Inorganic Nanoparticles and the Immune System: Detection, Selective Activation and Tolerance

The immune system is the responsible for body integrity and prevention of external invasion. On one side, nanoparticles are no triggers that the immune system is prepared to detect, on the other side it is known that foreign bodies, not only bacteria, viruses and parasites, but also inorganic matter, can cause various pathologies such as silicosis, asbestosis or inflammatory reactions. Therefore, nanoparticles entering the body, after interaction with proteins, will be either recognized as self-agents or detected by the immune system, encompassing immunostimulation or immunosuppression responses. The nature of these interactions seems to be dictated not specially by the composition of the material but by modifications of NP coating (composition, surface charge and structure). Herein, we explore the use of gold nanoparticles as substrates to carry multifunctional ligands to manipulate the immune system in a controlled manner, from undetection to immunostimulation. Murine bone marrow macrophages can be activated with artificial nanometric objects consisting of a gold nanoparticle functionalized with peptides. In the presence of some conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines were induced. The biochemical type of response depended on the type of conjugated peptide and was correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical NP conjugate design to either activate the immune system or hide from it, in order to reach their targets before being removed by phagocytes. Additionally, it opens up the possibility to modulate the immune response in order to suppress unwanted responses resulting from autoimmunity, or allergy or to stimulate protective responses against pathogens.

Fatigue analysis of brackets with and without scallop for bridge and other deck structure using effective notch stress.

Due to functional requirement of a structural detail brackets with and without scallop are frequently used in bridges, decks, ships and offshore structure. Scallops are designed to serve as passage way for fluids, to reduce weld length and plate distortions. Moreover, scallops are used to avoid intersection of two or more welds for the fact that there is the presence of inventible inherent initial crack except for full penetrated weld and the formation of multi-axial stress state at the weld intersection. Welding all around the scallop corner increase the possibility of brittle fracture even for the case the bracket is not loaded by primary load. Avoiding of scallop will establish an initial crack in the corner if bracket is welded by fillet welds. If the two weld run pass had crossed, this would have given a 3D residual stress situation. Therefore the presences and absence of scallop necessitates the 3D FEA fatigue resistance of both types of brackets using effective notch stress approach ( ). FEMAP 10.1 with NX NASTRAN was used for the 3D FEA. The first and main objective of this research was to investigate and compare the fatigue resistance of brackets with and without scallop. The secondary goal was the fatigue design of scallops in case they cannot be avoided for some reason. The fatigue resistance for both types of brackets was determined based on approach using 1 mm fictitiously rounded radius based on IIW recommendation. Identical geometrical, boundary and loading conditions were used for the determination and comparison of fatigue resistance of both types of brackets using linear 3D FEA. Moreover the size effect of bracket length was also studied using 2D SHELL element FEA. In the case of brackets with scallop the flange plate weld toe at the corner of the scallop was found to exhibit the highest and made the flange plate weld toe critical for fatigue failure. Whereas weld root and weld toe at the weld intersections were the highly stressed location for brackets without scallop. Thus weld toe for brackets with scallop, and weld root and weld toe for brackets without scallop were found to be the critical area for fatigue failure. Employing identical parameters on both types of brackets, brackets without scallop had the highest except for full penetrated weld. Furthermore the fatigue resistance of brackets without scallop was highly affected by the lack of weld penetration length and it was found out that decreased as the weld penetration was increased. Despite the fact that the very presence of scallop reduces the stiffness and also same time induce stress concentration, based on the 3D FEA it is worth concluding that using scallop provided better fatigue resistance when both types of brackets were fillet welded. However brackets without scallop had the highest fatigue resistance when full penetration weld was used. This thesis also showed that weld toe for brackets with scallop was the only highly stressed area unlike brackets without scallop in which both weld toe and weld root were the critical locations for fatigue failure when different types of boundary conditions were used. Weld throat thickness, plate thickness, scallop radius, lack of weld penetration length, boundary condition and weld quality affected the fatigue resistance of both types of brackets. And as a result, bracket design procedure, especially welding quality and post weld treatment techniques significantly affect the fatigue resistance of both type of brackets.

