Implementation of Signaling Gateway between Telephone Network and the Internet

Tässä diplomityössä perehdytään verkkoelementiltä, joka yhdistää H.323, MEGACO- ja ISUP-protokollia käyttävät tietoliikenneverkot toisiinsa, vaadittaviin ominaisuuksiin ja toiminnallisuuksiin. Tyypillisesti tällaista toiminnallisuutta tarvitaan IP- ja PSTN-verkkojen yhdistämisessä. Tarkastelu aloitetaan kuvaamalla PSTN-verkon signalointi ja rakenne, jatketaan kuvaamalla internet-protokollia käyttävä verkkoympäristö ja lopuksi perehdytään verkkoelementiltä vaadittaviin toiminnallisuuksiin, jotta PSTN ja MEGACO-pohjaiset verkot toimivat yhteen. Työn käytännöllisenä osuutena kuvataan osa viestisekvenssikaavioista, joita verkkoelementti toteuttaa puuttumatta kuitenkaan eri protokollien toimintaan viestien parametrien tasolla.

Design and Analysis of Forward Error Control Coding and Signaling for Guaranteeing QoS in Wireless Broadcast Systems

Broadcasting systems are networks where the transmission is received by several terminals. Generally broadcast receivers are passive devices in the network, meaning that they do not interact with the transmitter. Providing a certain Quality of Service (QoS) for the receivers in heterogeneous reception environment with no feedback is not an easy task. Forward error control coding can be used for protection against transmission errors to enhance the QoS for broadcast services. For good performance in terrestrial wireless networks, diversity should be utilized. The diversity is utilized by application of interleaving together with the forward error correction codes. In this dissertation the design and analysis of forward error control and control signalling for providing QoS in wireless broadcasting systems are studied. Control signaling is used in broadcasting networks to give the receiver necessary information on how to connect to the network itself and how to receive the services that are being transmitted. Usually control signalling is considered to be transmitted through a dedicated path in the systems. Therefore, the relationship of the signaling and service data paths should be considered early in the design phase. Modeling and simulations are used in the case studies of this dissertation to study this relationship. This dissertation begins with a survey on the broadcasting environment and mechanisms for providing QoS therein. Then case studies present analysis and design of such mechanisms in real systems. The mechanisms for providing QoS considering signaling and service data paths and their relationship at the DVB-H link layer are analyzed as the first case study. In particular the performance of different service data decoding mechanisms and optimal signaling transmission parameter selection are presented. The second case study investigates the design of signaling and service data paths for the more modern DVB-T2 physical layer. Furthermore, by comparing the performances of the signaling and service data paths by simulations, configuration guidelines for the DVB-T2 physical layer signaling are given. The presented guidelines can prove useful when configuring DVB-T2 transmission networks. Finally, recommendations for the design of data and signalling paths are given based on findings from the case studies. The requirements for the signaling design should be derived from the requirements for the main services. Generally, these requirements for signaling should be more demanding as the signaling is the enabler for service reception.

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.

Functional Study of Oncogenic Transcription Factor ERG and its Signaling in Prostate Cancer

