Field Instrumentation, Testing, and Long-Term Monitoring of High-Mast Lighting Towers in the State of Iowa, TR-518, 2006

As a result of the collapse of a 140 foot high-mast lighting tower in Sioux City, Iowa in November of 2003, a thorough investigation into the behavior and design of these tall, yet relatively flexible structures was undertaken. Extensive work regarding the root cause of this failure was carried out by Robert Dexter of The University of Minnesota. Furthermore, a statewide inspection of all the high-mast towers in Iowa revealed fatigue cracks and loose anchor bolts on other existing structures. The current study was proposed to examine the static and dynamic behavior of a variety of towers in the State of Iowa utilizing field testing, specifically long-term monitoring and load testing. This report presents the results and conclusions from this project. The field work for this project was divided into two phases. Phase 1 of the project was conducted in October 2004 and focused on the dynamic properties of ten different towers in Clear Lake, Ames, and Des Moines, Iowa. Of those ten, two were also instrumented to obtain stress distributions at various details and were included in a 12 month long-term monitoring study. Phase 2 of this investigation was conducted in May of 2005, in Sioux City, Iowa, and focused on determining the static and dynamic behavior of a tower similar to the one that collapsed in November 2003. Identical tests were performed on a similar tower which was retrofitted with a more substantial replacement bottom section in order to assess the effect of the retrofit. A third tower with different details was dynamically load tested to determine its dynamic characteristics, similar to the Phase 1 testing. Based on the dynamic load tests, the modal frequencies of the towers fall within the same range. Also, the damping ratios are significantly lower in the higher modes than the values suggested in the AASHTO and CAN/CSA specifications. The comparatively higher damping ratios in the first mode may be due to aerodynamic damping. These low damping ratios in combination with poor fatigue details contribute to the accumulation of a large number of damage-causing cycles. As predicted, the stresses in the original Sioux City tower are much greater than the stresses in the retrofitted towers at Sioux City. Additionally, it was found that poor installation practices which often lead to loose anchor bolts and out-of-level leveling nuts can cause high localized stresses in the towers, which can accelerate fatigue damage.

The 3' half of the mouse mammary tumor virus orf gene is not sufficient for its superantigen function in transgenic mice.

The Mouse Mammary Tumor Virus (MMTV) long terminal repeat contains an open reading frame (orf) of 960 nucleotides encoding a 36 kDa polypeptide with a putative transmembrane domain and five N-glycosylation sites in the N-terminal part of the protein. Transgenic mice bearing either the complete or the 3' terminal half of the orf sequence of MMTV-GR under the control of the SV40 promoter were raised. As shown previously by FACS analysis transgenic mice which express the complete orf gene have a significant deletion of V beta 14 expressing T cells at 6 weeks of age. Here we show that no clonal deletion of V beta 14 bearing T cells takes place in transgenic mice that contain orf sequences from the fifth ATG to the termination codon. The pattern of tissues expressing the truncated transgene was studied by the Polymerase Chain Reaction (PCR) and was very similar to the one obtained in the V beta 14 deleting animals. These data suggest that the amino-terminal portion of the ORF protein (pORF) is required for a superantigen function, while our previous data indicated that determinants from the carboxy-terminus play an important role for TCR V beta specificity.

Modeling and control of electromechanical systems

The material presented in the these notes covers the sessions Modelling of electromechanical systems, Passive control theory I and Passive control theory II of the II EURON/GEOPLEX Summer School on Modelling and Control of Complex Dynamical Systems.We start with a general description of what an electromechanical system is from a network modelling point of view. Next, a general formulation in terms of PHDS is introduced, and some of the previous electromechanical systems are rewritten in this formalism. Power converters, which are variable structure systems (VSS), can also be given a PHDS form.We conclude the modelling part of these lectures with a rather complex example, showing the interconnection of subsystems from several domains, namely an arrangement to temporally store the surplus energy in a section of a metropolitan transportation system based on dc motor vehicles, using either arrays of supercapacitors or an electric poweredflywheel. The second part of the lectures addresses control of PHD systems. We first present the idea of control as power connection of a plant and a controller. Next we discuss how to circumvent this obstacle and present the basic ideas of Interconnection and Damping Assignment (IDA) passivity-based control of PHD systems.

