The role of cathepsin K in lung development and newborn chronic lung injury.

Chronic lung diseases, specifically bronchopulmonary dysplasia (BPD), are still causing mortality and morbidity amongst newborn infants. High protease activity has been suggested to have a deleterious role in oxygen-induced lung injuries. Cathepsin K (CatK) is a potent protease found in fetal lungs, degrading collagen and elastin. We hypothesized that CatK may be an important modulator of chronic lung injury in newborn infants and neonatal mice. First we measured CatK protein levels in repeated tracheal aspirate fluid samples from 13 intubated preterm infants during the first two weeks of life. The amount of CatK at 9-13 days was low in infants developing chronic lung disease. Consequently, we studied CatK mRNA expression in oxygen-exposed wild-type (WT) rats at postnatal day (PN) 14 and found decreased pulmonary mRNA expression of CatK in whole lung samples. Thereafter we demonstrated that CatK deficiency modifies lung development by accelerating the thinning of alveolar walls in newborn mice. In hyperoxia-exposed newborn mice CatK deficiency resulted in increased number of pulmonary foam cells, macrophages and amount of reduced glutathione in lung homogenates indicating intensified pulmonary oxidative stress and worse pulmonary outcome due to CatK deficiency. Conversely, transgenic overexpression of CatK caused slight enlargement of distal airspaces with increased alveolar chord length in room air in neonatal mice. While hyperoxic exposure inhibited alveolarization and resulted in enlarged airspaces in wild-type mice, these changes were significantly milder in CatK overexpressing mice at PN7. Finally, we showed that the expression of macrophage scavenger receptor 2 (MSR2) mRNA was down-regulated in oxygen-exposed CatK-deficient mice analyzed by microarray analysis. Our results demonstrate that CatK seems to participate in normal lung development and its expression is altered during pulmonary injury. In the presence of pulmonary risk factors, like high oxygen exposure, low amount of CatK may contribute to aggravated lung injury while sustained or slightly elevated amount of CatK may even protect the newborn lungs from excessive injury. Besides collagen degrading and antifibrotic function of CatK in the lungs, it is obvious that CatK may affect macrophage activity and modify oxidative stress response. In conclusion, pulmonary proteases, specifically CatK, have distinct roles in lung homeostasis and injury development, and although suggested, broad range inhibition of proteases may not be beneficial in newborn lung injury.

The interplay of JNK and SCG10 during cortical development and in excitotoxic responses in brain

Initially identified as stress activated protein kinases (SAPKs), the c-Jun Nterminal kinases (JNKs) are currently accepted as potent regulators of various physiologically important cellular events. Named after their competence to phosphorylate transcription factor c-Jun in response to UVtreatment, JNKs play a key role in cell proliferation, cell death or cell migration. Interestingly, these functions are crucial for proper brain formation. The family consists of three JNK isoforms, JNK1, JNK2 and JNK3. Unlike brain specific JNK3 isoform, JNK1 and JNK2 are ubiquitously expressed. It is estimated that ten splice variants exist. However, the detailed cellular functions of these remain undetermined. In addition, physiological conditions keep the activities of JNK2 and JNK3 low in comparison with JNK1, whereas cellular stress raises the activity of these isoforms dramatically. Importantly, JNK1 activity is constitutively high in neurons, yet it does not stimulate cell death. This suggests a valuable role for JNK1 in brain development, but also as an important mediator of cell wellbeing. The aim of this thesis was to characterize the functional relationship between JNK1 and SCG10. We found that SCG10 is a bona fide target for JNK. By employing differential centrifugation we showed that SCG10 co-localized with active JNK, MKK7 and JIP1 in a fraction containing endosomes and Golgi vesicles. Investigation of JNK knockout tissues using phosphospecific antibodies recognizing JNK-specific phosphorylation sites on SCG10 (Ser 62/Ser 73) showed that phosphorylation of endogenous SCG10 was dramatically decreased in Jnk1-/- brains. Moreover, we found that JNK and SCG10 co-express during early embryonic days in brain regions that undergo extensive neuronal migration. Our study revealed that selective inhibition of JNK in the cytoplasm significantly increased both the frequency of exit from the multipolar stage and radial migration rate. However, as a consequence, it led to ill-defined cellular organization. Furthermore, we found that multipolar exit and radial migration in Jnk1 deficient mice can be connected to changes in phosphorylation state of SCG10. Also, the expression of a pseudo-phosphorylated mutant form of SCG10, mimicking the JNK1- phopshorylated form, brings migration rate back to normal in Jnk1 knockout mouse embryos. Furthermore, we investigated the role of SCG10 and JNK in regulation of Golgi apparatus (GA) biogenesis and whether pathological JNK action could be discernible by its deregulation. We found that SCG10 maintains GA integrity as with the absence of SCG10 neurons present more compact fragmented GA structure, as shown by the knockdown approach. Interestingly, neurons isolated from Jnk1-/- mice show similar characteristics. Block of ER to GA is believed to be involved in development of Parkinson's disease. Hence, by using a pharmacological approach (Brefeldin A treatment), we showed that GA recovery is delayed upon removal of the drug in Jnk1-/- neurons to an extent similar to the shRNA SCG10-treated cells. Finally, we investigated the role of the JNK1-SCG10 duo in the maintenance of GA biogenesis following excitotoxic insult. Although the GA underwent fragmentation in response to NMDA treatment, we observed a substantial delay in GA disintegration in neurons lacking either JNK1 or SCG10.

