Future perspectives in melanoma research: meeting report from the "Melanoma Bridge": Napoli, December 3rd-6th 2014.

The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma.

FGFR1 and PROKR2 rare variants found in patients with combined pituitary hormone deficiencies.

The genetic aetiology of congenital hypopituitarism (CH) is not entirely elucidated. FGFR1 and PROKR2 loss-of-function mutations are classically involved in hypogonadotrophic hypogonadism (HH), however, due to the clinical and genetic overlap of HH and CH; these genes may also be involved in the pathogenesis of CH. Using a candidate gene approach, we screened 156 Brazilian patients with combined pituitary hormone deficiencies (CPHD) for loss-of-function mutations in FGFR1 and PROKR2. We identified three FGFR1 variants (p.Arg448Trp, p.Ser107Leu and p.Pro772Ser) in four unrelated patients (two males) and two PROKR2 variants (p.Arg85Cys and p.Arg248Glu) in two unrelated female patients. Five of the six patients harbouring the variants had a first-degree relative that was an unaffected carrier of it. Results of functional studies indicated that the new FGFR1 variant p.Arg448Trp is a loss-of-function variant, while p.Ser107Leu and p.Pro772Ser present signalling activity similar to the wild-type form. Regarding PROKR2 variants, results from previous functional studies indicated that p.Arg85Cys moderately compromises receptor signalling through both MAPK and Ca(2) (+) pathways while p.Arg248Glu decreases calcium mobilization but has normal MAPK activity. The presence of loss-of-function variants of FGFR1 and PROKR2 in our patients with CPHD is indicative of an adjuvant and/or modifier effect of these rare variants on the phenotype. The presence of the same variants in unaffected relatives implies that they cannot solely cause the phenotype. Other associated genetic and/or environmental modifiers may play a role in the aetiology of this condition.

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4.

Background: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle tocause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP14 were assayed on the activated mast cells. Betahexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogenactivated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by betahexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase,and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions: Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.

Functional characterization of proteins required for mitotic progression and the spindle assembly checkpoint

During mitotic cell division, the genetic material packed into chromosomes is divided equally between two daughter cells. Before the separation of the two copies of a chromosome (sister chromatids), each chromosome has to be properly connected with microtubules of the mitotic spindle apparatus and aligned to the centre of the cell. The spindle assembly checkpoint (SAC) monitors connections between microtubules and chromosomes as well as tension applied across the centromere. Microtubules connect to a chromosome via kinetochores, which are proteinaceous organelles assembled onto the centromeric region of the sister chromatids. Improper kinetochore-microtubule attachments activate the SAC and block chromosome segregation until errors are corrected and all chromosomes are connected to the mitotic spindle in a bipolar manner. The purpose of this surveillance mechanism is to prevent loss or gain of chromosomes in daughter cells that according to current understanding contributes to cancer formation. Numerous proteins participate in the regulation of mitotic progression. In this thesis, the mitotic tasks of three kinetochore proteins, Shugoshin 1 (Sgo1), INCENP, and p38 MAP kinase (p38 MAPK), were investigated. Sgo1 is a protector of centromeric cohesion. It is also described in the tension-sensing mechanism of the SAC and in the regulation of kinetochore-microtubule connections. Our results revealed a central role for Sgo1 in a novel branch of kinetochore assembly. INCENP constitutes part of the chromosomal passenger complex (CPC). The other members of the core complex are the Aurora B kinase, Survivin and Borealin. CPC is an important regulatory element of cell division having several roles at various stages of mitosis. Our results indicated that INCENP and Aurora B are highly dynamic proteins at the mitotic centromeres and suggested a new role for CPC in regulation of chromosome movements and spindle structure during late mitosis. The p38 MAPK has been implicated in G1 and G2 checkpoints during the cell cycle. However, its role in mitotic progression and control of SAC signaling has been controversial. In this thesis, we discovered a novel function for p38γ MAPK in chromosome orientation and spindle structure as well as in promotion of viability of mitotic cells.

Preservação do inóculo de Plasmodiophora brassicae utilizando o método de congelamento

A preservação das estruturas de resistência de Plasmodiophora brassicae, em condições laboratoriais, é dificultada pelo fato de se tratar de um parasita obrigatório. O método de congelamento, utilizando freezer, comum foi testado com o objetivo de viabilizar a sobrevivência e a preservação de suas características infectivas. Raízes de diferentes brássicas, naturalmente infectadas por P. brassicae, contendo sintomas típicos de hérnia, de uma mesma propriedade localizada no município de Pardinho, Estado de São Paulo, foram coletadas em diferentes épocas e imediatamente congeladas, em freezer, a aproximadamente -20ºC. Os tratamentos foram divididos da seguinte maneira: T1: hérnias congeladas por 389 dias (rúcula); T2: hérnias congeladas por 242 dias (brócolis); T3: hérnias congeladas por 21 dias (couve chinesa) e T4: testemunha (sem inóculo). Os testes de patogenicidade, após diferentes períodos de armazenamento, foram realizados em condições de casa de vegetação (25±2ºC). Cada planta de uma variedade suscetível de couve-chinesa (Pak choi) foi inoculada com 2mL da suspensão de esporos de cada tratamento, na concentração de 10(7) esporos.mL-1. Cada tratamento contou com seis repetições distribuídas em blocos ao acaso. Passadas cinco semanas após a inoculação, as raízes das plantas foram lavadas e avaliadas. Houve diferença significativa entre os tratamentos. Os materiais congelados, entre 21 a 242 dias preservaram suas características infectivas, mostrando que o método de congelamento em freezer, nesse período, pode ser uma boa opção para a preservação das estruturas de resistência deste patógeno.

Efeito da temperatura sobre a severidade de Plasmodiophora brassicae

A temperatura é um dos parâmetros importantes para que ocorra a infecção, processo primordial para que haja doença, visando-se verificar a influência deste parâmetro sobre a severidade de Plasmodiophora brassicae em plantas de couve chinesa Pak choi, montou-se testes de infecção em temperaturas variando de 5 em 5ºC, indo de 10 a 40ºC, e observou-se uma redução da severidade da doença nas mudas de 28 dias de idade, nas temperaturas acima de 30ºC, verificando-se que nas temperaturas de 20 a 25ºC a ocorrência da condição ótima para o desenvolvimento da doença.

Kenttävarustuksessa suoritetun kävelymarssin vaikutus taistelijan maksimaaliseen fyysiseen suorituskykyyn

Tämän tutkimuksen tarkoituksena oli selvittää voimaominaisuuksissa tapahtuvat muutokset kenttävarustuksessa suoritetun kävelymarssin jälkeen. Kävelymarssin pituus oli 21km ja aikaa siihen kului 4h 20min. Kohdejoukkona olivat maavoimien jalkaväkilinjan kadetit 90. kadettikurssilta (n=12). Voimaominaisuuksien muutosta tarkasteltiin alku- ja lopputestillä, jotka olivat Pekoul-os:n PAK A 04:03.01/1.5.1999. mukaiset lihaskuntotestit. Testiin lisättiin vauhditon pituushyppy, jolla mitattiin ala-raajojen räjähtävää voimaa. Muut testit olivat etunojapunnerrus, istumaannousu, puristusvoima ja toistokyykky. Kohdejoukolle tehtiin myös antropometriset mittauk-set (pituus, paino, rasva %) ja mitattiin hapenottokyky polkupyöräergometritestillä. Tutkimus on luonteeltaan kvantitatiivinen kuormitusfysiologinen tutkimus. Tutkimuksen teoreettinen sisältö jakaantuu kahteen erilaiseen analysointi ja tarkastelu osioon: Teoria- ja empiiristen tulosten analysointiosioon. Tutkielman teoriaosassa selvitetään tutkielman käsitteelliset ja teoreettiset periaatteet ja lähtökohdat ihmisen fyysiseltä, sekä fysiologiselta kannalta tarkasteltuna. Tutkimuksen empiiristä tuloksista saatuja arvoja käsiteltiin ja laskettiin käyttämällä Excel -taulukkolaskentaohjelmaa ja SPSS 11.5 for windows – tilastointiohjelmaa. Tutkimuksen pääongelma Millaisia muutoksia taistelijan voimaominaisuuksissa tapahtuu kävelymarssin jälkeen? Tutkimuksen alaongelma Mikä on tällä hetkellä jalkaväen kadetin fyysinen suorituskyky? Tulokset osoittivat, että 4 tuntia 20 minuuttia kestäneen marssin aiheuttama kuormi-us kohdistui enemmän ylävartaloon, kun alavartaloon. Vauhdittoman pituushypyn tulos heikkeni 3,7 % ja toistokyykky 4,4 %. Vastaavasti etunojapunnerruksen tulos heikkeni 14 %, joka oli suurin pudotus lihaskuntotestissä. Syynä tähän on 25 kg:n lisätaakka, mikä aiheuttaa ylävartalon kuormittumista. Puristusvoimatestissä tulos heikkeni 2,5 % ja istumaannousussa 4,3 %. Kokonaisuutena 90. kadettikurssin jalkaväkilinjan fyysinen suorituskyky on hyvällä tasolla. Kadetit jaksavat marssia yli neljä tuntia 25 kg:n kantamusten kanssa erittäin hyvin. Lihaskunto on hyvä. Hapenottokyky oli 49,5 ml/kg/min, joten se on hyvin lähellä taistelijalta vaadittavaa 50 ml/kg/min rajaa.

