Diagnostic techniques for EHD and MHD interaction

The impact of plasma technologies is growing both in the academic and in the industrial fields. Nowadays, a great interest is focused in plasma applications in aeronautics and astronautics domains. Plasma actuators based on the Magneto-Hydro-Dynamic (MHD) and Electro- Hydro-Dynamic (EHD) interactions are potentially able to suitably modify the fluid-dynamics characteristics around a flying body without utilizing moving parts. This could lead to the control of an aircraft with negligible response time, more reliability and improvements of the performance. In order to study the aforementioned interactions, a series of experiments and a wide number of diagnostic techniques have been utilized. The EHD interaction, realized by means of a Dielectric Barrier Discharge (DBD) actuator, and its impact on the boundary layer have been evaluated by means of two different experiments. In the first one a three phase multi-electrode flat panel actuator is used. Different external flow velocities (from 1 to 20m/s) and different values of the supplied voltage and frequency have been considered. Moreover a change of the phase sequence has been done to verify the influence of the electric field existing between successive phases. Measurements of the induced speed had shown the effect of the supply voltage and the frequency, and the phase order in the momentum transfer phenomenon. Gains in velocity, inside the boundary layer, of about 5m/s have been obtained. Spectroscopic measurements allowed to determine the rotational and the vibrational temperature of the plasma which lie in the range of 320 ÷ 440°K and of 3000 ÷ 3900°K respectively. A deviation from thermodynamic equilibrium had been found. The second EHD experiment is realized on a single electrode pair DBD actuator driven by nano-pulses superimposed to a DC or an AC bias. This new supply system separates the plasma formation mechanism from the acceleration action on the fluid, leading to an higher degree of the control of the process. Both the voltage and the frequency of the nano-pulses and the amplitude and the waveform of the bias have been varied during the experiment. Plasma jets and vortex behavior had been observed by means of fast Schlieren imaging. This allowed a deeper understanding of the EHD interaction process. A velocity increase in the boundary layer of about 2m/s had been measured. Thrust measurements have been performed by means of a scales and compared with experimental data reported in the literature. For similar voltage amplitudes thrust larger than those of the literature, had been observed. Surface charge measurements led to realize a modified DBD actuator able to obtain similar performances when compared with that of other experiments. However in this case a DC bias replacing the AC bias had been used. MHD interaction experiments had been carried out in a hypersonic wind tunnel in argon with a flow of Mach 6. Before the MHD experiments a thermal, fluid-dynamic and plasma characterization of the hypersonic argon plasma flow have been done. The electron temperature and the electron number density had been determined by means of emission spectroscopy and microwave absorption measurements. A deviation from thermodynamic equilibrium had been observed. The electron number density showed to be frozen at the stagnation region condition in the expansion through the nozzle. MHD experiments have been performed using two axial symmetric test bodies. Similar magnetic configurations were used. Permanent magnets inserted into the test body allowed to generate inside the plasma azimuthal currents around the conical shape of the body. These Faraday currents are responsible of the MHD body force which acts against the flow. The MHD interaction process has been observed by means of fast imaging, pressure and electrical measurements. Images showed bright rings due to the Faraday currents heating and exciting the plasma particles. Pressure measurements showed increases of the pressure in the regions where the MHD interaction is large. The pressure is 10 to 15% larger than when the MHD interaction process is silent. Finally by means of electrostatic probes mounted flush on the test body lateral surface Hall fields of about 500V/m had been measured. These results have been used for the validation of a numerical MHD code.

Structure of the Southern Tyrrhenian subduction system: insights from seismological analysis of anisotropy and attenuation

The Southern Tyrrhenian subduction system shows a complex interaction among asthenospheric flow, subducting slab and overriding plate. To shed light on the deformations and mechanical properties of the slab and surrounding mantle, I investigated seismic anisotropy and attenuation properties through the subduction region. I used both teleseisms and slab earthquakes, analyzing shear-wave splitting on SKS and S phases, respectively. The fast polarization directions φ, and the delay time, δt, were retrieved using the method of Silver and Chan [1991. SKS and S φ reveal a complex anisotropy pattern across the subduction zone. SKS-rays sample primarily the sub-slab region showing rotation of fast directions following the curved shape of the slab and very strong anisotropy. S-rays sample mainly the slab, showing variable φ and a smaller δt. SKS and S splitting reveals a well developed toroidal flow at SW edge of the slab, while at its NE edge the pattern is not very clear. This suggests that the anisotropy is controlled by the slab rollback, responsible for about 100 km slab parallel φ in the sub-slab mantle. The slab is weakly anisotropic, suggesting the asthenosphere as main source of anisotropy. To investigate the physical properties of the slab and surrounding regions, I analyzed the seismic P and S wave attenuation. By inverting high-quality S-waves t* from slab earthquakes, 3D attenuation models down to 300 km were obtained. Attenuation results image the slab as low-attenuation body, but with heterogeneous QS and QP structure showing spot of high attenuation , between 100-200 km depth, which could be due dehydration associated to the slab metamorphism. A low QS anomaly is present in the mantle wedge beneath the Aeolian volcanic arc and could indicate mantle melting and slab dehydration.

Investigation on the Navigation Anomaly of Pioneer 10 and 11 probes

The present work consists of the investigation of the navigation of Pioneer 10 and 11 probes becoming known as the “Pioneer Anomaly”: the trajectories followed by the spacecrafts did not match the ones retrieved with standard navigation software. Mismatching appeared as a linear drift in the Doppler data received by the spacecrafts, which has been ascribed to a constant sunward acceleration of about 8.5×10-10 m/s2. The study presented hereafter tries to find a convincing explanation to this discrepancy. The research is based on the analysis of Doppler tracking data through the ODP (Orbit Determination Program), developed by NASA/JPL. The method can be summarized as: seek for any kind of physics affecting the dynamics of the spacecraft or the propagation of radiometric data, which may have not been properly taken into account previously, and check whether or not these might rule out the anomaly. A major effort has been put to build a thermal model of the spacecrafts for predicting the force due to anisotropic thermal radiation, since this is a model not natively included in the ODP. Tracking data encompassing more than twenty years of Pioneer 10 interplanetary cruise, plus twelve years of Pioneer 11 have been analyzed in light of the results of the thermal model. Different strategies of orbit determination have been implemented, including single arc, multi arc and stochastic filters, and their performance compared. Orbital solutions have been obtained without the needing of any acceleration other than the thermal recoil one indicating it as the responsible for the observed linear drift in the Doppler residuals. As a further support to this we checked that inclusion of additional constant acceleration as does not improve the quality of orbital solutions. All the tests performed lead to the conclusion that no anomalous acceleration is acting on Pioneers spacecrafts.

Noble-metal nanoparticles produced with colloidal lithography: fabrication, optical properties and applications

In this work, metal nanoparticles produced by nanosphere lithography were studied in terms of their optical properties (in connection to their plasmon resonances), their potential application in sensing platforms - for thin layer sensing and bio-recognition events -, and for a particular case (the nanocrescents), for enhanced spectroscopy studies. The general preparation procedures introduced early in 2005 by Shumaker-Parry et al. to produce metallic nanocrescents were extended to give rise to more complex (isolated) structures, and also, by combining colloidal monolayer fabrication and plasma etching techniques, to arrays of them. The fabrication methods presented in this work were extended not only to new shapes or arrangements of particles, but included also a targeted surface tailoring of the substrates and the structures, using different thiol and silane compounds as linkers for further attachment of, i.e. polyelectrolyte layers, which allow for a controlled tailoring of their nanoenvironment. The optical properties of the nanocrescents were studied with conventional transmission spectroscopy; a simple multipole model was adapted to explain their behaviour qualitatively. In terms of applications, the results on thin film sensing using these particles show that the crescents present an interesting mode-dependent sensitivity and spatial extension. Parallel to this, the penetrations depths were modeled with two simplified schemes, obtaining good agreement with theory. The multiple modes of the particles with their characteristic decay lengths and sensitivities represent a major improvement for particle-sensing platforms compared to previous single resonance systems. The nanocrescents were also used to alter the emission properties of fluorophores placed close to them. In this work, green emitting dyes were placed at controlled distances from the structures and excited using a pulsed laser emitting in the near infrared. The fluorescence signal obtained in this manner should be connected to a two-photon processes triggered by these structures; obtaining first insight into plasmon-mediated enhancement phenomena. An even simpler and faster approach to produce plasmonic structures than that for the crescents was tested. Metallic nanodiscs and nanoellipses were produced by means of nanosphere lithography, extending a procedure reported in the literature to new shapes and optical properties. The optical properties of these particles were characterized by extinction spectroscopy and compared to results from the literature. Their major advantage is that they present a polarization-dependent response, like the nanocrescents, but are much simpler to fabricate, and the resonances can be tailored in the visible with relative ease. The sensing capabilities of the metallic nanodiscs were explored in the same manner as for the nanocrescents, meaning their response to thin layers and to bio-recognition events on their surface. The sensitivity of these nanostructures to thin films proved to be lower than that of the crescents, though in the same order of magnitude. Experimental information about the near field extension for the Au nanodiscs of different sizes was also extracted from these measurements. Further resonance-tailoring approaches based on electrochemical deposition of metals on the nanodiscs were explored, as a means of modifying plasmon resonances by changing surface properties of the nanoparticles. First results on these experiments would indicate that the deposition of Ag on Au on a submonolayer coverage level can lead to important blue-shifts in the resonances, which would open a simple way to tailor resonances by changing material properties in a local manner.

