An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays

Evidence that some of the fungal metabolites present in food and feed may act as potential endocrine disruptors is increasing. Enniatin B (ENN B) is among the emerging Fusarium mycotoxins known to contaminate cereals. In this study, the H295R and neonatal porcine Leydig cell (LC) models, and reporter gene assays (RGAs) have been used to investigate the endocrine disrupting activity of ENN B. Aspects of cell viability, cell cycle distribution, hormone production as well as the expression of key steroidogenic genes were assessed using the H295R cell model. Cell viability and hormone production levels were determined in the LC model, while cell viability and steroid hormone nuclear receptor transcriptional activity were measured using the RGAs. ENN B (0.01–100 μM) was cytotoxic in the H295R and LC models used; following 48 h incubation with 100 μM. Flow cytometry analysis showed that ENN B exposure (0.1–25 μM) led to an increased proportion of cells in the S phase at higher ENN B doses (>10 μM) while cells at G0/G1 phase were reduced. At the receptor level, ENN B (0.00156–15.6 μM) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6 μM. Measurement of hormone levels in H295R cells revealed that the production of progesterone, testosterone and cortisol in exposed cells were reduced, but the level of estradiol was not significantly affected. There was a general reduction of estradiol and testosterone levels in exposed LC. Only the highest dose (100 μM) used had a significant effect, suggesting the observed inhibitory effect is more likely associated with the cytotoxic effect observed at this dose. Gene transcription analysis in H295R cells showed that twelve of the sixteen genes were significantly modulated (p < 0.05) by ENN B (10 μM) compared to the control. Genes HMGR, StAR, CYP11A, 3βHSD2 and CYP17 were downregulated, whereas the expression of CYP1A1, NR0B1, MC2R, CYP21, CYP11B1, CYP11B2 and CYP19 were upregulated. The reduction of hormones and modulation of genes at the lower dose (10 μM) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.

The novel pyrrolo-1,5-benzoxazepine, PBOX-21, potentiates the apoptotic efficacy of STI571 (imatinib mesylate) in human chronic myeloid leukaemia cells

The Bcr-Abl kinase inhibitor, STI571, is the first line treatment for chronic myeloid leukaemia (CML), but the recent emergence of STI571 resistance has led to the examination of combination therapies. In this report, we describe how a novel non-toxic G1-arresting compound, pyrrolo-1,5-benzoxazepine (PBOX)-21, potentiates the apoptotic ability of STI571 in Bcr-Abl-positive CML cells. Co-treatment of CML cells with PBOX-21 and STI571 induced more apoptosis than either drug alone in parental (K562S and LAMA84) and STI571-resistant cells lines (K562R). This potentiation of apoptosis was specific to Bcr-Abl-positive leukaemia cells with no effect observed on Bcr-Abl-negative HL-60 acute myeloid leukaemia cells. Apoptosis induced by PBOX-21/STI571 resulted in activation of caspase-8, cleavage of PARP and Bcl-2, upregulation of the pro-apoptotic protein Bim and a downregulation of Bcr-Abl. Repression of proteins involved in Bcr-Abl transformation, the anti-apoptotic proteins Mcl-1 and Bcl-(XL) was also observed. The combined lack of an early change in mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9 suggests that this pathway is not involved in the initiation of apoptosis by PBOX-21/STI571. Apoptosis was significantly reduced following pre-treatment with either the general caspase inhibitor Boc-FMK or the chymotrypsin-like serine protease inhibitor TPCK, but was completely abrogated following pre-treatment with a combination of these inhibitors. This demonstrates the important role for each of these protease families in this apoptotic pathway. In conclusion, our data highlights the potential of PBOX-21 in combination with STI571 as an effective therapy against CML.