Miehistön johtajakoulutus : keinoja vuorovaikutuksen kehittämiseksi

Miehistöntehtäviin sijoitettavat varusmiehet eivät tällä hetkellä saa minkäänlaista koulutusta liittyen johtajuuteen, johtamiskäyttäytymiseen tai vuorovaikutukseen. Tutkimuksessa tuodaan esille kuitenkin tärkeitä seikkoja miehistön johtajakoulutuksen puolesta, jotka parantaisivat tutkijan näkemyksen mukaan esimerkiksi ryhmäkiinteyttä. Tutkielman pääongelmana on tulisiko miehistölle kouluttaa asioita johtamisesta liittyen varusmiesten johtaja- ja kouluttajakoulutusohjelmaan (PEKOUL-OS PAK A 1:5.1.8)? Tutkimuksessa edetään vuorovaikutuksen näkökulmasta ja käydään läpi Puolustusvoimien johtaja- ja kouluttajakoulutus, sen taustalla vaikuttava syväjohtaminen, ryhmään liittyvät tekijät sekä lopuksi tiimien muodostuminen ja tiimitoiminnan mahdollisuudet. Tutkitun aineiston perusteella lopputulokseksi on saatu se, että miehistölle pitäisi kertoa varusmiesjohtajien johtamiskäyttäytymisen perustana olevasta johtaja- ja kouluttajakoulutuspaketista sekä siihen liittyvästä syväjohtamisen mallista. Vuorovaikutustaitojen opettaminen ja tiimityöskentely varusmieskoulutuksessa nousivat voimakkaasti esille tutkitusta aineistosta ja niiden kouluttaminen vaatii perehdyttämistä myös kouluttajina toimivalle henkilökunnalle. Alaisen tulisi myös tietää syväjohtamisen kysymyssarjaan perustuvasta johtaja-arvioinnista sekä sen vaikutuksista johtajan johtamiskäyttäytymiseen. Tutkimusmenetelmä on systemoitu kirjallisuuskatsaus ja analysointimenetelmänä on käytetty tekstianalyysiä. Valitut tekstit edustavat kattavasti tutkittavaa aihetta ja näkökulmia on otettu mukaan monen eri tieteenalan tutkimuksista.

Henkilöstövoimavarojen johtaminen joukkoyksikössä organisaatiorakenteen näkökulmasta

Tässä tutkimuksessa tutkitaan henkilöstövoimavarojen johtamista joukkoyksikössä organisaatiorakenteen näkökulmasta. Erityisessä tarkastelussa on joukkoyksikön organisaatiorakenteen ja –toimintojen välinearvo ja sopivuus kehitettäessä joukkoyksikköä oppivaksi organisaatioksi.

