Platelet-derived growth factor (PDGF) isoforms, which bind to closely related a- and b-tyrosine kinase receptors, induce migration, proliferation, survival and differentiation of mesenchymal cells. They signal by the active receptor attracting Src homology 2 (SH2) domain containing proteins, which subsequently initiate a set of signaling pathways. The aim of this thesis was to elucidate regulatory mechanisms involved in PDGFRb signaling. In the first two projects we investigated the roles in downregulation of PDGFRb of two related adaptor proteins, i.e. ALG-2 interacting protein X (Alix) and His-domain containing protein tyrosine phosphatase (HD-PTP) functions of. We found that Alix and HD-PTP influence ubiquitination of PDGFRb following PDGF stimulation, by affecting the E3 ligase c-Cbl. Alix enhances complex formation between c-Cbl and PDGFRb, increases c-Cbl phosphorylation and decreases its stability. Interestingly, while both HD-PTP and Alix participate in degradation of PDGFRb, only Alix affects receptor internalization. Moreover, we demonstrated that absence of HD-PTP promotes cell proliferation. In conclusion, we suggest that both Alix and HD-PTP are important adaptor proteins in regulation of PDGFRb downregulation, although the observed differences between their actions suggest that Alix and HD-PTP exert their functions via different mechanisms. The third study explored the importance of tyrosine residue 857 in the activation loop of PDGFRb. We report that, in vitro the tyrosine residue 857 to phenylalanine (Y857F) mutant receptor kinase activity is diminished while in vivo it does not affect the phosphorylation of PDGFRb. The phosphorylation pattern of PDGFRb revealed that most sites in the Y857F mutant receptor were phosphorylated similarly as in the wild-type receptor. However, tyrosine residue 771 was found to be hyperphosphorylated in the Y857F mutant receptor. This may be due to defective phosphorylation and activation of SHP-2, since it has been shown to dephosphorylate the receptor at Y771. In addition, activation of the Erk1/2 and Akt pathways was defective downstream of the Y857F mutant receptor. Interestingly, the Y857F mutant receptor was able to mediate cell migration, but not proliferation. The last study investigated a role of the tyrosine kinase Fer in PDGF signaling. We showed that Fer interacted with and was activated by PDGFRb in a ligand-dependent manner. In cells depleted of Fer, receptor phosphorylation was decreased and phosphorylation of Stat3 was abolished, whereas Stat5, Erk1/2 and Akt were activated normally. Colony formation in soft agar was abolished in cells depleted of Fer, but no effect was seen on cell proliferation and migration. Since Stat3 has been shown to be involved in transformation, we speculate that phosphorylation of Stat3 in Fer-depleted cells, affects the ability of cells to form colonies.

The topic of my diploma thesis is the statistical evaluation of biological sequences with the help of phylogenic trees. In the theoretical part we will create a literary recherche of estimation methodology concerning the course of phylogeny on the basis of the similarity of biological sequences (DNA and proteins) and we will focus on the inaccuracies of the estimation, their causes and the possibilities of their elimination. Afterwards, we will compare the methods for the statistical evaluation of the correctness of the course of phylogeny. In the practical part of the thesis we will suggest algorithms that will be used for testing the correctness of the phylogenic trees on the basis of bootstrapping, jackknifing, OTU jackknifing and PTP test which are able to the capture phylogenic tree with the method neighbor joining from the biological sequences in FASTA code. It is also possible to change the distance model and the substitution matrix. To be able to use these algorithms for the statistical support of phylogenic trees we have to verify their right function. This verification will be evaluated on the theoretical sequences of the amino acids. For the verification of the correct function of the algorithms, we will carry out single statistical tests on real 10 sequences of mammalian ubiquitin. These results will be analysed and appropriately discussed.

The master's thesis deals with design of a solution for time synchronization in computer networks that is a crucial problem of many network applications. Based on analysis of protocols for time synchronization, PTP protocol was chosen as an appropriate candidate. The thesis describes the implementation of the design for a special network interface card and demonstrates features of the solution in several tests. A part of the solution processing precise timestamps was implemented in FPGA chip on the network card while PTP messages are processed in a software application. Values of configurable parameters of the application were determined based on analysis of the network card properties and results of particular tests. It was achieved accuracy in order of tens of nanoseconds.

