Signal transduction pathways mediated by G protein-coupled receptors (GPCRs) and their intracellular coupling partners, the heterotrimeric G proteins, are crucial for several physiological functions in eukaryotes, including humans. This thesis describes a broad genomic survey and extensive comparative phylogenetic analysis of GPCR and G protein families from a wide selection of eukaryotes. A robust mining of GPCR families in fungal genomes (Paper I) provides the first evidence that homologs of the mammalian families of GPCRs, including Rhodopsin, Adhesion, Glutamate and Frizzled are present in Fungi. These findings further support the hypothesis that all main GPCR families share a common origin. Moreover, we clarified the evolutionary hierarchy by showing for the first time that Rhodopsin family members are found outside metazoan lineages. We also characterized the GPCR superfamily in two important model organisms (Amphimedon queenslandica and Saccoglossus kowalevskii) that belong to different metazoan phyla and which differ greatly in morphological characteristics. Curation of the GPCR superfamily (Paper II) in Amphimedon queenslandica (an important model to understand evolution of animal multicellularity) reveals the presence of four of the five GRAFS families and several other GPCR gene families. However, we find that the sponge GPCR subset is divergent from GPCRs in other studied bilaterian and eumetazoan lineages. Mapping of the GPCR superfamily (Paper III) in a hemichordate Saccoglossus kowalevskii (an essential model to understand the evolution of the chordate body plan) revealed the presence of all major GPCR GRAFS families. We find that S. kowalevskii encodes local expansions of peptide and somatostatin- like GPCRs. Furthermore, we delineate the overall evolutionary hierarchy of vertebrate-like G protein families (Paper IV) and provide a comparative perspective with GPCR repertoires. The study also maps the individual gene gain/loss events of G proteins across holozoans with more expanded invertebrate taxon sampling than earlier reports. In addition, Paper V describes a broad survey of nematode chemosensory GPCR families and provides insights into the evolutionary events that shaped the GPCR mediated chemosensory system in protostomes. Overall, our findings further illustrate the evolutionary hierarchy and the diversity of the major components of the G protein-coupled receptor signaling system in eukaryotes.

Oncogenic activating mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI 3-K), are highly prevalent in breast cancer. The protein kinase Akt is considered to be the primary effector of PIK3CA, though the mechanisms by which PI 3-K mediates tumorigenic signals in an Akt-independent manner remain obscure. My studies show that the serum and glucocorticoid-regulated kinases (SGKs) can function as effectors of PI 3-kinase and transduce signals to phenotypes associated with malignancy. We show that SGK3 is amplified in breast cancer and identify the mechanism by which SGK3 is activated downstream of PIK3CA, specifically through the catalytic activity of the phosphoinositide phosphatase INPP4B. Expression of INPP4B promotes SGK3 activation and in turn inhibits Akt phosphorylation. In breast cancer cell lines with elevated levels of INPP4B, SGK3 is required for proliferation in 3D and also for invasive migration. SGK3 phenotypes are in part mediated by phosphorylation of the substrate protein N-myc downstream regulated 1 (NDRG1), an established metastasis suppressor. The phosphorylation of NDRG1 leads to recruitment by F-box and WD repeat domain-containing 7 (FBW7), the substrate recognition domain of the Skp, Cullin, F-box containing (SCF) complex. Binding of Fbw7 to NDRG1 promotes its polyubiquitination and subsequent degradation by the 26S proteasome. By contrast, our studies also show that the related SGK1 isoform is polyubiquitinated by the functional E3 ubiquitin ligase Rictor-Cullin-1 complex, leading to SGK1 degradation. Proteasomal degradation of SGK1 by Rictor-Cullin-1is the first identified mTORC2-independent function of the Rictor protein. Moreover, the deregulation of SGK1 ubiquitination highlights a mechanism of SGK1 overexpression in breast cancers.