Yhdysvaltain ja Venäjän kehitteillä olevien viidennen sukupolven hävittäjien kehittämisaikataulu

Suomen F-18 Hornet -hävittäjät on määrä korvata uusilla hävittäjillä 2020-luvun puolivälin jälkeen. Suomi on osoittanut mielenkiintoa kehitteillä olevaan viidennen sukupolven F-35 Lightning II :een, joka on kiinnostuksen kohteena ympäri maailman. Myös Venäjän kehittelemä T-50 on maailmanlaajuisen mielenkiinnon kohteena. Tässä tutkimuksessa vertailtiin neljännen sukupolven hävittäjien kehittämisaikatauluja F- 35:n ja T-50:n kehittämisaikatauluihin. Vertailun perusteella laadittiin arvio F-35:n ja T- 50:n kehittämisaikataulujen tilasta sekä molempien alustavan operatiivisen käytettävyyden (Initial Operating Capability, IOC) saavuttamisajankohdasta. Tutkimuksen pääkysymys oli: Mikä on Yhdysvaltain ja Venäjän kehitteillä olevien viidennen sukupolven hävittäjien tilanne suhteessa edellisen sukupolven hävittäjien kehittämisaikajanaan? Tutkimus on kirjallisuustutkimukseen perustuva deskriptiivinen tulevaisuudentutkimus. Tutkimuksessa havaittiin, että neljännen sukupolven hävittäjien kehittäminen kesti noin 7½ vuodesta 26½ vuoteen. Neljännen sukupolven hävittäjien kehittämisen keskiarvoon nähden F-35A ja F-35C ovat kahdeksan vuotta, F-35B noin kuusi vuotta ja T-50 viisi vuotta jäljessä. Tutkimuksen perusteella voidaan arvioida, että F-35A ja F-35C saavuttavat IOC:n aikaisintaan vuonna 2018, F-35B vuonna 2016 ja T-50 vuonna 2019, mutta todennäköisesti vasta vuonna 2020.

A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.

Pressão arterial e concentração plasmática do peptídeo atrial natriurético e do peptídeo natriurético tipo B, em gestações complicadas pela pré-eclâmpsia

OBJETIVO: o estudo busca determinar a existência de associação entre a elevação da pressão arterial e os níveis plasmáticos dos peptídeos natriuréticos ANP e BNP, na gestação complicada pela pré-eclâmpsia. MÉTODOS: em estudo transversal caso-controle, pareado por idade gestacional, 25 grávidas normotensas e 61 portadoras de pré-eclâmpsia foram avaliadas quanto ao nível da pressão arterial e às concentrações plasmáticas dos peptídeos natriuréticos ANP e BNP. Exames clínico e laboratoriais foram realizados para o diagnóstico de pré-eclâmpsia, sendo a pressão arterial medida de forma não invasiva. As dosagens hormonais foram obtidas por radioimunoensaio, após extração em colunas Sep-pak C18. Os valores médios das concentrações plasmáticas do ANP e BNP foram comparados entre grupos com pressão arterial progressivamente maiores. A correlação entre os valores da pressão arterial e os níveis plasmáticos do ANP e BNP no sangue materno foi também investigada pela de análise de regressão no grupo completo de gestantes e em grupos específicos excluindo-se a hipertensão anterior à gestação e, em seguida, excluindo-se aquelas sem hipertensão prévia. RESULTADOS: os valores plasmáticos de ANP foram 41.5±7.3, 78.4±13.1 e 89.2±13.4 pg/mL (p<0,00001) e os de BNP plasmático foram 79.5±15.8, 176.7±42.2 e 208.3±63.5 pg/mL (p=0,005), respectivamente, para os grupos de pressão arterial média =107 mmHg, 107-139 mmHg e =140 mmHg. Verificou-se correlação positiva entre as concentrações plasmáticas do ANP e os níveis pressóricos na pré-eclâmpsia, independente da existência de estado hipertensivo prévio à gestação (p<0,0001 para pré-eclâmpsia e p<0,01 para pré-eclampsia sobreposta à hipertensão arterial crônica), ao passo que as dosagens de BNP não se mostraram associadas à pressão arterial no grupo com hipertensão arterial prévia à gestação (p=0,004 para pré-eclâmpsia e p=0,18 para pré-eclampsia sobreposta à hipertensão arterial crônica). CONCLUSÃO: o agravamento da hipertensão na pré-eclâmpsia correlacionou-se com as concentrações séricas do ANP e BNP, embora os valores do BNP possam ser influenciados pela existência de estado hipertensivo prévio.

