Stanniocalcin-1 in cell stress and differentiation

Stanniocalcin-1 (STC-1) is a 56 kD homodimeric protein which was originally identified in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. STC-1 was considered unique to fish until the cloning of cDNA for human STC-1 in 1995 and mouse Stc-1 in 1996. STC-1 is conserved through evolution with human and salmon STC-1 sharing 60% identity and 80% similarity. The surprisingly high homology between mammalian and fish STC-1 and the protective actions of STC-1 in terminally differentiated neurons, originally reported by my colleagues, prompted me to further study the role of STC-1 in cell stress and differentiation. One purpose was to determine whether there is an inter-relationship between terminally differentiated cells and STC-1 expression. The study revealed an accumulation of STC-1 in mature megakaryocytes and adipocytes, i.e. postmitotic cells with limited or lost proliferative capacity. Still proliferating uninduced cells were negative for STC-1 mRNA and protein, whereas differentiating cells accumulated STC-1 in their cytoplasm. Interestingly, in liposarcomas the grade inversely correlated with STC-1 expression. Another aim was to study how STC-1 gene expression is regulated. Given that IL-6 is a cytokine with neuroprotective actions, by unknown mechanisms, we examined whether IL-6 regulates STC-1 gene expression. Treatment of human neural Paju cells with IL-6 induced a dose-dependent upregulation of STC-1 mRNA levels. This induction of STC-1 expression by IL-6 occurred mainly through the MAPK signaling pathway. Furthermore, I studied the role of IL-6-mediated STC-1 expression as a mechanism of cytoprotection conferred by hypoxic preconditioning (HOPC) in brain and heart. My findings show that Stc-1 was upregulated in brain after hypoxia treatment. In the brain of IL-6 deficient mice, however, no upregulation of Stc-1 expression was evident. After induced brain injury the STC-1 response in brains of IL-6 transgenic mice, with IL-6 overexpression in astroglial cells, was stronger than in brains of WT mice. These results indicate that IL-6-mediated expression of STC-1 is one molecular mechanism of HOPC-induced tolerance to brain ischemia. The protection conferred by HOPC in heart occurs during a bimodal time course comprising early and delayed preconditioning. Interestingly, my results showed that the expression of Stc-1 in heart was upregulated in a biphasic manner during HOPC. IL-6 deficient mice did not, however, show a similar biphasic manner of Stc-1 upregulation as did WT mice. Instead, only an early upregulation of Stc-1 expression was evident. The results suggest that the upregulation of Stc-1 during the delayed preconditioning is IL-6-dependent. The upregulated expression of Stc-1 during the early preconditioning, however, is only partly IL-6-dependent and possibly also directly mediated by HIF-1. These findings suggest that STC-1 is a pro-survival protein for terminally differentiated cells and that STC-1 expression may in fact be regulated by stress. In addition, I show that STC-1 gene upregulation, mediated in part by IL-6, is a new mechanism of protection conferred by HOPC in brain and heart. Because of its importance for fundamental biological processes, such as differentiation and cytoprotection, STC-1 may have therapeutic implications for management of stroke, neurodegenerative diseases, cancer, and obesity.

Molecular profiling of malignant pleural mesothelioma and pulmonary fibrosis

Malignant mesothelioma (MM) is a rare, usually incurable, disease mainly caused by former exposure to asbestos. Even though MM has a strong etiological link, genetic factors may play a role, since not all cases can be linked to former asbestos exposure. This thesis focuses on lung diseases, mainly malignant mesothelioma (MM), and idiopathic pulmonary fibrosis (IPF), which resembles asbestosis. The specific asbestos-related pathways associated with malignant as well as non-malignant lung diseases, still need to be clarified. Since most patients diagnosed with MM or asbestosis/fibrosis have a dismal prognosis and few therapeutic options are available, early diagnosis and better understanding of the disease pathogenesis are of the utmost importance. The first objective of this thesis was to identify asbestos specific differentially expressed genes. This was approached by using high-resolution gene expression arrays, and three different human lung cell lines, as well as with three different bioinformatics approaches. Since the first study aimed to elucidate potential early changes, the second study was used to screen DNA copy number changes in MM tumour samples. This was performed using genome wide microarrays for identification of DNA copy number changes characterstic for MM. Study III focused on the role of gremlin in the regulation of bone morphogenetic protein (BMPs) in IPF. Further studies were conducted in asbestos-exposed cell cultures as well as in an asbestos-induced mouse model. Furthermore, GATA-6 was studied in MM and metastatic pleural adenocarcinoma. The GATA transcription factors are important during embryonic development, but their role in cancer is still unclear. GATA-6 is a co-factor/target of thyroid transcription factor 1 (TTF-1), which is used in differential diagnostics of pleural MM and adenocarcinoma. Bioinformatics probed the genes and biological processes ordered in terms of significance, clusters, and highly enriched chromosomal regions. The study revealed several already identified targets, produced new ideas about genes which are central for asbestos exposure, as well as provided supplementary data for researchers to check their own novel findings or ideas. The analysis revealed DNA copy number changes characteristic for MM tumors. The most common regions of loss were detected in 1p, 3p, 6q, 9p, 13, 14, and 22, and gains at 17q. The histological features in asbestosis and IPF are very similar, wherefore IPF can be studied in asbestos models. The BMP antagonist gremlin was up-regulated by asbestos exposure in human epithelial cell lines, which was also observed in Study I. The transforming growth factor (TGF) -β and BMP expression and signaling activities were measured from murine and human fibrotic lungs. BMP-7 signaling was down-regulated in response to up-regulation of gremlin, and restoration of BMP-7 signaling prevented progression of fibrosis in mice. Therefore, the study suggests that the restoration of BMP-7 signaling in fibrotic lung could potentially aid in the treatment of IPF patients. Study IV revealed that GATA-6 was strongly expressed in the majority of the MM cases, and correlated statistically significant with longer survival in subgroups of MM.

Molecular Pathways of Disturbed Sleep and Depression: Studies on Adenosine and Gene Expression Patterns

Background: Adenosine is a potent sleep-promoting substance, and one of its targets is the basal forebrain. Fairly little is known about its mechanism of action in the basal forebrain and about the receptor subtype mediating its regulating effects on sleep homeostasis. Homeostatic deficiency might be one of the causes of the profoundly disturbed sleep pattern in major depressive disorder, which could explain the reduced amounts of delta-activity-rich stages 3 and 4. Since major depression has a relatively high heritability, and on the other hand adenosine regulates sleep homeostasis and might also be involved in mood modulation, adenosine-related genes should be considered for their possible contribution to a predisposition for depression and disturbed sleep in humans. Depression is a complex disorder likely involving the abnormal functioning of several genes. Novel target genes which could serve as the possible common substrates for depression and comorbid disturbed sleep should be identified. In this way specific brain areas related to sleep regulation should be studied by using animal model of depression which represents more homogenous phenotype as compared to humans. It is also important to study these brain areas during the development of depressive-like features to understand how early changes could facilitate pathophysiological changes in depression. Aims and methods: We aimed to find out whether, in the basal forebrain, adenosine induces recovery non-rapid eye movement (NREM) sleep after prolonged waking through the A1 or/and A2A receptor subtype. A1 and A2A receptor antagonists were perfused into the rat basal forebrain during 3 h of sleep deprivation, and the amount of NREM sleep and delta power during recovery NREM sleep were analyzed. We then explored whether polymorphisms in genes related to the metabolism, transport and signaling of adenosine could predispose to depression accompanied by signs of disturbed sleep. DNA from 1423 individuals representative of the Finnish population and including controls and cases with depression, depression accompanied by early morning awakenings and depression accompanied by fatigue, was used in the study to investigate the possible association between polymorphisms from adenosine-related genes and cases. Finally to find common molecular substrates of depression and disturbed sleep, gene expression changes were investigated in specific brain areas in the rat clomipramine model of depression. We focused on the basal forebrain of 3-week old clomipramine-treated rats which develop depressive-like symptoms later in adulthood and on the hypothalamus of adult female clomipramine-treated rats. Results: Blocking of the A1 receptor during sleep deprivation resulted in a reduction of the recovery NREM sleep amount and delta power, whereas A2A receptor antagonism had no effect. Polymorphisms in adenosine-related genes SLC29A3 (equilibrative nucleoside transporter type 3) in women and SLC28A1 (concentrative nucleoside transporter type 1) in men associated with depression alone as well as when accompanied by early morning awakenings and fatigue. In Study III the basal forebrain of postnatal rats treated with clomipramine displayed disturbances in gamma-aminobutyric acid (GABA) receptor type A signaling, in synaptic transmission and possible epigenetic changes. CREB1 was identified as a common transcription denominator which also mediates epigenetic regulation. In the hypothalamus the major changes included the expression of genes in GABA-A receptor pathway, K+ channel-related, glutamatergic and mitochondrial genes, as well as an overexpression of genes related to RNA and mRNA processing. Conclusions: Adenosine plays an important role in sleep homeostasis by promoting recovery NREM sleep via the A1 receptor subtype in the basal forebrain. Also adenosine levels might contribute to the risk of depression with disturbed sleep, since the genes encoding nucleoside transporters showed the strongest associations with depression alone and when accompanied by signs of disturbed sleep in both women and men. Sleep and mood abnormalities in major depressive disorder could be a consequence of multiple changes at the transcriptional level, GABA-A receptor signaling and synaptic transmission in sleep-related basal forebrain and the hypothalamus.