All of the members of the microsporidia possess a unique, highly specialized invasion mechanism that involves the polar tube and spore wall. This chapter reviews the data on the organization, structure, and function of this invasion organelle. The application of immunological and molecular techniques and recent genome sequencing data has resulted in the identification of multiple polar tube and spore wall proteins (SWPs). The interactions of these identified proteins in the formation and function of the polar tube and spore wall remain to be determined. Inside the spore, the polar tube is filled with material and is often termed the polar filament; however, this chapter uses the term polar tube to refer to this structure when it is within the spore as well as when it forms a hollow tube after germination and is found outside the spore. The chapter presents details on the spore activation and discharge.

Although combination inhaled corticosteroids (ICs) are known to cause voice disorders in otherwise vocally healthy individuals, it is unknown whether those adverse effects can be reversed by the cessation of treatment. Quantitative aerodynamic measures such as phonation threshold pressure (PTP) and phonation threshold flow (PTF) can be used to identify the development of, and recovery from, vocal pathologies. We examined the effects and reversibility of ICs on laryngeal aerodynamics. This study was a mid-project investigation as part of a larger ongoing project. The 18 larynges were from rabbits that received ICs, a control condition, or no treatment. Experimental group rabbits received ICs twice per day until inflammatory changes (e.g., erythema, edema) became visible through endoscopic observation. One experimental group (i.e., the induction group) received treatment until symptoms were observed and then larynges were harvested. The other experimental group (i.e., the reversibility group) had ICs withdrawn once visible changes were detected via endoscopy; larynges were harvested only after these changes no longer differed from baseline. Both experimental groups had corresponding control rabbits that received twice-daily nebulized saline and followed the same withdrawal and larynx harvest schedule. A final group received no treatment. During benchtop phonation trials, PTP and PTF values were determined. All data were analyzed using descriptive and parametric statistics. No significant between-group differences were observed. Descriptively, however, average PTP and PTF values for the reversibility group were lower than the induction group. Additionally, average PTP and PTF values for the reversibility group were slightly lower than the induction group. Both experimental groups had higher PTP and PTF values than the control larynges. The results of this study indicate a trend in recovery for larynges afforded a recovery period from ICs. Further testing is needed to substantiate these preliminary findings.

The existence of distributed real-time systems calls for protocols for high accuracy time synchronization between devices. One such protocol, the Precision Time Protocol (PTP) reaches sub microsecond synchronization precision. PTP can be implemented both in hardware and software. This study aimed to analyze how system stress could affect the accuracy and precision of software implemented PTP between two devices. This was done using two Intel Galileo Generation 2 running Linux systems. Software was used to simulate CPU, I/O, network, and OS usage. Data was extracted from software logs and summarized in charts and then analyzed. The results showed that PTP synchronization accuracy and precision does suffer under certain types of system load, most notably under heavy I/O load. However the results might not be applicable to real-world scenario due to limitations in hardware and the synthetic stress tests do not correspond to real-world usage. Further research is required to analyze why and how different types of system load affects PTPs accuracy and precision. / Förekomsten av distribuerade realtidssystem kräver protokoll för noggrann tidssynkronisering mellan enheter. Ett sådant protokoll, Precision Time Protocol (PTP), kan uppnå en precision på under mikrosekunden under synkronisering. PTP kan implementeras i både hårdvara och mjukvara. Den här rapporten fokuserar på att analysera hur systembelastning kan påverka precision och noggrannheten hos mjukvaruimplementerad PTP mellan två enheter. Testen utfördes på två stycken Intel Galileo Generation 2 kö- randes Linux. Mjukvara användes sedan för att simulera belastning på olika system såsom CPU, I/O, nätverk och på operativsystemet. Data extraherades ifrån loggar från mjukvaran, vilken sammanfattades i grafer för att sedan analyseras. Resultaten visade att precisionen och noggrannheten hos PTP försämras under vissa typer av systembelastningar, mest märkbart under tung I/O belastning. Resultaten är dock potentiellt inte applicerbara på verklighetscenarion på grund av begränsingar i hårdvaran samt att syntetiska stresstest inte motsvarar normal belastning. Ytterligare forskning krävs för att analysera hur och varför olika typer av systembelastning påverkar PTPs precision och noggrannhet.

La protéine tyrosine phosphatase 1B (PTP 1B) et la glycogène phosphorylase (GP) sont des nouveaux cibles thérapeutiques pour le traitement du diabète (type II) et l'obésité. Dans l'objective de trouver des inhibiteurs actives et sélectives de ces enzymes, des aryl C glycosides tels que les C glycosyl 1,4 naphthaquinones, les 6 O benzoyl C glycosyl benzoquinone /naphthaquinones, les dérivés quinone d'acides glycuroniques et d'amides carboxylés ainsi que les inhibiteurs bidentates C glycosyl quinones ont été conçus et synthétisés. En tout, 178 composés ont été préparés. Le mécanisme et les facteurs d'influence de la réaction de C aryl glycosylation ont également été étudiés. Les essais préliminaires montrent que certaines molécules présentent des activités biologiques très intéressantes. Des études biologiques plus approfondies seront réalisées par la suite.

Studien syftade till att undersöka hur psykologer upplever skapandet av sin profession under den praktiska tjänstgöringsperioden. Vi använde oss av grundad teori som kvalitativ metod för datainsamling och databearbetning. Vi intervjuade 14 psykologer och fann ett antal centrala teman som var återkommande i intervjuberättelserna. Dessa handlade om hur sociala interaktioner med omgivningen påverkade den egna processen i att bli psykolog. Datamaterialet genererade en teori som visar hur psykologen under PTP-året är en del i ett möte och interaktion med organisation, erfarenheter, utveckling och den egna individen. Dessutom framkom en processmodell som påvisade fem processer som samverkar under PTP-året. Dessa processer är reflektionsprocess, känsloprocess, yrkesrollprocess, lärandeprocess samt yrkesidentitetsprocess. Slutligen använde vi oss av respondentvalidering för att kvalitetssäkra de upptäckta fynden. / The aim of this study was to investigate how psychologists experience the creation of their profession during the first year of practice. We used “Grounded Theory” as research method. We interviewed 14 psychologists and found some central themes which were frequently described. The material from the interviews let us know that the social interaction with others had an effect on their own processes in becoming a psychologist. The material generated a theory that shows how the psychologist, during the first year of practice, is part in a meeting and an interaction with organization, experiences, professional development and the individual person. We also exposed a process model that revealed five processes that interact during the first year of practice. These processes were a reflection process, an emotional process, a professional role process, a learning process and a professional identity process. Finally, we used the quality assessment method of respondent validation to confirm the findings discovered.

