Investigation on the general activity and uncoupling of the catalytic cycle of different cytochrome P450 monooxygenase

Cytochrom P450 Monooxygenasen wurden in den 1940er Jahren entdeckt und wurden seither intensiv erforscht. Obwohl sie sehr interessante Reaktionen katalysieren, die Anwendung in der Produktion von Feinchemikalien und Pharmazeutika finden könnten, wird ihre Wirtschaftlichkeit meist durch ihre Aktivität und Stabilität negativ beeinflusst. Beide Eigenschaften, die Aktivität und die Stabilität, werden durch die Entkopplung des katalytischen Zyklus beeinflusst. Im Rahmen dieser Doktorarbeit, wurde ein Assay für das „screening“ auf Aktivität und Entkopplung erfolgreich entwickelt. Nachdem optimale Reaktionsbedingungen für das Assay identifiziert waren, wurde die Entkopplung von Cytochrom P450 BM3 und fünf Mutanten (F87Y, R47L, Y51F, A82L and T268A) mit verschiedenen Substraten untersucht. Obwohl bereits viele andere Wasserstoffperoxid Detektionsmethoden beschrieben wurden, war die Kombination einer parallelen Bestimmung der NADPH Abnahme und der Wasserstoffperoxid Bildung neu. Dieser parallele Ansatz stellt eine gute Basis für ein „pre- screening“ großer Mutantenbibliotheken dar, worauf eine detaillierte Untersuchung von ausgewählten Varianten folgt. Um die Aktivität des CYP11A1 Systems, bestehend aus CYP11A1, Adx und AdR als Redox Partner System, untersuchen zu können, wurde zunächst die Expression und Aufreinigung aller drei Proteine untersucht. Für die Proteine CYP11A1 und Adx, wurden gute Expression Levels erreicht, wohingegen die Konzentration für AdR sehr gering war. Für alle drei Proteine wurde nach Aufreinigungsversuchen eine konzentrierte Lösung erhalten, die jedoch noch andere Proteine enthielt. Daher wurde in der Masterarbeit von Christopher Grimm, die pH und Temperaturstabilität aller drei Proteine untersucht, um die Bedingungen während der Ionenaustauschchromatographie als Aufreinigungsmethode optimieren zu können und optimale Bedingungen für in vitro Biokatalysen zu finden. Leider wurde trotz weiterer Expressionsversuche mit AdR keine Verbesserung erzielt, wodurch ein Ganzzellsystem als nächstes untersucht wurde. Für dieses System konnte die Produktbildung auf 2.2%, achtfach erhöht werden, im Vergleich zu den publizierten 0.27% Umsatz nach 24h durch die Verwendung eines anderen Detergenz zur Lösung des Substrates Cholesterol, wodurch vermutlich die Bereitstellung des Substrates für das Enzym erleichtert wurde. Auf Grund der beobachteten geringen Aktivität und Stabilität, wurde anschließend ein weiteres P450 System untersucht, das CYP17A1 Enzym. Zunächst wurden in vitro Biokatalysen mit dem humanen CYP17A1 durchgeführt, welches in E. coli exprimiert wurde. Nachdem verschiedene Redox-Partner Systeme getestet wurden, erzielte das Pdx/PdR System von P. putida in Kombination mit dem CYP17A1 Enzym den höchsten Umsatz, mit 91% nach 24 h. Um die Aktivität des Enzyms näher zu untersuchen, wurden alle Aminosäuren in einem Abstand von 4 Å zum gebundenen Substrat durch Alanine ersetzt. Alle Varianten wurden exprimiert, jedoch konnte kaum richtig gefaltetes Protein erhalten werden. Auch nachdem verschiedene Puffer untersucht wurden, zur eventuellen Stabilitätserhöhung, konnte keine Verbesserung festgestellt werden. Deshalb wurde auch hier auf einen Ganzzellansatz zurückgegriffen. Dieser bestand aus dem Enzym aus Rind welches in S. cerevisiae exprimiert wurde. Mit diesem System, wurde die Bedeutung der Positionen N202, R239, G297, E305 und T306A bestätigt, die in der Literatur als wichtig für die katalytische Aktivität beschrieben werden. Am wichtigsten waren drei Positionen, die die Regioselektivität des Enzyms verändern. Die Reaktion der V483A-Mutante wurde daher auch durch präparative Biokatalyse weiter untersucht. Danach wurde das neue Produkt durch präparative HPLC getrennt und als 16α-Hydroxyprogesteron identifiziert, was mittels NMR-Spektroskopie bestätigt werden konnte. Im letzten Teil der Arbeit wurde ein weiterer Screening-Ansatz für ein mögliches Hochdurchsatz-Screening untersucht. Im Gegensatz zum bereits beschriebenen Screening- Ansatz wurde hier die Untersuchung der Substratumwandlung und der Wasserstoffperoxidbildung für den Einsatz in „droplets“ optimiert. Nachdem festgestellt wurde, dass DCFH-DA gegenüber Wasserstoffperoxid nicht empfindlich genug war, wurde das AmplifluTM Red / HRP System verwendet. Da beide fluoreszierenden Produkte oberhalb von pH 7,4 in der wässrigen Phase blieben, wurden die für den AmplifluTM Red Assay untersuchten Bedingungen angewendet. Das NADPH-Substrat-Verhältnis wurde mit einer Entkopplungsvariante, einer aktiven Variante aus der Literatur und dem Cytochrom P450 BM3 Wildtyp-Enzym untersucht. Nach Ermittlung eines guten Verhältnisses wurden die fünf für die Untersuchung des AmplifluTM Red Assays verwendeten Varianten in der gleichen Konzentration untersucht, die später in den „droplets“ gefunden wird (1 Zelle pro 4 pL). Hier zeigte eine Variante eine verbesserte Produktbildung im Vergleich