Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk with skin flora, such as Staphylococcus epidermidis and Staphylococcus capitis, being the predominant contaminants. These bacteria are able to form surface-attached aggregates or biofilms, which are present in the skin of healthy blood donors and can subsequently be isolated from contaminated PCs. Disinfection of the venipuncture area before donation with a combination of 2% chlorhexidine-gluconate and 70% isopropanol is used at Canadian Blood Services. However, not all bacteria are eliminated during skin disinfection since contaminated PCs are still captured during routine PC screening. In this thesis, the ability of biofilm-forming S. epidermidis and S. capitis to resist the currently used disinfectants was explored. It was demonstrated that although a combination of chlorhexidine and isopropanol has a bactericidal effect, it is unable to completely eradicate skin flora biofilms. Several countries have implemented Pathogen Inactivation Technologies (PITs) as a measure to help control transfusing bacterially-contaminated PCs by exposing PC units to ultra violet light. However, no investigations have been done to evaluate the ability of PITs against bacterial biofilms, which was one of the objectives of this thesis. Data revealed that the efficacy of a currently used PIT, the Mirasol® system, is similar for S. epidermidis present in PCs produced from whole blood inoculated with biofilm or non-biofilm cells. However, treatment effectiveness was strain dependent. In conclusion, further investigation to improve donor skin disinfection and PITs should be considered. Surveillance at Canadian Blood Services shows that contamination rates in single-donor apheresis PCs (Aph-PCs) is generally higher than in four-donor buffy coat platelet pools (BC-PCs). This study investigated whether the BC-PC production method contributes to this observation as BC-PCs are derived from WB that is left to rest overnight while Aph-PCs are collected directly from the donor. Data showed that WB hold during BC-PC production does not have a broad-spectrum bactericidal effect and therefore other factors contribute to low rates of contamination in BC-PCs. The work presented in this thesis provides an insight to bacterial residence and persistence during blood product manufacturing and makes suggestions for PC safety improvements.

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22124号 / 工博第4654号 / 新制||工||1726(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 跡見 晴幸, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

This diploma thesis is focused on the study of the effect of ultraviolet radiation and titanium dioxide on the yeast Candida vini. Photocatalytic inactivation of this yeast was performed on various types of titanium dioxide films, which were immobilized on soda lime glasses. Acridine orange was used as a dye to distinguish live and dead yeast cells after certain irradiation time. Live and dead cells emit different light in colour after staining in acridine orange. Candida vini photocatalytic inactivation depends on the amount of titanium dioxide immobilized on glass as well as on the structure of its surface if the lamp intensity remains the same. Kinetics of this photocatalytic process was studied, too.

In this diploma thesis, photocatalytic effect of titanium dioxide with addition of silver was described and demonstrated on inactivation of the yeast Candida vini. Titanium dioxide was prepared by sol-gel process and deposited by printing metod on soda-lime glasses. On the deposit of titanium dioxide was added silver in various concentrations which increased effect of TiO2. Inactivation of yeasts was examined by effectiveness of UV light with intensity 170 and 100 W/m2.

Klinefelter Syndrome (KS) is a multisystemic disorder associated with a plethora of phenotypic features including mental retardation, cardiac abnormalities, osteoporosis, infertility, gynecomastia, type two diabetes and increased cancer risk. KS is the most common aneuploidy in humans (with a prevalence of 1:500 to 1:1000 born males) and is characterized by one or more supernumerary X-chromosomes (47-XXY, 48-XXXY, and 49-XXXXY karyotypes). While X-chromosome inactivation (XCI) represses extra Xs, few genes called “escape genes” elude the XCI mechanism and are actively transcribed from X inactive. The overdosage of escape genes has been considered the molecular landscape that underlies KS clinical features. In this project, we exploit an integration-free reprogramming method to generate the largest described cohort of iPSCs from seven patients with KS and healthy donor fibroblasts from two relatives. The unicity of this cohort relies on the derivation of 47-XXY iPSCs and their isogenic 46-XY healthy counterparts, along with multiple rare 48-XXXY and 49-XXXXY iPSC lines. Through X chromosome inactivation (XCI) assessment, we show consistent retention of n-1 XCI in all derived KS-iPSCs. We identify the genes within the PAR1 region as the most susceptible to dosage-dependent transcriptional dysregulation and therefore putatively responsible for the progressively worsening phenotype in higher grade X aneuploidies. Moreover, we explore the transcriptional impact of X overdosage on autosomes and identify that the X-dosage-sensitive autosomal transcription factor NRF1 is a master regulator of the X-linked escape gene ZFX. Finally, we dissect the potential pathological impact of the escape gene KDM6A on low- and high-grade supernumerary X iPSCs and differentiated derivatives. We highlight a considerable proportion of KDM6A targets that could be responsible for paradigmatic clinical manifestations of KS.

We report a de novo dup(X)(q23→q26) in a 3-year-old girl with growth retardation, developmental delay, and minor anomalies. X-inactivation in lymphocytes by BRDU labeling showed the abnormal X was late replicating. The androgen receptor assay (HAR) demonstrated a skewed methylation (88.8%) of the paternal allele and a 11.2% methylation of the maternal allele. These data, which suggest the duplication was paternally inherited, are the first parental-origin identification of a duplication Xq. The mild phenotype of the patient may be related to the size and region of the duplication, the low percentage of a dup(X) active detected by the HAR assay, or a combination of these mechanisms. .

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+]e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+]e. It was also much slower in the presence of [Ca2+]e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797 , were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.

Pavlovian conditioned stimuli can acquire incentive motivational properties, and this phenomenon can be measured in animals using Pavlovian conditioned approach behavior. Drugs of abuse can influence the expression of this behavior, and nicotine in particular exhibits incentive amplifying effects. Both conditioned approach behavior and drug abuse rely on overlapping corticolimbic circuitry. We hypothesize that the orbitofrontal cortex (OFC) regulates conditioned approach, and that one site of nicotine action is in the OFC where it reduces cortical output. To test this, we repeatedly exposed rats to 0.4 mg/kg nicotine (s.c.) during training and then pharmacologically inactivated the lateral OFC or performed in vivo electrophysiological recordings of lateral OFC neurons in the presence or absence of nicotine. In Experiment 1, animals were trained in a Pavlovian conditioning paradigm and behavior was evaluated after inactivation of the OFC by microinfusion of the GABA agonists baclofen and muscimol. In Experiment 2, we monitored phasic firing of OFC neurons during Pavlovian conditioning sessions. Nicotine reliably enhanced conditioned responding to the conditioned cue, and inactivation of the OFC reduced conditioned responding, especially the sign-tracking response. OFC neurons exhibited phasic excitations to cue presentation and during goal tracking, and nicotine acutely blunted this phasic neuronal firing. When nicotine was withheld, both conditioned responding and phasic firing in the OFC returned to the level of controls. These results suggest that the OFC is recruited for the expression of conditioned responses, and that nicotine acutely influences this behavior by reducing phasic firing in the OFC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20264号 / 医博第4223号 / 新制||医||1021(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 椛島 健治, 教授 松田 秀一, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM