Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies

This work was supported by a Knowledge Transfer Network BBSRC Industrial Case (#414 BB/L502467/1) studentship in association Zoetis Inc.

A Multi-Criteria GIS-Based Route Selection Tool for Hazardous Material Transport: Consideration of Environmental Consequence, Traffic Congestions and Costs

Hazardous materials are substances that, if not regulated, can pose a threat to human populations and their environmental health, safety or property when transported in commerce. About 1.5 million tons of hazardous material shipments are transported by truck in the US annually, with a steady increase of approximately 5% per year. The objective of this study was to develop a routing tool for hazardous material transport in order to facilitate reduced environmental impacts and less transportation difficulties, yet would also find paths that were still compelling for the shipping carriers as a matter of trucking cost. The study started with identification of inhalation hazard impact zones and explosion protective areas around the location of hypothetical hazardous material releases, considering different parameters (i.e., chemicals characteristics, release quantities, atmospheric condition, etc.). Results showed that depending on the quantity of release, chemical, and atmospheric stability (a function of wind speed, meteorology, sky cover, time and location of accidents, etc.) the consequence of these incidents can differ. The study was extended by selection of other evaluation criteria for further investigation because health risk as an evaluation criterion would not be the only concern in selection of routes. Transportation difficulties (i.e., road blockage and congestion) were incorporated as important factor due to their indirect impact/cost on the users of transportation networks. Trucking costs were also considered as one of the primary criteria in selection of hazardous material paths; otherwise the suggested routes would have not been convincing for the shipping companies. The last but not least criterion was proximity of public places to the routes. The approach evolved from a simple framework to a complicated and efficient GIS-based tool able to investigate transportation networks of any given study area, and capable of generating best routing options for cargos. The suggested tool uses a multi-criteria-decision-making method, which considers the priorities of the decision makers in choosing the cargo routes. Comparison of the routing options based on each criterion and also the overall suitableness of the path in regards to all the criteria (using a multi-criteria-decision-making method) showed that using similar tools as the one proposed by this study can provide decision makers insights in the area of hazardous material transport. This tool shows the probable consequences of considering each path in a very easily understandable way; in the formats of maps and tables, which makes the tradeoffs of costs and risks considerably simpler, as in some cases slightly compromising on trucking cost may drastically decrease the probable health risk and/or traffic difficulties. This will not only be rewarding to the community by making cities safer places to live, but also can be beneficial to shipping companies by allowing them to advertise as environmental friendly conveyors.

A Metabolomic Protocol For Plant Systematics By Matrix-assisted Laser-desorption/ionization Time-of Flight Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) has been widely used for the identification and classification of microorganisms based on their proteomic fingerprints. However, the use of MALDI-TOF MS in plant research has been very limited. In the present study, a first protocol is proposed for metabolic fingerprinting by MALDI-TOF MS using three different MALDI matrices with subsequent multivariate data analysis by in-house algorithms implemented in the R environment for the taxonomic classification of plants from different genera, families and orders. By merging the data acquired with different matrices, different ionization modes and using careful algorithms and parameter selection, we demonstrate that a close taxonomic classification can be achieved based on plant metabolic fingerprints, with 92% similarity to the taxonomic classifications found in literature. The present work therefore highlights the great potential of applying MALDI-TOF MS for the taxonomic classification of plants and, furthermore, provides a preliminary foundation for future research.

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

Background: During mating, insect males eject accessory gland proteins (Acps) into the female genital tract. These substances are known to affect female post-mating behavior and physiology. In addition, they may harm the female, e. g., in reducing its lifespan. This is interpreted as a consequence of sexual antagonistic co-evolution. Whereas sexual conflict abounds in non-social species, the peculiar life history of social insects (ants, bees, wasps) with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. Harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. Indeed, mating appears to increase female longevity in at least one ant species. Acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. In this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant Leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. Results: By a suppression subtractive hybridization protocol we obtained 20 unique sequences (USs). Twelve had mutual best matches with genes predicted for Apis mellifera and Nasonia vitripennis. Functional information (Gene Ontology) was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. The remaining eight USs did not match sequences from other species. Six genes were further analyzed by quantitative RT-PCR in three life cycle stages of male ants. A gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. Conclusions: Our study is the first one to investigate differential gene expression in ants in a context related to mating. Our findings indicate that male accessory glands of L. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. Identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial Hymenoptera. While the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females.

A novel method for selection of chymotrypsin inhibitors from a phage peptide library

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

Homeopathic practice in Intensive Care Units: objective semiology, symptom selection and a series of sepsis cases

Homeopathy has been used for more than two hundred years to treat chronic disease using various approaches in a wide range of diseases. However, for acute disease and critical illness, application has been limited by inadequate training of homeopathic physicians and the small number of pertinent clinical studies. In view of the difficulty of practising homeopathy in Intensive Care Units (ICU), a protocol was developed to facilitate description of objective homeopathic symptoms with a ranking of symptoms appropriate for these situations (Protocol for Objective Homeopathic Semiology). Examples of favorable results with individualized homeopathic treatments for a series of cases of Systemic Inflammatory Response Syndrome (sepsis) are described. Homeopathy (2008) 97, 206-213.

A rapid PCR protocol for marker assisted detection of heterozygotes in segregating generations involving 1BL/1RS translocation and normal wheat lines

A rapid and reliable polymerase chain reaction (PCR)-based protocol was developed for detecting zygosity of the 1BL/1RS translocation in hexaploid wheat. The protocol involved a multiplex PCR with 2 pairs of oligonucleotide primers, rye-specific Ris-1 primers, and consensus 5S intergenic spacer (IGS) primers, and digestion of the PCR products with the restriction enzyme, MseI. A small piece of alkali-treated intact leaf tissue is used as a template for the PCR, thereby eliminating the necessity for DNA extraction. The test is simple, highly sensitive, and rapid compared with the other detection systems of 1BS1RS heterozygotes in hexaploid wheat. PCR results were confirmed with AFLP analyses. Diagnostic tests for 1BL/1RS translocation based on Sec-1-specific ELISA, screening for chromosome arm 1RS controlled rust resistance locus Yr9, and the PCR test differed in their ability to detect heterozygotes. The PCR test and rust test detected more heterozygotes than the ELISA test. The PCR test is being used to facilitate S1 family recurrent selection in the Germplasm Enhancement Program of the Australian Northern Wheat Improvement Program. A combination of the PCR zygosity test with other markers currently being implemented in the breeding program makes this test economical for 1BL/1RS characterisation of S1 families.

Road drivers performance appraisal using a MCDA method: a case study in the road transport company

Performance evaluation increasingly assumes a more important role in any organizational environment. In the transport area, the drivers are the company’s image and for this reason it is important to develop and increase their performance and commitment to the company goals. This evaluation can be used to motivate driver to improve their performance and to discover training needs. This work aims to create a performance appraisal evaluation model of the drivers based on the multi-criteria decision aid methodology. The MMASSI (Multicriteria Methodology to Support Selection of Information Systems) methodology was adapted by using a template supporting the evaluation according to the freight transportation company in study. The evaluation process involved all drivers (collaborators being evaluated), their supervisors and the company management. The final output is a ranking of the drivers, based on their performance, for each one of the scenarios used.

Supporting NAT traversal and secure communications in a protocol implementation framework

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection.

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor approximately 18-25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

Agents mòbils i DTN: disseny i implementació d'un protocol d'encaminament en l'entorn PROSES

En aquest projecte s’ha treballat en l’entorn PROSES, on aeroports i avions de l’espai aeri són mules de transport sobre una xarxa DTN. L’objectiu principal és estudiar i simular dos escenaris concrets: l’enviament de notícies des de les torres de control als avions, i l’enviament de canvis de rutes de vol dels avions a un aeroport en qüestió. S’ha simulat el comportament de dos protocols d’encaminament diferents sobre els escenaris creats. Per a realitzar les proves s’ha utilitzat el simulador The ONE, s’ha implementat un nou protocol d’encaminament, s’ha creat un Generador de Mapes i Rutes, i s’han realitzat amb èxit les simulacions.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.