The capillary microextraction of volatiles (CMV) device was equipped with a novel Peltier cooler to investigate cryofocused extraction of organic gunshot residue (OGSR) for the first time. Prior research demonstrated the CMV’s capabilities for detecting nitroglycerin, 2,4-dinitrotoluene, diphenylamine, and ethyl centralite on shooters’ hands via gas chromatography-mass spectrometry. Further method development increased the recoveries of these four target compounds with an optimal 20-minute equilibrium time at 80˚C followed by extracting 3 L at a 1 L/min flow rate. The Cryo-CMV was evaluated for detection of semi-volatile OGSR compounds. The unique challenges presented with sampling of semi-volatiles were overcome by sample heating, applying high (>1 L/min) sampling flow rates and heating the transfer line between the container and cooled CMV. Cryofocusing at -10˚C provided increased recoveries for smokeless powders and OGSR compounds and therefore demonstrates excellent potential for other forensic applications with analysis of VOCs from fire debris and illicit drugs.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22359号 / 医博第4600号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長尾 美紀, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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INFECTIOUS COMPLICATIONS IN CHRONIC RENAL FAILURE Infections represent a serious problem in chronic kidney disease (cohort and they are) associated with signifficant morbidity and mortality. The thesis originated in the nephrology division of the Department of Internal Medicine I., Charles University Teaching Hospital and Medical Faculty in Pilsen, an institution with a long standing research activity in the field. In the theoretical part of this work, a general summary of infectious complications in chronic kidney disease population is presented. The other part of this work presents results of our research dealing with pharmacoeconomical aspects of cytomegalovirus infection and finally our results in the field of influenza vaccination. The Aim of the first presented work was to evaluate the cost impact of four different strategies for prevention of cytomegalovirus infection after renal transplantation. We provide post hoc analysis of 2 randomized studies performed in our department and calculating direct CMV-related expenses using valacyclovir prophylaxis, ganciclovir prophylaxis, preemptive vlaganciclovir treatment and deferred therapy. To simulate ihe impact of varying prices of prharmacotherapy or diagnostic procedures, a sensitivity analysis was performed. With respect to our results, valacyclovir...

Genom en digital enkätundersökning har kunskapsläget om medfödd Cytomegalovirus (CMV) i Sverige undersökts. Virusinfektionen är en av de vanligaste i världen och kan innebära skador på foster om smitta överförs från moder till foster under en graviditet. Av de 304 individer som besvarade enkäten hade 21 % hört talas om CMV men endast 10 % hade kunskap om hur man skyddar sig mot CMV. Att denna information inte nått ut till fler stämmer överens med andra undersökningar som genomförts i världen, men det preliminära genomsnittliga kunskapsläget för svenskar är lågt jämfört med övriga Europa. Blivande föräldrar behöver få veta riskerna med medfödd CMV och sambandet det har med rutiner i vardagen, som att dela bestick eller pussa syskon i yngre förskoleålder på munnen under en graviditet. Även i nära anslutning till COVID-19-pandemin tycks hygienrutiner inte blivit tillräckligt etablerade och säkra för att skydda även de svagaste i samhället mot onödig risk att smittas av medfödd CMV infektion som kan medföra allvarliga och bestående skador. / Through a digital survey, the level of knowledge about congenital Cytomegalovirus (CMV) has been investigated in Sweden. The virus infection is one of the most common worldwide and can cause damage to the fetus if the infection is transmitted from mother to fetus during pregnancy. Out of the 304 individuals who responded to the survey, 21% had heard of CMV, but only 10% had knowledge of how to protect themselves from CMV. The fact that this information has not reached more people is consistent with other studies conducted globally, but the preliminary knowledge level among Swedes is low compared to the rest of Europe. Parents need to be aware of the risks associated with congenital CMV infection and its connection to daily routines, such as sharing utensils or kissing younger siblings on the mouth during pregnancy. Even in the immediate aftermath of the COVID-19 pandemic, hygiene practices do not seem to have been sufficiently established and secure to protect the most vulnerable in society also from unnecessary risks of CMV transmission, which can lead to permanent and serious injuries.

Cytomegalovirus (CMV) is a significant cause of mortality and morbidity in immunocompromised patients and an important cause of birth defects if acquired in utero. The licensed CMV antivirals, ganciclovir, cidofovir and foscarnet, all target the viral DNA polymerase. For each drug prolonged use is associated with significant toxicities and development of drug resistance. None are approved for use during pregnancy. Therefore, development of new anti-CMV drugs that target different pathways would be beneficial. All herpesviruses encode an alkaline nuclease. That genetic disruption of the CMV alkaline nuclease, UL98, reduces CMV replication by 1,000-fold suggests that UL98 may be a useful target for development of novel anti-CMV drugs. Moreover, using herpes simplex virus type 1 Hsiang and Ho found that the anthraquinone emodin inhibits activity of the viral alkaline nuclease, blocks viral replication in cell culture, and reduces viral pathogeneses in a mouse model (Brit. J. of Pharm., 2008). Earlier studies also showed that anthraquinone derivatives including emodin have anti-CMV activity (Barnard et al., Antiviral Research 1992 & 1995), although the mechanism of CMV inhibition has not been further studied. We therefore sought to confirm the anti-CMV activities of emodin and related anthraquinone derivatives, to characterize their mechanisms of action, and to determine specifically if they act through inhibition of UL98. Using a luciferase-based CMV yield reduction assay emodin inhibited CMV replication (IC50 = 4.9 μM); however, that the TD50 for cytotoxicity (determined using an luciferase-based cell viability assay) was only 2-fold higher suggested that emodin may act non-specifically. Two additional anthraquinone derivatives (acid blue 40 and alizarin violet R) inhibited CMV only at high concentrations (IC50 = 238; 265 μM) that were also cytotoxic. Atanyl blue PRL, however, exhibited anti-CMV activity (IC50 = 6.3 μM) with low cytotoxicity (TD50 = 216 μM). Thus, characterization of atanyl blue PRL (impact on gene expression, GFP expression, viral spread, infectivity, time of addition studies, and inhibition of UL98 nuclease activity) should be informative. Atanyl blue PRL appears to block immediate-early gene expression and reduce early and late gene expression. Atanyl blue PRL also blocked GFP expression, reduced viral spread, and also lowered the infectivity of CMV. Finally, atanyl blue PRL inhibits UL98 alkaline nuclease activity at an IC50 of 5.7 μM. This suggests that atanyl blue PRL may inhibit CMV through inhibition of UL98. Thus, atanyl blue PRL represents a novel class of anti-herpesvirals and provides a lead structure for structure based drug discovery.