TMPRSS2–ERG is the most frequent type of genomic rearrangement present in prostate tumors, in which the 5- prime region of the TMPRSS2 gene is fused to the ERG oncogene. TMPRSS2, containing androgen response elements (AREs), is regulated by androgens in the prostate. The truncated TMPRSS2-ERG fusion transcript is overexpressed in half of the prostate cancer patients. The formation of TMPRSS2-ERG transcript is an early event in prostate carcinogenesis and previous in vivo and in vitro studies have shown ectopic ERG expression to be associated with increased cell invasion. However, the molecular function of ERG and its role in cell signaling is poorly understood. In this study, genomic rearrangement of ERG with TMPRSS2 was studied by using comparative genomic hybridization (CGH) in prostate cancer samples. The biological processes associated with the ERG oncogene expression in prostate epithelial cells were studied, and the results were compared with findings observed in clinical prostate tumor samples. The gene expression data indicated that increased WNT signaling and loss of cell adhesion were a characteristic of TMPRSS2- ERG fusion positive prostate tumor samples. Up- regulation of WNT pathway genes were present in ERG positive prostate tumors, with frizzled receptor 4 (FZD4) presenting with the highest association with ERG overexpression, as verified by quantitative reverse transcription-PCR, immunostaining, and immunoblotting in TMPRSS2-ERG positive VCaP prostate cancer cells. Furthermore, ERG and FZD4 silencing increased cell adhesion by inducing active β1-integrin and E-cadherin expression in VCaP cells. Furthermore, we found a novel inhibitor, 4-(chloromethyl) benzoyl chloride which inhibited the WNT signaling and induced similar phenotypic effects as observed after ERG or FZD4 down regulation in VCaP cells. In conclusion, this work deepens our understanding on the complex oncogenic mechanisms of ERG in prostate cancer that may help in developing drugs against TMPRSS2-ERG positive tumors.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Modulation of Signaling Molecules by Human Leucocyte Antigen B27; Special Reference to STAT1

Reactive arthritis (ReA) is an inflammatory joint disease, which belongs to the group of Spondyloarthritis (SpA). It may occur after infections with certain gram-negative bacteria such as Salmonella and Yersinia. SpAs are strongly associated with the human leucocyte antigen (HLA)-B27. Despite active research, the mechanism by which HLA-B27 causes disease susceptibility is still unknown. However, HLA-B27 has a tendency to misfold during assembly. It is possible that the misfolding of HLA-B27 could alter signaling pathways and/or molecules involved in inflammatory response in cells. We have earlier discovered that in HLA-B27-positive cells the interaction between the host and causative bacteria is disturbed. Our recent studies indicate that the expression of HLA-B27 may alter certain signaling molecules by disturbing their activation. The aim of this study was to investigate whether the expression of HLA-B27 disturbs the signaling molecules, especially the phosphorylation of transcription factor STAT1. STAT1 is an important mediator of inflammatory responses. Our results show that the phosphorylation of the STAT1 is significantly altered in HLA-B27-expressing U937 monocytic cells compared with control cells. STAT1 tyrosine 701 is more strongly phosphorylated in HLAB27- expressing cells; whereas the phosphorylation of STAT1 serine 727 is prolonged. Phosphorylation of STAT1 was discovered to be dependent on protein kinase PKR. Furthermore, we found out that the expression of posttranscriptional gene regulator HuR was altered in HLA-B27-expressing cells. We also detected that HLA-B27-positive cells secrete more interleukin 6, which is an important mediator of inflammation. These results help to understand how HLA-B27 may confer susceptibility to SpAs.

The expression of HLA-B27 modulates intracellular signaling in human monocytic macrophages

Reactive arthritis (ReA) is an inflammatory joint disease triggered by certain bacterial infections e.g. gastroenteritis caused by Salmonella. ReA is strongly associated to HLA-B27. However, the mechanism behind this association is unknown but it is suggested that the bacteria or bacterial compartments persist in the body. In this study, it was investigated whether the intracellular signaling is altered in HLA-B27- transfected U937 monocytic macrophages. Moreover, the contribution of HLA–B27 heavy chain (HC) misfolding was of interest. The study revealed that p38 activity plays a crucial role in controlling intracellular Salmonella Enteritidis in U937 cells. The replication of intracellular bacteria was dependent on p38 kinase and the activity of p38 was dysregulated in HLA-B27- transfected cells expressing misfolding heavy chains (HCs). Also the double-stranded RNA -dependent kinase (PKR) that modifies p38 signaling was overexpressed and hypophosphorylated upon infection and lipopolysaccharide stimulation. The expression of CCAAT enhancer binding protein beta (C/EBPβ) was found to be increased after infection and stimulation. Increased amount of full length human antigen R (HuR), disturbed HuR cleavage and reduced dependence on PKR after infection were observed. All the findings were linked to HLA-B27 HCs containing misfoldingassociated glutamic acid 45 (Glu45) at the peptide binding groove. The results indicate that the expression of HLA-B27 modulates the intracellular environment of U937 monocytic macrophages by altering signaling. This phenomenon is at least partially associated to the HLA-B27 misfolding. These observations offer a novel explanation how HLA-B27 may modulate inflammatory response induced by ReA-triggering bacteria.