Characterization of the TRIM37 gene and mutations underlying mulibrey nanism

Mulibrey nanism is a hereditary developmental disorder, characterized by prenatal onset growth failure without postnatal catch-up growth, distinctive craniofacial features, progressive cardiopathy and failure of sexual maturation. In addition, the patients develop insulin resistance syndrome and type 2 diabetes and they have an increased risk of developing tumors. The TRIM37 gene that underlies mulibrey nanism encodes for a member of the tripartite motif (TRIM) protein family. The physiological function of TRIM37 and the pathogenetic mechanisms leading from TRIM37 dysfunction to the mulibrey nanism phenotype are unknown. However, TRIM37 localizes at least partially to peroxisomes, and possesses ubiquitin E3-ligase activity. Thus, it may mediate ubiquitin dependent protein degradation, suggesting that accumulation of yet unknown substrate proteins may underlie the disease pathogenesis. In this study, the TRIM37 gene was characterized in detail. A transcription initiation window, with several separate transcription start sites, was identified and the putative promoter region immediately upstream from the transcription initiation window was shown to possess basal promoter activity. Further, several alternative splice variants of the gene were identified, including a highly expressed testis specific variant, encoding for an identical protein product with the main transcript. Expression of TRIM37 mRNA was detected in several different tissues, with highest expression seen in testis and in brain, when the expression patterns of the two major transcripts in different human tissues were studied by quantitative real-time PCR. Several mulibrey nanism patients were studied and thirteen novel mutations in TRIM37 were found, including three mutations (p.Gly322Val, p.Cys109Ser, p.Glu271_Ser287), that are likely to express mutant TRIM37 proteins. These mutations were further shown to alter the subcellular localization of the mutant proteins. Most of the mulibrey nanism associated mutations however, lead to premature termination codons and degradation of mRNA. All the TRIM37 mutations identified to date predict loss-of-function alleles, and thus no phenotype-genotype correlation is seen among the patients. In order to understand the pathogenetic mechanisms underlying mulibrey nanism, an animal model for the disorder is needed. For the development of a Trim37 knock-out mouse, the mouse Trim37 gene was characterized. Alternative splice variants, were identified, including a testis specific variant predicting a longer protein product. Further, a strictly tissue and cell-specific pattern of Trim37 expression was observed in developing and adult mouse tissues, when studied by immunohistochemical methods. This distribution of Trim37 expression in mouse tissues is in agreement with the clinical findings in human mulibrey nanism patients. This thesis work gives new tools for the diagnostics of mulibrey nanism as well as for studying the molecular pathogenesis behind this interesting disorder.