COMPARISON OF ALBUTEROL DELIVERY BETWEEN HIGH FREQUENCY OSCILLATORY VENTILATION AND CONVENTIONAL MECHANICAL VENTILATION IN A SIMULATED ADULT LUNG MODEL USING DIFFERENT COMPLIANCE LEVELS By Waleed A. Alzahrani, BSRT BACKGROUND: Delivery of aerosol by pMDI has been described with conventional mechanical ventilation (CMV) but not with high frequency oscillatory ventilation (HFOV). The purpose of this study was to compare aerosol delivery to a simulated 75 kg adult with low compliance during both CMV and HFOV. Since actuation of pMDI with inspiration is not feasible with HFOV, we investigated the impact of actuation timing only during CMV. METHOD: CMV (Respironics Esprit) and HFOV (Sensor Medics 3100B) ventilators with passover humidifiers and heated circuits were connected by 8 mm ID ETT and filter (Respirgard II, Vital Signs) to a test lung (TTL) with compliance settings of 20 and 40 ml/cm H2O in order to simulate a non compliant lung. Settings for CMV (VT 6 ml/kg, I:E 1:1, PEEP 20 cm H2O, and RR 25/min), and HFOV (RR 5 Hz, IT 33%, ∆P 80 cm H2O and mPaw 35 cm H2O) were used, with similar mPaw on CMV and HFOV. Parameters were selected based on ARDSnet protective lung strategy (Fessler and Hess, Respiratory Care 2007) Eight actuations of albuterol from pMDI (ProAir HFA, Teva Medical) with double nozzle small volume spacer (Mini Spacer, Thayer Medical) placed between the “Y” adapter and ETT at more than 15 sec intervals for each condition (n=3). During CMV, pMDI actuations were synchronized (SYNC) with the start of inspiration at more than 15 s, and nonsynchronized (NONSYNC) with actuations at 15 s intervals. Drug was eluted from the filter and analyzed by spectrophotometry (276 nm). Repeated measures ANOVA, pairwise comparisons and independent t- tests were performed at the significance level of 0.05. RESULTS: In all cases, aerosol delivery was greater with HFOV than CMV (p<0.05). Synchronizing pMDI actuations with the beginning of inspiration increased aerosol deposition significantly at compliance levels 20 ml/cm H2O and 40 ml/cm H2O (p=0.011 and p=0.02, respectively). Lung compliance and aerosol delivery are directly related. Increasing lung compliance to 40 ml/cmH2O improved aerosol delivery during CMV and HFOV (p<0.05). CONCLUSION: Albuterol deposition with pMDI was more than two fold greater with HFOV than CMV in this in-vitro lung model. Changing lung compliance has almost 2 fold impact on aerosol delivery during both modes of ventilation. Furthermore, synchronizing pMDI actuations during CMV improved aerosol delivery up to 4 fold.

The quality of immune responses develops from birth into adulthood and in the context of the host microbial environment. The aim of this work was to study immune maturation during childhood, and how this process can be affected by the common herpesviruses; Epstein-Barr virus (EBV) and cytomegalovirus (CMV). In paper I we studied monocytes, an important cell type for immunity in the newborn. We showed that the neonatal monocyte subsets exist in similar frequencies as adult subsets, and have a potent capacity for pro-inflammatory cytokine production. In paper II, III and IV we studied the effects of EBV and CMV infections on immune cell function in children. In paper II we found that monocyte-induced NK-cell production of IFN-γ, and plasma IFN-γ levels, were decreased in 2-year old EBV- and/or CMV-seropositive children and mostly so in co-infected children. In paper III we found that in 5-year old children, EBV and CMV co-infection was associated with the highest levels of differentiated NKG2C+ NK cells. CMV+ children had higher plasma IFN-γ and IL-15 levels and higher NK-cell cytotoxic capacity. In vitro PBMC systems showed elevated frequencies of NKG2C+ NK cells in the presence of EBV-infected cells. In paper IV we showed that a child’s age and subsequent capacity for anti-viral cytokine production affects in vitro EBV infection in terms of B-cell proliferation and B-cell acquisition of memory phenotype. PBMC from CMV+ children had lower EBV-induced accumulation of switched memory B cells, which was connected to high prevalence of CD57+CD8+ T cells and IFN-γ production. Taken together, this thesis work shows that monocyte subsets at birth can give potent functional responses and that latency with EBV and CMV has a significant effect on the differentiation process and functional capacity of anti-viral effector cells during childhood. This in turn could affect responses to related or unrelated infections or even to non-invasive antigens such as allergens. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

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