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

PIAS1 is increased in human prostate cancer and enhances proliferation through inhibition of p21

Prostate cancer development and progression are associated with alterations in expression and function of elements of cytokine networks, some of which can activate multiple signaling pathways. Protein inhibitor of activated signal transducers and activators of transcription (PIAS)1, a regulator of cytokine signaling, may be implicated in the modulation of cellular events during carcinogenesis. This study was designed to investigate the functional significance of PIAS1 in models of human prostate cancer. We demonstrate for the first time that PIAS1 protein expression is significantly higher in malignant areas of clinical prostate cancer specimens than in normal tissues, thus suggesting a growth-promoting role for PIAS1. Expression of PIAS1 was observed in the majority of tested prostate cancer cell lines. In addition, we investigated the mechanism by which PIAS1 might promote prostate cancer and found that down-regulation of PIAS1 leads to decreased proliferation and colony formation ability of prostate cancer cell lines. This decrease correlates with cell cycle arrest in the G0/G1 phase, which is mediated by increased expression of p21(CIP1/WAF1). Furthermore, PIAS1 overexpression positively influences cell cycle progression and thereby stimulates proliferation, which can be mechanistically explained by a decrease in the levels of cellular p21. Taken together, our data reveal an important new role for PIAS1 in the regulation of cell proliferation in prostate cancer.

O papel do regulador transcricional CITED2 na leucemia linfoblástica aguda de linfócitos T

A leucemia linfoblástica aguda de Linfócitos T (LLA-T) é uma neoplasia agressiva de precursores de células T do timo, que afeta principalmente crianças e adolescentes. A identificação de fatores moleculares que controlam a iniciação e progressão da LLA-T é fundamental para desenvolvimento de terapêuticas mais específicas e eficientes. Devido a atividade de fatores de transcrição estar muitas vezes desregulada na LLA-T e o regulador transcricional CITED2 controla diversos processos no desenvolvimento e oncogénese, levando assim ao objetivo principal, verificar se o CITED2 está envolvido na iniciação e/ou progressão da LLA-T, para se cumprir este iremos verificar se o CITED2está expresso nas linhas celulares leucémicas, quais os seus efeitos e por último verificar se o seu promotor é influenciada por três vias de sinalização: JAK-STAT, NFkB e NOTCH1. Analisou-se a expressão de CITED2 em linhas celulares de leucemia linfoblástica das células T e de células T normais, realizando o PCR quantitativo. Verificou-se que todas as células expressavam o CITED2, algumas com elevada expressão, por exemplo a linha celular EL4.2 expressa sete vezes mais Cited2 que os timócitos normais. Selecionou-se duas linhas celulares, EL4.2 e as DND41, para efetuar a subexpressão de CITED2, utilizando um plasmídeo lentiviral que expressa um RNA de interferência contra o CITED2. Conseguimos obter transdução estável deste plasmídeo para a linha celular DND41, tendo então efetuado análises de proliferação celular em meio de cultura normal ou meio com redução de soro fetal bovino. Verificou-se que a linha celular DND41/shCITED2 apresentava um maior crescimento celular comparando com a linha DND41/pLKO.1, sendo significativo, a partir das 48 horas, mas análise das fases do ciclo celular não demonstrou qualquer diferença. Analisou-se a resistência destas linhas celulares à apoptose, tratando as células com dexametasona, um agente quimioterápico para leucemias LLA-T. Observou-se que existe uma ligeira tendência para a linha celular DND41/shCITED2 ser mais resistente à apoptose. Mas nas linhas celulares sem tratamento, verificou-se diferenças na fase G0/G1 e G2/M, sendo que a linha celular DND41 sh CITED2 apresenta mais células com ciclo celuar ativo. Analisou-se no presente trabalho, se o promotor do CITED2 seria influenciando pela ativação das vias de sinalização JAK-STAT, NF-kB e NOTCH1. Os nossos resultados indicam que a ativação destas vias regulam negativamente o promotor do CITED2. Tendo em conta o conjunto dos nossos resultados, podemos concluir que a inativação do regulador transcricional CITED2 pode contribuir para o desenvolvimento da LLA-T. No entanto, será necessário desenvolver mais estudos para compreender os mecanismos subjacentes.