JNK, a versatile regulator of kinesin-1 transport and cytoskeleton dynamics

C-Jun N-terminal kinase (JNK) is traditionally recognized as a crucial factor in stress response and inducer of apoptosis upon various stimulations. Three isoforms build the JNK subfamily of MAPK; generally expressed JNK1 and JNK2 and brain specific JNK3. Degenerative potency placed JNK in the spotlight as potential pharmacological option for intervention. Unfortunately, adverse effects of potential drugs and observation that expression of only JNK2 and JNK3 are induced upon stress, restrained initial enthusiasm. Notably, JNK1 demonstrated atypical high constitutive activity in neurons that is not responsive to cellular stresses and indicated existence of physiological activity. This thesis aimed at revealing the physiological functions of JNK1 in actin homeostasis through novel effector MARCKS-Like 1 (MARCKSL1) protein, neuronal trafficking mediated by major kinesin-1 motor protein and microtubule (MT) dynamics via STMN2/SCG10. The screen for novel physiological JNK substrates revealed specific phosphorylation of C-terminal end of MARCKSL1 at S120, T148 and T183 both ex vivo and in vitro. By utilizing site-specific mutagenesis, various actin dynamics and migrations assays we were able to demonstrate that JNK1 phosphorylation specifically facilitates F-actin bundling and thus filament stabilisation. Consecutively, this molecular mechanism was proved to enhance formation of filopodia; cell surface projections that allow cell sensing surrounding environment and migrate efficiently. Our results visualize JNK dependent and MARCKSL1 executed induction of filopodia in neurons and fibroblast indicating general mechanism. Subsequently, inactivation of JNK action on MARCKSL1 shifts cellular actin machinery into lamellipodial dynamic arrangement. Tuning of actin cytoskeleton inevitably melds with cell migration. We observed that both active JNK and JNK pseudo-phosphorylated form of MARCKSL1 reduce actin turnover in intact cells leading to overall diminished cell motility. We demonstrate that tumour transformed cells from breast, prostate, lung and muscle-derived cancers upregulate MARCKSL1. We showed on the example of prostate cancer PC-3 cell line that JNK phosphorylation negatively controls MARCKSL1 ability to induce migration, which precedes cancer cell metastasis. The second round of identification of JNK physiological substrates resulted in detection of predominant motor protein kinesin-1 (Kif5). Mass spectrometry detailed analysis showed evident endogenous phosphorylation of kinesin-1 on S176 within motor domain that interacts with MT. In vitro phosphorylation of bacterially expressed kinesin heavy chain by JNK isoforms displayed higher specificity of JNK1 when compared to JNK3. Since, JNK1 is constitutively active in neurons it signified physiological aspect of kinesin-1 regulation. Subsequent biochemical examination revealed that kinesin-1, when not phosphorylated on JNK site, exhibits much higher affinity toward MTs. Expression of the JNK non-phosphorable kinesin-1 mutant in intact cells as well as in vitro single molecule imaging using total internal reflection fluorescence microscopy indicated that the mutant loses normal speed and is not able to move processively into proper cellular compartments. We identify novel kinesin-1 cargo protein STMN2/SCG10, which along with known kinesin-1 cargo BDNF is showing impaired trafficking when JNK activity is inhibited. Our data postulates that constitutive JNK activity in neurons is crucial for unperturbed physiologically relevant transport of kinesin-1 dependant cargo. Additionally, my work helps to validate another novel physiological JNK1 effector STMN2/SCG10 as determinant of axodendritic neurites dynamics in the developing brain through regulation of MT turnover. We show successively that this increased MT dynamics is crucial during developmental radial migration when brain layering occurs. Successively, we are able to show that introduction of JNK phosphorylation mimicking STMN2/SCG10 S62/73D mutant rescues completely JNK1 genetic deletion migration phenotype. We prove that STMN2/SCG10 is predominant JNK effector responsible for MT depolymerising activity and neurite length during brain development. Summarizing, this work describes identification of three novel JNK substrates MARCKSL1, kinesin-1 and STMN2/SCG10 and investigation of their roles in cytoskeleton dynamics and cargo transport. This data is of high importance to understand physiological meaning of JNK activity, which might have an adverse effect during pharmaceutical intervention aiming at blocking pathological JNK action.

Viidennen sukupolven hävittäjien häiveteknologia

Tämän tutkielman tarkoituksena on selvittää viidennen sukupolven hävittäjien häiveteknologian toteutusratkaisuja. On käsitelty miksi häiveteknologiaa tarvitaan sekä vertailtu miten häiveteknologia toteutetaan viidennen sukupolven hävittäjissä. Suomen ilmavoimien päähävittäjäkalusto F/A-18 Hornet edustaa neljättä sukupolvea ja saavuttaa elinkaarensa lopun 2020-luvun lopussa, joten seuraajasta on lähivuosina tehtävä päätös. On siis mahdollista, että seuraajaksi hankitaan jokin viidennen sukupolven hävittäjä häiveteknologialla varustettuna. Viidennen sukupolven hävittäjiä kehitellään ainakin Yhdysvalloissa, Venäjällä, Kiinassa, Intiassa ja Japanissa, mutta tässä tutkielmassa on vertailtu vain Yhdysvaltojen, Venäjän ja Kiinan viidennen sukupolven hävittäjiä. Yhdysvalloilla on tällä hetkellä ainut operaatiokäytössä oleva viidennen sukupolven hävittäjä, Lockheed Martin F-22 Raptor. Raptorin lisäksi Yhdysvalloilla on kehitysvaiheessa oleva Lockheed Martin F-35 Lightning II. Venäjällä on myös kehitysvaiheessa oleva Suhoi PAK FA samoin kuin Kiinalla on Chengdu J-20. Nämä kolme kehitysvaiheessa olevaa viidennen sukupolven hävittäjää on tarkoitus ottaa palveluskäyttöön ennen 2020-lukua. Tutkielman päätutkimuskysymys on, miten häiveteknologia toteutetaan viidennen sukupolven hävittäjissä. Lisäksi tutkielmassa on kaksi alakysymystä. Nämä ovat seuraavat: Miksi häiveteknologiaa tarvitaan? Miten häiveteknologian toteutus eroaa viidennen sukupolven hävittäjissä? Tutkimusmenetelmänä on käytetty kvalitatiivista kirjallisuustutkimusta ja vertailua julkisista lähteistä. Häiveteknologia ei ole mikään uusi innovaatio. Teknologiaa on kehitelty aina 1940-luvulta asti ja yksi tunnetuimmista häivepommikoneista, Lockheed Martinin F-117 Nighthawk, on jo poistunut palvelukäytöstä. Tuolloin häiveominaisuuksien ylläpito ja huolto aiheuttivat merkittäviä kustannuksia tutkasignaalia absorboivien maalien ja materiaalien takia. Viidennen sukupolven hävittäjissä on pyritty vähentämään näiden maalien ja materiaalien käyttöä, jotta käyttökustannukset saataisiin pienennettyä. On panostettu enemmän hävittäjien muodon antamaan suojaan tutkasignaalia vastaan maalien ja materiaalien sijaan. Tämä on nykyaikana mahdollista kehittyneiden tietokoneiden avulla, jotka pystyvät laskemaan optimaalisen muodon hävittäjälle sekä häive- että aerodynaamisten ominaisuuksien kannalta. Tutkasignaalia absorboivia maaleja ja materiaaleja on kuitenkin pakko käyttää erilaisten luukkujen ympärillä sekä muissa reunoissa, joissa muodolla ei saada riittävää vaikutusta.

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.