From Unspecific Quenching to Specific Signaling: Functional GTPase Assays Utilizing Quenching Resonance Energy Transfer (QRET) Technology

The aim of the work presented in this study was to demonstrate the wide applicability of a single-label quenching resonance energy transfer (QRET) assay based on time-resolved lanthanide luminescence. QRET technology is proximity dependent method utilizing weak and unspecific interaction between soluble quencher molecule and lanthanide chelate. The interaction between quencher and chelate is lost when the ligand binds to its target molecule. The properties of QRET technology are especially useful in high throughput screening (HTS) assays. At the beginning of this study, only end-point type QRET technology was available. To enable efficient study of enzymatic reactions, the QRET technology was further developed to enable measurement of reaction kinetics. This was performed using proteindeoxyribonuclei acid (DNA) interaction as a first tool to monitor reaction kinetics. Later, the QRET was used to study nucleotide exchange reaction kinetics and mutation induced effects to the small GTPase activity. Small GTPases act as a molecular switch shifting between active GTP bound and inactive GDP bound conformation. The possibility of monitoring reaction kinetics using the QRET technology was evaluated using two homogeneous assays: a direct growth factor detection assay and a nucleotide exchange monitoring assay with small GTPases. To complete the list, a heterogeneous assay for monitoring GTP hydrolysis using small GTPases, was developed. All these small GTPase assays could be performed using nanomolar protein concentrations without GTPase pretreatment. The results from these studies demonstrated that QRET technology can be used to monitor reaction kinetics and further enable the possibility to use the same method for screening.

Networking Notch : regulation of Notch traffic and signaling crosstalk

Utvecklingen av flercelliga organismer är en mångfacetterad process som kräver kommunikation celler emellan. Under utvecklingen av en organism måste cellerna göra vissa val, vilket bestämmer riktningen för deras fortsatta utveckling. Utgående från dessa val erhåller cellerna egenskaper som är karaktäristiska för olika celltyper. Notch-signalräckan är en viktig reglerare av valet mellan olika cellöden. Notch-signalräckan aktiveras när Notch-receptorer som uttrycks på ytan av en cell binder till Notch-ligander som uttrycks på ytan av en annan närliggande cell. Denna avhandling belyser mekanismerna som reglerar omsättningen av såväl Notch-receptorer som -ligander till och från cellmembranen, samt ökar förståelsen för hur dessa mekanismer påverkar Notch-medierade cellöden i stamceller. Internalisering av Notch receptorer anses nödvändigt för fullständig aktivering av Notch-signalvägen. De bakomliggande molekylära mekanismerna är dock fortfarande oklara. Vi har upptäckt att atypiskt protein kinas Cζ (aPKCζ) reglerar internaliseringen av Notch-receptorer. aPKCζ fosforylerar Notch, vilket leder till receptorns internalisering, men effekten är beroende av receptorns signaleringsstatus. Vi visar att aPKCζ reglerar Notch-signaleringen och styr både neuroners och muskelcellers differentiering. Ytterligare har vi analyserat samspelet mellan cellskelettet och Notch-signalvägen. Våra resultat visar att intermediärfilamenten, en del av cellskelettet, är viktiga reglerare av Notch-signaleringen både under neuronal och vaskulär utveckling. Intermediärfilamenten vimentin och GFAP reglerar uttrycket av Notch-ligander vid cellmembranen i hjärnans stödceller, astrocyterna, och påverkar därmed neuronala stamcellers cellödesbeslut. Vimentin är även viktigt reglerare av Notch-signalräckan vid angiogenesen. Celler som saknar vimentin uppvisar avvikande Notch-signalering emedan möss som saknar vimentin påvisar en fördröjd utveckling av vaskulaturen under embryonalstadiet. ------------------------------------------------- Monisoluisten organismien kehittyminen on monimutkainen prosessi, joka vaatii viestintää solujen välillä. Kehittymisen aikana solut joutuvat tiettyjen valintojen eteen, mitkä tulevat määrittämään niiden erilaistumisen suunnan. Solut omaksuvat solutyypille ominaisia ominaisuuksia näihin valintoihin perustuen Notch-signalointireitti säätelee solujen erilaistumista eri suuntiin. Notch-signalointireitti aktivoituu, kun Notch-reseptori yhden solun pinnalla sitoo Notch-ligandin toisen, viereisen solun solukalvolla. Tutkimukseni lisää tuntemusta Notch-reseptoreiden ja ligandien solun sisäisestä liikennöinnistä ja sitä säätelevistä mekanismeista, sekä tämän säätelyn vaikutuksista kantasulojen erilaistumiseen. Notch-signalointireitin aktivoituminen vaatii reseptoreiden ja ligandien sisäistämisen solukalvolta, mutta taustalla olevat ja sisäistymistä säätelevät mekanismit ovat vielä epäselviä. Tutkimukseni osoittaa, että atyyppinen proteiinikinaasi Cζ (aPKCζ) säätelee Notch-reseptoreiden endosytoosia. Endosytoosin lopputulos riippuu siitä onko reseptori aktivoitunut ligandin välityksellä vai ei. Tuloksemme osoittavat aPKCζ säätelevän Notch-signalointia ja kontrolloivan sekä hermosolujen, että lihassolujen erilaistumista. Analysoimme myös Notch-signaloinnin ja solun tukirangan vuorovaikutuksia. Välikokoiset filamentit, jotka ovat osa tukirankaa, säätelevät Notch-signalointia neuronaalisen erilaistumisen sekä verisuonten uudismuodostumisen aikana. Vimentiini ja GFAP ovat välikokoisia säikeitä, jotka säätelevät Notch-ligandien ekspressiota astrosyyttien, eli aivojen hermotukisolujen solukalvolla. Vimentiini säätelee myös Notch-signalointireittiä angiogeneesin aikana. Vimentiiniä vailla olevilla soluilla ilmenee heikentynyttä Notch-signalointia, joka voidaan liittää hiirillä ilmenevään vimenttiinin puutteesta johtuvaan viivästyneeseen verisuonien kehitykseen.

Modulation of TGF-β signaling by Hypoxia

A tumor is a fast-growing malignant tissue. This creates areas inside the tumor that are distant from local blood vessels to be able to get enough oxygen. This hypoxic condition activates a transcription factor called hypoxia inducible factor (HIF). HIF responses help a cell to adapt to decreased oxygen by activating glycolytic and angiogenesis pathways and by regulating apoptotic responses. Hypoxia drives the upregulation of a growth factor called transforming growth factor beta (TGF-beta). Similar to a hypoxia response, TGF is an important regulator of cell fate. TGF-β and HIF pathways regulate partially overlapping target genes. This regulation can also be cooperative. The TGF-beta signal is initiated by activation of plasma membrane receptors that then activate effector proteins called small mothers against decapentaplegic (Smad) homologs. In healthy tissue, TGF-β keeps cell proliferation and growth under control. During cancer progression, TGF-beta has shown a dual role, whereby it inhibits initial tumor formation but, conversely, in an existent tumor, TGF-beta drives malignant progression. Along with HIF and TGF-beta also protein dephosphorylation is an important regulatory mechanism of cell fate. Protein dephosphorylation is catalyzed by protein phosphatases such as Protein phosphatase 2A (PP2A). PP2A is a ubiquitous phosphatase that can exist in various active forms. PP2A can specifically regulate TGF-beta signaling either by enhancing or inhibiting the receptor activity. This work demonstrates that during hypoxia, PP2A is able to fine-tune TGF-beta signal by specifically targeting Smad3 effector in a Smad7-dependent manner. Inactivation of Smad3 in hypoxia leads to malignant conversion of TGF-beta signaling.

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.