This diploma thesis provides an overview of gene conversion, its role in the pathogenesis of human diseases and the use of methods based on next-generation sequencing (NGS) for detection rare variants of DNA sequence. Labeling of target DNA molecules by random nucleotides in primer and NGS were used for detection point mutations arising de novo in the β-glucocerebrosidase gene by gene conversion between it and its pseudogene in meiotic and mitotic cells of control subjects. Primers specific for the active gene were used to selectively amplify the ninth and tenth exon of the gene where "recombinant" variants occur most frequently. Sequences generated from 20 genomic DNA samples on Illumina MiSeq platform were quality filtered, sorted by unique labels and consensus sequences were created from alignments of sequences carrying the same DNA tag. The number of potential point mutations in the samples ranged between 12 and 48. The mutations were manually re-evaluated from the alignments. The number of alignments with unique labeling was in the range of 7-15 thousand per sample. Only three samples carried possible recombinant mutations, suggesting a lower frequency of conversion in the region than reported by other techniques. Analysis of unique sequences in primer indicated possible ways to improve the...

Quinones have been the subject of much interest due to their various biological activities, mainly as antitumor and as trypanocidal agents. Quinones are cytotoxic by two main mechanisms: the generation of ROS resulting in oxidative stress and alkylation of cellular nucleophiles, such as DNA and some enzymes such as topoisomerases. Their activity depends on bioreduction, similarly to what happens to nitroaromatic compounds. They also catalyze electron transfer reactions in biological processes and, after reduction generate radical anions (semiquinone radical anion and nitro), which depending on stability, can furnish their free electrons to acceptor molecules. In the present study, compounds with mixed functionalities derived from nor-β-lapachone, including a nitroaniline group were electrochemically studied in protic (acetate buffer) and aprotic (DMF+TBABF4, DMSO+TBAP and Acetonitrile+TBABF4) media, using glassy carbon and mercury as working electrodes. The compounds showed a complex redox behavior and the mechanism was elucidated using electron spin resonance. The electroreduced products of nor-β-lapachone and of the nitroquinones reacted with oxygen, indicative of the generation of reactive oxygen species, reactivity in the order of 2 > nor-β-lapachone > 3 > 1. We investigated their interaction with DNA, which was shown to be positive for nitroquinones and negative for the precursor nor-β-lapachone, in agreement with biological assays which had also shown that the nitroquinones cause DNA damage. The stability of the nitrosemiquinones, their half-life times were measured using mercury electrode, and the reaction rates for the electrochemical process-following-up-isproportionation reaction were measured. From these studies, a lower stability for the meta-substituted nitrophenylaniline (k2 = 5.188 x 103 L mol-1 s-1 and t1/2 = 0.06 s) was evidenced. Upon spectroelectrochemical reduction studies of the nitroquinones, the generation of radicalar intermediates (semiquinone radical anion and nitro radical anion) was observed, with differences between o- and m-derived compounds and the p-substituted one. To increase the solubility of the nitroquinones, in order to allow in vivo studies, the formation of inclusion complexes with β-cyclodextrin were evaluated. Positive results were obtained, leading to a viable formulation alternative for further biological studies with the compounds. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Quinonas despertam muito interesse devido às suas diversas atividades biológicas, principalmente como agentes antitumoral e tripanossomicida. A citotoxicidade de quinonas decorre de dois mecanismos principais: geração de EROs resultando no estresse oxidativo e alquilação de nucleófilos celulares, como o DNA e algumas enzimas, como as topoisomerases. Sua atividade depende de biorredução, similarmente ao que acontece com compostos nitroaromáticos. Eles também catalisam reações de transferência de elétrons biológicas e, após a redução geram ânions radicais (ânions radicais semiquinona e nitro), que dependendo da estabilidade, podem transferir seus elétrons livres a moléculas aceptoras. Neste trabalho, compostos de funcionalidade mista derivados da nor-β-lapachona, com um grupo