Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Veronika Ackermannová Supervisor: prof. Radomír Hrdina, MD, CSc. Title of diploma thesis: Topic and systemic treatment of acne vulgaris Acne vulgaris is a skin disease affecting the hair follicles and sebaceous glands. The disease manifests itself by increased sebum production, non-inflammatory (comedones) and inflammatory lesions (papules, pustules, nodules, cysts). It occurs predominantly in adolescents, but may persist into adulthood. It is a multifactorial disease, which is caused by several factors (internal and external stimuli). The major pathogenetic factors include increased sebum production, hyperkeratosis, P. acnes colonization and inflammation present. First, it is necessary to diagnose the type of acne in order to choose the right and effective therapy, because there is not only one type of acne. There are many types and variants of acne, and although they show similar symptoms (affecting the follicles of sebaceous glands), their cause often differs. There is no uniform classification system for acne vulgaris and it varies between authors. Some authors classify acne vulgaris according to severity into mild, moderate and severe, others into comedonic, papulopustular, nodulocystic...

Waste management of vulcanized rubber is a serious environmental problem as the molecular structure formed during the process complicates recycling. Due to this, researchers have developed a process that can break the strong bonds and return the rubber to its original state. This process is called devulcanization and is developed for the tire industry due to the large amount of waste formed from this sector. This rubber is vulcanized with sulfurs like 90 % of other rubber products. However, there exist other products that are vulcanized with peroxides where much less research has been done. Due to this, it is unclear if devulcanization is possible as a different type of bond is formed in the vulcanization process. This work therefore investigates the possibilities with recycling by devulcanization of peroxide cured rubber where a collaboration is done with Roxtec International AB. The work also investigates the possibilities with reuse of the cooperating partner’s particular rubber and how everything can be implemented to understand the efficiency of these two waste management strategies and to share knowledge.  The used method for this work is a combination of the Participatory Action Research (PAR) and the Design thinking (DT) framework. A systematic literature review has also been conducted to collect relevant material for one of the research questions. The result showed that there exist many methods and possibilities with devulcanization of peroxides if sufficient energy is provided. However, the quality of the reclaimed rubber will be lacking as the bonds in the polymers main chain are degraded which affects the mechanical properties. Due to this only a small percentage can be reused by mixing it with virgin rubber. The efficiency for peroxides is therefore lower than for sulfurs. Regarding reuse many alternative usage areas were brainstromed but once the concepts were evaluated against the limitations with the material, the needs and the sustainability factor, only few solutions survived. The winning concept became a punching bag filled with granulated rubber crumbs. When investigating how everything can be implemented it turned out that a lot of effort is needed. For example, to get favorable results, the solution will require the punching bag company to also want to work towards sustainability by adapting take back systems. Devulcanization will then require that the waste is sorted which requires a big investment in both time and money to achieve. The conclusion of this work is that devulcanization and reuse is possible and the developed system solution can take care of parts of the waste. However, the possibilities are limited and to get rid of all the waste and develop a long-term solution that is both sustainable and further prepared for the future, more actions are needed. It is therefore of high interest to start investigating the possibilities with a new material that is easy to recycle or are degradable. / Avfallshantering av vulkaniserat gummi är ett allvarligt miljöproblem eftersom den molekylära strukturen som bildas under processen komplicerar återvinning. På grund av detta har forskare utvecklat en process som kan bryta de starka bindningarna och återställa gummit till sitt ursprungliga tillstånd. Denna process kallas devulkanisering och är främst utvecklad för däck-industrin på grund av den stora mängden avfall som bildas från denna sektor. Detta gummi vulkaniseras med svavel som 90% av andra gummiprodukter. Det finns dock andra produkter som vulkaniseras med peroxider där mycket mindre forskning har gjorts. På grund av detta är det oklart om devulkanisering är möjlig eftersom en annan typ av bindning bildas i vulkaniseringsprocessen. Detta arbete undersöker därför möjligheterna med återvinning genom devulkanisering av peroxid härdat gummi där ett samarbete görs med Roxtec International AB. Arbetet undersöker också möjligheterna med återanvändning av samarbetspartners specifika gummi spill och hur allt kan implementeras för att förstå effektiviteten i dessa två avfallshanterings strategier och för att sprida kunskap. Den använda metoden för detta arbete var en kombination av PAR (Participatory Action Research) och design thinking (DT). En systematisk litteraturstudie har också genomförts för att samla in relevant material för en av forskningsfrågorna. Resultatet visade att det finns många metoder och möjligheter för devulkanisering av peroxider om tillräcklig energi tillhandahålls. Dock kommer kvalitén på det återvunna gummit att försämras markant då bindningarna i polymerens huvudkedja bryts vilket påverkar de mekaniska egenskaperna. På grund av detta kan endast en liten andel återanvändas genom att blanda med jungfruligt gummi. Effektiviteten för peroxider är därför lägre än för svavel. När det gäller återanvändning, brainstormades många alternativa användningsområden, men när koncepten utvärderades mot begränsningarna med materialet, behoven och hållbarhetsfaktorer överlevde bara ett fåtal lösningar. Det vinnande konceptet blev en slagsäck fylld med granulerat gummi. När det undersöktes hur allt kan implementeras visade det sig att det krävs mycket ansträngningar. Till exempel, för att få gynnsamma resultat kommer lösningen att kräva att boxsäck företaget också vill arbeta för hållbarhet genom att anpassa take back system. Devulkaniserings-processen kommer även kräva att avfallet sorteras vilket kräver en stor investering i både tid och pengar för att uppnå. Slutsatserna av detta arbete är att devulkanisering och återanvändning är möjligt och den utvecklade systemlösningen kan ta hand om delar av avfallet. Men möjligheterna är begränsade och för att bli av med allt avfall och utveckla en långsiktig lösning som både är hållbar och ytterligare förberedd för framtiden, kommer fler åtgärder att behövas. Det är därför av stort intresse att börja undersöka möjligheterna med ett nytt material som är lätt att återvinna eller är nedbrytbart.

This work is focused on study of electrical discharges in liquids, especially in water solutions. Generation of the discharge in water solutions leads to simultaneous effect of UV radiation, shock waves, electrical field and most importantly, chemically reactive species. This can be utilized in many applications such as sterilization, degradation of organic waste products, lithotripsy or other medical applications. The experimental part is concentrated on a diaphragm arrangement of the reaction system. This means that the reactor is divided into two electrode reservoirs connected only through a small orifice in a dielectric barrier. This barrier is made of Macor® non-porous ceramics with thickness of 1 mm, with the diameter of the orifice 0.6 mm, in the first part of work. In the second part, ShapalTM-M ceramics of thickness 1.0 mm and orifice diameter 0.6 mm was used. The experimental part is divided into two sections. For both, NaCl is chosen as an electrolyte to set the initial conductivity of the tested solutions to the value of 400 S/cm. Supplied direct voltage is regulated to attain power of 100 W in the system. In the first part, effect of addition of chosen alcohols (ethanol, isopropylalcohol and glycerol) on the efficiency of the discharge in their water solutions is studied. For this purpose, a special glass reactor was designed and constructed. The efficiency of the discharge is measured by a spectroscopic determination of concentration of complex formed by a titanium reagent and hydrogen peroxide, which is generated during the discharge. The results show no positive effect of addition of extra OH group to the reaction through the alcohols. The use of isopropylalcohol causes even a significant decrease in the amount of hydrogen peroxide generated. The subject of the second part is a comparison of effect of different electrode materials on the discharge. The efficiency is measured by the same method as in the first part. Materials chosen were stainless steel, platinum, aluminium, copper and carbon. Each material shows different hydrogen peroxide production rate under the same parameters. The most perspective material seems to be carbon, as an inert material, that can be expected not to initiate any decomposition of hydrogen peroxide. The least favourable appears to be copper. When used, no production of hydrogen peroxide was observed in one of the electrode parts of the reactor.

Disertační práce se zabývá detailním studiem vlastností různých elektrických výbojů generovaných ve vodných roztocích. Tyto výboje se staly v posledním desetiletí velmi populárním tématem, a to zejména díky mnoha praktickým využitím jako například v biomedicíně, čištění odpadních vod, ekologii nebo nanoinženýrství. Studium je zaměřeno na generaci peroxidu vodíku, jakožto jednu z nejvýznamnějších částic generovaných právě elektrickými výboji v kapalinách. Pro první část této práce byla využita speciální výbojová komora zkonstruovaná na Fakultě chemické Vysokého Učení Technického v Brně. Komora byla rozdělena tenkou diafragmovou přepážkou na dvě poloviny, přičemž uvnitř přepážky se nacházela malá dírka. V každé části komory se nacházela jedna elektroda, a obě dvě části komory byly vyplněny kapalinou. Ze zdroje bylo do kapaliny aplikováno vysokofrekvenční napětí (1 a 2 kHz), které tak vlastně upravovalo roztok chloridu sodného (1.5 l). Bylo zjištěno, že tento druh napětí, v porovnání s DC, nezpůsobuje nežádoucí přehřívání roztoku (počáteční vodivost 100 - 800 S/cm) během jeho úpravy při zachování účinnosti produkce peroxidu. Experimentální aparatura pro druhou část práce byla sestavena na Gentské Univerzitě v Belgii. Stejnosměrný výboj byl generován v bublinách plynů (He, Ar, N2, vzduch) v prostředí vodných roztoků. Byla studována generace peroxidu vodíku a odbourávání organických barviv přítomných ve zkoumaném roztoku. Ke generaci peroxidu vodíku byl použit roztok NaH2PO4 . 2H2O (5 microS/cm, V= 750 ml), ke zkoumání rozkladu barviv byly použity roztoky organických barviv Direct Red 79 (20 mg/l) a Direct Blue 106 (20 mg/l, V= 750 ml). Minimální koncentrace peroxidu vodíku byla naměřena při aplikaci proudu 10 mA, zatímco maximální koncentrace peroxidu vodíku byla zaznamenána při použití proudu 30 mA. Rozklad organických barviv vykazoval stejné vlastnosti. Čím vyšší byla dodávaná energie, tím více barviva se odbouralo. Třetí část práce probíhala ve spolupráci s Queen's University of Belfast, Centrum for Plasma Physics, UK. K realizaci experimentů bylo využito vysokofrekvenčního plazmového skalpelu (Arthrocar). Bylo zjištěno, že hodnota koncentrace peroxidu vodíku dosahovala maxima v roztocích s nepatrným přídavkem alkoholu (0.25 %). Celkem byly studovány čtyři 0.15 M roztoky BaCl2, Na2CO3, KCl a NaCl (V= 20 ml), jejichž počáteční vodivost se pohybovala kolem 13 mS/cm. Z výsledků bylo patrné, že největší rozdíl hodnot pH byl zaznamenán u roztoků s přídavkem ethanolu. V optických emisních spektrech byly identifikovány především radikály OH, které jsou prekurzory peroxidu vodíku. Výsledky ukázaly, že plazma v takovémto roztoku je stále tvořeno, což může být považováno za první krok generace plazmatu v organických sloučeninách. Poslední část práce byla zaměřena na tzv. mikroplazmatický jet v přímém kontaktu s kapalnou fází. Tato experimentální práce byla realizována na pracovišti Nanotechnology & Integrated Bio-Engineering Centre (NIBEC), University of Ulster, UK během studijní stáže. Jako vodivé médium byl použit roztok trihydrátu kyseliny chlorozlatité s různou počáteční vodivostí. Zajímavým zjištěním je fakt, že při tomto druhu měření bylo generováno stabilní plazma i při velmi malém výbojovém proudu (0.05 a 0.2 mA), a tedy i peroxid vodíku vznikal při velmi malé vstupní energii, což může být považováno za velmi dobrý výsledek.

Die vorliegende Arbeit beschreibt die Herstellung und Charakterisierung der ersten Antikörper gegen Triacetontriperoxid (TATP), einem hoch empfindlichen und unkonventionellen (nicht-kommerziellen) Initialsprengstoff. Entscheidend dafür war die Synthese eines TATP-imitierenden Haptens, welches die typische nonagonale Struktur des TATP mit seinen drei Peroxid- und sechs Methylgruppen nahezu perfekt nachbildet, aber den Vorzug einer zusätzlichen Carboxygruppe zur kovalenten Kopplung an Proteine aufweist. Dadurch konnte das TATP-Hapten an Rinderserumalbumin (BSA) gebunden werden, um ein immunogenes Konjugat zu erzeugen, welches die erfolgreiche Immunisierung zweier Säugetierarten, Maus und Kaninchen, ermöglichte. Der Verlauf der In vivo-Immunisierungen wurde durch die Analyse der Tierseren in regelmäßigen Abständen mittels enzymgekoppeltem Immunoassay (ELISA) verfolgt. Die polyklonalen Antikörper beider Spezies waren ungewöhnlich selektiv gegenüber TATP. Jedoch unterschied sich die Affinität der Antikörper der zwei Spezies um das 5000-fache, wobei die Kaninchenseren den Mausseren überlegen waren. Entsprechend war auch die mit Kaninchenserum erreichbare TATP-Nachweisgrenze von 0.01 µg/L deutlich besser im Vergleich zu 50 µg/L, die mit Mausserum erzielt wurden. Der Messbereich des TATP-ELISA mit Kaninchenserum deckte zudem mehr als vier Zehnerpotenzen ab, wie mittels Präzisionsprofil bestimmt wurde. Die erhaltenen TATP-Antikörper aus Kaninchen stehen damit Anwendungen in Nachweissystemen für die sehr empfindliche Detektion von TATP zur Verfügung, die u. a. in sicherheitsrelevanten Bereichen zum Einsatz kommen könnten. Als erste Anwendung wurde ein TATP-ELISA realisiert, der im Rahmen dieser Arbeit ausführlich optimiert