Molecular biology of progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by age of onset at 6-15 years, stimulus-sensitive myoclonus, tonic-clonic epileptic seizures and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common mutation underlying EPM1 is a dodecamer repeat expansion in the promoter region of CSTB. In addition, nine other mutations have been identified. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed inhibitor of cathepsins, but its physiological function is unknown. The purpose of this study was to investigate CSTB gene expression and CSTB protein function in normal and pathological conditions. The basal CSTB promoter was mapped and characterized using different promoter-luciferase gene constructs. The binding activity of transcription factors to one ARE half, five Sp1 and four AP1 sites in the CSTB promoter was demonstrated. The CSTB promoter activity was clearly decreased using a CSTB promoter with "premutation" repeat expansions and in individuals with alike expansions. The expression of CSTB mRNA and protein was markedly reduced in patient cells. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes, and in differentiated cells merely to the cytoplasm. This suggests that the subcellular distribution of CSTB is dependent on the differentation status of the cells. The proteins representing patient missense mutations failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. Several alternatively spliced CSTB isoforms were identified. Of these CSTB2 was widely expressed with very low levels whereas the other alternatively spliced forms seemed to have limited tissue expression. In patients CSTB2 expression was reduced similarly to that of CSTB. The physiological relevance of CSTB alternative splicing remains unknown. The mouse Cstb transcript was shown to be present in all embryonic stages and adult tissues examined. The expression was highest at embryonic day 7 and in thymus, as well as in postnatal brain in the cortex, caudate putamen, thalamus, hippocampus, and in the Purkinje cell layer of the cerebellum. Our data implies that CSTB expression is tightly temporally and spatially regulated. The data presented in my thesis lay the basis for further understanding of the role of CSTB in health and disease.

Differential thermal analysis of potassium perchlorate

A study has been made of the differential thermal analysis of (i) potassium perchlorate in powdered form, (ii) potassium perchlorate in pelletized form, (iii) potassium perchlorate recrystallized from liquid NH3, and (iv) potassium perchlorate preheated for 24 hours at 375°. Pretreatment of potassium perchlorate leads to a desensitization of both endothermic and exothermic processes. Additionally, the pretreatment tends to convert the symmetric exotherm into an asymmetric exotherm due to merging of the two exotherms. An analysis of the factors causing asymmetry in the exotherm has thrown fresh light on the mechanism of thermal decomposition of potassium perchlorate.

The virulence of Finnish Pyrenophora teres f. teres isolates and its implications for resistance breeding

In Finland, barley, Hordeum vulgare L., covers 50 % of the total acreage devoted to cereal cultivation. The most common disease of barley in Finland is net blotch, a foliar disease caused by the ascomycete Pyrenophora teres Drechsler. Disease resistance based on plant genes is an environmentally friendly and economical way to manage plant diseases caused by biotic stresses. Development of a disease resistance breeding programme is dependent on knowledge of the pathogen. In addition to information on the epidemiology and virulence of a pathogen, knowledge on how the pathogen evolves and the nature of the risks that might arise in the future are essential issues that need to be taken into account to achieve the final breeding aims. The main objectives of this study were to establish reliable and efficient testing methods for Pyrenophora teres f. teres virulence screening, and to understand the role of virulence of P. teres f. teres in Finland from a disease resistance breeding point of view. The virulence of P. teres was studied by testing 239 Finnish P. teres f. teres isolates collected between 1994 2007 originating from 19 locations, and 200 P. teres progeny isolates originating from artificially produced P. teres matings. According to the results of this study, screening for P. teres f. teres isolates on barley seedlings under greenhouse conditions is a feasible and cost efficient method to describe the virulence spectrum of the pathogen. Inoculum concentration and the seedling leaf used to gauge virulence had significant effects. Barley grain size, morphological traits of P. teres isolates, spore production and growth rate on agar did not affect the expression of virulence. A common barley differential set to characterize the P. teres virulence was developed and is recommended to be used globally. The virulence spectrum of Finnish P. teres f. teres isolates collected in 1994-2007 was constant both within and between the years. The results indicated differences in the pathogen s aggressiveness and in barley genotypes resistance. However, differences in virulence were rarely significant. Unlike in laboratory conditions, no indications of changes in virulence caused by the sexual reproduction have been observed in Finnish barley fields. In Finland, durable net blotch resistance has been achieved by introducing resistance from other barley varieties using traditional crossing methods, including wide crossing, and testing the breeding material at early generations at several sites under natural infection pressure. Novel resistance is available, which is recommended to minimize the risk of selection of virulent isolates and breakdown of currently deployed resistance.

