The Rap1 Guanine Nucleotide Exchange Factor C3G Is Required for Preservation of Larval Muscle Integrity in Drosophila melanogaster

C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, we have observed that loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize βPS integrins to muscle attachment sites. Moreover, we show that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, we conclude that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages

Deciphering the Alk signaling pathway in Drosophila

In Drosophila melanogaster the visceral mesoderm (VM) develops during embryogenesis in a process where myoblasts become specified to generate two distinct cell types, the founder cells (FCs) and the fusion competent myoblasts (FCMs) that consequently fuses. The cell specification is dependent on cell signaling mediated by the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb), how this further sets up different identity programs that drive myoblasts to differentiate into FCs and FCMs is still not well understood. We have analysed whether the Midkine (MDK)/Pleiotrophin (PTN) homologues in Drosophila, Miple1 and Miple2 activate the Alk RTK in vivo. Earlier results from cell culture experiments suggested that vertebrate MDK/PTN is capable of activating ALK, findings that have become controversial with other studies showing contradictory results. We wanted to use Drosophila that have conserved homologues of both MDK/PTN and ALK, to address the question in vivo. We analysed the contribution of Miple in Alk dependent developmental processes such as visceral mesoderm (VM) specification during embryogenesis and in body size regulation of adult flies. Specification of VM as well as body size are not effected by loss of Miple proteins, and over expression of Miple proteins do not effect VM specification or body size. All together we conclude that there is no evidence that Miple1 or Miple2 can activate Alk in vivo. We found that loss of Miple protein effect the median lifespan of the fly which is reduced, interestingly the over expression of Miple proteins can promote an increased median life span in Drosophila. We have also analysed how Alk RTK signaling regulates the Gli-like transcription factor Lame duck (Lmd) in vivo on a post-translational level. It has already been reported that Lmd plays an essential role in specification of FCMs in the somatic mesoderm during embryogenesis. We detect Lmd protein exclusively in FCMs of VM in control embryos, but in Alk mutants Lmd protein is present in all cells of VM and opposite to this when Alk is activated in all cells in VM by over expression of Jeb this results in total loss of Lmd protein. This suggests that Alk signaling is regulating Lmd, and we additionally show that Lmd persist in FCMs in mutants where VM is specified but where myoblast fusion do not occur, supporting that Alk activity in FCs is regulating the downregulation of Lmd in FCMs upon fusion. Finally we have characterised the Rap1GEF C3G in vivo in Drosophila. In cell culture systems, the GTPase Rap1 has been identified to mediate Alk signaling and that this is regulated by the GEF C3G and interestingly the Drosophila C3G is expressed in the FCs of VM. We generated deletion mutants of C3G which exhibit semi-lethality and reduced life span, but no defects in visceral mesoderm development during embryogenesis. Instead we detected distinct phenotypes in somatic muscles of 3rd instar mutant larvae, with detachment and mistargeting of muscles, which effect localisation of integrins. We suggest that Drosophila C3G regulates Rap1 via inside out signaling of integrins which in turn effects cell adhesion in vivo in Drosophila larval muscles

Online data privacy as a driving factor for customer loyalty : A qualitative study

In recent years, there has been an increase in the focus on online data privacy by companies worldwide. Data has become an increasingly valuable resource for companies to use in their marketing efforts. As the company use of data increases, more and more online users are becoming aware of problems with online data privacy and the integrity of their data. The proper balance between protecting the integrity of the users and the value of data collection is difficult to predict, as seen with recent scandals where tech giants have been accused of abusing their user’s data in such a way that it violates their personal integrity. A company that has seen media attention for their battle for online data privacy is Apple, where they have put an emphasis on the data privacy in their marketing. But how does this new frontier of privacy affect the loyalty of customers? The purpose of this thesis is to explore how a focus on online data privacy is a driving factor in customer. The study was conducted using an exploratory approach to better understand the connection between online data privacy and customer loyalty, and to examine its relationship. The research was conducted through qualitative semi-structured interviews where the respondents were Swedish university students aged between 20 – 29 years old who owned at least one Apple product. The answers were then compared and analysed using a theoretical framework based on existing online data privacy and customer loyalty literature. As a conclusion of the thesis, the empirical data suggests that a focus on online data privacy is not a driving factor for customer loyalty, and that consumers are unaware of the focus to protect their privacy online. Customers do not seem to view online data privacy as a field of importance to them when making a purchase decision, their loyalty may instead be influenced more by other criteria

The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo

A novel in vivo system to study coral biomineralization in the starlet sea anemone, Nematostella vectensis

Summary: Coral conservation requires a mechanistic understanding of how environmental stresses disrupt biomineralization, but progress has been slow, primarily because corals are not easily amenable to laboratory research. Here, we highlight how the starlet sea anemone, Nematostella vectensis, can serve as a model to interrogate the cellular mechanisms of coral biomineralization. We have developed transgenic constructs using biomineralizing genes that can be injected into Nematostella zygotes and designed such that translated proteins may be purified for physicochemical characterization. Using fluorescent tags, we confirm the ectopic expression of the coral biomineralizing protein, SpCARP1, in Nematostella. We demonstrate via calcein staining that SpCARP1 concentrates calcium ions in Nematostella, likely initiating the formation of mineral precursors, consistent with its suspected role in corals. These results lay a fundamental groundwork for establishing Nematostella as an in vivo system to explore the evolutionary and cellular mechanisms of coral biomineralization, improve coral conservation efforts, and even develop novel biomaterials

Association of surgeon and hospital volume with short-term outcomes after robot-assisted radical prostatectomy : Nationwide, population-based study

Background and objective: Few studies have investigated the association between surgical volume and outcome of robot-assisted radical prostatectomy (RARP) in an unselected cohort. We sought to investigate the association between surgical volume with peri-operative and short-term outcomes in a nation-wide, population-based study group. Methods: 9,810 RARP's registered in the National Prostate Cancer Register of Sweden (2015-2018) were included. Associations between outcome and volume were analyzed with multivariable logistic regression including age, PSA-density, number of positive biopsy cores, cT stage, Gleason score, and extent of lymph node dissection. Results: Surgeons and hospitals in the highest volume group compared to lowest group had shorter operative time; surgeon (OR 9.20, 95% CI 7.11-11.91), hospital (OR 2.16, 95% CI 1.53-3.06), less blood loss; surgeon (OR 2.58. 95% CI 2.07-3.21) hospital (no difference), more often nerve sparing intention; surgeon (OR 2.89, 95% CI 2.34-3.57), hospital (OR 2.02, 95% CI 1.66-2.44), negative margins; surgeon (OR 1.90, 95% CI 1.54-2.35), hospital (OR 1.28, 95% CI 1.07-1.53). There was wide range in outcome between hospitals and surgeons with similar volume that remained after adjustment. Conclusions: High surgeon and hospital volume were associated with better outcomes. The range in outcome was wide in all volume groups, which indicates that factors besides volume are of importance. Registration of surgical performance is essential for quality control and improvement

Combined expression of the human Miple homologues (MDK and PTN) and human ALK does not affect eye morphology in <i>Drosophila</i>.

<p>(A-H) Ectopic expression of human wildtype ALK (A) in developing eye using <i>GMR-Gal4</i> does not affect eye morphology as no functional ligand is present in the fly. Ectopic expression of human MDK and PTN in the developing eye, alone (G, H) and in combination with wildtype human ALK (B, C), using <i>GMR-Gal4</i> does not affect adult eye morphology. Neither MDK (E) nor PTN (F) modify the eye phenotype caused by expressing a constitutively active human ALK (hALK F1174L gain-of-function mutation observed in human neuroblastoma) (D).</p