Not available / text

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

A fundamental problem in developmental and evolutionary biology is understanding the developmental genetic basis of morphological diversity. The current paradigm holds that a genetic and developmental program, or developmental genetic "toolkit", conserved across hundreds of millions of years patterns development in all metazoans. However, outside of a few well-characterized signal transduction pathways and developmental processes, overly broad strokes have been used to paint this "toolkit" metaphor as a hypothesis. Arthropoda, one of the largest groups of metazoans, represent the most morphologically diverse groups of metazoans, making them of particular interest for studies of morphological diversity and its evolution. Arthropoda is also home to one of the most well-understood model systems for developmental and genetic studies, the fruit fly Drosophila melanogaster. However, Drosophila is highly derived among arthropods with respect to the molecular genetic mechanisms that function during its development. As it is expected that all arthropods have access to the same development "toolkit", some changes are expected based on the observable differences in morphology, making arthropods extremely powerful tools for comparative genomic and molecular genetic studies. In this dissertation I characterize how modifications to the developmental "toolkit" contribute to the evolution of morphological diversity using emerging model arthropod systems. First, as part of a collaboration, I show that several genes expected to be conserved in all arthropods, belonging to the Hox family of transcription factors, have been lost from the genome of a phylogenetically basal arthropod, the two-spotted spider mite Tetranychus urticae. Second, I perform a genomic survey and find an overall reduction in the conservation of Drosophila orthologs from several major signal transduction pathways in the Tetranychus genome in comparison with findings from previous insect surveys. Third, I show that arthropod Hox genes, expected to be found in a tightly linked genomic cluster in most arthropod genomes, are not as tightly clustered as previously thought. Fourth, I show that changes in the genomic arrangement of Tetranychus Hox genes correspond with shifts in their expression and morphological change. Finally, I show the terminal Hox gene Abdominal-B is required for proper axial elongation and segment formation (both segment identity and number) during embryogenesis and metamorphosis in the red-flour beetle Tribolium castaneum. Taken together, these findings advance our knowledge of the evolution of morphological change, with a primary focus on Hox genes and their contribution to axial patterning during development.

