Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)

Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50-mu-g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.

Dielectric Function of Undoped and Doped Poly2-methoxy-5-(3 `,7 `-dimethyloctyloxy)-1,4-phenylene-vinylene] by Ellipsometry in a Wide Spectral Range

Ellipsometric measurements in a wide spectral range (from 0.05 to 6.5 eV) have been carried out on the organic semiconducting polymer, poly2-methoxy-5-(3',7'-dimethyloctyloxy)-1,4-phenylene-vinylene] (MDMO-PPV), in both undoped and doped states. The real and imaginary parts of the dielectric function and the refractive index are determined accurately, provided that the layer thickness is measured independently. After doping, the optical properties show the presence of new peaks, which could be well-resolved by spectroscopic ellipsometry. Also for the doped material, the complex refractive index, with respect to the dielectric function, has been determined. The broadening of the optical transitions is due to the delocalization of polarons at higher doping level. The detailed information about the dielectric function as well as refractive index function obtained by spectroscopic ellipsometry allows not only qualitative but also quantitative description of the optical properties of the undoped/doped polymer. For the direct characterization of the optical properties of MDMO-PPV, ellipsometry turns out to be advantageous compared to conventional reflection and transmission measurements.

Evaluation of the Genotoxic and Antigenotoxic Potential of the Alkaloid Punarnavine from Boerhaavia diffusa

Boerhaavia diffusa is a traditional herbal medicine extensively used in the Ayurveda and Unani forms of medicine in India and many parts of the world. Different parts of the plant are used as an appetizer, alexiteric, eye tonic, for flushing out the renal system, and to treat blood pressure. This study was conducted to evaluate the in vivo genotoxic and/or antigenotoxic potential of punarnavine, a separated alkaloid from the root of B. diffusa using toxicity studies (OECD guideline 474, 1997). The genotoxic and antigenotoxic potential of punarnavine was assayed using the comet assay on lymphocytes, liver, spleen, brain, and bone marrow as well as using the micronucleus test in bone marrow cells including the in vitro chromosomal aberration test. The results demonstrated that none of the tested doses of punarnavine showed genotoxic effects by the comet assay, or clastogenic effects in the micronucleus test. On the other hand, for all cells evaluated, the three tested doses of punarnavine promoted inhibition of DNA damage induced by cyclophosphamide. Based on these results, we concluded that punarnavine, an alkaloid from the Boerhaavia diffusa root, has no genotoxic or clastogenic effects in our experimental conditions. However, it caused a significant decrease in DNA damage induced by cyclophosphamide. It is suggested that the antigenotoxic properties of this alkaloid may be of great pharmacological importance and beneficial for cancer prevention.

Magmas functions as a ROS regulator and provides cytoprotection against oxidative stress-mediated damages

Redox imbalance generates multiple cellular damages leading to oxidative stress-mediated pathological conditions such as neurodegenerative diseases and cancer progression. Therefore, maintenance of reactive oxygen species (ROS) homeostasis is most important that involves well-defined antioxidant machinery. In the present study, we have identified for the first time a component of mammalian protein translocation machinery Magmas to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues and cancer cells that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance toward oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of electron transport chain (ETC) complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as a ROS sensor was found to be independent of its role in protein import. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cell tolerance to oxidative stress even in yeast model organism. The cytoprotective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress-related diseases.

A thermodynamic correlation between damage and fracture as applied to concrete fatigue

Fatigue damage in concrete is characterized by the simultaneous presence of micro and macrocracks. The theory of fracture mechanics conveniently handles the propagation of macrocracks, whereas damage mechanics precisely describes the state of microcracking. This paper provides a platform to correlate fracture mechanics and damage mechanics theories through an energy equivalence within a thermodynamic framework by equating the energy dissipated according to each theory. Through this correlation, damage corresponding to a given crack length could be obtained, and alternatively a discrete crack could be transformed into an equivalent damage zone. The results are validated using available experimental data on concrete fatigue including stiffness degradation and acoustic emission. (C) 2015 Elsevier Ltd. All rights reserved.

A thermodynamic correlation between damage and fracture as applied to concrete fatigue

Fatigue damage in concrete is characterized by the simultaneous presence of micro and macrocracics. The theory of fracture mechanics conveniently handles the propagation of macrocracks, whereas damage mechanics precisely describes the state of microcracking. This paper provides a platform to correlate fracture mechanics and damage mechanics theories through an energy equivalence within a thermodynamic framework by equating the energy dissipated according to each theory. Through this correlation, damage corresponding to a given crack length could be obtained, and alternatively a discrete crack could be transformed into an equivalent damage zone. The results are validated using available experimental data on concrete fatigue including stiffness degradation and acoustic emission. (C) 2015 Elsevier Ltd. All rights reserved.

Spallation analysis with a closed trans-scale formulation damage evolution

A closed, trans-scale formulation of damage evolution based on the statistical microdamage mechanics is summarized in this paper. The dynamic function of damage bridges the mesoscopic and macroscopic evolution of damage. The spallation in an aluminium plate is studied with this formulation. It is found that the damage evolution is governed by several dimensionless parameters, i.e., imposed Deborah numbers De* and De, Mach number M and damage number S. In particular, the most critical mode of the macroscopic damage evolution, i.e., the damage localization, is deter-mined by Deborah number De+. Deborah number De* reflects the coupling and competition between the macroscopic loading and the microdamage growth. Therefore, our results reveal the multi-scale nature of spallation. In fact, the damage localization results from the nonlinearity of the microdamage growth. In addition, the dependence of the damage rate on imposed Deborah numbers De* and De, Mach number M and damage number S is discussed.

Modeling and mesoscopic damage constitutive relation of brittle short-fiber-reinforced composites

Aimed at brittle composites reinforced by randomly distributed short-fibers with a relatively large aspect ratio, stiffness modulus and strength, a mesoscopic material model was proposed. Based on the statistical description, damage mechanisms, damage-induced anisotropy, damage rate effect and stress redistribution, the constitutive relation were derived. By taking glass fiber reinforced polypropylene polymers as an example, the effect of initial orientation distribution of fibers, damage-induced anisotropy, and damage-rate effect on macro-behaviors of composites were quantitatively analyzed. The theoretical predictions compared favorably with the experimental results.

Synchronous Multi-Scale Observations on Rock Damage and Rupture

This paper reports a multi-scale study on damage evolution process and rupture of gabbro under uniaxial compression with several experimental techniques, including MTS810 testing machine, white digital speckle correlation method, and acoustic emission technique. In particular, the synchronization of the three experimental systems is realized for the study of relationship of deformation and damage at multiple scales. It is found that there are significant correlation between damage evolution at small and large length scales, and rupture at sample scale, especially it displays critical sensitivity at multiple scales and trans-scale fluctuations.

Damage detection in civil structures using high-frequency seismograms

The dynamic properties of a structure are a function of its physical properties, and changes in the physical properties of the structure, including the introduction of structural damage, can cause changes in its dynamic behavior. Structural health monitoring (SHM) and damage detection methods provide a means to assess the structural integrity and safety of a civil structure using measurements of its dynamic properties. In particular, these techniques enable a quick damage assessment following a seismic event. In this thesis, the application of high-frequency seismograms to damage detection in civil structures is investigated.

Two novel methods for SHM are developed and validated using small-scale experimental testing, existing structures in situ, and numerical testing. The first method is developed for pre-Northridge steel-moment-resisting frame buildings that are susceptible to weld fracture at beam-column connections. The method is based on using the response of a structure to a nondestructive force (i.e., a hammer blow) to approximate the response of the structure to a damage event (i.e., weld fracture). The method is applied to a small-scale experimental frame, where the impulse response functions of the frame are generated during an impact hammer test. The method is also applied to a numerical model of a steel frame, in which weld fracture is modeled as the tensile opening of a Mode I crack. Impulse response functions are experimentally obtained for a steel moment-resisting frame building in situ. Results indicate that while acceleration and velocity records generated by a damage event are best approximated by the acceleration and velocity records generated by a colocated hammer blow, the method may not be robust to noise. The method seems to be better suited for damage localization, where information such as arrival times and peak accelerations can also provide indication of the damage location. This is of significance for sparsely-instrumented civil structures.

The second SHM method is designed to extract features from high-frequency acceleration records that may indicate the presence of damage. As short-duration high-frequency signals (i.e., pulses) can be indicative of damage, this method relies on the identification and classification of pulses in the acceleration records. It is recommended that, in practice, the method be combined with a vibration-based method that can be used to estimate the loss of stiffness. Briefly, pulses observed in the acceleration time series when the structure is known to be in an undamaged state are compared with pulses observed when the structure is in a potentially damaged state. By comparing the pulse signatures from these two situations, changes in the high-frequency dynamic behavior of the structure can be identified, and damage signals can be extracted and subjected to further analysis. The method is successfully applied to a small-scale experimental shear beam that is dynamically excited at its base using a shake table and damaged by loosening a screw to create a moving part. Although the damage is aperiodic and nonlinear in nature, the damage signals are accurately identified, and the location of damage is determined using the amplitudes and arrival times of the damage signal. The method is also successfully applied to detect the occurrence of damage in a test bed data set provided by the Los Alamos National Laboratory, in which nonlinear damage is introduced into a small-scale steel frame by installing a bumper mechanism that inhibits the amount of motion between two floors. The method is successfully applied and is robust despite a low sampling rate, though false negatives (undetected damage signals) begin to occur at high levels of damage when the frequency of damage events increases. The method is also applied to acceleration data recorded on a damaged cable-stayed bridge in China, provided by the Center of Structural Monitoring and Control at the Harbin Institute of Technology. Acceleration records recorded after the date of damage show a clear increase in high-frequency short-duration pulses compared to those previously recorded. One undamage pulse and two damage pulses are identified from the data. The occurrence of the detected damage pulses is consistent with a progression of damage and matches the known chronology of damage.