zum Wildtyp. Dieser Befund zeigt deutlich die Anwendbarkeit des Assays für Hochdurchsatzscreening in „droplets“.In the 1940s cytochrome P450 monooxygenases have been discovered and have been the focus of many studies ever since. Although they catalyze very interesting reactions that might find applications in the production of fine chemicals or pharmaceuticals, their low activity and stability often reduces their economic value. Both properties, the activity and the stability, are influenced by the uncoupling of the catalytic cycle. In this PhD thesis, an assay for the screening of activity and uncoupling of cytochrome P450 enzymes was successfully developed. After finding optimal conditions for the assay, concerning pH and enzyme concentration, the uncoupling of cytochrome P450 BM3 and five mutants (F87Y, R47L, Y51F, A82L and T268A) was investigated. With the results obtained, a comparison of data from literature was possible and revealed similarities. Additionally, through negative controls, the reliability of the assay could be further demonstrated. Although other methods have been described for the detection of hydrogen peroxide formation, the combination of NADPH consumption measurement and hydrogen peroxide formation in parallel was new and represents a very good basis for a pre-screening of large mutant libraries, followed by closer investigation of selected variants. For the investigation of the activity of the CYP11A1 system, consisting of CYP11A1 and Adx and AdR as redox partner system, the expression and purification for all three proteins was investigated first. For the protein CYP11A1 and Adx, good expression levels were achieved, whereas for AdR the protein concentration obtained was very low. The purification of all three proteins was partially accomplished but left room for improvement. Therefore, in the Master thesis of Christopher Grimm, the pH and temperature stability of all three proteins was further investigated in order to improve conditions used for ion exchange chromatography and to investigate possible conditions for in vitro biocatalysis. As unfortunately even with further investigation of the expression of AdR, no improvement was achieved, a whole-cell system was further investigated. Here, the product formation could be increased 8-fold in comparison to the published data, from 0.27% conversion to 2.2% conversion over 24 h by using a different detergent for substrate solubilization, which might have led to a better substrate supply to the enzyme. Due to the low activity and stability, a different P450 system, the CYP17A1 enzyme, was subsequently investigated, first by in vitro biocatalysis with the human CYP17A1 expressed in E. coli. Therefore, a suitable redox partner system needed to be found for efficient electron supply of the enzyme. In in vitro biocatalysis, in combination with the Pdx/PdR system of P. putida the CYP17A1 enzyme showed the highest conversion with 91% after 24 h. To investigate the activity of the enzyme further, all active site residues in 4 Å proximity to the bound substrate were exchanged with alanine. After expression of the variants, almost no correctly folded protein was obtained for the variants. Also, after investigating different buffers to possibly enhance the stability, no improvements were achieved. Therefore, a whole-cell approach with the bovine enzyme was chosen in order to investigate the activity of the alanine variants. Here the importance of positions N202, R239, G297, E305, and T306A, described in literature to be important for catalytic activity, was confirmed. Most importantly, three positions that alter the regioselectivity of the enzyme were identified. The reaction of the V483A mutant was therefore also further investigated by preparative biocatalysis. Afterwards the new product was separated by preparative HPLC and identified as 16α- hydroxyprogesterone as confirmed by NMR spectroscopy analysis. In the last part of the thesis, another screening approach for possible high-throughput screening was investigated. In contrast to the other screening approach, here the investigation of the substrate conversion and the hydrogen peroxide formation were optimized for application in droplets. After finding that DCFH-DA was not sensitive enough towards hydrogen peroxide, the AmplifluTM Red probe was used. As both fluorescent products were found to stay in the aqueous phase above pH 7.4, the conditions investigated for the AmplifluTM Red assay were applied and only NADPH to substrate ratio was investigated by using an uncoupling variant, an active variant from literature and the cytochrome P450 BM3 wild-type enzyme. After finding a good ratio, the five variants used for the investigation of the AmplifluTM Red assay were investigated in the same concentration later on found in the droplets (1 cell per 4 pL), and one variant showed improved product formation compared to wild-type. This finding clearly shows the applicability of the assay for high-throughput screening in droplets