Lipase-catalyzed Approaches towards Secondary Alcohols: Intermediates for Enantiopure Drugs

The use of enantiopure intermediates for drug synthesis is a trend in pharmaceutical industry. Different physiological effects are associated with the enantiomers of chiral molecules. Thus, the safety profile of a drug based on an enantiopure active pharmaceutical ingredient is more reliable. Biocatalysis is an important tool to access enantiopure molecules. In biocatalysis, the advantage of selectivity (chemo-, regio- and stereoselectivity) is combined with the benefits of a green synthesis strategy. Chemoenzymatic syntheses of drug molecules, obtained by combining biocatalysis with modern chemical synthesis steps usually consists of fewer reaction steps, reduced waste production and improved overall synthetic efficiency both in yields and enantio- and/or diastereoselectivities compared with classical chemical synthesis. The experimental work together with the literature review clearly indicates that lipase catalysis is highly applicable in the synthesis of enantiopure intermediates of drug molecules as the basis to infer the correct stereochemistry. By lipase catalysis, enantiopure secondary alcohols used as intermediates in the synthesis of Dorzolamide, an antiglaucoma drug, were obtained. Enantiopure _-hydroxy nitriles as potential intermediates for the synthesis of antidepressant drugs with 1-aryl-3- methylaminopropan-1-ol structure were also obtained with lipases. Kinetic resolution of racemates was the main biocatalytic approach applied. Candida Antarctica lipase B, Burkholderia cepacia lipase and Thermomyces lanuginosus lipase were applied for the acylation of alcohols and the alcoholysis of their esters in organic solvents, such as in diisopropyl ether and tert-butyl methyl ether. Candida Antarctica lipase B was used under solvent free conditions for the acylation of ethyl 3-hydroxybutanoate.

Diameter structure of stratum and ecological groups of a Semideciduous Forest area in Dionísio-MG

The objective of this study was to obtain homogeneous groups of species and information on their density, dominance and volume, in terms of ecological group and diameter structure of an area of Submontane Semideciduous forest (Mata do Mumbaça) in Dionísio, MG. This work was conducted with data of the diameter distribution per species from floristic and phytosociological (Mata do Mumbaça) survey of 120 plots with 10 x 10 m each one. The 120 plots were contiguous and corresponding to a total sample area of 12,000 m² distributed over the topographic units (Low Ramp, Lower Slope, Upper Slope and Hill Top). The topographic units Low Ramp, Lower Slope and Upper Slope were in the middle stage of succession as they presented incipient stratification into two strata (canopy and understory) i.e. canopy ranging from 5 to 12 m high. However, the stratum Hill Top was classified as intermediate/advanced succession because it had a total height equal to or greater than 12 m. The distribution of individual trees of the four strata on diameter classes showed a typical J-inverted pattern that is, high concentration of individuals in smaller diameter classes and a sharp reduction towards the larger classes. In relation to absolute dominance and total volume of species, the ecological group that stood out in the four strata (Low Ramp, Lower Slope, Upper Slope and Hill Top) was the initial secondary, which were in the intermediate stage of secondary, rapidly developing into the mature phase.

Landscape structure in the northern coast of Paraná state, a hotspot for the brazilian Atlantic Forest conservation

The "Serra do Mar" region comprises the largest remnant of the Brazilian Atlantic Forest. The coast of the Paraná State is part of the core area of the "Serra do Mar" corridor and where actions for biodiversity conservation must be planned. In this study we aimed at characterizing the landscape structure in the APA-Guaraqueçaba, the largest protected area in this region, in order to assist environmental policies of this region. Based on a supervised classification of a mosaic of LANDSAT-5-TM satellite images (from March 2009), we developed a map (1:75,000 scale) with seven classes of land use and land cover and analyzed the relative quantities of forests and modified areas in slopes and lowlands. The APA-Guaraqueçaba is comprised mainly by the Dense Ombrophilous Forest (68.6% of total area) and secondary forests (9.1%), indicating a forested landscape matrix; anthropogenic and bare soil areas (0.8%) and the Pasture/Grasslands class (4.2%) were less representative. Slopes were less fragmented and more preserved (96.3% of Dense Ombrophilous Forest and secondary forest) than lowlands (71.3%), suggesting that restoration initiatives in the lowlands must be stimulated in this region. We concluded that most of the region sustains well-conserved ecosystems, highlighting the importance of Paraná northern coast for the biodiversity maintenance of the Atlantic Forest.

Litter fall production and decomposition in a fragment of secondary Atlantic Forest of São Paulo, sp, southeastern Brazil

Litter fall consists of all organic material deposited on the forest floor, being of extremely important for the structure and maintenance of the ecosystem through nutrient cycling. This study aimed to evaluate the production and decomposition of litter fall in a secondary Atlantic forest fragment of secondary Atlantic Forest, at the Guarapiranga Ecological Park, in São Paulo, SP. The litter samples were taken monthly from May 2012 to May 2013. To assess the contribution of litter fall forty collectors were installed randomly within an area of 0.5 ha. The collected material was sent to the laboratory to be dried at 65 °C for 72 hours, being subsequently separated into fractions of leaves, twigs, reproductive parts and miscellaneous, and weighed to obtain the dry biomass. Litterbags were placed and tied close to the collectors to estimate the decomposition rate in order to evaluate the loss of dry biomass at 30, 60, 90, 120 and 150 days. After collection, the material was sent to the laboratory to be dried and weighed again. Total litter fall throughout the year reached 5.7 Mg.ha-1.yr-1 and the major amount of the material was collected from September till March. Leaves had the major contribution for total litter fall (72%), followed by twigs (14%), reproductive parts (11%) and miscellaneous (3%). Reproductive parts had a peak during the wet season. Positive correlation was observed between total litter and precipitation, temperature and radiation (r = 0.66, p<0.05; r = 0.76, p<0.05; r = 0.58, p<0.05, respectively). The multiple regression showed that precipitation and radiation contributed significantly to litter fall production. Decomposition rate was in the interval expected for secondary tropical forest and was correlated to rainfall. It was concluded that this fragment of secondary forest showed a seasonality effect driven mainly by precipitation and radiation, both important components of foliage renewal for the plant community and that decomposition was in an intermediate rate.

The Structure and Function of αI Domains in Collagen Receptor and Leukocyte Integrins

Integrins are heterodimeric, signaling transmembrane adhesion receptors that connect the intracellular actin microfilaments to the extracellular matrix composed of collagens and other matrix molecules. Bidirectional signaling is mediated via drastic conformational changes in integrins. These changes also occur in the integrin αI domains, which are responsible for ligand binding by collagen receptor and leukocyte specific integrins. Like intact integrins, soluble αI domains exist in the closed, low affinity form and in the open, high affinity form, and so it is possible to use isolated αI domains to study the factors and mechanisms involved in integrin activation/deactivation. Integrins are found in all mammalian tissues and cells, where they play crucial roles in growth, migration, defense mechanisms and apoptosis. Integrins are involved in many human diseases, such as inflammatory, cardiovascular and metastatic diseases, and so plenty of effort has been invested into developing integrin specific drugs. Humans have 24 different integrins, four of which are collagen receptor (α1β1, α2β1, α10β1, α11β1) and five leukocyte specific integrins (αLβ2, αMβ2, αXβ2, αDβ2, αEβ7). These two integrin groups are quite unselective having both primary and secondary ligands. This work presents the first systematic studies performed on these integrin groups to find out how integrin activation affects ligand binding and selectivity. These kinds of studies are important not only for understanding the partially overlapping functions of integrins, but also for drug development. In general, our results indicated that selectivity in ligand recognition is greatly reduced upon integrin activation. Interestingly, in some cases the ligand binding properties of integrins have been shown to be cell type specific. The reason for this is not known, but our observations suggest that cell types with a higher integrin activation state have lower ligand selectivity, and vice versa. Furthermore, we solved the three-dimensional structure for the activated form of the collagen receptor α1I domain. This structure revealed a novel intermediate conformation not previously seen with any other integrin αI domain. This is the first 3D structure for an activated collagen receptor αI domain without ligand. Based on the differences between the open and closed conformation of the αI domain we set structural criteria for a search for effective collagen receptor drugs. By docking a large number of molecules into the closed conformation of the α2I domain we discovered two polyketides, which best fulfilled the set structural criteria, and by cell adhesion studies we showed them to be specific inhibitors of the collagen receptor integrins.

Detection of anthraquinones and identification of 1,4-naphthohydroquinone in cell suspension cultures of Rudgea jasminoides (Cham.) Müll. Arg. (Rubiaceae)

In Rubiaceae, anthraquinones and naphthoquinones are secondary metabolites characteristic of the subfamily Rubioideae, in which Rudgea jasminoides is included. Thin-layer chromatography using specific solvent systems and spray reagents indicated the presence of anthraquinones constitutively produced by cell suspension cultures of R. jasminoides. GC/MS analysis detected 1,4-naphthohydroquinone as a product of biosynthesis only after elicitation of the cells with yeast extract (Saccharomyces cerevisiae). The latter compound is probably a phytoalexin produced by suspension cultures of R. jasminoides.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 ± 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 ± 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 ± 3.8 and 15.3 ± 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Photic driving in the electroencephalogram of children and adolescents: harmonic structure and relation to the resting state

In order to identify latent bioelectrical oscillators, 15 normal subjects (aged 9-17 years, 8 males, 7 females) were subjected to intermittent photic stimulation. The EEG amplitude spectra corresponding to the 11 fixed frequencies of stimulation presented (3-24 Hz) were combined to form "profiles" of the driving reaction in the right occipital area. The driving response varied with frequency, and was demonstrable in 70-100% of cases (using as criterion peak amplitudes 20% larger than those of the neighbors). The strongest responses were observed at the frequency closest to the alpha peak of the resting EEG. A secondary profile maximum was in the theta band. In 10 subjects, this maximum exceeded half the alpha peak (with an average of 72.4% of the alpha peak), while in the resting spectra, theta amplitudes were much lower than the alpha maxima. This responsiveness in theta activity seems to be characteristic of prepubertal and pubertal subjects. The profiles and resting EEG spectra showed a highly significant Pearson's correlation, with the peak in the theta band of the profiles being the main difference observed between them. The correlation coefficient was significantly correlated with the ratio of the maxima in the theta and alpha bands (R = -0.77, P<0.001). The correlation coefficient between profile and resting spectrum may be a useful indicator in screening methods used to reveal latent cerebral oscillators. Profiles for the second and third harmonics were correlated with those of the first harmonic (fundamental frequency), when considering the corresponding EEG frequencies. Peak frequencies in all three profiles were close to those of the individual's background alpha rhythm, and peak amplitudes in higher harmonics were not much lower than those of the fundamental frequency (mean values of 84 and 63%, for second and third harmonics, respectively).

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4% and decreased its ß-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

The P48T germline mutation and polymorphism in the CDKN2A gene of patients with melanoma

CDKN2A has been implicated as a melanoma susceptibility gene in some kindreds with a family history of this disease. Mutations in CDKN2A may produce an imbalance between functional p16ink4a and cyclin D causing abnormal cell growth. We searched for germline mutations in this gene in 22 patients with clinical criteria of hereditary cancer (early onset, presence of multiple primary melanoma or 1 or more first- or second-degree relatives affected) by secondary structural content prediction, a mutation scanning method that relies on the propensity for single-strand DNA to take on a three-dimensional structure that is highly sequence dependent, and sequencing the samples with alterations in the electrophoretic mobility. The prevalence of CDKN2A mutation in our study was 4.5% (1/22) and there was a correlation between family history and probability of mutation detection. We found the P48T mutation in 1 patient with 2 melanoma-affected relatives. The patient descends from Italian families and this mutation has been reported previously only in Italian families in two independent studies. This leads us to suggest the presence of a mutational "hotspot" within this gene or a founder mutation. We also detected a high prevalence (59.1%) of polymorphisms, mainly alleles 500 C/G (7/31.8%) or 540 C/T (6/27.3%), in the 3' untranslated region of exon 3. This result reinforces the idea that these rare polymorphic alleles have been significantly associated with the risk of developing melanoma.

Griscelli syndrome-type 2 in twin siblings: case report and update on RAB27A human mutations and gene structure

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutation in the MYO5A (GS1, Elejalde), RAB27A (GS2) or MLPH (GS3) genes. Typical features of all three subtypes of this disease include pigmentary dilution of the hair and skin and silvery-gray hair. Whereas the GS3 phenotype is restricted to the pigmentation dysfunction, GS1 patients also show primary neurological impairment and GS2 patients have severe immunological deficiencies that lead to recurrent infections and hemophagocytic syndrome. We report here the diagnosis of GS2 in 3-year-old twin siblings, with silvery-gray hair, immunodeficiency, hepatosplenomegaly and secondary severe neurological symptoms that culminated in multiple organ failure and death. Light microscopy examination of the hair showed large, irregular clumps of pigments characteristic of GS. A homozygous nonsense mutation, C-T transition (c.550C>T), in the coding region of the RAB27A gene, which leads to a premature stop codon and prediction of a truncated protein (R184X), was found. In patient mononuclear cells, RAB27A mRNA levels were the same as in cells from the parents, but no protein was detected. In addition to the case report, we also present an updated summary on the exon/intron organization of the human RAB27A gene, a literature review of GS2 cases, and a complete list of the human mutations currently reported in this gene. Finally, we propose a flow chart to guide the early diagnosis of the GS subtypes and Chédiak-Higashi syndrome.

Institutional environment in North Korea: market structure and overcoming institutional voids

Tutkimus sai innoituksensa, kun tutkija huomasi tarpeen liiketaloudelliselle, ajantasaiselle ja realistiselle tutkimukselle Pohjois-Korean markkinoista, joka kuvailisi markkinoiden olemassaolevia ja puuttuvia rakenteita sekä tutkisi mahdollisuuksia ylittää puuttuvat rakenteet. Institutionaalinen teoria valittiin sopivaksi viitekehykseksi kuvailla ja tutkia markkinarakennetta. Tutkimuskysymys muotoiltiin seuraavasti: “Miten ulkomaiset yritykset voivat reagoida puuttuviin markkinarakenteisiin Pohjois-Koreassa?”. Tutkimuskysymys jaettiin kolmeen osakysymykseen: (1) Millainen on Pohjois-Korean markkinoiden institutionaalinen ympäristö? (2) Mitkä ovat merkittävimmät puuttuvat markkinarakenteet Pohjois-Koreassa? (3) Mitä mahdollisuuksia ulkomaisilla yrityksillä voisi olla reagoida puuttuviin markkinarakenteisiin? Tutkimus toteutettiin kvalitatiivisena, koska tutkimuskysymys on deskriptiivinen. Aineisto kerättiin asiantuntijahaastattelun ja kvalitatiivisen sisällönanalyysin keinoin. Primääriaineiston muodostavat 2 asiantuntijahaastattelua ja sekundääriaineiston muodostavat 95 artikkelia, jotka kerättiin 40 lähteestä. Aineisto analysoitiin kvalitatiivisen sisällönanalyysin keinoin. Aineisto koodattiin, luokiteltiin ja esitettiin kokonaisuuksina luokittelurungon avulla, joka laadittiin tutkimusta varten muodostetun teoreettisen viitekehyksen mukaan. Tulokset ja johtopäätökset voidaan tiivistää seuraavasti. (1) Pohjois-Korean markkinan instituutioihin vaikuttaa kaksoisrakenne, jossa muodollinen, sosialistinen rakenne ja epämuodollinen, markkinalähtöinen rakenne toimivat päällekkäin. (2) Puuttuvia rakenteita on sekä markkinan kontekstissa että markkinatasolla. Puutteet ovat osittain seurausta vanhojen rakenteiden korvaantumisesta uusilla, jotka eivät ole institutionalisoituneet. (3) Yritykset voivat käyttää samoja mahdollisuuuksia reagoida puuttuviin markkinarakenteisiin Pohjois-Koreassa, joita kehittyvien markkinoiden yhteydessä on esitetty. Sen tulkittiin vähentävän käsitystä, jonka mukaan Pohjois-Korean markkina on liian erikoinen yritystoiminnalle. (4) Kasvava keskiluokka sekä yrittäjyyden ja naisten yhä merkittävämpi rooli liike-elämässä aiheuttavat alhaalta ylöspäin suuntautuvaa kehitystä markkinoilla. Nämä ovat merkkejä viimeaikaisesta kehityksestä, jotka eivät ole saaneet laajaa huomiota länsimaisessa mediassa. Se korostaa tarvetta liiketaloudelliselle, ajantasaiselle jatkotutkimukselle Pohjois-Korean markkinoista.

Institutional environment in North Korea: market structure and overcoming institutional voids

The research was sparked by an exchange in South Korea, as the author identified a gap in research that provides economic, up to date and realistic information about the North Korean market in English language. A need for a research was identified that would describe the market’s existing and missing market structures and explore possibilities to overcome the missing market structures. Institutional theory was chosen as a suitable framework to describe and explore the market. The research question was formulated as follows: “How can foreign companies overcome institutional voids in the North Korean market?”. To answer the research question, it was divided into three sub-questions as follows: (1) What is the institutional environment in North Korea like? (2) What are the major institutional voids in the North Korean market? (3) What possibilities do foreign companies have to overcome institutional voids? The research is qualitative by nature due to the descriptive and exploratory nature of the research question. Data collection consisted of expert interview and content analysis, resulting in primary data of two interviews and secondary data of 95 articles from 40 different sources. The data was analyzed with the systematical technique of content analysis. The data was coded, classified and presented as concepts with the help of a classification system that was build following the theoretical framework adapted for this study. The findings can be summarized as follows. (1) The market institutions are characterized by an overlapping dual system of formal, socialist structures and informal, market-oriented structures. (2) Institutional voids prevail on both the market’s contextual and on the market level. They are partly result of old institutions being replaced by new institutions that lack institutionalization. (3) Identified possibilities to overcome institutional voids correspond with possibilities drawn from previous research. This decreases the image of North Korea as an impossibly unique market to operate in. (4) Emerging middle class, rapidly growing entrepreneurial activities and women’s increasing role in business drive a down-to-up change in the market. This signals the recent development of the market, yet has been overlooked in the Western media. Thus there is a need for further economic, up to date research concerning North Korea.