nitroanilina, foram estudados eletroquimicamente nos meios prótico (tampão acetato) e aprótico (DMF + TBABF4, DMSO + TBAP e acetonitrila + TBABF4), utilizando carbono vítreo e mercúrio como eletrodos de trabalho. Os compostos apresentaram um comportamento redox complexo e o mecanismo elucidado por espectroeletroquímica. Os produtos da eletro-redução da nor-β-lapachona e nitroquinonas reagiram com oxigênio, indicativo da geração de EROs, na ordem de reatividade 2 > nor-β-lapachona > 3 > 1. Foi investigada a interação com DNA, que se mostrou positiva para as nitroquinonas e negativa para o precursor nor-β-lapachona, concordantes com ensaios biológicos que também evidenciou que as nitroquinonas causam dano ao DNA. A estabilidade do nitrosemiquinona, tempos de meia-vida e as taxas reacionais referentes à reação química de desproporcionamento acoplada ao processo eletroquímico para cada derivado foram determinadas usando eletrodo de mercúrio. A partir desses estudos, foi evidenciada a menor estabilidade para o nitrofenilamina meta-substituída (k2 = 15,188 x 103 L mol-1 s-1 and t1/2 = 0,06 s). Nos estudos por espectroeletroquímica das nitroquinonas, observou-se a geração de intermediários radicalares (ânions radicais semiquinona e nitro), com diferenças entre os nitroderivados o- e m-, assim como para o p- derivado. Com objetivo de aumentar a solubilidade das nitroquinonas, para posteriores estudos in vivo, foi avaliada a formação de complexos de inclusão com β-ciclodextrina. Foram obtidos resultados positivos, refletindo em alternativa viável para formulações posteriores com estes compostos e ensaios biológicos.

Les entérocoques sont des bactéries commensales de l'Homme majoritairement rencontrées dans le tractus digestif. En dépit du caractère bénéfique pour leur hôte, ces microorganismes sont retrouvés au deuxième rang des bactéries responsables d'infection nosocomiales en France ces dernières décennies. Diverses études tendent à montrer que le métabolisme énergétique constitue un facteur crucial pour le processus infectieux des microorganismes. Lors de ce travail, nous nous sommes intéressés à l'étude des métabolismes de différents polymères de glucoses chez Enterococcus faecalis : les maltodextrines et le gentiobiose. L'utilisation du maltose et des maltodextrines est, chez cette bactérie, directement coordonnée au niveau transcriptionnel par le répresseur MalR. L'activité de ce régulateur est rapidement modulée par le maltose qui représente l'inducteur du système et par un corépresseur protéique : la protéine P Ser HPr qui, à l'inverse, favorise la répression exercée par MalR. Le métabolisme des maltodextrines complexes, mais pas le métabolisme du maltose, est également réprimé par le régulateur pléiotrope CcpA en coordination avec son cofacteur P Ser HPr en présence de glucose. La répression catabolique de l'opéron genBA, impliqué dans le métabolisme du β glycoside gentiobiose, est aussi assurée par ce régulateur CcpA en présence de glucose. Cet opéron genBA est responsable de l'import du gentiobiose par un PTS ainsi que de son catabolisme grâce à une hydrolase. L'expression de cette structure opéronique nécessite la présence de l'activateur transcriptionnel GenR actif en présence de l'inducteur gentiobiose 6' P. / Enterococci are commensal bacteria of Humans predominantly encountered in the digestive tract. Despite their beneficial activity for their host, these microorganisms became the second leading bacterial cause of hospital acquired infections in France for last decades. Some studies showed that the central metabolism is a critical factor for microorganisms infection process. In this study, we worked on the characterisation of metabolisms of the different glucose polymers maltodextrins and gentiobiose in Enterococcus faecalis. The maltose and maltodextrins utilization is coordinated in this bacterium transcriptionally by the MalR repressor. The MalR activity is rapidly modulated by the inducer maltose and by the co repressor P Ser HPr which strengthens the MalR DNA binding. The metabolism of long maltodextrins is also repressed by the pleiotropic regulator CcpA in complex with its essential cofactor P Ser HPr in presence of glucose. The Catabolite repression of the operon genBA, involved in metabolism of the β glycoside gentiobiose, is assumed by CcpA in presence of glucose. This operon genBA allows the gentiobiose uptake with a PTS and its catabolism by a hydrolase. The expression of this latter operon requires both the GenR transcriptional activator and the inducer gentiobiose 6' P.

The discovery of new β-haematin inhibitors has become one focus for researches in response to the resistance of P. falciparum malaria parasites that emerged towards well-known antimalarials. While hundreds of new β-haematin inhibitors have been discovered using detergent mediated high-throughput screening methods, a crucial aspect is understanding exactly how these β-haematin inhibitors behave in the malaria parasite and inhibit the formation of haemozoin. What is known, is that well-known β-haematin inhibitors like chloroquine cause increased amounts of exchangeable haem in the parasite digestive vacuole and form a Fe(III)PPIX-inhibitor complex by accumulating at high concentrations which consequently inhibits parasite growth. Another important focus is on understanding the digestion of haemoglobin and its role in haemozoin formation. This research investigates the in vitro modelling of cellular haemozoin and inhibition by various β-haematin inhibitors across different scaffolds and the role of haemoglobin degradation in P. falciparum malaria parasites. The investigated β-haematin inhibitors resulted in micromolar IC50 (NF54) values and decreased parasite growth with increases in concentration. Using a pyridine-based parasite haem fractionation plate method, these β-haematin inhibitors were shown to target haemozoin formation by causing increased amounts of exchangeable haem that corresponded to decreasing amounts of haemozoin in chloroquine-sensitive parasites. The amounts of exchangeable haem were shown to be inversely proportional to the percentage of parasite growth in the presence of these β-haematin inhibitors. It was apparent that there was a tendency for parasite growth inhibition activity to decrease as the amount of exchangeable haem present in chloroquine sensitive parasites increased, although, the trend was not statistically significant. Moreover, it was observed that experimental cellular accumulation ratio values were low in comparison to chloroquine and amodiaquine. Based on the experimental cellular accumulation ratio values, it was deduced that the accumulation of these β-haematin inhibitors was not primarily due to pH trapping and more complex than previously proposed. Further investigations into the exchangeable haem amounts as a function of intracellular test compound amounts at the IC50 values of these β-haematin inhibitors highlighted that there was an apparent 1:1 relationship with the amount of intracellular exchangeable haem, indicative of complex formation. Transmission electron microscopy images were obtained for untreated parasites that showed intact parasites inside red blood cells with clearly visible haemozoin crystals dispersed throughout the parasite digestive vacuole, whilst, treated parasites showed less defined haemozoin crystals as a result of inhibition. Moreover, electron energy-loss spectroscopy revealed that untreated parasites exhibited a strong iron signal which was associated with haemozoin in the parasite digestive vacuole with a weaker signal attributed to the red blood cell cytoplasm. Similarly, a strong iron signal was shown in the digestive vacuole of treated parasites which was associated with less defined haemozoin crystals. A halo around these haemozoin crystals was observed and was suggested to be indicative of the build-up of exchangeable haem. Additionally, a strong bromine signal attributed to a bromine-containing β-haematin inhibitor, test compound 1, was also observed in the same region as the haemozoin crystals. Overlaid signal distribution maps for iron and bromine showed direct evidence of Fe(III)PPIX and test compound 1, suggesting complexation. High-quality Raman spectra were obtained for the Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetically prepared samples for the Fe(III)PPIX porphyrin dominated spectral region of 1700-500 cm-1 at an excitation wavelength of 532 nm. From the spectra, a putative Fe(III)PPIX-test compound 1 complex was identified and shown to be similar to the synthetically prepared counterpart, haematin-test compound 1 mixture. It was highlighted that a unique peak at 1080 cm-1 indicated π- π interactions between the pyrrole-imidazole ring and thus confirming that the formation of this putative Fe(III)PPIX-inhibitor complex occurs. The confocal Raman