Entomological monitoring in the cultural heritage facilities as prerequisite for a successful IPM programme

The European Committee for Standardization is working on a standard for the application of IPM (Integrated Pest Management) in museums and cultural heritage facilities. Since one of the aims of this research was to verify the approach against pests adopted in Italian conservation facilities, a survey was conducted. The results show that for the Italian museums, archives, libraries and historical houses pests are a problem, but IPM is unknown and prevention programmes to avoid damages caused by them, are not applied. In the most of cases pests problems are solved only when the risk is high and damages are visible. Also entomological monitoring, which represents a crucial part of IPM and could be very useful, is not included among the ordinary prevention activities. In addition, at present, the scientific researches on entomological traps, whether light or pheromones, for “cultural heritage pests” is extremely poor and only recently the behaviour and/or the physiology of the insects “of museums” have been investigated. For these reasons, tests to increase the traps using are performed. In particular, S. paniceum behaviour towards different attraction systems was investigated and the results indicate that the light traps efficiency could be improved using specific wavelengths and light sources.

New analytical LC-mass spectrometry methodologies for the quali-quantitative determination of natural substances and drugs in complex matrices

This thesis reports an integrated analytical and physicochemical approach for the study of natural substances and new drugs based on mass spectrometry techniques combined with liquid chromatography. In particular, Chapter 1 concerns the study of Berberine a natural substance with pharmacological activity for the treatment of hepatobiliary and intestinal diseases. The first part focused on the relationships between physicochemical properties, pharmacokinetics and metabolism of Berberine and its metabolites. For this purpose a sensitive HPLC-ES-MS/MS method have been developed, validated and used to determine these compounds during their physicochemical properties studies and plasma levels of berberine and its metabolites including berberrubine(M1), demethylenberberine(M3), and jatrorrhizine(M4) in humans. Data show that M1, could have an efficient intestinal absorption by passive diffusion due to a keto-enol tautomerism confirmed by NMR studies and its higher plasma concentration. In the second part of Chapter 1, a comparison between M1 and BBR in vivo biodistribution in rat has been studied. In Chapter 2 a new HPLC-ES-MS/MS method for the simultaneous determination and quantification of glucosinolates, as glucoraphanin, glucoerucin and sinigrin, and isothiocyanates, as sulforaphane and erucin, has developed and validated. This method has been used for the analysis of functional foods enriched with vegetable extracts. Chapter 3 focused on a physicochemical study of the interaction between the bile acid sequestrants used in the treatment of hypercholesterolemia including colesevelam and cholestyramine with obeticolic acid (OCA), potent agonist of nuclear receptor farnesoid X (FXR). In particular, a new experimental model for the determination of equilibrium binding isotherm was developed. Chapter 4 focused on methodological aspects of new hard ionization coupled with liquid chromatography (Direct-EI-UHPLC-MS) not yet commercially available and potentially useful for qualitative analysis and for “transparent” molecules to soft ionization techniques. This method was applied to the analysis of several steroid derivatives.

The biological basis of autism spectrum disorders: evaluation of oxidative stress and erytrocyte membrane alterations

This case-control study involved a total of 29 autistic children (Au) aged 6 to 12 years, and 28 gender and age-matched typically developing children (TD). We evaluated a high number of peripheral oxidative stress parameters, erythrocyte and lymphocyte membrane functional features and membrane lipid composition of erythrocyte. Erythrocyte TBARS, Peroxiredoxin II, Protein Carbonyl Groups and urinary HEL and isoprostane levels were elevated in AU (confirming an imbalance of the redox status of Au); other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma Total antioxidant capacity and plasma carbonyl groups, erythrocyte SOD and catalase activities) were unchanged, whilst peroxiredoxin I showed a trend of elevated levels in red blood cells of Au children. A very significant reduction of both erythrocyte and lymphocyte Na+, K+-ATPase activity (NKA), a reduction of erythrocyte membrane fluidity, a reduction of phospatydyl serine exposition on erythrocyte membranes, an alteration in erythrocyte fatty acid membrane profile (increase in MUFA and in ω6/ω3 ratio due to decrease in EPA and DHA) and a reduction of cholesterol content of erythrocyte membrane were found in Au compared to TD, without change in erythrocyte membrane sialic acid content and in lymphocyte membrane fluidity. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity, and ADOS and CARS score are inversely related to peroxiredoxin II levels. Oxidative stress and erythrocyte structural and functional alterations may play a role in the pathogenesis of Autism Spectrum Disorders and could be potentially utilized as peripheral biomarkers.

The role of SENP1 in B cell development and differentiation

SUMOylation is a highly dynamic and reversible posttranslational protein modification closely related to ubiquitination. SUMOylation regulates a vast array of different cellular functions, such as cell cycle, nuclear transport, DNA damage response, proliferation and transcriptional activation. Several groups have shown in in vitro studies how important SUMOylation is for early B cell development and survival as well as for later plasma cell differentiation. This thesis focuses on the deSUMOylation protease SENP1 and its in vivo effects on B cell development and differentiation. For this a conditional SENP1 knockout mouse model was crossed to the CD19-Cre mouse strain to generate a B cell specific SENP1 knockout mouse.rnIn our conditional SENP1ff CD19-Cre mouse model we observed normal numbers of all B cell subsets in the bone marrow. However in the spleen we observed an impairment of B cell survival, based on a 50% reduction of the follicular B cell compartment, whereas the marginal zone B cell compartment was unchanged. T cell numbers were comparable to control mice. rnFurther, impairments of B cell survival in SENP1ff CD19-Cre mice were analysed after in vivo blocking of IL7R signalling. The αIL7R treatment in mature mice blocked new B cell formation in the bone marrow and increased apoptosis rates could be observed in splenic SENP1 KO B cells. Additionally, a higher turnover rate of B cells was measured by in vivo BrdU incorporation.rnSince it is known that the majority of transcription factors that are important for the maintenance of the germinal centre reaction or for induction of plasma cell development are SUMOylated, the question arose, how defective deSUMOylation will manifest itself in these processes. The majority of in vitro cultured splenic B cells, stimulated to undergo class switch recombination and plasma cell differentiation underwent activation induced cell death. However, the surviving cells increasingly differentiated into IgM expressing plasma cells. Class switch recombination to IgG1 was reduced. These observations stood in line with observation made in in vivo sheep red blood cell immunization experiments, which showed increased amounts of germinal centres and germinal centre B cells, as well as increased amounts of plasma cells differentiation in combination with decreased class switch to IgG1.rnThese results lead to the conclusion that SENP1 KO B cells increasingly undergo apoptosis, however, B cells that survive SENP1 deficiency are more prone to undergo plasma cell differentiation. Further, the precursors of these plasma cells either are not as capable of undergoing class switch recombination or they do switch to IgG1 and succumb to activation induced cell death. One possible explanation for both scenarios could be a defective DNA damage response mechanisms during class switch recombination, caused by impaired deSUMOylation. rn

Na +-coupled betaine symporters involved in osmotic stress response in prokaryotes and eukaryotes

The betaine/GABA transporter BGT1 is one of the most important osmolyte transporters in the kidney. BGT1 is a member of the neurotransmitter sodium symporter (NSS) family, facilitates Na+/Cl--coupled betaine uptake to cope with hyperosmotic stress. Betaine transport in kidney cells is upregulated under hypertonic conditions by a yet unknown mechanism when increasing amounts of intracellular BGT1 are inserted into the plasma membrane. Re-establishing isotonicity results in ensuing depletion of BGT1 from the membrane. BGT1 phosphorylation on serines and threonines might be a regulation mechanism. In the present study, four potential PKC phosphorylation sites were mutated to alanines and the responses to PKC activators, phorbol 12-myristate acetate (PMA) and dioctanoyl-sn-glycerol (DOG) were determined. GABA-sensitive currents were diminished after 30 min preincubation with these PKC activators. Staurosporine blocked the response to DOG. Three mutants evoked normal GABA-sensitive currents but currents in oocytes expressing the mutant T40A were greatly diminished. [3H]GABA uptake was also determined in HEK-293 cells expressing EGFP-tagged BGT1 with the same mutations. Three mutants showed normal upregulation of GABA uptake after hypertonic stress, and downregulation by PMA was normal compared to EGFP-BGT1. In contrast, GABA uptake by the T40A mutant showed no response to hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells, grown on glass or filters, revealed that T40A was present in the cytoplasm after 24 h hypertonic stress while the other mutants and EGFP-BGT1 were predominantely present in the plasma membrane. All four mutants co-migrated with EGFP-BGT1 on Western blots suggesting they are full-length proteins. In conclusion, T235, S428, and S564 are not involved in downregulation of BGT1 due to phosphorylation by PKC. However, T40 near the N-terminus may be part of a hot spot important for normal trafficking or insertion of BGT1 into the plasma membrane. Additionally, a link between substrate transport regulation, insertion of BGT1 into the plasma membrane and N-glycosylation in the extracellular loop 2 (EL2) could be revealed. The functional importance of two predicted N-glycosylation sites, which are conserved in EL2 within the NSS family were investigated for trafficking, transport and regulated plasma membrane insertion by immunogold-labelling, electron microscopy, mutagenesis, two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radioactive-labelled substrate into MDCK cells. Trafficking and plasma membrane insertion of BGT1 was clearly promoted by proper N-glycosylation in both, oocytes and MDCK cells. De-glycosylation with PNGase F or tunicamycin led to a decrease in substrate affinity and transport rate. Mutagenesis studies revealed that in BGT1 N183 is the major N-glycosylation site responsible for full protein activity. Replacement of N183 with aspartate resulted in a mutant, which was not able to bind N-glycans suggesting that N171 is a non-glycosylated site in BGT1. N183D exhibited close to WT transport properties in oocytes. Surprisingly, in MDCK cells plasma membrane insertion of the N183D mutant was no longer regulated by osmotic stress indicating unambiguously that association with N-glycans at this position is linked to osmotic stress-induced transport regulation in BGT1. The molecular transport mechanism of BGT1 remains largely unknown in the absence of a crystal structure. Therefore investigating the structure-function relationship of BGT1 by a combination of structural biology (2D and 3D crystallization) and membrane protein biochemistry (cell culture, substrate transport by radioactive labeled GABA uptake into cells and proteoliposomes) was the aim of this work. While the functional assays are well established, structure determination of eukaryotic membrane transporters is still a challenge. Therefore, a suitable heterologous expression system could be defined, starting with cloning and overexpression of an optimized gene. The achieved expression levels in P. pastoris were high enough to proceed with isolation of BGT1. Furthermore, purification protocols could be established and resulted in pure protein, which could even be reconstituted in an active form. The quality and homogeneity of the protein allowed already 2D and 3D crystallization, in which initial crystals could be obtained. Interestingly, the striking structural similarity of BGT1 to the bacterial betaine transporter BetP, which became a paradigm for osmoregulated betaine transport, provided information on substrate coordination in BGT1. The structure of a BetP mutant that showed activity for GABA was solved to 3.2Å in complex with GABA in an inward facing open state. This structure shed some light into the molecular transport mechanisms in BGT1 and might help in future to design conformationally locked BGT1 to enforce the on-going structure determination.

Chronic impact of topiramate on acid-base balance and potassium in childhood

Topiramate, which is commonly prescribed for seizure disorders and migraine prophylaxis, sometimes causes metabolic acidosis and hypokalemia. Since the effects of topiramate on acid-base balance and potassium levels have not been well explored in children, acid-base balance, anion gap and potassium were assessed in 24 patients (8 females and 16 males) aged between 4.6 and 19 years on topiramate for more than 12 months and in an age-matched control group. Plasma bicarbonate (21.7 versus 23.4 mmol/L; P<0.03), carbon dioxide pressure (39.7 versus 43.2mm Hg; P<0.05), and potassium (3.7 versus 4.0 mmol/L; P<0.03) were on the average lower and chloride (109 versus 107 mmol/L; P<0.03) higher in patients treated with topiramate than in controls. Blood pH, plasma sodium and the anion gap were similar in patients on topiramate and in controls. In patients on topiramate no significant correlation was observed between the dosage of this agent and plasma bicarbonate or potassium as well as between topiramate blood level and the mentioned electrolytes. In conclusion long-term topiramate treatment is associated with a mild, statistically significant tendency towards compensated normal anion gap metabolic acidosis and hypokalemia.

Eculizumab in atypical hemolytic uremic syndrome: long-term clinical course and histological findings

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with defective regulation of the alternative complement pathway. The prognosis for patients with aHUS is poor, and plasma exchange represents the first-line therapy. Eculizumab is a humanized monoclonal anti-C5 antibody that prevents the activation of the terminal complement pathway. Here, we report the case of a 9-year-old girl with frequent relapsing aHUS due to heterozygous factor H mutation who was initially treated with plasma exchange three times per week with 150% plasma exchange volume. This treatment frequently caused allergic reactions and school absences. Because any reduction in the frequency of plasma exchange immediately induced relapses of the aHUS, treatment with eculizumab, 600 mg every 2 weeks, was started and plasma exchange completely stopped. On this drug regimen the patient showed no evidence of disease activity during a period of more than 24 months. Renal function improved, proteinuria disappeared, the number of antihypertensive medications could be decreased, and the quality of life increased substantially. The inhibition of the terminal complement pathway by eculizumab was also confirmed by renal biopsy, which showed the absence of thrombotic microangiopathy 2 months after the initiation of eculizumab therapy. This case illustrates the long-term favorable outcome of aHUS with eculizumab treatment.

Association of macular pigment density with plasma ω-3 fatty acids: the PIMAVOSA study

To assess the correlation between macular pigment optical density and plasma levels of lutein, zeaxanthin, and fatty acids, especially omega-3 polyunsaturated fatty acids (PUFAs).

Line focusing for soft x-ray laser-plasma lasing

A computational study of line-focus generation was done using a self-written ray-tracing code and compared to experimental data. Two line-focusing geometries were compared, i.e., either exploiting the sagittal astigmatism of a tilted spherical mirror or using the spherical aberration of an off-axis- illuminated spherical mirror. Line focusing by means of astigmatism or spherical aberration showed identical results as expected for the equivalence of the two frames of reference. The variation of the incidence angle on the target affects the line-focus length, which affects the amplification length such that as long as the irradiance is above the amplification threshold, it is advantageous to have a longer line focus. The amplification threshold is physically dependent on operating parameters and plasma-column conditions and in the present study addresses four possible cases.

Quantitative assessment of physiological and behavioural parameters in healthy dairy cows evoked by transcutaneous electrical nerve stimulation of the udder

The pain and distress associated with transcutaneous electrical nerve stimulation (TENS) of the udder was evaluated by treating 20 healthy dairy cows with an electrical udder stimulator. This generated a sequence of pulses (frequency: 160+/-10% impulses per second, duration 250 mus) and provided voltage ranges from 0 to 10 volts (+/-10%). Trials took place on three consecutive days, twice daily after morning and evening milking. Daily sessions were divided into two periods: (1) control (sham treatment) and (2) treatment (real treatment). Physiological (heart rate, respiratory rate, and plasma cortisol concentration) as well as ethological parameters (kicking, weight shifting, and looking backwards to udder) were defined as pain-indicating parameters and observed. Evaluation of data showed that only one parameter (kicking) was significantly increased during real treatment compared to sham treatment. It is concluded that the TENS therapy tested in this study can evoke changes in behaviour (increased kicking) consistent with an experience of pain in some cows.

Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function

The histidine triad nucleotide-binding (Hint2) protein is a mitochondrial adenosine phosphoramidase expressed in liver and pancreas. Its physiological function is unknown. To elucidate the role of Hint2 in liver physiology, the Hint2 gene was deleted. Hint2(-/-) and Hint2(+/+) mice were generated in a mixed C57Bl6/J x 129Sv background. At 20 weeks, the phenotypic changes in Hint2(-/-) relative to Hint2(+/+) mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycaemia, an increase in plasma interprandial insulin but a decrease in glucose stimulated insulin secretion and defective thermoregulation upon fasting. Leptin mRNA in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2(-/-) hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II to III electron transfer was decreased in Hint2(-/-) mitochondria, which was accompanied by a lower content of coenzyme Q. HIF-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2(-/-) mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-CoA dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2(-/-) vs. 83% in Hint2(+/+) ). This suggests a greater extent of acetylation in Hint2(-/-) than in Hint2(+/+) . Conlusions: Hint2 positively regulates mitochondrial lipid metabolism and respiration, and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins. (HEPATOLOGY 2012.).