Semi-Automated Methods for Measuring Practice Conformance for Capital Projects

The goal of this thesis is to explore semi-automated methods for measuring practice conformance for capital projects. Thorough measurement of practice conformance for capital projects typically requires manual audits. Surveys that may assist can often be subjective, non-repeatable and unverifiable, since they are self-reported. However, some of the tasks assigned to auditors are also non-repeatable, and they may be costly, time-consuming, tedious, and error-prone. Tools for assisting practice conformance measurements are in high demand in the construction domain. In response, various information technology-based and web deployed Benchmarking and Metrics (BM&M) programs have been introduced to reduce time and costs, to assist in providing repeatable and accurate results, and to increase efficiency and productivity of reporters and auditors. Moreover, moves toward automated practice conformance measurement are expected to reduce time and cost. Past studies have also resulted in significant advances in data mining, natural language processing, machine learning, computer vision and other artificial intelligence-based approaches toward complete automation, but technical limitations exist that constrain complete automation or make it impractical. An approach is needed to support practical, net beneficial, incremental steps toward automation of practice conformance measurement for capital projects that would assist capital project participants to improve project performance over time. To address this need, a new approach is proposed in this thesis. Additionally, a framework to beneficially increase automation is presented. Toolsets are explored that may make practice conformance measurement cheaper, faster, easier, repeatable, and more accurate for capital project participants. This framework and the toolsets are validated through the development of a practice conformance model, case studies on real project data, and application experiments. It is concluded that the proposed semi-automated framework for measuring practice conformance for capital projects is practical to implement in the near term. These results provide a basis on which capital project participants can implement efficacious practice conformance measurement to support capital project performance improvement programs

Ex vivo promoter analysis of antiviral heat shock cognate 70B gene in Anopheles gambiae

<p>Abstract</p> <p>Background</p> <p>The <it>Anopheles gambiae </it>heat shock cognate gene (<it>hsc70B</it>) encodes a constitutively expressed protein in the <it>hsp70 </it>family and it functions as a molecular chaperone for protein folding. However, the expression of <it>hsc70B </it>can be further induced by certain stimuli such as heat shock and infection. We previously demonstrated that the <it>An. gambiae hsc70B </it>is induced during o'nyong-nyong virus (ONNV) infection and subsequently suppresses ONNV replication in the mosquito. To further characterize the inducibility of <it>hsc70B </it>by ONNV infection in <it>An. gambiae</it>, we cloned a 2.6-kb region immediately 5' upstream of the starting codon of <it>hsc70B </it>into a luciferase reporter vector (pGL3-Basic), and studied its promoter activity in transfected Vero cells during infection with o'nyong-nyong, West Nile and La Crosse viruses.</p> <p>Results</p> <p>Serial deletion analysis of the <it>hsc70B </it>upstream sequence revealed that the putative promoter is likely located in a region 1615–2150 bp upstream of the <it>hsc70B </it>starting codon. Sequence analysis of this region revealed transcriptional regulatory elements for heat shock element-binding protein (HSE-bind), nuclear factor κB (NF-κB), dorsal (Dl) and fushi-tarazu (Ftz). Arbovirus infection, regardless of virus type, significantly increased the <it>hsc70B </it>promoter activity in transfected Vero cells.</p> <p>Conclusion</p> <p>Our results further validate the transcriptional activation of <it>hsc70B </it>during arbovirus infection and support the role of specific putative regulatory elements. Induction by three taxonomically distinct arboviruses suggests that the HSC70B protein may be expressed to cope with cellular stress imposed during infection.</p

High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

Background: Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti

Identification of anti-flaviviral drugs with mosquitocidal and anti-Zika virus activity in Aedes aegypti.

Zika virus (ZIKV), an emerging arbovirus belonging to the genus Flavivirus, is transmitted by Aedes mosquitoes. ZIKV infection can cause microcephaly of newborn babies and Guillain-Barré syndrome in adults. Because no licensed vaccine or specific antiviral treatment is available for ZIKV infection, the most commonly used approach to control the spread of ZIKV is suppression of the mosquito vector population. A novel proposed strategy to block arthropod virus (arbovirus) transmission is based on the chemical inhibition of virus infection in mosquitoes. However, only a few drugs and compounds have been tested with such properties. Here we present a comprehensive screen of 55 FDA-approved anti-flaviviral drugs for potential anti-ZIKV and mosquitocidal activity. Four drugs (auranofin, actinomycin D (Act-D), bortezomib and gemcitabine) were toxic to C6/36 cells, and two drugs (5-fluorouracil and mycophenolic acid (MPA)) significantly reduced ZIKV production in C6/36 cells at 2 μM and 0.5 μM, respectively. Three drugs (Act-D, cyclosporin A, ivermectin) exhibited a strong adulticidal activity, and six drugs (U18666A, retinoic acid p-hydroxyanilide (4-HPR), clotrimazole, bortezomib, MPA, imatinib mesylate) significantly suppressed ZIKV infection in mosquito midguts. Some of these FDA-approved drugs may have potential for use for the development of ZIKV transmission-blocking strategies

Profiling Transcripts of Vector Competence between Two Different Aedes aegypti Populations in Florida

A Chikungunya virus (CHIKV) outbreak in Italy in 2007 spread to include the islands of the Caribbean and most of the Americas and still circulates in Europe and Africa. Florida being close in distance to the Caribbean islands experienced a CHIKV outbreak in 2014 and continues to have a few travel-related cases each year. It is known that different environmental conditions in different regions can result in genetic variation that favor changes in competence to arbovirus. We evaluated the vector competence of Florida Aedes aegypti for CHIKV and determined if there is a geographic component that influences genes involved in CHIKV competence. We utilized a genomic approach to identify the candidate genes using RNA sequencing. The infection and dissemination results showed that field populations were more competent vectors for CHIKV than a lab population. The differentially expressed genes in the two field-collected CHIKV-infected populations, compared to the Rockefeller strain, were related to the Wnt/Notch signaling pathway, with similarity to genes scattered throughout the signaling pathway. This result suggested the possibility of identifying genes involved in the determination of vector competence in different gene pools of Ae. aegypti

Suppressing dengue-2 infection by chemical inhibition of Aedes aegypti host factors.

Dengue virus host factors (DENV HFs) that are essential for the completion of the infection cycle in the mosquito vector and vertebrate host represent potent targets for transmission blocking. Here we investigated whether known mammalian DENV HF inhibitors could influence virus infection in the arthropod vector A. aegypti. We evaluated the potency of bafilomycin (BAF; inhibitor of vacuolar H+-ATPase (vATPase)), mycophenolic acid (MPA; inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH)), castanospermine (CAS; inhibitor of glucosidase), and deoxynojirimycin (DNJ; inhibitor of glucosidase) in blocking DENV infection of the mosquito midgut, using various treatment methods that included direct injection, ingestion by sugar feeding or blood feeding, and silencing of target genes by RNA interference (RNAi). Injection of BAF (5 µM) and MPA (25 µM) prior to feeding on virus-infected blood inhibited DENV titers in the midgut at 7 days post-infection by 56% and 60%, and in the salivary gland at 14 days post-infection by 90% and 83%, respectively, while treatment of mosquitoes with CAS or DNJ did not affect susceptibility to the virus. Ingestion of BAF and MPA through a sugar meal or together with an infectious blood meal also resulted in various degrees of virus inhibition. RNAi-mediated silencing of several vATPase subunit genes and the IMPDH gene resulted in a reduced DENV infection, thereby indicating that BAF- and MPA-mediated virus inhibition in adult mosquitoes most likely occurred through the inhibition of these DENV HFs. The route and timing of BAF and MPA administration was essential, and treatment after exposure to the virus diminished the antiviral effect of these compounds. Here we provide proof-of-principle that chemical inhibition or RNAi-mediated depletion of the DENV HFs vATPase and IMPDH can be used to suppress DENV infection of adult A. aegypti mosquitoes, which may translate to a reduction in DENV transmission

Culex quinquefasciatus (Diptera: Culicidae) From Florida Transmitted Zika Virus

We report a laboratory colony of Culex quinquefasciatus mosquitoes were experimentally able to salivate Zika virus (ZIKV, Flaviviridae; Flavivirus) at 16 days post infection (dpi). ZIKV RNA was detected in bodies and in saliva deposited on filter paper cards with subsequent studies demonstrating the presence of live ZIKV in saliva

Co-silencing of vATPase (vATP-V0B) and IMPDH.

<p>Co-silencing of vATPase (vATP-V0B) and IMPDH reduced DENV titers more than single silencing, although the difference between the co-silenced and vATPase single-silenced mosquito cohorts was not significant (p = 0.0535, Mann-Whitney test). ***; p<0.001, **; p<0.01, Mann-Whitney test. Descriptive statistics for DENV infection assays are presented in supplementary table S2.</p

Mixing Simulation for Powder Injection Moulding Feedstock: Quantification and Sensitivity Analysis

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Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein.

Dengue virus (DENV) is the most prevalent and burdensome arbovirus transmitted by Aedes mosquitoes, against which there is only a limited licensed vaccine and no approved drug treatment. A Chromobacterium species, C. sp. Panama, isolated from the midgut of A. aegypti is able to inhibit DENV replication within the mosquito and in vitro. Here we show that C. sp. Panama mediates its anti-DENV activity through secreted factors that are proteinous in nature. The inhibitory effect occurs prior to virus attachment to cells, and is attributed to a factor that destabilizes the virion by promoting the degradation of the viral envelope protein. Bioassay-guided fractionation, coupled with mass spectrometry, allowed for the identification of a C. sp. Panama-secreted neutral protease and an aminopeptidase that are co-expressed and appear to act synergistically to degrade the viral envelope (E) protein and thus prevent viral attachment and subsequent infection of cells. This is the first study characterizing the anti-DENV activity of a common soil and mosquito-associated bacterium, thereby contributing towards understanding how such bacteria may limit disease transmission, and providing new tools for dengue prevention and therapeutics