP) i palliativ vård - en litteraturstudieBakgrund: De senaste åren har det rapporterats mycket kring LCP, mestadelsnegativt. I några länder där vårdplanen LCP använts, ändras nu vårdplaner förpalliativ vård, däribland Sverige.Syfte: Denna litteraturstudie syftar till att beskriva vårdpersonalens erfarenheter avatt använda vårdplanen LCP.Metod: Åtta artiklar med kvalitativ ansats användes till litteraturstudien. Artiklarnasresultat lästes, analyserades och kategoriserades.Resultat: I litteraturstudiens resultat framkom två kategorier med sexunderkategorier: Möjligheter i användandet av LCP och Hinder i användandet avLCP.Konklusion: Det råder delade meningar om dokumentationen inom LCP.Vårdplanen upplevs fungera som ett bra stöd i palliativ vård, speciellt förnyutexaminerad vårdpersonal. Den förbättrade kommunikationen inom vårdteametbidrar till stärkt patientsäkerhet, medan bristande utbildning inom LCP kan leda tillatt patientsäkerheten inte kan garanteras.

Thesis advisor: Charles Hoffman / The study of mammalian cAMP signaling has often been confounded by the fact that ten different genes encode adenylyl cyclases (ACs) that produce cAMP from ATP and 16 different genes encode phosphodiesterases (PDEs) that hydrolyze cAMP to AMP. In this study, mammalian AC cDNAs were cloned and integrated into strains of the fission yeast Schizosaccharomyces pombe that lack their endogenous AC to determine the basal activity of all ten AC isoforms. In addition, response to the stimulatory mammalian Gsα was determined by co-expression of a mutationally-activated form of the human GNAS1 gene. AC activity was assessed using an fbp1-GFP reporter that is repressed by cAMP production and PKA activity. Results confirm that all ten isoforms have detectable basal activity, and AC1-9 definitively respond to Gsα stimulation. When matched with a sufficiently potent mammalian phosphodiesterase (PDE), strains expressing mammalian ACs make good candidates for small molecule high throughput screening (HTS) to detect AC inhibitors. A 100,000 compound screen was recently performed to detect AC and Gsα inhibitors as well as PDE activators. A promising “hit” was progesterone, which has been previously suggested to inhibit ACs in Xenopus. Initial results suggest that progesterone inhibits AC1 and the closely-related AC3. These data demonstrate the utility of using S. pombe as an effective platform for identifying inhibitors of both basal and GNAS1-stimulated AC activity. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Dysregulated Hippo pathway signaling promotes the onset of aggressive cancers through the induced nuclear activity of yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) (YAP/TAZ). Uncontrolled nuclear YAP/TAZ activity evokes tumor-initiating properties in a range of epithelial-derived cancers, including oral and breast cancers, but their downstream targets and mechanisms of action are unclear. Recent studies have suggested that the pro-tumorigenic roles for YAP/TAZ relate to their convergence with growth factor signaling pathways. Based on these previous studies, I hypothesized that YAP/TAZ driven transcription contributes to carcinoma progression, and that cooperation with transforming growth factor β (TGFβ)-induced signals promotes aggressive oncogenic traits. In this thesis I show that dysregulated YAP localization precedes oral squamous cell carcinoma (OSCC) development, and that nuclear YAP/TAZ activity drives cell proliferation, survival, and migration in vitro, and is required for tumor growth and metastasis in vivo. Global gene expression studies in OSCC cells revealed that YAP/TAZ-mediated gene expression correlates with expression changes that occur in human OSCCs identified by “The Cancer Genome Atlas” (TCGA), many of which encode cell cycle and survival regulators. By exploring the relationship with growth factor signaling, I found that YAP/TAZ induce pro-tumorigenic events by converging with TGFβ-induced signals, particularly in breast cancer cells where TGFβ is known to promote metastatic properties. My observations indicated that YAP/TAZ are necessary for maintaining and promoting TGFβ-induced tumorigenic phenotypes in breast cancer cells, and that these phenotypes result from the cooperative activity of YAP/TAZ, the TEA domain family of transcription factors (TEADs), and TGFβ-activated SMAD2/3 in the nucleus. Genome-wide expression analyses indicated that YAP/TAZ, TEADs, and TGFβ-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by these complexes, such as the novel targets neuronal growth regulator 1 (NEGR1) and urothelial cancer associated 1 (UCA1), are necessary to maintain tumorigenic activity in metastatic breast cancer cells. Nuclear YAP/TAZ also cooperate with TGFβ signaling to promote phenotypic and transcriptional changes in non-tumorigenic cells to overcome TGFβ-mediated growth inhibition. This work thus defines novel roles for YAP/TAZ in cancer, offering molecular mechanisms that may be useful for identifying and targeting YAP/TAZ-driven cancers.

