Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

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

Combustion properties of biomass residues rich in phosphorus

Tillgången på traditionella biobränslen är begränsad och därför behöver man ta fram nya, tidigare outnyttjade biobränslen för att möta de uppställda CO2 emissionsmålen av EU och det ständigt ökande energibehovet. Under de senare åren har intresset riktats mot termisk energiutvinning ur olika restfraktioner och avfall. Vid produktion av fordonsbränsle ur biomassa är den fasta restprodukten ofta den största procesströmmen i produktionsanläggningen. En riktig hantering av restprodukterna skulle göra produktionen mera lönsam och mer ekologiskt hållbar. Ett alternativ är att genom förbränning producera elektricitet och/eller värme eftersom dessa restprodukter anses som CO2-neutrala. Målsättningen med den här avhandlingen var att studera förbränningsegenskaperna hos några fasta restprodukter som uppstår vid framställning av förnybara fordonsbränslen. De fyra undersökta materialen är rapskaka, palmkärnskaka, torkad drank och stabiliserat rötslam. I studien används ett stort urval av undersökningsmetoder, från laboratorieskala till fullskalig förbränning, för att identifiera de huvudsakliga utmaningarna förknippade med förbränning av restprodukterna i pannor med fluidiserad bäddteknik. Med hjälp av detaljerad bränslekarakterisering kunde restprodukterna konstateras vara en värdefull källa för värme- och elproduktion. Den kemiska sammansättningen av restprodukterna varierar stort jämfört med mera traditionellt använda biobränslen. En gemensam faktor för alla de studerade restprodukterna är en hög fosforhalt. På grund av de låga fosforkoncentrationerna i de traditionella biobränslena har grundämnet hittills inte ansetts spela någon större roll i askkemin. Experimenten visade nu att fosfor inte mera kan försummas då man studerar kemin i förbränningsprocesser, då allt flera fosforrika bränslen tränger in på energimarknaden.

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

Characterization Of Process Efficiency Improvement In Laser Additive Manufacturing

Laser additive manufacturing (LAM), known also as 3D printing, has gained a lot of interest in past recent years within various industries, such as medical and aerospace industries. LAM enables fabrication of complex 3D geometries by melting metal powder layer by layer with laser beam. Research in laser additive manufacturing has been focused in development of new materials and new applications in past 10 years. Since this technology is on cutting edge, efficiency of manufacturing process is in center role of research of this industry. Aim of this thesis is to characterize methods for process efficiency improvements in laser additive manufacturing. The aim is also to clarify the effect of process parameters to the stability of the process and in microstructure of manufactured pieces. Experimental tests of this thesis were made with various process parameters and their effect on build pieces has been studied, when additive manufacturing was performed with a modified research machine representing EOSINT M-series and with EOS EOSINT M280. Material used was stainless steel 17-4 PH. Also, some of the methods for process efficiency improvements were tested. Literature review of this thesis presents basics of laser additive manufacturing, methods for improve the process efficiency and laser beam – material- interaction. It was observed that there are only few public studies about process efficiency of laser additive manufacturing of stainless steel. According to literature, it is possible to improve process efficiency with higher power lasers and thicker layer thicknesses. The process efficiency improvement is possible if the effect of process parameter changes in manufactured pieces is known. According to experiments carried out in this thesis, it was concluded that process parameters have major role in single track formation in laser additive manufacturing. Rough estimation equations were created to describe the effect of input parameters to output parameters. The experimental results showed that the WDA (width-depth-area of cross-sections of single track) is correlating exponentially with energy density input. The energy density input is combination of the input parameters of laser power, laser beam spot diameter and scan speed. The use of skin-core technique enables improvement of process efficiency as the core of the part is manufactured with higher laser power and thicker layer thickness and the skin with lower laser power and thinner layer thickness in order to maintain high resolution. In this technique the interface between skin and core must have overlapping in order to achieve full dense parts. It was also noticed in this thesis that keyhole can be formed in LAM process. It was noticed that the threshold intensity value of 106 W/cm2 was exceeded during the tests. This means that in these tests the keyhole formation was possible.

The developmental anatomy of the subterranean system in Mandevilla illustris (Vell.) Woodson and M. velutina (Mart. ex Stadelm.) Woodson (Apocynaceae)

Two species of Mandevilla from the savanna area of São Paulo State, Brazil were studied. These species have been prescribed as folk medicine as infusions or alcoholic extracts of the underground system for treatment of venomous snake bites. To explain the morphological nature of such a system, its ontogeny was described to determine which parts are involved in its formation. In both Mandevilla species examined, the underground system consists of a xylopodium whose basal region joins a tuberous root.

Communicating trustworthiness in online stores

Even though e-commerce systems are expected to have many advantages compared to the traditional ways of doing business, it is not always the reality. Lack of trust is still said to be one of the most important barriers to online shopping. In traditional stores, trust has usually been established in a direct contact between the customer and the company or its personnel. In online stores, there is no direct interaction. The purpose of this thesis is to identify the key antecedents to online trust and to distinguish between effective and ineffective practices. A model on how consumers establish initial trust towards an unknown online vendor was proposed based on previous theories. The model was tested empirically by targeting an online survey at higher degree students in Finland and in Germany. The data confirmed the proposed view that trusting intentions are affected by individual characteristics, characteristics of the company as well as characteristics of the website. Additionally national differences were found between Finnish and German respondents. The data suggested that online vendors can convey a message of trustworthiness by improving information quality and overall usefulness of the website. Perceived risk of online shopping was found to depend especially on general trust in the Internet, service quality and ease of use. A trustworthy online store should include several payment methods as well as means to access and modify given data. The vendors should also make sure that inquiries are addressed quickly, transactions are confirmed automatically and that customers have a possibility to track their order. A model that includes three different sources of trust should contribute to the theoretical understanding of trust formation in online stores. The resulting list of trust antecedents can also be used as a checklist when e-commerce practitioners wish to optimize the trust building.

Floral resources used by insects in a grassland community in Southern Brazil

The goal of the present study was to identify plant species used as food source, the floral resources utilized, and the insects that visit flowers in a grassland community in southern Brazil. The study was carried out in an area of one hectare, located in a grassland formation in the Parque Estadual de Itapuã, State of Rio Grande do Sul, Brazil. The flowering pattern was seasonal, and richness and abundance of insects was higher during the period of high resource availability. Flowers of 106 species of angiosperms (73 genera and 34 families) were used as source of floral resources for 219 species (2,767 specimens) of insects. A total of 91.5% of plant species were visited by bees, 53.8% by flies, 34.9% by wasps, 22.6% by butterflies, and 12.3% by beetles. Nectar was the main resource consumed by the visitors (41.1%). Asteraceae was the richest (38 spp.) and most visited family, with 63.1% of the species and 49.5% of all specimens of recorded insects. Bees were the most representative insects (33.2% spp., 65% indiv.), followed by flies (26.9% spp., 16.5% indiv.), wasps, butterflies and beetles. 40 plant species were considered important resources for the floral visitors' community, due to high number of, both, species and individuals recorded in their flowers. The family Asteraceae as a species set was the main floral resource used by insect visitors through the year and has great importance for the maintenance of populations of many species of bees, flies, wasps and butterflies in the studied area.

Reduction of nitrogen oxide emissions in lime kiln

Different nitrogen oxide removal technologies for rotary lime kiln are studied in this thesis, the main focus being in commercial technologies. Post-combustion methods are investigated in more detail as potential possible NOx removal with combustion methods in rotary lime kiln is more limited or primary methods are already in use. However, secondary methods as NOx scrubber, SNCR or SCR technologies are not listed as the Best Available Technologies defined by European Union. BAT technologies for NOx removal in lime kiln are (1) Optimised combustion and combustion control, (2) Good mixing of fuel and air, (3) Low-NOx burner and (4) Fuel selection/low-N fuel. SNCR method is the most suitable technique for NOx removal in lime kiln when NOx removal from 50 % to 70 % is required in case primary methods are already in use or cannot be applied. In higher removal cases ammonia slip is an issue in SNCR. By using SCR better NOx reduction can be achieved but issues with catalyst materials are expected to arise because of the dust and sulphur dioxide which leads to catalyst poison formation in lower flue gas temperatures. NOx scrubbing has potential when simultaneous NOx and SO2 removal is required. The challenge is that NO cannot be scrubbed directly, but once it is oxidized to NO2 or further scrubbing can be performed as the solubility of NO2 is higher. Commercial installations have not been made regarding SNCR, SCR or NOx scrubbing regarding rotary lime kiln. For SNCR and SCR the closest references come from cement industry.