true mapping technique proved to be efficient and reliable for imaging the signal distribution of haemozoin at the Raman peak of 754 cm-1 and 1080 cm-1 for the Fe(III)PPIX-test compound 1 complex which co-localized in the digestive vacuole of chloroquine sensitive parasites. Moreover, oxy- and deoxy-haemoglobin was observed to be localized to the red blood cell, where, deoxy-haemoglobin was located on the outer parts of the parasite. Principle component analysis, based on the Raman peak positions, exhibited significant differences in the spectra for Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetic samples where clusters were observed to separate mainly along principle component 1. These data proved that the spectra of the Fe(III)PPIX-test compound 1 complex was the same as its synthetically prepared counterpart but different from the remaining Fe(III)PPIX species. In comparison to the Fe(III)PPIX-test compound 1 complex, the cluster separations were observed to be significant, where, no significant separation was observed for the Fe(III)PPIX-test compound 1 complex and the haematin-test compound 1 mixture. Based on this, it was evident that a Fe(III)PPIX-test compound 1 complex existed in the digestive vacuole of treated chloroquine sensitive parasites. To fully understand the inhibition of haemozoin, the development of a haem pathway model is necessary, but, requires certain prerequisites. Bioinformatics data from PAXdb and ExPASy revealed that chloroquine resistance (Dd2) parasites, containing 1337 previously identified proteins with an average abundance-weighted molecular weight of 40,483 ± 77 g/mol. With this, the protein mass per cell for red blood cells, chloroquine-sensitive and - resistant parasites were consistent across three protein quantification methods was measured and revealed that chloroquine resistant parasites had a significantly higher protein mass per cell than chloroquine sensitive parasites and in turn a higher total number of protein molecules per cell. Aspartic proteases are 4-fold higher in concentration than cysteine proteases with histo-aspartic protease having the highest concentration in chloroquine resistant parasites. Along with these data, a time point quantification for chloroquine sensitive parasites throughout the blood-stage showed that the amount of haemoglobin decreased in a sigmoidal manner and corresponded to a linear increase in the amount of haemozoin and relatively constant exchangeable haem amount. This was consistent with Giemsa smears that showed that for early time points, large initial decreases in the amount of haemoglobin were observed between the early trophozoite to late trophozoite stage.

Ractopamine hydrochloride (RAC) has been used in the pig production industry for over 30 years. RAC is a beta-adrenergic agonist which is supplemented in the feed during the last 28 days prior to harvesting in finisher pigs to modify the pig's metabolism such that nutrients are redirected to favour muscle accretion rather than adipose deposition, and hence improve growth efficiencies, feed utilisation and carcass revenues. The objective of this study was to investigate the effect of dietary ractopamine (Paylean®) supplementation at levels of 0, 5, and 10 mg.kg-1 (hereafter referred to as 0-RAC, 5-RAC, and 10-RAC respectively) on animal growth performance, efficiency and carcass characteristics including daily voluntary feed intake, feed efficiency, absolute daily growth rate (ADG) and daily live weight gain, and backfat thickness for the last 27 days in finishing gilts. In this 27-day study, a homogenous group of 71 grower gilts (LW = 43 ± 1 kg) were pre-selected at a source farm. The gilts were then housed in similar and equally sized group pens at the Hatfield experimental facility of the University of Pretoria and fed a standard maize-soya oilcake based grower ration formulated to contain 0.94% standardised ileal digestible Lys (1.05% total Lys) and 14.01 MJ ME kg-1 during the 28 day pre-adaptation phase. From these gilts, individuals were weighed and 58 gilts selected (average LW = 68.7 ± 4.3 kg), and placed into individual pens and the same diet for 7 days (adaptation phase), afterwhich they were assigned to 1 of 3 treatments in a completely randomized block design with 19, 19, and 20 replicate pens