Structure-Function Analysis of Insect Olfactory Receptors

Organisms use their senses to transform external stimuli into an internal representation of the world. Insects employ their keen sense of smell for a variety of tasks including location of food sources, which can vary from yeast growing on ripe fruits for the vinegar fly Drosophila melanogaster to mammals for blood-feeding insects such as the mosquito Anopheles gambiae. The first informational relay between the external environment and the organism is the olfactory sensory neuron (OSN), whose activation translates the intensity, quality, and temporal features of volatile chemicals into spike trains. This dissertation focuses on understanding how the insect olfactory system functions at the periphery, shedding light on the molecular players involved and the interactions between environmental chemicals and OSNs. In Drosophila, most of the ~1,200 OSNs express members of the olfactory receptor (OR) protein family (Stocker, 1994; Vosshall et al., 1999). The functional OR complex comprises at least one variable odorant-binding subunit and one constant subunit named OR83b (Benton et al., 2006). Insect ORs have historically been grouped with mammalian and nematode ORs, both of which are G protein coupled receptors (GPCRs), whose activation leads to increased concentrations of intracellular second messengers and opening of cyclic nucleotide-gated channels (CNG; Buck and Axel, 1991; Colbert et al., 1997; Firestein et al., 1991; Nakamura and Gold, 1987; Troemel et ii al., 1995). Insect ORs lack similarity to GPCRs (Benton et al., 2006; Vosshall et al., 1999), and we hypothesized that they function as odorant-gated ion channels. We showed that expression of insect ORs in heterologous cells generates odorant-evoked currents that are resistant to G protein inhibitors, independent of cyclic nucleotides, and whose properties change based on OR subunit composition (Sato et al., 2008). This surprising discovery supports our hypothesis that insect ORs are indeed odorant-gated ion channels. Concurrently with these findings, we investigated the mode of action of DEET, the most widely used topical insect repellent, and showed that ORs are among its molecular targets. We demonstrated that DEET suppresses Drosophila food-seeking behavior, modulates OSN activity, and decreases OR-mediated currents in heterologous cells (Ditzen et al., 2008). Moreover, we showed that a missense polymorphism in a ligand-binding OR subunit leads to pharmacological resistance to the repellent in vivo. This is the first finding that identifies a molecular target of DEET. Within the OR complex, OR83b plays an essential role. Ligand-binding subunits fail to localize properly at the OSN dendrite in the absence of OR83b, resulting in almost complete loss of sense of smell (Benton et al., 2006; Larsson et al., 2004). We identified a putative localization motif in the OR83b protein, and showed that mutations in conserved residues abolish proper OR trafficking and impair odorant-evoked responses. This discovery defines critical amino acids that might be used as possible targets of future repellents to modulate the activity of insect OSNs. The discoveries described in this thesis will have an impact on the design of better and safer insect repellents and the control of insect-borne diseases

Effect of the numbers of slots and barriers on the optimal design of synchronous reluctance machines

This paper analyzes the impact of the numbers of stator slots and rotor layers on the optimal design of synchronous reluctance (SyR) machines. Eighteen SyR machine examples have been designed by means of a multi-objective optimization algorithm and finite element analysis so to maximize torque and minimize torque ripple. Twelve, twenty-four and forty-eight slot stators are considered, associated to rotors with fourpoles and one to six flux barriers per pole. The results of the comparative analysis show that high numbers of slots and layers are beneficial for maximizing the torque and the power factor, and that torque ripple and iron loss minimization require precise matches between the slots and the layers, which are not necessarily the same for the two purposes. Finally, for some slot/layer combinations the optimization algorithm produces nonconventional barrier distributions, very promising is some cases. A fast finite element evaluation is used for the evaluation of thousands of candidate machines during the optimization, whereas an accurate transient with motion finite element analysis stage is used for the off-line characterization of the final designs

Deepening inside the pictorial layers of Etruscan sarcophagus of Hasti Afunei: An innovative micro-sampling technique for Raman/SERS analyses

The Hasti Afunei sarcophagus is a large Etruscan urn, made up of two chalky alabaster monoliths. Dated from the last quarter of the third century BC, it was found in 1826 in the small town of Chiusi (Tuscany- Il Colle place) by a landowner, Pietro Bonci Casuccini, who made it part of his private collection. The noble owner’s collection was sold in 1865 to the Royal Museum of Palermo (today under the name of Antonino Salinas Regional Archaeological Museum), where it is still displayed. The sarcophagus is characterized by a complex iconography that is meticulously illustrated through an excellent sculptural technique, despite having subjected to anthropic degradation and numerous restorative actions during the last century. During the restoration campaign carried out between 2016 and 2017, a targeted diagnostic campaign was carried out to identify the constituent materials of the artefact, the pigments employed and the executive technique, in order to get an overall picture of conservation status and conservative criticalities. In particular, this last intervention has allowed the use of the innovative micro-sampling technique, patented by the Cultural Heritage research group of Sapienza, in order to identify the employee of lake pigments through SERS analyses. Together with this analysis, Raman and NMR technique have completed the information requested by restorers, for what concerns the wax employed as protective layers, and allowed to rebuild the conservation history of the sarcophagus. In fact, together with the identification of red ocher and yellow ocher, carbon black, Egyptian blue and madder lake, pigments compatible with the historical period of the work, modern pigments (probably green Paris, chrome orange, barium yellow, blue phtalocyanine) have been recognized, attributable with not documented intervention during the eighteenth and twentieth centuries. © 2019 by the authors

Association Between Periodontal Disease and Left Ventricle Mass in Essential Hypertension

Chronic periodontitis has been associated with an increased risk for cardiovascular disease. Left ventricular mass is an established independent predictor of cardiovascular disease. In the present cross-sectional study, we tested the association between periodontitis and left ventricular mass in subjects with essential hypertension. One hundred four untreated subjects with essential hypertension underwent clinical examinations, including echocardiographic study, laboratory tests, and assessment of periodontal status according to the community periodontal index of treatment needs (CPITN). With increasing severity of periodontitis, there was a progressive increase in left ventricle mass. Mean values (g/height2.7) were 39.0 (±2.7) in CPITN 0 (periodontal health), 40.2 (±6.4) in CPITN 1 (gingival bleeding), 42.7 (±6.8) in CPITN 2 (calculus), 51.4 (±11.7) in CPITN 3 (pockets 4 to 5 mm), and 76.7 (±11.3) in CPITN 4 (pockets ≥6 mm) (overall F 51.2;P<0.0001). Body surface area (P=0.04), systolic (P<0.0001) and diastolic (P<0.01) blood pressure, and left ventricular mass (P<0.0001) were determinants of a composite of CPITN 3 and 4. In a multivariate logistic analysis, left ventricular mass was the sole determinant (P<0.0001) of CPITN stages 3 and 4. Our findings suggest a direct association between severity of periodontitis and left ventricular mass in subjects with essential hypertension. Periodontal evaluation might contribute to refine cardiovascular risk assessment in hypertensive subjects

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7

CLOE: Identification of putative functional relationships among genes by comparison of expression profiles between two species

BACKGROUND: Public repositories of microarray data contain an incredible amount of information that is potentially relevant to explore functional relationships among genes by meta-analysis of expression profiles. However, the widespread use of this resource by the scientific community is at the moment limited by the limited availability of effective tools of analysis. We here describe CLOE, a simple cDNA microarray data mining strategy based on meta-analysis of datasets from pairs of species. The method consists in ranking EST probes in the datasets of the two species according to the similarity of their expression profiles with that of two EST probes from orthologous genes, and extracting orthologous EST pairs from a given top interval of the ranked lists. The Gene Ontology annotation of the obtained candidate partners is then analyzed for keywords overrepresentation. RESULTS: We demonstrate the capabilities of the approach by testing its predictive power on three proteomically-defined mammalian protein complexes, in comparison with single and multiple species meta-analysis approaches. Our results show that CLOE can find candidate partners for a greater number of genes, if compared to multiple species co-expression analysis, but retains a comparable specificity even when applied to species as close as mouse and human. On the other hand, it is much more specific than single organisms co-expression analysis, strongly reducing the number of potential candidate partners for a given gene of interest. CONCLUSIONS: CLOE represents a simple and effective data mining approach that can be easily used for meta-analysis of cDNA microarray experiments characterized by very heterogeneous coverage. Importantly, it produces for genes of interest an average number of high confidence putative partners that is in the range of standard experimental validation techniques

Harshlight: a "corrective make-up" program for microarray chips

BACKGROUND: Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans do show similar artifacts, which might affect subsequent analysis. Although all but the starkest blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs), few tools are available to help with the detection of those defects. RESULTS: We develop a novel tool, Harshlight, for the automatic detection and masking of blemishes in HDONA microarray chips. Harshlight uses a combination of statistic and image processing methods to identify three different types of defects: localized blemishes affecting a few probes, diffuse defects affecting larger areas, and extended defects which may invalidate an entire chip. CONCLUSION: We demonstrate the use of Harshlight can materially improve analysis of HDONA chips, especially for experiments with subtle changes between samples. For the widely used MAS5 algorithm, we show that compact blemishes cause an average of 8 gene expression values per chip to change by more than 50%, two of them by more than twofold; our masking algorithm restores about two thirds of this damage. Large-scale artifacts are successfully detected and eliminated

Low Latency Protocols Investigation for Event-Driven Wireless Body Area Networks

Nowadays distributed electronic health and fitness monitoring are hot-topics in bio-engineering, however common solutions for Wireless Body Area Networks (WBANs) featuring high-density sampled data transmission still stumbles over the trade-off among data rate, application throughput, and latency. Therefore, the Bluetooth Low Energy (BLE) and the IEEE 802.15.4 protocols are here investigated, with the aim of developing an event-driven WBAN to support a threshold-crossing surface ElectroMyoGraphy (sEMG) acquisition approach. We then implemented a custom protocol to overcome their limitations and fulfil all the requirements, resulting in a transmission latency of 0.856 ms ± 1 µs and enabling a functional operating time up to 110 h