No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. METHODOLOGY/PRINCIPAL FINDINGS: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. CONCLUSIONS/SIGNIFICANCE: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Activation of human meiosis-specific recombinase Dmc1 by Ca2+.

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.

05028-010 Urban Watershed of Dickinson Cty Lakes Final Report

The Dickinson SWCD is applying for $486,800 over three years from the Watershed Improvement Fund to enhance water quality in Dickinson County through an impairment-based, locally directed watershed improvement project dealing specifically with storm water runoff. The LID Project will provide a cost share incentive and technical expertise to individual and business owners in specially targeted districts who are willing to implement low impact development techniques such as rain gardens, bioswales, pervious paving to reduce storm water runoff from their properties. Goals for the project include: 1) Defining and prioritizing urban watersheds in the Iowa Great Lakes region for implementation of Low Impact Development Practices; 2) Providing technical expertise in the form of a graduate assistant/project manager to design and oversee construction; 3) Continuing public education of such practices and their local existence through project kiosk, brochures, County Naturalist programs, local cable television shows, tours and other interactions of the Clean Water Alliance with its 50 partners in the area concerned about water quality; and 4) Completing 125 separate projects over a three year period.

Structure and function of RecA-DNA complexes.

While the E. coli RecA protein has been the most intensively studied enzyme of homologous recombination, the unusual RecA-DNA filament has stood alone until very recently. It now appears that this protein is part of a universal family that spans all of biology, and the filament that is formed by the protein on DNA is a universal structure. With RecA's role in recombination given new and greatly increased significance, we focus in this review on the energetics of the RecA-mediated strand exchange and the relation between the energetics and recombination spanning heterologous inserts.

Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

Probing Rad51-DNA interactions by changing DNA twist.

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

RDM1, a novel RNA recognition motif (RRM)-containing protein involved in the cell response to cisplatin in vertebrates.

A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis.

Ennakoiva vikojen havaitseminen liityntäverkosta

Tässä diplomityössä on tutkittu mahdollisuuksia ennakoida ja havaita kaapelivikoja mahdollisimman varhaisessa vaiheessa teleoperaattorin liityntäverkosta, joka käsittää optisen rajapinnan ja asiakkaan välisen parikaapeleilla toteutetun osan verkkoa. Liityntäverkon ikääntyessä kaapeleiden ja liitosten viat yleistyvät sekä niiden korjauskustannukset kasvavat. Mahdollisimmanvarhaisessa vaiheessa vikoja havaitsemalla ehkäistään vikojen laajentumista sekä asiakkaiden palveluiden katkeamista. Kannattavin tapa toteuttaa liityntäverkon vikoja ennakoiva järjestelmä Elisa Oyj:n tapauksessa on hyödyntää yrityksessä uudistettua DSL-tekniikkaa. DSLAM:eilla päivittäin suoritetuilla mittauksilla voidaan havaita liityntäverkon kaapeleiden kunnon heikkeneminen sekä vikaantumiset. Työssä on tutkittu vikatapausten aiheuttamia muutoksia linjojen sähköisiin ominaisuuksiin sekä niiden näkymistä mittaustuloksissa. Työssä löydettiin menetelmät toteuttaa liityntäverkon kuntoa mittaava ja tarkkaileva järjestelmä. Ennakoivalla toiminnalla voidaan aikaansaada merkittäviä kustannussäästöjä viankorjausprosessin eri vaiheissa sekä parantaa palveluiden laatua.

The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose.

Pob1 participates in the Cdc42 regulation of fission yeast actin cytoskeleton.

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1(+) as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745-2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1DeltaN) or the SAM domain (pob1DeltaSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1DeltaN, and pob1DeltaSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.