per treatment. The pigs were then fed a standard maize-soya oilcake finisher (treatment) diet containing either 0-RAC, 5-RAC or 10-RAC for 27 d before harvesting. All treatment diets were formulated to contain 1.02% standardised ileal digestible Lys (1.13% total Lys) and 13.96 MJ ME kg-1. Individual pig LW, P2 thickness and pen feed disappearance were recorded weekly to determine LW changes, ADG, ADFI, and G:F. After 27 d on trial, gilts were slaughtered and carcass measurements were recorded at 24 h post-mortem. Overall, RAC supplementation did not affect ADFI or P2 (P > 0.05) but did influence LW (P = 0.049) and overall G:F (P = 0.012) after d27. At d15-d21 and d22-27, only a tendency (P = 0.169, 0.104 respectively) for a linear decrease in G:F with RAC supplementation was found. RAC also affected HCY (P= 0.045) and CCY (P = 0.045) but not fat depth, meat depth or fat % (P > 0.05). These results indicate that RAC may have small but beneficial effects in modern pig production, but further research is required to optimize concentrations and duration of supplementation in modern lean pig genotypes. / Dissertation (MSc (Agric))--University of Pretoria, 2016. / Animal and Wildlife Sciences / MSc (Agric) / Unrestricted

Betaxolol, a long-acting β-adrenergic blocker that enters the central nervous system, was examined for therapeutic effects on the persistent anxiety of anxiety disorders. Prior studies of β-blockers examined only agents that were short-acting or did not enter the brain. Betaxolol was administered to 31 patients in open trials. Of 13 outpatients, 11 had generalized anxiety disorder (GAD) and 2 had adjustment disorder with anxiety. Five with GAD had concurrent panic disorder. Of 18 inpatients, 16 had GAD and 2 had adjustment disorder with anxiety. Betaxolol doses were increased until the patient responded or declined further dosage. Severity was rated on a 4-point global scale. Before betaxolol, all were moderately or severely ill. In all patients with panic disorder panic attacks stopped within 2 days (p < 0.001). Anxiety decreased to no more than marginally ill in 85% of outpatients (p < 0.0001) and all inpatients (p < 0.0001). Betaxolol doses were usually 5 mg once or twice daily; four inpatients took 10 to 20 mg twice daily. In sum, betaxolol administration was rapidly followed by improvements that were easily noticed by the doctor, even in patients with longstanding anxiety and obsessive-compulsive personality disorder. Preliminary observations in posttraumatic stress disorder are similar.

Background: Candida albicans is an opportunistic fungal pathogen that induces strong proinflammatory responses, such as IL-1β production. Much less is known about the induction of immune modulatory cytokines, such as the IL-1 receptor antagonist (IL-1Ra) that is the main natural antagonist of IL-1, by C. albicans. Methods: Peripheral blood mononuclear cells (PBMC) of healthy individuals were stimulated with C. albicans and different components of the fungal cell wall. The role of pathogen recognition receptors (PRRs) for the induction of IL-1β and IL-1Ra was investigated by using specific blockers or in PBMC from Dectin-1 deficient patients. Results: C. albicans induced a strong IL-1Ra response, and this induction was primarily induced by the cell-wall component β-glucan. Blocking IL-1Ra significantly increased C. albicans β-glucan hyphae induced IL-1β and IL-6 production. Surprisingly, blocking the β-glucan receptor Dectin-1 or the downstream Syk or Raf-1 pathways only marginally reduced C. albicans-induced IL-1Ra production, while blocking of the complement receptor 3 (CR3), TLR2 or TLR4 had no effect. In line with this, blocking MAP kinases had little effect on Candida-induced IL-1Ra production. PBMC isolated from Dectin-1 deficient patients produced normal IL-1Ra amounts in response to C. albicans stimulation. Interestingly, the IL-1Ra synthesis induced by β-glucan was blocked by inhibitors of the Akt/PI3. K pathway. Conclusions: β-glucan of C. albicans induces a strong IL-1Ra response, which is independent of the β-glucan receptors dectin-1 and CR3. These data strongly argue for the existence of an unknown β-glucan receptor that specifically induces an Akt/PI3. K-dependent anti-inflammatory IL-1Ra response upon recognition of C. albicans.

Recognition of the fungal cell wall carbohydrate β-glucan by the host receptor Dectin-1 elicits broad immunomodulatory responses, such as phagocytosis and activation of oxidative burst. These responses are essential for engulfing and killing fungal pathogens. Phagocytic monocytes are key mediators of these early host inflammatory responses to infection. Remarkably, whether phagocytosis of fungal β-glucan leads to an inflammatory response in human monocytes remains to be established. Here, we show that phagocytosis of heat-killed Candida albicans is essential to trigger inflammation and cytokine release. By contrast, inhibition of actin-dependent phagocytosis of particulate (1-3,1-6)-β-glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal β-glucan particles upon actin cytoskeleton disruption, relies on Dectin-1 and results in the classical caspase-1 inflammasome formation through NLRP3, generation of an oxidative burst, NF-κB activation, and increased inflammatory cytokine release. PI3K and NADPH oxidase were crucial for both cytokine secretion and ROS generation, whereas Syk signaling mediated only cytokine production. Our results highlight the mechanism by which phagocytosis tightly controls the activation of phagocytes by fungal pathogens and strongly suggest that actin cytoskeleton dynamics are an essential determinant of the host's susceptibility or resistance to invasive fungal infections.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12918号 / 論医博第2093号 / 新制||医||1009(附属図書館) / 32128 / (主査)教授 羽賀 博典, 教授 中川 一路, 教授 上杉 志成 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Atherosclerosis is a multifactorial inflammatory disease of the arterial wall and its clinical manifestations, including myocardial infarction and stroke, are the leading causes of death in western societies. Recent data has suggested that disruption of protein homeostasis in a cell's endoplasmic reticulum (ER), a condition known as ER stress, is associated with the progression of atherosclerosis. Furthermore, signaling by the serine/threonine kinase glycogen synthase kinase (GSK)-3α/β mediates pro-atherogenic processes. This thesis examines the role of ER stress and GSK3α/β signaling in atherosclerosis. Initially, three apolipoprotein-E deficient (ApoE-/-) mouse models of accelerated atherosclerosis were established. Relative to ApoE-/- mice fed a chow diet, pro-atherogenic conditions promoted hepatic steatosis, atherosclerosis, ER stress and GSK3β activity. A subset of mice from each group were given the GSK3α/β inhibitor valproate. Valproate supplementation suppressed hepatic steatosis, atherosclerosis and GSK3β activity in each mouse model without altering ER stress levels. This study revealed a role for ER stress and GSK3α/β in multiple murine models of atherosclerosis. Next, we investigated ER stress and GSK3α/β signaling in macrophage foam cell formation. In macrophages, ER stress induced GSK3α/β activity in a protein kinase R-like endoplasmic reticulum kinase (PERK) dependent manor. GSK3α/β inhibition attenuated ER stress induced lipid accumulation and the expression of distal components of the PERK pathway. Overexpression of constitutively active GSK3β induced foam cell formation. In mice, valproate supplementation attenuated PERK signaling in peritoneal macrophages and macrophages within atherosclerotic lesions. Together, these results point to GSK3α/β being a downstream component of the PERK pathway and that PERK-GSK3α/β signaling mediates ER stress induced foam cell formation. Lastly, we investigated the tissue and homolog specific functions of GSK3α and GSK3β in atherosclerosis. In high fat diet (HFD) fed low-density lipoprotein receptor deficient (LDLR-/-) mice, deletion of GSK3α or GSK3β in hepatocytes did not alter liver lipid content or atherosclerosis. Myeloid cell deletion of GSK3α, but not GSK3β, attenuated HFD induced atherosclerosis. Mechanistically, deletion of GSK3α in macrophages promotes the anti-atherogenic M2 macrophage phenotype by modulating signal transducer and activator of transcription (STAT)-3 and STAT6 phosphorylation and activation. Together, the data presented in this thesis suggest; 1) GSK3α/β inhibition attenuates atherosclerosis in multiple mouse models, 2) PERK-GSK3α/β signaling regulates macrophage foam cell formation and 3) myeloid cell GSK3α mediates atherosclerosis and macrophage phenotype. / Thesis / Doctor of Philosophy (PhD)