Perinatal development and adult blood pressure

A growing body of evidence supports the concept of fetal programming in cardiovascular disease in man, which asserts that an insult experienced in utero exerts a long-term influence on cardiovascular function, leading to disease in adulthood. However, this hypothesis is not universally accepted, hence animal models may be of value in determining potential physiological mechanisms which could explain how fetal undernutrition results in cardiovascular disease in later life. This review describes two major animal models of cardiovascular programming, the in utero protein-restricted rat and the cross-fostered spontaneously hypertensive rat. In the former model, moderate maternal protein restriction during pregnancy induces an increase in offspring blood pressure of 20-30 mmHg. This hypertensive effect is mediated, in part, by fetal exposure to excess maternal glucocorticoids as a result of a deficiency in placental 11-ß hydroxysteroid dehydrogenase type 2. Furthermore, nephrogenesis is impaired in this model which, coupled with increased activity of the renin-angiotensin system, could also contribute to the greater blood pressure displayed by these animals. The second model discussed is the cross-fostered spontaneously hypertensive rat. Spontaneously hypertensive rats develop severe hypertension without external intervention; however, their adult blood pressure may be lowered by 20-30 mmHg by cross-fostering pups to a normotensive dam within the first two weeks of lactation. The mechanisms responsible for this antihypertensive effect are less clear, but may also involve altered renal function and down-regulation of the renin-angiotensin system. These two models clearly show that adult blood pressure is influenced by exposure to one of a number of stimuli during critical stages of perinatal development.

The role of reelin in the development and evolution of the cerebral cortex

Reelin is an extracellular matrix protein that is defective in reeler mutant mice and plays a key role in the organization of architectonic patterns, particularly in the cerebral cortex. In mammals, a "reelin signal" is activated when reelin, secreted by Cajal-Retzius neurons, binds to receptors of the lipoprotein receptor family on the surface of cortical plate cells, and triggers Dab1 phosphorylation. As reelin is a key component of cortical development in mammals, comparative embryological studies of reelin expression were carried out during cortical development in non-mammalian amniotes (turtles, squamates, birds and crocodiles) in order to assess the putative role of reelin during cortical evolution. The data show that reelin is present in the cortical marginal zone in all amniotes, and suggest that reelin has been implicated in the evolution of the radial organization of the cortical plate in the synapsid lineage leading from stem amniotes to mammals, as well as in the lineage leading to squamates, thus providing an example of homoplastic evolution (evolutionary convergence). The mechanisms by which reelin instructs radial cortical organization in these two lineages seem different: in the synapsid lineage, a drastic amplification of reelin production occurred in Cajal-Retzius cells, whereas in squamates, in addition to reelin-secreting cells in the marginal zone, a second layer of reelin-producing cells developed in the subcortex. Altogether, our results suggest that the reelin-signaling pathway has played a significant role in shaping the evolution of cortical development.

Visual maps in the adult primate cerebral cortex: some implications for brain development and evolution

In this paper, the topology of cortical visuotopic maps in adult primates is reviewed, with emphasis on recent studies. The observed visuotopic organisation can be summarised with reference to two basic rules. First, adjacent radial columns in the cortex represent partially overlapping regions of the visual field, irrespective of whether these columns are part of the same or different cortical areas. This primary rule is seldom, if ever, violated. Second, adjacent regions of the visual field tend to be represented in adjacent radial columns of a same area. This rule is not as rigid as the first, as many cortical areas form discontinuous, second-order representations of the visual field. A developmental model based on these physiological observations, and on comparative studies of cortical organisation, is then proposed, in order to explain how a combination of molecular specification steps and activity-driven processes can generate the variety of visuotopic organisations observed in adult cortex.

Innovation process development and business process integration - a multiple case study

The main objective of this study was to find out the bases for innovation model formulation in an existing organization based on cases. Innovation processes can be analyzed based on their needs and based on their emphasis on the business model development or R&D. The research was conducted in energy sector within one company by utilizing its projects as cases for the study. It is typical for the field of business that development is slow, although the case company has put emphasis on its innovation efforts. Analysis was done by identifying the cases’ needs and comparing them. The results were that because of the variances in the needs of the cases, the applicability of innovation process models varies. It was discovered that by dividing the process into two phases, a uniform model could be composed. This model would fulfill the needs of the cases and potential future projects as well.

Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

Lead (Pb2+) poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g) before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM) to the bath. Changes in rate of stimulation (0.1-1.5 Hz), relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM), and the effect of isoproterenol (20 ng/mL) were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz) and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

Severity score system for progressive myelopathy: development and validation of a new clinical scale

Progressive myelopathies can be secondary to inborn errors of metabolism (IEM) such as mucopolysaccharidosis, mucolipidosis, and adrenomyeloneuropathy. The available scale, Japanese Orthopaedic Association (JOA) score, was validated only for degenerative vertebral diseases. Our objective is to propose and validate a new scale addressing progressive myelopathies and to present validating data for JOA in these diseases. A new scale, Severity Score System for Progressive Myelopathy (SSPROM), covering motor disability, sphincter dysfunction, spasticity, and sensory losses. Inter- and intra-rater reliabilities were measured. External validation was tested by applying JOA, the Expanded Disability Status Scale (EDSS), the Barthel index, and the Osame Motor Disability Score. Thirty-eight patients, 17 with adrenomyeloneuropathy, 3 with mucopolysaccharidosis I, 3 with mucopolysaccharidosis IV, 2 with mucopolysaccharidosis VI, 2 with mucolipidosis, and 11 with human T-cell lymphotropic virus type-1 (HTLV-1)-associated myelopathy participated in the study. The mean ± SD SSPROM and JOA scores were 74.6 ± 11.4 and 12.4 ± 2.3, respectively. Construct validity for SSPROM (JOA: r = 0.84, P < 0.0001; EDSS: r = -0.83, P < 0.0001; Barthel: r = 0.56, P < 0.002; Osame: r = -0.94, P < 0.0001) and reliability (intra-rater: r = 0.83, P < 0.0001; inter-rater: r = 0.94, P < 0.0001) were demonstrated. The metric properties of JOA were similar to those found in SSPROM. Several clinimetric requirements were met for both SSPROM and JOA scales. Since SSPROM has a wider range, it should be useful for follow-up studies on IEM myelopathies.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles culturedin vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Development and applications of the SWAN rating scale for assessment of attention deficit hyperactivity disorder: a literature review

This study reviewed the use of the Strengths and Weaknesses of Attention-Deficit/Hyperactivity-symptoms and Normal-behaviors (SWAN) rating scale in diagnostic and evolutive approaches to attention deficit hyperactivity disorder (ADHD) and in correlational studies of the disorder. A review of articles published in indexed journals from electronic databases was conducted and 61 articles on the SWAN scale were analyzed. From these, 27 were selected to a) examine use of SWAN in research on attention disorders and b) verify evidence of its usefulness in the areas of genetics, neuropsychology, diagnostics, psychiatric comorbidities, neuroimaging, pharmacotherapy, and to examine its statistical reliability and validity in studies of diverse populations. This review of articles indicated a growing use of the SWAN scale for diagnostic purposes, for therapy, and in research on areas other than ADHD, especially when compared with other reliable scales. Use of the scale in ADHD diagnosis requires further statistical testing to define its psychometric properties.

Development and characterization of edible films based on gluten from semi-hard and soft Brazilian wheat flours (development of films based on gluten from wheat flours)

Edible films based on gluten from four types of Brazilian wheat gluten (2 "semi-hard" and 2 "soft") were prepared and mechanical and barrier properties were compared with those of wheat gluten films with vital gluten. Water vapor, oxygen permeability, tensile strength and percent elongation at break, solubility in water and surface morphology were measured. The films from "semi-hard" wheat flours showed similar water vapor permeability and solubility in water to films from vital gluten and better tensile strength than the films from "soft" and vital gluten. The films from vital gluten had higher elongation at break and oxygen permeability and also lower solubility in water than the films from the Brazilian wheat "soft" flours. In spite of the vital gluten showed greater mechanical resistance, desirable for the bakery products, for the purpose of developing gluten films Brazilian "semi-hard" wheat flours can be used instead of vital gluten, since they showed similar barrier and mechanical properties.

Structural dynamics in the global pulp and paper industry

The pulp and paper industry is currently facing broad structural changes due to global shifts in demand and supply. These changes have significant impacts on national economies worldwide. Planted forests (especially eucalyptus) and recovered paper have quickly increased their importance as raw material for paper and paperboard production. Although advances in information and communication technologies could reduce the demand for communication papers, and the growth of paper consumption has indeed flattened in developed economies, particularly in North America and Western Europe, the consumption is increasing on a global scale. Moreover, the focal point of production and consumption is moving from the Western world to the rapidly growing markets of Southeast Asia. This study analyzes how the so-called megatrends (globalization, technological development, and increasing environmental awareness) affect the pulp and paper industry’s external environment, and seeks reliable ways to incorporate the impact of the megatrends on the models concerning the demand, trade, and use of paper and pulp. The study expands current research in several directions and points of view, for example, by applying and incorporating several quantitative methods and different models. As a result, the thesis makes a significant contribution to better understand and measure the impacts of structural changes on the pulp and paper industry. It also provides some managerial and policy implications.