Protein Tyrosine Phosphatases (PTPs) initiate, modulate and terminate key cellular processes by dephosphorylating phosphotyrosine (pY) residues on signaling proteins. The coordinated action of PTPs with their cognate tyrosine kinases is crucial for the maintenance of cellular homeostasis. Five Receptor Tyrosine Phosphatases (RPTPs) DLAR, PTP99A, PTP69D,PTP10D and PTP52F are involved in the axon guidance process of the fruit-fly Drosophila melanogaster. The receptors in these RPTPs comprise of Cell Adhesion Molecules (CAMs) whilethe cytosolic region contains the catalytic PTP domains. Extensive studies on the genetic interactions between these RPTPs reveal that these five RPTPs collaborate, compete or are partially redundant in some developmental contexts. While the genetic interactions between these RPTPs are well characterized, the role of domain-domain interactions and the mechanism(s) of substrate recognition are poorly understood. The aim of this study was to understand the molecular basis for these interactions using a combination of biophysical, biochemical and structural biology tools. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis highlights the mechanistic issues in signal transduction with an emphasis on the role of the RPTPs in the neuro-development of Drosophila melanogaster. The first part of this chapter describes the structural features and the catalytic mechanism of the PTP domain. This is followed by a description of the mechanisms that modulate the activity of a PTP domain. The latter part of the chapter summarizes the role ofthese RPTPs in axon guidance of Drosophila melanogaster. The interactions between the RPTPsbased on genetic data provide a mechanistic hypothesis that could be examined in vitro. The studies described in the subsequent chapters of this thesis were performed to evaluate this hypothesis. Chapter 2: This chapter reports our observations on the so-called construct dependence on the expression of recombinant PTP domains in Escherichia coli. This chapter details the strategies used to obtain recombinant PTP domains in a soluble form suitable for biochemical and structural studies. This study involved substantial optimization in the size of the protein and overexpression strategies to avoid inclusion-body formation. Five strains of E. coli as well as three variations in purification tags viz., poly-histidine peptide attachments at the N-and C-termini and a construct with Glutathione-S-transferase at the N-terminus were examined. In this study, we observed that inclusion of a 45 residue stretch at the N-terminus was crucial for the over-expression of the PTP domains, influencing both the solubility and the stability of these recombinant proteins. While the addition of negatively charged residues in the N-terminal extension could partially rationalize the improvement in the solubility of these constructs, conventional parameters like the proportion of order-promoting residues or the aliphatic index did not correlate with the improved biochemical characteristics. The findings in this chapter suggest that the inclusion of additional parameters like secondary structure propensities apart from rigid domain predictions could play a crucial role in obtaining a soluble recombinant protein upon expression in E. coli. Chapter 3: This chapter reports the crystal structure of the PTP domain of PTP10D and PTP10Dsubstrate/inhibitor complexes. These structural studies revealed aromatic ring stacking interactions that mediate substrate recruitment into the PTP active site. In particular, these studies revealed the role of conserved aromatic residue in Motif 1 (Phenylalaline 76 in case ofPTP10D). Mutation of Phenylalanine 76 residue to a Leucine (similar to the mutation found in the inactive distal PTP domains in other bi-domain PTPs) resulted in a sixty-fold decrease in the catalytic efficiency of the enzyme. Fluorescence kinetic measurements to monitor ligand binding showed a three fold increase in the half time of enzyme-ligand complex formation. These studies highlight the role of the KNRY loop in substrate recruitment at the active of the PTP domain and the role of this segment in modulating the kinetics of the enzyme-substrate complex formation. Chapter 4: This chapter describes a strategy to utilize protein-protein interaction data to identify putative peptide substrates for a given protein. This study was performed in collaboration with Shameer Khader and Prof. R. Sowdhamini at the National Center for Biological Sciences (NCBS).This integrated search approach, called ‘PeptideMine’ was developed into a web-server for experimental and computational biologists. The Peptide Mine strategy combines sequence searches in the 'interacting sequence space' of a protein using sequence patterns or functional motifs. A compilation of indices that describe the chemical and solubility properties of potential peptide substrates to facilitate investigation by in vitro or in silico studies is also obtained from this server. The biological significance of such a design-strategy was examined in the context of protein-peptide interactions in the case of RPTPs of Drosophila melanogaster. Chapter 5: In this chapter, we report an analysis of the influence of the membrane distal (D2) domain on the catalytic activity and substrate specificity of the membrane proximal (D1) domain using two bi-domain RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of the Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A revealed a conformational rationale for the experimental observations. These studies suggested that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain. Chapter 6: This chapter describes biochemical studies to understand the role of the D2 domain of PTP99A. While the catalytic activity of PTP99A is localized to its membrane proximal (D1)domain, the inactive membrane distal (D2) domain influences the catalytic activity of the D1domain. Phosphatase activity, monitored using small molecule as well as peptide substrates, suggested that the D2 domain activates D1. Thermodynamic measurements on the bi-domain(D1-D2 protein) as well as single domain PTP99A protein constructs suggest that the presence of the inactive D2 domain influences the stability of the bi-domain protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by a few interactions at the inter-domain interface. In particular, we note that mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent position to the so-called allosteric site of a canonical PTP, PTP1B. These observations suggest functional optimization in bi-domain RPTPs wherein the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme. Chapter 7: This chapter summarizes the experimental and computational studies on the Drosophila melanogaster PTP domains. The salient features of the experimental data that revealed hitherto uncharacterized sequence-structure relationships in the conserved PTP domain are highlighted. The latter part of this chapter briefly suggests the scope of future research in this area based on some of the findings reported in this thesis. Appendix : This thesis has an appendix section with four parts. These comprise of technical details and auxiliary work that was not included in the main text of the thesis. Appendix I describes cloning strategies, purification protocols and a list of all recombinant proteins used in this study. Appendix II describes the standardization of the ‘Three Phase partitioning’ protocol for refolding and solubilization of protein from inclusion bodies. Appendix III includes theimmunochemical work performed to elucidate the localization of PTP10D in Drosophila embryos. Appendix IV describes the work on a Quercetin 2,3 Dioxygenase from Bacillus subtilis with an emphasis on the role of metal ions in modulating catalytic activity in this class of proteins.