Organization and function of the phage Lambda chromosome

The process of prophage integration by phage λ and the function and structure of the chromosomal elements required for λ integration have been studied with the use of λ deletion mutants. Since att^φ, the substrate of the integration enzymes, is not essential for λ growth, and since att^φ resides in a portion of the λ chromosome which is not necessary for vegetative growth, viable λ deletion mutants were isolated and examined to dissect the structure of att^φ.

Deletion mutants were selected from wild type populations by treating the phage under conditions where phage are inactivated at a rate dependent on the DNA content of the particles. A number of deletion mutants were obtained in this way, and many of these mutants proved to have defects in integration. These defects were defined by analyzing the properties of Int-promoted recombination in these att mutants.

The types of mutants found and their properties indicated that att^φ has three components: a cross-over point which is bordered on either side by recognition elements whose sequence is specifically required for normal integration. The interactions of the recognition elements in Int-promoted recombination between att mutants was examined and proved to be quite complex. In general, however, it appears that the λ integration system can function with a diverse array of mutant att sites.

The structure of att^φ was examined by comparing the genetic properties of various att mutants with their location in the λ chromosome. To map these mutants, the techniques of heteroduplex DNA formation and electron microscopy were employed. It was found that integration cross-overs occur at only one point in att^φ and that the recognition sequences that direct the integration enzymes to their site of action are quite small, less than 2000 nucleotides each. Furthermore, no base pair homology was detected between att^φ and its bacterial analog, att^B. This result clearly demonstrates that λ integration can occur between chromosomes which have little, if any, homology. In this respect, λ integration is unique as a system of recombination since most forms of generalized recombination require extensive base pair homology.

An additional study on the genetic and physical distances in the left arm of the λ genome was described. Here, a large number of conditional lethal nonsense mutants were isolated and mapped, and a genetic map of the entire left arm, comprising a total of 18 genes, was constructed. Four of these genes were discovered in this study. A series of λdg transducing phages was mapped by heteroduplex electron microscopy and the relationship between physical and genetic distances in the left arm was determined. The results indicate that recombination frequency in the left arm is an accurate reflection of physical distances, and moreover, there do not appear to be any undiscovered genes in this segment of the genome.

Efeitos benéficos da rosuvastatina na estrutura renal de ratos espontaneamente hipertensos (SHR)

A incidência de doenças renais crônicas está aumentando no mundo, e há uma grande necessidade de identificar as terapias capazes de deter ou reduzir a progressão da doença. Há crescente evidência clínica e experimental de que as estatinas poderiam desempenhar um papel terapêutico. Recentes estudos clínicos e experimentais têm mostrado que as estatinas têm "efeitos pleiotrópicos", além da modulação lipídica. Estudos têm avaliado os efeitos das estatinas sobre a progressão da doença renal crônica, mas os resultados são controversos. Estudos ultra-estruturais em humanos e em ratos demonstraram a presença de junções GAP dentro de todas as células do glomérulo e os podocitos demonstraram conter principalmente conexina-43 (Cx-43). O presente estudo tem como objetivo observar os efeitos da rosuvastatina na estrutura e ultra-estrutura renal e a expressão glomerular de Cx-43 em ratos normotensos (WKY) e em ratos espontaneamente hipertensos (SHR). O foco do estudo foi avaliar os efeitos pleiotrópicos da rosuvastatina em rins de animais hipertensos normocolesterolêmicos. Os ratos foram divididos aleatoriamente em quatro grupos: WKY-C: animais normotensos que não receberam rosuvastatina; WKY-ROS: animais normotensos que receberam rosuvastatina 20mg/kg/dia por gavagem orogástrica; SHR-C: animais hipertensos que não receberam rosuvastatina; SHR-ROS: animais hipertensos que receberam rosuvastatina, como descrito no grupo WKY-ROS. Os animais dos grupos SHR-C e SHR-ROS apresentaram níveis de pressão arterial maiores que os animais dos grupos WKY-C e WKY-Ros. A massa corporal dos grupos de animais não diferiram significativamente durante o experimento. Não houve diferença nos níveis sanguíneos de uréia, creatinina, ácido úrico e creatinafosfoquinase entre os animas dos grupos estudados. No entanto, houve um aumento da excreção de proteína de 24 horas nos animais do grupo SHR-C. Houve um aumento na área capsular nos animais do grupo SHR-C. Por microscopia eletrônica de transmissão observou-se que nos animais SHR-C a barreira de filtração glomerular, o diafragma de fenda e os podócitos estão alterados exibindo os vacúolos nos podócitos e pedicelos mais curtos e mais espessos. Por microscopia eletrônica de varredura, os animais SHR-C exibiram pedicelos mais afilados, curtos e tortuosos. Um aumento da imunofluorescência para Cx-43 foi observada em células epiteliais viscerais dos glomérulos dos animais do grupo WKY-ROS e nas células parietais e viscerais dos glomérulos dos animais do grupo SHR-ROS, se comparado com os grupos WKY-C e SHR-C. Em conclusão, podemos supor que o efeito pleiotrópico renal da rosuvastatina pode ser uma ferramenta terapêutica para melhorar a estrutura e conseqüentemente a função renal em indivíduos hipertensos.

Efeitos da rosuvastatina e da atividade física de baixa intensidade na morfologia renal de ratos normotensos e espontaneamente hipertensos (SHR)

Desordens do sistema renal podem ser as causas da hipertensão arterial, a qual pode, por sua vez, causar doenças renais. A pressão sanguínea elevada é muito comum também nas doenças crônicas dos rins, e é, além disso, um conhecido fator de risco para uma mais rápida progressão da falha renal. A incidência de doenças renais crônicas está aumentando no mundo, e há uma grande necessidade de identificar as terapias capazes de deter ou reduzir a progressão da doença. Há crescente evidência de que as estatinas poderiam desempenhar um papel terapêutico. Além disso, tem sido demonstrado que a atividade física melhora a função renal em pacientes. Estudos ultra-estruturais em humanos e em ratos demonstraram a presença de junções gap dentro de todas as células do glomérulo e os podócitos demonstraram conter principalmente conexina-43 (Cx-43). O presente estudo tem como objetivo observar os efeitos da rosuvastatina e da atividade física de baixa intensidade na estrutura e ultra-estrutura renal e na expressão glomerular de Cx-43 em ratos normotensos (WKY) e em ratos espontaneamente hipertensos (SHR). Os ratos foram divididos aleatoriamente em oito grupos: WKY-C: animais normotensos que não receberam rosuvastatina; WKY-ROS: animais normotensos que receberam rosuvastatina 20mg/kg/dia por gavagem orogástrica; SHR-C: animais hipertensos que não receberam rosuvastatina; SHR-ROS: animais hipertensos que receberam rosuvastatina, como descrito no grupo WKY-ROS; SED-WKY: animais normotensos sedentários; EX-WKY: animais normotensos exercitados; SED-SHR: animais hipertensos sedentários; e, EX-SHR: animais hipertensos exercitados. Os animais dos grupos SHR-C, SHR-ROS e SED-SHR apresentaram níveis de pressão arterial maiores que os animais dos grupos WKY-C, WKY-ROS, SED-WKY, EX-WKY e EX-SHR. A massa corporal dos grupos de animais não diferiram significativamente durante o experimento. Não houve diferença nos níveis sanguíneos de uréia, creatinina, ácido úrico e creatinafosfoquinase entre os animas dos grupos estudados. No entanto, houve um aumento da excreção de proteína de 24 horas nos animais do grupo SHR-C. Houve um aumento na área capsular nos animais do grupo SHR-C. Por microscopia eletrônica de transmissão observou-se que nos animais SHR-C e SED-SHR a barreira de filtração glomerular, o diafragma de fenda e os podócitos estão alterados exibindo os vacúolos nos podócitos e pedicelos mais curtos e mais espessos. Por microscopia eletrônica de varredura, os animais SHR-C e SED-SHR exibiram pedicelos mais afilados, curtos e tortuosos. Um aumento da imunofluorescência para Cx-43 foi observada em células epiteliais viscerais dos glomérulos dos animais do grupo WKY-ROS e nas células parietais e viscerais dos glomérulos dos animais do grupo SHR-ROS, se comparado com os grupos WKY-C e SHR-C. Por outro lado, os animais dos grupos SED-SHR e EX-SHR exibiram diminuição da expressão de Cx-43, comparados aos animais SED-WKY e EX-WKY. Em conclusão, podemos supor que os efeitos renais da rosuvastatina e da atividade física de baixa intensidade podem ser ferramentas terapêuticas para melhorar a estrutura e conseqüentemente a função renal em indivíduos hipertensos

Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

Background: Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. Methods: In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. Results: GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. Conclusions: This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms.