Particle flux characterisation and sedimentation patterns of protistan plankton during the iron fertilisation experiment LOHAFEX in the Southern Ocean

The taxonomic composition and types of particles comprising the downward particle flux were examined during the mesoscale artificial iron fertilisation experiment LOHAFEX. The experiment was conducted in low-silicate waters of the Atlantic Sector of the Southern Ocean during austral summer (January-March 2009), and induced a bloom dominated by small flagellates. Downward particle flux was low throughout the experiment, and not enhanced by addition of iron; neutrally buoyant sediment traps contained mostly faecal pellets and faecal material apparently reprocessed by mesozooplankton. TEP fluxes were low, <5 mg GX eq/m**2/day, and a few phytodetrital aggregates were found in the sediment traps. Only a few per cent of the POC flux was found in the traps consisting of intact protist plankton, although remains of taxa with hard body parts (diatoms, tintinnids, thecate dinoflagellates and foraminifera) were numerous, far more so than intact specimens of these taxa. Nevertheless, many small flagellates and coccoid cells, belonging to the pico- and nanoplankton, were found in the traps, and these small, soft-bodied cells probably contributed the majority of downward POC flux via mesozooplankton grazing and faecal pellet export. TEP likely played an important role by aggregating these small cells, and making them more readily available to mesozooplankton grazers.

Pico Cell Integration to Operator’s Incident Management Process

Mobile network coverage is traditionally provided by outdoor macro base stations, which have a long range and serve several of customers. Due to modern passive houses and tightening construction legislation, mobile network service is deteriorated in many indoor locations. Typically, solutions for indoor coverage problem are expensive and demand actions from the mobile operator. Due to these, superior solutions are constantly researched. The solution presented in this thesis is based on Small Cell technology. Small Cells are low power access nodes designed to provide voice and data services.. This thesis concentrates on a specific Small Cell solution, which is called a Pico Cell. The problem regarding Pico Cells and Small Cells in general is that they are a new technological solution for the mobile operator, and the possible problem sources and incidents are not properly mapped. The purpose of this thesis is to figure out the possible problems in the Pico Cell deployment and how they could be solved within the operator’s incident management process. The research in the thesis is carried out with a literature research and a case study. The possible problems are investigated through lab testing. Pico Cell automated deployment process was tested in the lab environment and its proper functionality is confirmed. The related network elements were also tested and examined, and the emerged problems are resolvable. Operators existing incident management process can be used for Pico Cell troubleshooting with minor updates. Certain pre-requirements have to be met before Pico Cell deployment can be considered. The main contribution of this thesis is the Pico Cell integrated incident management process. The presented solution works in theory and solves the problems found during the lab testing. The limitations in the customer service level were solved by adding the necessary tools and by designing a working question pattern. Process structures for automated network discovery and pico specific radio parameter planning were also added for the mobile network management layer..

Étude de mortalité de cinq cancers : mélanome cutané, cancer broncho-pulmonaire, leucémie myéloïde chronique, maladie de Hodgkin et cancer de l’endomètre.

Pour chacun des cinq cancers, nous avons fait un rappel de l’épidémiologie en Amérique du Nord, des classifications et des facteurs pronostics, la description des études, l’étude commentée de la mortalité, et enfin la conclusion. L’étude du mélanome cutané a montré que les mélanomes sont assurables dès les premières années aux stades IA, IB, IIA et IIIA, aux stades IIB, IIC et IIIB après cinq ans et au stade IIIC après 15ans. L’étude du cancer broncho-pulmonaire a montré que le cancer à petites cellules n’est pas assurable et que les cancers broncho-pulmonaires non à petites cellules pourraient être assurables chez les moins de 65 ans aux stades IA à IIIA après dix ans, et chez les 65 ans et plus au stade IA dès les premières années, aux stades IB et IIA après cinq ans et aux stades IIB et IIIA après dix ans L’étude de la leucémie myéloïde chronique a montré l’assurabilité seulement des sujets de plus de 65 ans dès les premières années et des sujets de 60 à 65 ans après 5 ans. L’étude du lymphome de Hodgkin a montré que chez les sujets de moins de 45 ans le stade IA est assurable dès les premières années, les stades IB et IIA le sont après 5 ans et les stades IIB à IVA le sont après 10 ans. Les sujets de 45 à 64 ans aux stades IA et IIA sont assurables dès les premières années et autres stades après 5 ans. Les sujets de 65 ans et plus sont assurables dès les premières années aux stades IA à IIIA et après 5 ans aux autres stades. L’étude du cancer de l’endomètre montre qu’il n’est assurable les cinq premières années que pour le type I au stade I chez les femmes âgées de 45 ans et plus, au stade II chez les femmes de 55 ans et plus et au stade III chez les femmes de 65 ans et plus ; pour le type II au stade I chez les 65 ans et plus, et au stade II chez les 75 ans et plus ; et pour les tumeurs mullériennes malignes mixtes au stade I chez les 65 ans et plus.

Étude de mortalité de cinq cancers : mélanome cutané, cancer broncho-pulmonaire, leucémie myéloïde chronique, maladie de Hodgkin et cancer de l’endomètre

Pour chacun des cinq cancers, nous avons fait un rappel de l’épidémiologie en Amérique du Nord, des classifications et des facteurs pronostics, la description des études, l’étude commentée de la mortalité, et enfin la conclusion. L’étude du mélanome cutané a montré que les mélanomes sont assurables dès les premières années aux stades IA, IB, IIA et IIIA, aux stades IIB, IIC et IIIB après cinq ans et au stade IIIC après 15ans. L’étude du cancer broncho-pulmonaire a montré que le cancer à petites cellules n’est pas assurable et que les cancers broncho-pulmonaires non à petites cellules pourraient être assurables chez les moins de 65 ans aux stades IA à IIIA après dix ans, et chez les 65 ans et plus au stade IA dès les premières années, aux stades IB et IIA après cinq ans et aux stades IIB et IIIA après dix ans L’étude de la leucémie myéloïde chronique a montré l’assurabilité seulement des sujets de plus de 65 ans dès les premières années et des sujets de 60 à 65 ans après 5 ans. L’étude du lymphome de Hodgkin a montré que chez les sujets de moins de 45 ans le stade IA est assurable dès les premières années, les stades IB et IIA le sont après 5 ans et les stades IIB à IVA le sont après 10 ans. Les sujets de 45 à 64 ans aux stades IA et IIA sont assurables dès les premières années et autres stades après 5 ans. Les sujets de 65 ans et plus sont assurables dès les premières années aux stades IA à IIIA et après 5 ans aux autres stades. L’étude du cancer de l’endomètre montre qu’il n’est assurable les cinq premières années que pour le type I au stade I chez les femmes âgées de 45 ans et plus, au stade II chez les femmes de 55 ans et plus et au stade III chez les femmes de 65 ans et plus ; pour le type II au stade I chez les 65 ans et plus, et au stade II chez les 75 ans et plus ; et pour les tumeurs mullériennes malignes mixtes au stade I chez les 65 ans et plus.

Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection. (C) 2011 Elsevier B.V. All rights reserved.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

High-dose sequential chemotherapy (ICE) under circulating progenitor cells (CPC) protection in patients with small cell lung carcinoma (SCLC). Preliminary report on a multicentric study

Starting in February 1994, 20 patients (pt) with a median age of 50 years(range 41-63) from 7 European centers have been included. Completedata were obtained in 16 patients so far. CPC were mobilized with chemo(Epirubicine 75 mg/m2 /d, 01 + 02) followed by G-CSF 5 p.gfkg/d for14 days. HD chemo consisted in 3 sequential courses of ICE regimen(UOs. 10 g/m2 , Carbo. 1200 mg/m2 and Etop. 1200 mg/m2 ) underCPC protection and G-CSF 5 p.g/kg/d. Out of the 16 pt, 12 completedfull program (3 cycles). One pt died of septic shock before receivingany ICE course. One pt died during the first ICE of renal insufficiency.Two pt had only 2 courses because of toxicity. Among the 16 pt, responserate (RR) was: 7 CR, 6 PR, 1 PO; 3 pt are not evaluable dueto early withdrawal (overall RR: 13/16 = 81 %). Thirty-nine cycles ofHD chemo were given with a median hematological recovery of 9 days(range 7-12) until neutro. counts> 1.0 x 109 /1 and 9 days (range 717)until thrombo. > 20 x 109 /1. No cumulative, hematological toxicitywas seen. Accrual of patients is still ongoing and updated results will bepresented.

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability

Small cell lung cancer (SCLC) is an aggressive disease, representing 15% of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80% sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.

Small bowel injury associated to allergy is triggered by platelet-activating factor, mast cells, neutrophils and protected by nitric oxide

Allergy to components of the diet is followed by gut inflammation which in children, sometimes progress to mucosal lesions and anaphylaxis. In newborns suffering of cow`s milk allergy, bloody stools, rectal. bleeding and ulcerations are found. The rat systemic anaphylaxis is a suitable model to study the intestinal lesions associated to allergy. In the present study we used this model to investigate some mechanisms involved. We found that 15 min after antigen challenge of sensitized rats, hemorrhagic lesions develop in the small intestine. The lesions were more severe in jejunum and ileum compared to duodenum. Pretreatment of the rats with a platelet-activating factor-receptor antagonist (WEB-2170) reduced the lesions whereas inhibition of endogenous nitric oxide by L-NAME, greatly increased the hemorrhagic lesions and mortality. Both, lesions and mortality were reversed by L-arginine. The hemorrhagic lesions were also significantly reduced by the mast cell stabilizers, disodium cromoglycate and ketotifen as well as by neutrophils depletion (with anti-PMN antibodies) or inhibition of selectin binding (by treatment with fucoidan). Thus, the intestinal hemorrhagic lesions in this model are dependent on ptatelet-activating factor, mast cell granule-derived mediators and neutrophils. Endogenous nitric oxide and supplementation with L-arginine has a protective role, reducing the lesions and preventing mortality. These results contributed to elucidate mechanisms involved in intestinal lesions which could be of relevance to human small bowel injury associated to allergy. (c) 2007 Elsevier B.V. All rights reserved.

Grafts of porcine small intestinal submucosa seeded with cultured homologous smooth muscle cells for bladder repair in dogs

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