Peroxisomes represent crucial subcellular compartments for human life and health. They are remarkably dynamic organelles which respond to stimulation by adapting their structure, abundance, and metabolic functions according to cellular needs. Peroxisomes can form from pre-existing organelles by membrane growth and division, which results in peroxisome multiplication/proliferation. Growth and division in mammalian cells follows a well-defined multi-step process of morphological alterations including elongation/remodeling of the peroxisomal membrane (by PEX11β), constriction and recruitment of division factors (e.g. Fis1, MFF), and final membrane scission (by the dynamin-related GTPase Drp1) (Chapter 1). Although our understanding of the mechanisms by which peroxisomes proliferate is increasing, our knowledge on how the division/multiplication process is linked to extracellular signals is limited, in particular in humans. The classical pathway involved in peroxisome proliferation is mediated by a family of ligand-activated transcription factors known as peroxisome proliferator activated receptors (PPARs) (Chapter 1). This project focused on identifying novel signaling pathways and associated factors involved in peroxisome proliferation in humans. In this study, a cell-based peroxisome proliferation assay using the HepG2 cell model with spherical peroxisomal forms has been developed to investigate different stimuli and their ability to induce peroxisome proliferation (Chapters 2 and 3). In this system, peroxisome elongation has been used as the read-out for peroxisome 4 proliferation. We also showed that the number of peroxisomes increased after division of elongated peroxisomes indicating peroxisome proliferation. Different stimuli, such as fatty acids, PPAR agonists and antagonists, have been used in this study. PPAR agonists and antagonists had no stimulatory or inhibitory effect on peroxisome elongation in our assay, suggesting PPAR-independent regulatory processes. However, arachidonic acid and linoleic acid were able to induce peroxisome elongation, whereas palmitic acid and oleic acid were not effective. These findings indicate that general stimulation of fatty acid β-oxidation is not sufficient to induce peroxisome elongation/proliferation in HepG2 cells. Moreover, mRNA expression levels of peroxismal genes have been monitored during a time course in the HepG2 cell-based assay by qPCR. This analysis shows a regulation of expression of peroxins during peroxisome proliferation in human cells and suggests differences in the regulation pattern of PEX11α and PEX11β. In Chapter 4, motif binding sites for transcription factors in peroxisomal genes were analyzed. An initial map of candidate regulatory motif sites across the human peroxisomal genes has been developed (Secondment at the University of Sevilla, Spain with Prof. D. Devos). This analysis also revealed the presence of different transcription factor binding sites in the promoter regions of PEX11α and PEX11β, supporting different regulatory mechanisms. Based on the computational analysis, PEX11β contained a putative SMAD2/3 binding site suggesting a novel link between the canonical TGFβ signaling pathway and expression of PEX11β, a key regulator of peroxisome dynamics and proliferation. 5 Addition of TGFβ to HepG2 cells cultured under serum-free conditions induced elongation/growth of peroxisomes as well as peroxisome proliferation supporting a role for TGFβ signalling in peroxisomal growth and division (Chapter 5). Furthermore, to demonstrate that this induction is through a direct effect of TFGβ on the SMAD binding site found in PEX11β, we performed functional studies using a dual luciferase reporter assay with PEX11β wild type and mutated promoter regions (Secondment at Amsterdam Medical Center, Netherlands with Prof. H. Waterham). Whereas luciferase activity was induced by TGFβ stimulation with the PEX11β wild type promoter, mutation of the SMAD binding site abolished activation. In summary, this study revealed a new signaling pathway involved in peroxisome proliferation in humans and provided a tool to monitor peroxisome morphology and gene expression upon treatment with defined stimuli. Furthermore, I contributed to a study revealing that ER-peroxisome contacts are important for peroxisome elongation (Chapter 6). Our group identified peroxisomal acyl-CoA binding domain protein 5 (ACBD5), ACBD4 and VABP as a molecular linker between peroxisomes and the ER (Costello et al., 2017). Motif analysis of ACBD4 and ACBD5 promoter regions revealed that unlike PEX11β, these genes do not contain a binding site for SMAD, suggesting they are not co-regulated. Also, ACBD4 and ACBD5 do not share any common transcription factor binding sites suggesting different regulation. An interesting binding motif within the ACBD4 promoter is a glucocorticoid receptor binding site. In our study, we found potential glucocorticoid response elements (GRE) in other peroxisomal genes encoding β-oxidation enzymes. This may suggest an important role for glucocorticoid receptors in activating expression of peroxisomal genes resulting in the stimulation of fatty acid breakdown and energy production.