Molecular and biotechnological approaches to essential oils production in thymus caespititius

Tese de doutoramento, Biologia (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2014

Effect of CDC25 and WEE1 on Plant Cell Cycle and Morphogenesis

The cell cycle comprise the four phases of, G1, S-phase, G2 and mitosis. Two critical transitions are G1/S and G2/M; the latter is regulated by WEE1 kinase and CDC25 phosphatases. The scope of this thesis was to investigate the regulation of the G2/M transition of the cell cycle by WEE1 and CDC25, and how these genes interface with plant growth regulators in Arabidopsis thaliana. In Arabidopsis roots, the frequency of lateral roots was found to be increased by ectopic expression of Schizosaccharomyces pombe (Sp)cdc25e and reduced by Arath;WEE1 expression. I examined the effect of Arath;WEE1 and Spcdc25 on induction of shoots and roots in Arabidopsis hypocotyls in vitro. Hypocotyl explants from two over-expressing WEE1 lines , three T-DNA insertion lines and two expressing cdc25 (Spcdc25e) lines together with wild type (WT) were cultured on two-way gradients of kinetin (Kin) and naphthyl acetic acid (NAA). Below a threshold concentration of NAA (100 ng ml-1), WEE1 repressed morphogenesis in vitro, whereas at all NAA/Kin combinations Spcdc25 promoted morphogenesis (particularly root formation) over and above that in WT. Loss of function wee1-1 cultures were very similar to WT. Quantitative data indicated a significant increase in the frequency of root formation in Spcdc25e cultures compared with WT particularly at low Kin concentrations, and WEE1oe’s repressive effect was overcome by NAA but not Kin. In conclusion, WEE1 has a repressive effect on morphogenesis in vitro that can be overcome by auxin whereas Spcd25 by-passes a cytokinin requirement for the induction of morphogenesis in vitro. The role of CDC25 and WEE1 in DNA damage responses was also analysed. Two over-expressing Arath;CDC25 lines and T-DNA mutants showed no difference to WT either in standard conditions or zeocin-supplemented treatments. However, root length was longer in Arath;CDC25oe lines treated with hydroxyurea (HU) and lateral root number was increased compared to WT. This suggests a differential response of Arath;CDC25oe in the DNA replication (HU-induced) and DNA damage (zeocin-induced) checkpoints (Chapter 5). Finally the roles of WEE1 and CDC25 in cell cycle regulation were examined using tobacco TBY-2 cell cultures expressing Arath;WEE1, Nicotiana tabacum (Nicta)WEE1 or Arath;CDC25. Whilst Nicta;WEE1 lengthened G2 of the cell cycle, Arath;WEE1 had an unusual effect of shortening G2 phase and Arath;CDC25 had no observable effect (Chapter 6).

Efeitos da ingestão de simbiótico e indol-3-carbinol sobre o processo de carcinogênese química de cólon em ratos Wistar alimentados com dieta contendo heme

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of PLK4 levels and activity to ensure centriole number control

Dissertation presented to obtain a Ph.D degree in Cellular Biology

The role of the TRAF-interacting protein in proliferation and differentiation.

Ubiquitination of proteins is a post-translational modification, which decides on the cellular fate of the protein. Addition of ubiquitin moieties to proteins is carried out by the sequential action of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3, ubiquitin ligase. The TRAF-interacting protein (TRAIP, TRIP, RNF206) functions as Really Interesting New Gene (RING)-type E3 ubiquitin ligase, but its physiological substrates are not yet known. TRAIP was reported to interact with TRAF [tumor necrosis factor (TNF) receptor-associated factors] and the two tumor suppressors CYLD and Syk (spleen tyrosine kinase). Ectopically expressed TRAIP was shown to inhibit nuclear factor-kappa B (NF-κB) signalling. However, recent results suggested a role for TRAIP in biological processes other than NF-κB regulation. Knock-down of TRAIP in human epidermal keratinocytes repressed cellular proliferation and induced a block in the G1/S phase of the cell cycle without affecting NF-κB signalling. TRAIP is necessary for embryonal development as mutations affecting the Drosophila homologue of TRAIP are maternal effect-lethal mutants, and TRAIP knock-out mice die in utero because of aberrant regulation of cell proliferation and apoptosis. These findings underline the tight link between TRAIP and cell proliferation. In this review, we summarize the data on TRAIP and put them into a larger perspective regarding the role of TRAIP in the control of tissue homeostasis.

Kriisinhallintaoperaatioista palaavien sotilaiden psykososiaalinen tuki puolustusvoimissa

Viimeisen vuosikymmenen aikana Suomi on osallistunut toistaiseksi vaarallisimpaan kriisinhallintaoperaatioon koko rauhanturvahistoriansa aikana. Tämä on johtanut henkisten ongelmien kasvuun kriisinhallintaveteraanien keskuudessa. Tästä johtuen Suomen puolustusvoimat aloitti vuonna 2008 kriisinhallintaveteraaneille suunnatun psykososiaalisen tukijärjestelmän. Tämän tutkimuksen tavoitteena on kartoittaa kriisinhallintaoperaatioista palaavien sotilaiden psykososiaalista tukea Puolustusvoimissa. Tutkimus pyrkii vastaamaan minkälaisen prosessin rauhanturvaajat käyvät läpi kotiutumisen yhteydessä. Tutkimuksen tulokset on kerätty vuosina 2013 ja 2014 pääasiassa kahdella asiantuntijahaastattelulla. Haastateltavat asiantuntijat olivat Porin prikaatin henkilökuntaa, jotka vastasivat psykososiaalisen tuen järjestämisestä. Keskeisimpänä tuloksena ja johtopäätöksenä voidaan todeta Puolustusvoimien järjestämän psykososiaalisen tukijärjestelmän olevan tarpeeseen ja resursseihin suhteutettuna tarkoituksenmukaisesti toteutettu. Rauhanturvaajat käyvät kotiutumisen jälkeen läpi tehokkaan prosessin, jonka päätarkoituksena on kouluttaa heille vertaistukea. Tämän lisäksi järjestelmä pyrkii seulomaan mahdollisesti tuen tarpeessa olevia henkilöitä ja saattamaan heidät tarvittavan jatkohoidon piiriin.

Aktiivinen omasuojajärjestelmä ja sitä vastaan toimiminen

Panssarialusta ja panssarintorjunta ovat kulkeneet kilpajuoksussa ensimmäisestä maailman-sodasta alkaen. Panssarivaunun suojausta on parannettu lisäämällä panssaroinnin paksuutta ja asentamalla lisäpanssarointia. Uusimpana trendinä on ollut kehittää aktiivisia omasuojajär-jestelmiä jotka pyrkivät estämään panssarintorjunta-aseen vaikuttamisen panssarivaunuun ennen kuin se ehtii osua kohteeseensa. Tutkielmassa selvitetään panssarintorjunta-asetta vastaan vasta-aseella toimivia hard-kill-omasuojajärjestelmiä vastaan toimimista jokaiselle jalkaväen sotilaalle koulutettavilla ker-tasingoilla. Tutkielma selvittää, miltä ampuma-etäisyyksiltä pitää toimia, jotta aktiivinen omasuojajärjestelmä ei ehdi reagoida sitä kohti tulevaan uhkaan. Tutkielma on tyyliltään kvalitatiivinen kirjallisuuskatsaus ja tutkielma on tyyliltään her-meneuttinen analyysi. Tilastollista menetelmää on käytetty verrattaessa aseiden ja aktiivisten omasuojajärjestelmien vuorovaikutusta. Tutkielman keskeisenä johtopäätöksenä on, että osaa järjestelmistä vastaan voidaan toimia niin, etteivät ne ehdi reagoida panssarintorjunta-aseeseen. Järjestelmiä jotka kykenevät toi-mimaan uhkaa vastaan, on käsitelty vaihtoehtoisia vaikuttamismenetelmiä.

Venäjän erikoisjoukkojen tarkkuuskiväärit ja raskaat tarkkuuskiväärit sekä niiden ominaisuudet

Erikoisjoukkojen sotilaat ovat aina olleet taistelukentällä pelätty vastustaja, johtuen niiden henkisistä ja fyysisistä ominaisuuksista sekä huippuluokan varustuksesta. Nykypäivänä taistelutilan jatkuva muuttuminen on vaikuttanut erikoisjoukkojen kalustoon ja käyttöön. Soluttautuminen vastustajan kohdealueelle on vaikeutunut nykyisten valvontasensoreiden kehittymisen myötä ja näin ollen erikoisjoukot joutuvat usein suorittamaan konventionaalisin asein toteutettavat tehtävät varsin pitkille etäisyyksille. Tällaisten tehtävien suorittamiseen tarkkuuskiväärit ovat yksi varteenotettavimmista vaikuttamisen välineistä. Tutkimuksen tavoitteena on selvittää millaisia tarkkuuskiväärejä ja raskaita tarkkuuskiväärejä Venäjän erikoisjoukoilla on käytössään ja millaisia ominaisuuksia niissä itse aseen ja patruunan osalta on käytettävissä. Aseita käsittelevät tiedot on kerätty kirjallisia lähteitä sekä internetlähteitä hyväksikäyttäen. Patruunoiden tarkastelussa tutkija on käyttänyt edellä mainittujen lähteiden lisäksi myös internetistä ladattua ballistiikkaohjelmaa, jolla on luotu taulukoita erilaisten luotien ominaisuuksista optimiolosuhteissa. Tutkimuksen perusteella Spetsnaz -joukkojen tarkkuuskiväärit ja raskaat tarkkuuskiväärit perustuvat pääosin varsin vanhoista asemalleista muokattuihin ja paranneltuihin versioihin. Asesuunnittelussa on kuitenkin havaittavissa muutosta aseiden toimintaperiaatteessa ja varusteissa, vaikka uusia asemalleja ei 2000 -luvun puolelta tässä tutkimuksessa löytynyt. Myös kivääreiden kaliiperit ovat pysyneet varsin samoina jo vuosikymmeniä, vaikka tutkimuksessa paljastui Venäjällä käytettävän nykyisin myös siellä harvoin esiintyneitä kaliipereja.

CISE-hanke ja rannikkovartiostotehtävät Suomessa

Euroopan komissio vahvisti kattavan meripolitiikan kehittämisen yhdeksi kauden 2005–2009 strategisista tavoitteistaan. Merten tehokas hallinnointi on EU:lle elinehto niin talouden kilpailukyvyn kuin ihmisten hyvinvoinninkin kannalta. Euroopan unionin yhdennetty meripolitiikka (Integrated Maritime Policy, IMP) kattaa kaikki meriin ja rannikkoalueisiin liittyvät hallinnonalat ja politiikanlohkot. Sen jalkauttamiseen pyritään hankkeilla jotka koordinoidaan EU-tasolla ja toteutetaan kansallisella tasolla. Tässä tutkimuksessa tarkastellaan Eurooppalaisen merivalvontaverkoston luomiseksi aloitet-tua CISE-hanketta (Common Information Sharing Environment), joka kuuluu tärkeäksi nostetun Eurooppalaisen merivalvontaverkosto-hankkeen alle. Tutkimustehtävänä tässä tutkielmassa on selvittää EU:n CISE-hankkeen toteutuessaan tuomat mahdollisuudet Rajavartiolaitoksen rannikkovartiostotehtävien täyttämisessä. Tutkielma on luonteeltaan laadullinen, analyysimenetelmä on induktiivinen lähdeaineistoanalyysi. Tutkielman keskeisen aineiston muodostavat EU:n CISE-hankkeen asiakirjat, asiantuntijahaastattelut, kansallinen ja kansainvälinen lainsäädäntö sekä Rajavartiolaitoksen julkinen materiaali. Tutkimuksen perusteella suurimmat hyötyjät CISE-hankkeesta tulevat todennäköisesti olemaan moniviranomaistehtävät ja kansainvälistä yhteistyötä vaativat tehtävät, kuten suuret ympäristöonnettomuudet tai meripelastustehtävät, sekä rajat ylittävän rikollisuuden torjunta rannikkoalueilla.