Tidigare forskning har visat på att övergången från studier till yrkesliv är en kritisk period där övergångschock kan förekomma. Detta har visat sig gälla även psykologer tidigt i karriären. Det saknas däremot en djupare förståelse kring övergången hos PTP-psykologer i Sverige. Denna studie syftade till att undersöka psykologers berättelser om övergången från studier till första tiden inom professionen och vad som karaktäriserar dessa berättelser. De 15 deltagarna, en man och 14 kvinnor, rekryterades via PTP-samordnare/-rektorer, sociala medier och privata kontakter. Studien genomfördes med en textbaserad datainsamling där materialet, som bestod av livshändelsekalender (LHC) och guidande frågor, skickades via post. Rådata analyserades med en narrativ analysmetod, inspirerad av Labov och Waletzkys indelning av berättelsers delelement. Därefter skapades prototypberättelser som är ett verktyg för att skapa sammanfattningar som blir jämförbara med det fenomen som undersöks. Slutligen skapades fem prototypberättelser utifrån gemensamma kontextuella omständigheter samt vad övergången i övrigt präglades av. Däribland psykiska ohälsa, sociala förändringar och tidigare erfarenhet av yrket. Prototypberättelserna kallas “Arbetslivets labyrint,” “En uppförsbacke,” “Den inre resan,” “Rustad och redo” och “Det sociala skiftet.” Analysen visar på att övergångschock även gäller psykologer i Sverige samt hur den påverkas av organisatoriska omständigheter och bristande eller fungerande socialt stöd. Sammanfattningsvis tycks förklaringen till motgångar flyttas från en själv till kontexten och omständigheterna. Hos samtliga deltagare har övergångschocken avtagit och i de flesta fall gått över, vilket tyder på att psykologers övergång från studier till yrkesliv är en kritisk men avgränsad period. / Previous research has shown that transitioning from studies to working life is a critical period, where the concept of transition-shock can appear. This can also be applied to early-career psychologists. However, there is a need for deeper understanding of the transition for psychologists in training in Sweden. The aim of this study was therefore to investigate psychologists’ stories about the transition from studies to the first years in practice and what characterized these stories. The 15 participants, one man and 14 women, were recruited via PTP-coordinators/-principals, social media platforms and private contacts. The study was conducted by a text-based data collection, consisting of a life history calendar (LHC) and guiding questions, which were sent by post. The data was analyzed with a narrative method, inspired by Labov and Waletzkys’ research on the narratives’ different components. Thereafter prototype stories were made, which is a tool to make the studied phenomenon comparable. Five prototype stories were then created from shared contexts and other circumstances, such as mental health, social changes and work-experience. These are called “The work-life labyrinth,” “The uphill,” “The inner journey,” “Equipped and ready” and “The social shift.” The analysis indicate that transition-shock applies to psychologists in Sweden with the influence of organizational contexts and social support. In summary, the explanation for the setbacks are switched from the individual to the context and circumstances. For all participants, the transition-shock has decreased or passed over, which indicates that psychologists’ transition from studies to working life is a critical yet limited period.