Group portrait, 10th Anniversary Dinner, Display Novelties Co. Ltd.; New York.

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Regulation of strawberry growth and development

Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.

Preparation and characterization of potato starch films plasticized with polyols

The present study investigated the potato starches and polyols which were used to prepare edible films. The amylose content and the gelatinization properties of various potato starches extracted from different potato cultivars were determined. The amylose content of potato starches varied between 11.9 and 20.1%. Onset temperatures of gelatinization of potato starches in excess water varied independently of the amylose content from 58 to 61°C determined using differential scanning calorimetry (DSC). The crystallinity of selected native starches with low, medium and high amylose content was determined by X-ray diffraction. The relative crystallinity was found to be around 10 13% in selected native potato starches containing 13 17% water. The glass transition temperature, crystallization melting behavior and relaxations of polyols, erythritol, sorbitol and xylitol, were determined using (DSC), dielectric analysis (DEA) and dynamic mechanical analysis (DMA). The glass transition temperatures of xylitol and sorbitol decreased as a result of water plasticization. Anhydrous amorphous erythritol crystallized rapidly. Edible films were obtained from solutions containing gelatinized starch, plasticizer (polyol or binary polyol mixture) and water by casting and evaporating water at 35°C. The present study investigated effects of plasticizer type and content on physical and mechanical properties of edible films stored at various relative water vapor pressures (RVP). The crystallinity of edible films with low, medium and high amylose content was determined by X-ray diffraction and they were found to be practically amorphous. Water sorption and water vapor permeability (WVP) of films was affected by the type and content of plasticizer. Water vapor permeability of films increased with increasing plasticizer content and storage RVP. Generally, Young's modulus and tensile strength decreased with increasing plasticizer and water content with a concurrent increase in elongation at break of films. High contents of xylitol and sorbitol resulted in changes in physical and mechanical properties of films probably due to phase separation and crystallization of xylitol and sorbitol which was not observed when binary polyol mixtures were used as plasticizers. The mechanical properties and the water vapor permeability (WVP) of the films were found to be independent of the amylose content.

Lost in Translation : Translation Mechanisms in Production of Cocksfoot Mottle Virus Proteins

The present study focuses on the translational strategies of Cocksfoot mottle virus (CfMV, genus Sobemovirus), which infects monocotyledonous plants. CfMV RNA lacks the 5'cap and the 3'poly(A) tail that ensure efficient translation of cellular messenger RNAs (mRNAs). Instead, CfMV RNA is covalently linked to a viral protein VPg (viral protein, genome-linked). This indicates that the viral untranslated regions (UTRs) must functionally compensate for the lack of the cap and poly(A) tail. We examined the efficacy of translation initiation in CfMV by comparing it to well-studied viral translational enhancers. Although insertion of the CfMV 5'UTR (CfMVe) into plant expression vectors improved gene expression in barley more than the other translational enhancers examined, studies at the RNA level showed that CfMVe alone or in combination with the CfMV 3'UTR did not provide the RNAs translational advantage. Mutation analysis revealed that translation initiation from CfMVe involved scanning. Interestingly, CfMVe also promoted translation initiation from an intercistronic position of dicistronic mRNAs in vitro. Furthermore, internal initiation occurred with similar efficacy in translation lysates that had reduced concentrations of eukaryotic initiation factor (eIF) 4E, suggesting that initiation was independent of the eIF4E. In contrast, reduced translation in the eIF4G-depleted lysates indicated that translation from internally positioned CfMVe was eIF4G-dependent. After successful translation initiation, leaky scanning brings the ribosomes to the second open reading frame (ORF). The CfMV polyprotein is produced from this and the following overlapping ORF via programmed -1 ribosomal frameshift (-1 PRF). Two signals in the mRNA at the beginning of the overlap program approximately every fifth ribosome to slip one nucleotide backwards and continue translation in the new -1 frame. This leads to the production of C-terminally extended polyprotein, which encodes the viral RNA-dependent RNA polymerase (RdRp). The -1 PRF event in CfMV was very efficient, even though it was programmed by a simple stem-loop structure instead of a pseudoknot, which is usually required for high -1 PRF frequencies. Interestingly, regions surrounding the -1 PRF signals improved the -1 PRF frequencies. Viral protein P27 inhibited the -1 PRF event in vivo, putatively by binding to the -1 PRF site. This suggested that P27 could regulate the occurrence of -1 PRF. Initiation of viral replication requires that viral proteins are released from the polyprotein. This is catalyzed by viral serine protease, which is also encoded from the polyprotein. N-terminal amino acid sequencing of CfMV VPg revealed that the junction of the protease and VPg was cleaved between glutamate (E) and asparagine (N) residues. This suggested that the processing sites used in CfMV differ from the glutamate and serine (S) or threonine (T) sites utilized in other sobemoviruses. However, further analysis revealed that the E/S and E/T sites may be used to cleave out some of the CfMV proteins.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Liquid bubble manometer for low differential heads

The development of a highly sensitive liquid bubble manometer which can measure low differential heads to an accuracy of 0.01 mm of water is reported in this paper. The liquid bubble consists of two miscible liquids,benzaldehyde and normal hexane (each of which is immiscible in water) in such a proportion that the bubble density is within ±2 % of the density of water. The movement of the liquid bubble, which occupies the full cross-sectional area of the glass tube containing water in the manometer, is indicative of the applied differential head to a magnified scale. The manometer is found to give excellent results in open channel flow and is recommended for use for differential heads up to 2 cm of water. The manometer is economical, simple in fabrication and with simple modifications the sensitivity of the manometer can be increased to more than 0.01 mm of water.

Tailoring polyolefin properties by metallocene catalysis

This doctoral thesis deals with the syntheses of olefin homo- and copolymers using different kind of metallocene catalyst. Ethene, propene, 1-hexene, 1-hexadecene, vinylcyclohexane and phenylnorbornene were homo- or copolymerized with the catalysts. The unbridged benzyl substituted zirconium dichloride catalysts (1-4), ansa- bridged acenaphtyl substituted zirconium dichloride catalysts, ( 5, 6), rac- and meso-ethylene-bis(1-indenyl)zirconium dichlorides, (rac- and meso-8), rac-ethylene-bis(1-indenyl)hafnium dichloride, ( 12), bis(9-fluorenyl)hafnium dichloride (14 ) enantiomerically pure (R)- phenylethyl[(9-fluorenyl-1-indenyl)]ZrCl2, (11), 14 and asymmetric dimethylsilyl[(3-benzylindenyl-(2-methylbenzen[e]indenyl)] zirconium dichloride, (13), were prepared in our laboratory. Dimethylsilyl-bis(1-indenyl)zirconium dichloride, (9), isopropylidene(9-fluorenyl-cyclopentadienyl)zirconium dichloride, (10), and were obtained commercially. The solid-state structures of the catalysts rac- and meso-1 were determined by X-ray crystallography. Computational methods were used for the structure optimization of the catalyst rac- and meso-1 in order to compare the theoretical calculations with the experimental results. Polymerization experiments were conducted in a highly purified autoclave system using low pressures (< 5 bar) of gaseous monomers. The experiments were designed to attain the optimal catalytic activity and a uniform copolymer composition. The prepared homo- and copolymers were characterized by the gel permeation chromatography, GPC, differential scanning calorimetry, DSC, nuclear magnetic resonance, NMR, and Fourier transform infrared spectrometry, FTIR . Molar mass (Mw, Mn), molar mass distribution (Mw/Mn), tacticity, comonomer content, melting temperature, glass transition temperature, and end group structures and content were determined. A special attention was paid on the correlation of the polymer properties with the catalyst structures and polymerization conditions. An intramolecular phenyl coordination was found in phenyl substituted benzyl zirconocenes 1-3 explaining the decreased activity of the catalysts. Novel copolymers poly(propene-co-phenylnorbornene) and poly(propene co-vinylcyclohexane), were synthesized and high molar mass poly(ethene-co-1-hexene) and poly(ethene-co-1-hexadecene) copolymers with elastic properties were prepared. Activation of a hafnocene catalyst was studied with UV-Vis spectrometry and activation process for the synthesis of ultra high molar mass poly(1-hexene) was found out.