Η Heparin affin regulatory peptide (HARP) είναι ένας αυξητικός παράγοντας με μοριακό βάρος 18 kDa, που έχει μεγάλη συγγένεια με την ηπαρίνη. Είναι συντηρημένη μεταξύ διαφόρων ειδών και παρουσιάζει 50% ομολογία με τη Midkine και την RI-HBP. Οι πρωτεΐνες αυτές συγκροτούν μια σχετικά νέα οικογένεια αυξητικών παραγόντων που έχουν συγγένεια με την ηπαρίνη. Η HARP απομονώθηκε για πρώτη φορά από τον εγκέφαλο νεογέννητου βοός ως ένα μόριο που μπορεί να επάγει την προέκταση των νευρικών κυττάρων. Επίσης, εκφράζεται στη μήτρα, στους χόνδρους και στα οστά. Αρκετές αναφορές αποδεικνύουν ότι υπάρχει μεγάλη συσχέτιση μεταξύ της έκφρασης της HARP και της ανάπτυξης καρκινικού όγκου και της αγγειογένεσης. Υψηλά επίπεδα της πρωτεΐνης έχουν ανιχνευθεί σε πολλούς καρκινικούς όγκους, αλλά και κυτταρικές σειρές που προέρχονται από διάφορους τύπους καρκίνου σε ανθρώπους. Η HARP αποτελεί μιτογόνο παράγοντα για διάφορους τύπους ενδοθηλιακών κυττάρων, ενώ μπορεί να επάγει την αγγειογένεση in vivo και in vitro. Ασκεί τη βιολογική της δράση μετά από αλληλεπίδραση με πρωτεογλυκάνες της επιφάνειας του κυττάρου, όπως η N-συνδεκάνη, ή μετά από δέσμευση σε πιο ειδικούς υποδοχείς. Η RPTPβ/ζ, η εκκρινόμενη μορφή της (φωσφακάνη), αλλά και η κινάση ALK, έχει αναφερθεί ότι μπορούν να δεσμεύουν τη HARP και να συμμετέχουν στη μεταγωγή του σήματός της. Παλαιότερες αναφορές έχουν δείξει ότι η HARP μπορεί να επάγει τις MAP-κινάσες και το μονοπάτι PI3K-Akt, ενώ αναστολείς των Erk½, ή της PI3K καταστέλλουν τη σύνθεση του DNA που επάγεται από τη HARP. Επιπλέον, η Shc και οι Erk ½ φωσφορυλιώνονται μετά από επώαση κυττάρων με HARP. Ωστόσο, τα ενδοκυτταρικά σήματα ειδικών υποδοχέων της HARP προς την PI3K ή τις MAPK δεν έχουν ακόμα χαρακτηριστεί ικανοποιητικά. Στην εργασία αυτή μελετήσαμε την επίδραση της HARP στη μετανάστευση κυττάρων HUVEC, στη δημιουργία αυλών σε υπόστρωμα matrigel, καθώς και το μονοπάτι μεταγωγής σήματος που ενεργοποιείται από τη HARP. Τα αποτελέσματά μας δείχνουν ότι η HARP επάγει τη μετανάστευση και τη διαφοροποίηση των ενδοθηλιακών κυττάρων HUVEC μετά από δέσμευσή της στην RPTPβ/ζ. Η δέσμευση αυτή οδηγεί σε ενεργοποίηση της Src, της FAK, της PI3K και των Erk ½. Το ορθοβαναδικό νάτριο, η θειική χονδροϊτίνη-C, το ΡΡ1, η wortmannin, το LY294002 και το U0126 αναστέλλουν τη μεταγωγή σήματος της HARP, καθώς και την επαγωγή της μετανάστευσης και διαφοροποίησης των HUVEC. Επιπλέον, η μείωση της έκφρασης της RPTPβ/ζ με τη χρησιμοποίηση παρεμβαλλόμενου RNA παρεμποδίζει τα ενδοκυτταρικά σήματα, καθώς και την επαγωγή της μετανάστευσης και της διαφοροποίησης που επάγεται από τη HARP. Τα αποτελέσματα αυτά δείχνουν ότι η RPTPβ/ζ αποτελεί υποδοχέα της HARP σε ενδοθηλιακά κύτταρα και αποσαφηνίζουν το μονοπάτι μεταγωγής σήματος της HARP στα κύτταρα αυτά. / Heparin affin regulatory peptide (HARP) is an 18 kDa growth factor that has a high affinity for heparin. HARP is highly conserved among species and shares 50% homology with Midkine and RI-HBP. The above proteins constitute a relatively new family of growth factors with high affinity for heparin. HARP has been originally purified from perinatal rat brain as a molecule that induces neurite outgrowth. HARP is also expressed in uterus, cartilage and bone extracts. Several reports have established a strong correlation between HARP expression and tumour growth and angiogenesis. High levels of this protein were found in many human cancers and cell lines derived from human tumours. HARP has been reported to be mitogenic for different types of endothelial cells and angiogenic in vivo and in vitro. HARP exerts its biological activity through interactions with cell surface proteoglycans, such as N-syndecan, or binding to more specific cell surface receptors. Receptor-type protein tyrosine-phosphatase β/ζ (RPTPβ/ζ) and its secreted variant phosphacan, as well as ALK, have been recently reported to bind HARP and to be implicated in its signalling.HARP has been previously shown to activate both the MAPK and PI3K - Akt signalling axes. Inhibitors of Erk½ or PI3K inhibit DNA synthesis stimulated by HARP. Additionally, analysis of tyrosine phosphorylated proteins following HARP stimulation, revealed induction of Shc and Erk ½ phosphorylation. Nevertheless, the signals from specific receptors to PI3K or MAPK are not well documented. In the present work, we examined the effect of HARP on migration and tube formation on matrigel of HUVEC and investigated the signalling pathway induced by HARP. We report that HARP induces migration and differentiation of endothelial cells through binding to RPTPβ/ζ, leading to activation of Src, FAK, PI3K and Erk½. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002 and U0126 inhibit HARP-mediated signalling and HARP-induced HUVEC migration and differentiation. In addition, RPTPβ/ζ suppression using siRNA technology, interrupts intracellular signals, as well as HUVEC migration and differentiation that are induced by HARP. These results establish the role of RPTPβ/ζ as a receptor of HARP in HUVEC and elucidate the HARP signalling pathway in endothelial cells.