Guided Reading Adaptations For Multilingual Learners

Elementary educators continue to face the challenge of providing an equitable education for all students. As more multilingual students are seated in the classroom, the challenge to support literacy and language development becomes increasingly important. This capstone seeks to answer the question: How can elementary educators improve guided reading instruction, and what adaptations support multilingual learners? The research concludes that guided reading modifications should be made through the additive approach to language, translanguaging, teacher and student participation, corrective feedback, and assessment. Using these modifications during guided reading can have many positive benefits on a student’s academic achievement and linguistic identity

Rehabilitation following a Distal Fibular Fracture in a 16 Year Old Female Athlete: A Case Report.

Background and Purpose: Distal fibular fractures are an uncommon injury in athletes, they are produced by repetitive stress to the fibula often due to overuse. The purpose of this case report is to discuss the rehabilitation following a distal fibular fracture of a young, female athlete and how the rehabilitation prepared the patient to return to her sports. Case Description: The patient is 16 year old Caucasian female who participated in both cheerleading and gymnastics. She sustained a left distal fibular fracture while performing a stunt in cheerleading. The patient was referred to physical therapy following six weeks of activity modification and rest. Intervention: The patient\u27s plan of care included interventions that focused on lower extremity strengthening focusing on the left ankle and bilateral hip musculature, balance, jumping and running. Outcome: The patient returned to her sports with no pain or difficulty following six weeks of intensive physical therapy. Discussion: The patient obtained great results and her results fit in with former literature on fibular fractures

Examining The Perceptions Of Bullying Between Certified And Non-Certified School Personnel

Bullying has been a widely researched topic. However, many areas are yet to be researched in regards to school personnels’ perceptions. This dissertation was focused around several of these missing research areas, as well as included non-certified staff. Non-certified staff have been left out of the vast majority of studies involving school personnel and bullying. That, or their perceptions have not been explored as a separate group of school personnel. This is problematic as non-certified staff are often part of supervisory roles in settings where bullying often occurs. This study addressed differences among school personnel perceptions in regards to bullying and media influence, student understanding and awareness, the need for school-wide training, bullying definition changes, and legal and employment consequences. Legal and employment consequences and the need for school-wide training were explored in depth, and differences among perceptions of school personnel were discussed

Letter from G. Morlock to Ogden Goelet

https://digitalcommons.salve.edu/goelet-miscellaneous/1021/thumbnail.jp

The borderlands : living between archetypes in young adult Chicana literature

This thesis focuses on two models for Chicana womanhood, which are the La Virgen de Guadalupe archetype and the La Malinche archetype. They are both mythic figures in Mexican culture that are diametrically opposed to one another. La Virgen is the good mother and La Malinche is the bad one. Some of the interventions include identifying the different degrees of liminality (YA lit, Borderlands Theory, and Adolescence). Additionally I close read scenes and look at how actions subvert language that reinforces the archetypes. I also argue that each Estrella and Esperanza seem to be each others' foils in that they are in different environments and have opposing female archetypes as their role model for womanhood, but that they are actually very similar. I look at how they each reject their archetype and end up in the same independent headspace. In chapter one, I ultimately make the argument that because Estrella has no opportunity for material or economic agency, sexual agency because an avenue to explore self sovereignty. And that, in Chapter 2, after being sexually assaulted, Esperanza decides to reject sexual sovereignty in favor of economic independence. She opts to leave the barrio and to pursue education in the hopes of coming back for those "who cannot out." In Chapter Three, I bring the two novels together to talk about hybridity and Anzaldua's "new Mestiza" -- that is, someone who lives in the in-between, who takes from both sides of the dichotomy and who lives for herself. Additionally, both girls straddle the line between girlhood and adulthood in both their ages and their responsibilities. Essentially, the two protagonists are representative of the "new mestiza." Each of them is "outsider within" -- someone who can navigate both the hegemonic culture and the non-hegemonic culture