Differential effects of lead and zinc on inhibitory avoidance learning in mice

We studied the effects of chronic intoxication with the heavy metals lead (Pb2+) and zinc (Zn2+) on memory formation in mice. Animals were intoxicated through drinking water during the pre- and postnatal periods and then tested in the step-through inhibitory avoidance memory task. Chronic postnatal intoxication with Pb2+ did not change the step-through latency values recorded during the 4 weeks of the test (ANOVA, P>0.05). In contrast, mice intoxicated during the prenatal period showed significantly reduced latency values when compared to the control group (day 1: q = 4.62, P<0.05; day 7: q = 4.42, P<0.05; day 14: q = 5.65, P<0.05; day 21: q = 3.96, P<0.05, and day 28: q = 6.09, P<0.05). Although chronic postnatal intoxication with Zn2+ did not alter a memory retention test performed 24 h after training, we noticed a gradual decrease in latency at subsequent 4-week intervals (F = 3.07, P<0.05), an effect that was not observed in the control or in the Pb2+-treated groups. These results suggest an impairment of memory formation by Pb2+ when the animals are exposed during the critical period of neurogenesis, while Zn2+ appears to facilitate learning extinction.

JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.

Building bone tissue: matrices and scaffolds in physiology and biotechnology

Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

Control of respiration in fish, amphibians and reptiles

Fish and amphibians utilise a suction/force pump to ventilate gills or lungs, with the respiratory muscles innervated by cranial nerves, while reptiles have a thoracic, aspiratory pump innervated by spinal nerves. However, fish can recruit a hypobranchial pump for active jaw occlusion during hypoxia, using feeding muscles innervated by anterior spinal nerves. This same pump is used to ventilate the air-breathing organ in air-breathing fishes. Some reptiles retain a buccal force pump for use during hypoxia or exercise. All vertebrates have respiratory rhythm generators (RRG) located in the brainstem. In cyclostomes and possibly jawed fishes, this may comprise elements of the trigeminal nucleus, though in the latter group RRG neurons have been located in the reticular formation. In air-breathing fishes and amphibians, there may be separate RRG for gill and lung ventilation. There is some evidence for multiple RRG in reptiles. Both amphibians and reptiles show episodic breathing patterns that may be centrally generated, though they do respond to changes in oxygen supply. Fish and larval amphibians have chemoreceptors sensitive to oxygen partial pressure located on the gills. Hypoxia induces increased ventilation and a reflex bradycardia and may trigger aquatic surface respiration or air-breathing, though these latter activities also respond to behavioural cues. Adult amphibians and reptiles have peripheral chemoreceptors located on the carotid arteries and central chemoreceptors sensitive to blood carbon dioxide levels. Lung perfusion may be regulated by cardiac shunting and lung ventilation stimulates lung stretch receptors.

5-Bromo-2’-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae)

5-Bromo-2’-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.

In vivo and in vitro anti-inflammatory and anti-nociceptive activities of lovastatin in rodents

Statins are among the most prescribed drugs in recent clinical practice. They are also known for their pleiotropic actions, which are independent of their lipid-lowering properties. The effect of lovastatin was investigated against carrageenan-induced paw edema in male Wistar rats (200-250 g) and on leukocyte migration, as measured by carrageenan-induced peritonitis in male Swiss mice (20-25 g), which are models of acute inflammation. Lovastatin (administered 1 h prior to carrageenan), at oral doses of 2, 5, and 10 mg/kg, markedly attenuated paw edema formation in rats at the 4th hour after carrageenan injection (25, 43, and 37% inhibition, respectively). Inhibitions of 20, 45 and 80% were observed in the leukocyte migration, as evaluated by carrageenan-induced peritonitis in mice with lovastatin doses of 0.5, 1 and 5 mg/kg, as compared to controls. Furthermore, lovastatin (administered 1 h before initiation) reduced the nociceptive effect of the formalin test in mice, at both phases, at doses of 2, 5, and 10 mg/kg: first phase (51, 65, and 70%, respectively) and second phase (73, 57, and 66% inhibition of licking time, respectively). The anti-nociceptive activity of lovastatin was inhibited by naloxone (3 mg/kg, sc). Lovastatin (0.01, 0.1, and 1 µg/mL) inhibited by 23, 79, and 86%, respectively, the release of myeloperoxidase from human neutrophils. Leukocyte (predominantly neutrophils) infiltration was almost completely reduced by lovastatin treatment, as observed in the model of acute paw edema with hematoxylin and eosin staining. In addition, lovastatin decreased the number of cells expressing tumor necrosis factor-α (TNF-α) and the inducible form of nitric oxide synthase (iNOS) activity. Therefore, the alterations in leukocyte activity and cytokine release could contribute to the anti-inflammatory activity of lovastatin.

BMP-2 and titanium particles synergistically activate osteoclast formation

A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.