Vienas esminių gyvybės bruožų – gebėjimas prisitaikyti prie kintančių aplinkos sąlygų. Dėl aplinkos poveikio kinta tiek prokariotiniai, tiek vienaląsčiai ir daugialąsčiai eukariotiniai organizmai, todėl nenuostabu, kad tam tikri gyvų ląstelių pasitelkiami atsako į aplinką ar poveikį būdai yra universalūs. Vienas tokių universalių su atsaku į aplinką susijusių reiškinių yra signalinių molekulių ir signalo perdavimo kelių egzistavimas įvairiausiose gyvybės formose, nuo bakterijų iki žinduolių ląstelių. Signalo perdavimo sistemos elementai kinta signalo perdavimo metu, todėl, analizuojant signalo perdavimo kelių elementus – signalines molekules, galima įvertinti ar ląstelė „pajuto“ aplinkos poveikį, ar aktyvintos atsako į poveikį sistemos. Taigi signalo perdavimo elementų analizė gali būti naudojama kaip ląstelės atsako į poveikį vertinimo įrankis. Šioje apžvalgoje nagrinėjama: • Eukariotų signalinių kelių komponentai – Ras šeimos baltymų funkcijos mejozėje; • Prokariotų signalinių kelių komponentai – bakterijų atsako į rūgštinį aplinkos stresą sistema; • Sutrikusių funkcijų (vėžinių) ląstelių atsakas į taikomą terapiją – signalinių elementų atsako į terapiją vertinimui paieška; • Signalinių kelių komponentų tyrimai pogenominėje eroje – kokybiškai naujas tyrimų etapas. Išvados 1. MAPK – universalus Ras aktyvinamo ląstelių dalijimosi signalinio kelio komponentas pagal kurio fosforilinimo laipsnį galima spręsti apie ląstelių aktyvinimą dalijimuisi; 2. asr RNR – universalus... [toliau žr. visą tekstą] / One of the fundamental features of the live species is the ability to adapt to the changing environment. Both prokaryotes and eukaryotes change due to the environmental stress, therefore it is obvious, that common mechanisms to cope the environmental stresses are universal among all the live organisms. One of such universal mechanisms related to the environmental stresses – signal transduction pathways and signal molecules existing among all kingdoms of live species. Elements of signal transduction systems change during the signal transduction, therefore the analysis of signal molecules allows the evaluation of the state of cellular response and the analysis of signaling elements could be employed as a tool to evaluate the cellular response. Following topics are discussed in this review: • Components of eukaryotic signal transduction system – function of Ras proteins in meiosis; • Components of prokaryotic signal transduction system – components of bacterial response to acid stress; • Response to the used therapy of transformed cells – search for the signal elements to evaluate the response to therapy; • Investigations of signal transduction components in post genomic era – qualitatively new stage of investigations. Conclusions 1. MAPK is a universal component of Ras signal transduction pathway to evaluate, according to the stage of phosphorylation of MAPK, the activation of the cell for division 2. asr RNA is a universal component of adaptation of enterobacteria to... [to full text]

The signal transducer and activator of transcription-3 (Stat3) is a latent cytoplasmic protein that is activated through phosphorylation of tyrosine-705 by a number of receptor and non-receptor tyrosine kinases. This leads to Stat3 dimerization by reciprocal SH2-ptyr interactions, followed by translocation to the nucleus to initiate transcription of genes involved in cell growth, survival, and differentiation. Many of these signaling molecules known to activate Stat3 concentrate in specialized plasma membrane microdomains called caveolae, and are sequestered in an inactive state to the caveolin scaffolding domain (CSD) of the main caveolae resident protein, caveolin-1 (cav1). Since many of these signaling molecules are known, potent Stat3 stimulators, we set out to examine the effect of cav1 upon Stat3 activity. To this effect, cav1 was downregulated using a cholesterol chelator (methylcyclodextrin), or an antisense approach. Since we previously found that cell density can dramatically activate Stat3, all experiments were conducted at several densities. The results show that cav1 downregulation causes an increase in Stat3-tyr705 phosphorylation at all densities examined. We next examined the effect of cav1 upregulation upon Stat3 activity by transfecting an EGFP-cav1 construct. The results revealed that cav1 overexpression using this construct reduces Stat3 activity and induces apoptosis, which can be overcome by expression of a constitutively active form of Stat3. Finally, by expressing a Stat3 shRNA with an adenovirus vector, we demonstrated that Stat3 downregulation leads to an increase in cav1 levels. These results reveal the presence of a potent, negative regulatory relationship between cav1 and Stat3 phosphorylation. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2008-11-09 20:53:54.787

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