wurde. Außerdem wurden erste Schritte zur Entwicklung eines TATP-Schnelltests (LFA) unternommen. Weitere Biosensoren auf Grundlage der neu entwickelten TATP-Antikörper sind denkbar. / The present work decribes the production and characterization of the first antibodies against triacetone triperoxide (TATP), a highly sensitive and improvised (non-commercial) primary explosive. Crucial to this work was the synthesis of a TATP-related hapten that mimics almost perfectly the typical nonagonal structure of TATP with its three peroxide and six methyl groups. Advantageously, it has an additional carboxylic acid group, which provides a conjugation site for covalent attachment to proteins. Thus, the TATP hapten could be linked to bovine serum albumin (BSA) to produce an immunogenic conjugate, allowing the successful immunization of two different mammalian species, mouse and rabbit. The in vivo immunization progress was followed by periodically analyzing the animals’ sera using enzyme-linked immunosorbent assay (ELISA). The polyclonal antibodies of both species were remarkably selective to TATP. The affinity of these TATP-antibodies was, however, different between the two species, with the rabbit sera showing an affinity about 5000-fold superior than the murine one. Consequently, the TATP detection limit of 0.01 µg/L was considerably better using the sera from rabbit in contrast to 50 µg/L when mouse serum was used. The working range of the TATP-ELISA with rabbit sera covers more than four decades, calculated from a precision profile. The obtained TATP antibodies from rabbit are now available for applications in highly sensitive detection systems for TATP, which could be employed, among others, in security-relevant areas. The first application was the realization of a TATP-ELISA, which was extensively optimized within the course of this work. Furthermore, the first steps towards the development of a lateral flow assay (LFA) targeting TATP were taken, making conceivable further biosensor platforms based on the newly developed TATP antibodies.

My diploma thesis is focused on a comparison of direct-current and high frequency (15-80 kHz) electric discharge, which generates non-thermal plasma in water solution of sodium chloride. Mainly current-voltage and Lissajous charts are discussed in the first part of this thesis. These charts describe different discharge phases: electrolysis, bubble formation, discharge breakdown and discharge regular operation in a pin-hole of a dielectric barrier. Influence of frequency, electrolyte conductivity, thickness of the diaphragm (or length of the capillary) and pin-hole diameter on discharge breakdown and bubble generation was studied, too. Measurements were realized in a polycarbonate reactor with total volume of 110 ml, which was divided by a changeable polyacetal insulating wall. This wall divided the reactor into two approximately equal spaces with one stainless steel planar electrode in each part. The Shapal-MTM ceramic discs (thickness of 0.3–1.5 mm and diameter of the central pin-hole of 0.3-0.9 mm) were mounted in the centre of the insulating wall. Initial conductivity of sodium chloride solution was chosen within the interval of 100900 S/cm. The second part of my thesis compares an influence of the direct-current (DC) and high frequency (HF) power sources on physical solution properties (conductivity, pH and temperature) and generation of hydrogen peroxide. A plasma reactor with total volume of 4 l and with mixing set up was divided into two equal spaces with one planar platinum electrode in each part. Diaphragm with thickness of 0.6 mm and pin-hole diameter of 0.6 mm was installed in the middle of the separating wall. Experiment was held at discharge operation of 45 W for 40 minutes with both power sources. Detection of hydrogen peroxide was realised by using a titanium reagent forming a yellow complex, which was analysed by absorption spectroscopy. If HF discharge power is plotted as a function of applied frequency, exponential decrease of frequency with increasing power can be observed. Higher breakdown voltage is necessary for thicker dielectric barriers, on the other hand for bigger diameter of the pin-hole lower breakdown voltage and higher power is needed in DC as well as in HF regime. Breakdown voltage is decreased by the increasing conductivity in both regimes; due to more charge carriers in the higher conductivity lower breakdown voltage is needed. However frequency in HF regime and DC discharge power increases. HF discharge power is decreased by the increasing conductivity. Solution conductivity and temperature are increased by initial conductivity value in both discharge regimes. Solution pH drops to acidic conditions when HF or DC positive regime is applied due to the generation of reactive species and electrolysis (in DC regime). However solution becomes alkaline when DC negative regime is applied. Concentration of hydrogen peroxide is produced linearly when HF or DC negative regime is applied and it depends on initial solution conductivity.

This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR.