Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH-dependent FabG into 3-hydroxyacyl-ACP, which is dehydrated by FabA to form enoyl-ACP. The NADH-dependent FabI (InhA) completes the cycle. Subsequent rounds of elongations in the pathways are catalyzed by the condensing enzyme FabF. For undecylprodiginine biosynthesis in S. coelicolor, homologues of the condensing enzymes (FabH and FabF) and the ACP (FabC) are encoded by redP, redR and redQ respectively in the red gene cluster. The genes encoding 3-ketoacyl-ACP reductase (FabG), 3-hydroxyacyl-ACP dehydratase (FabA), and enoyl-ACP reductase (FabI), are putatively shared between fatty acid and undecylprodigine biosynthesis, since the corresponding genes are not present within the red gene cluster of S. coelicolor. RedP is proposed to initiate biosynthesis of undecylprodiginine alkane chain by condensing an acetyl-CoA with a malonyl-RedQ, in contrast to FabH which process a broad range of acyl-CoA with malonyl-FabC. The 3-keto group of the resulting 3-ketoacyl-RedQ is then reduced to provide butyryl-RedQ, presumably by the type II FAS enzymes FabG, FabA and FabI. These enzymes would not differentiate between straight and branched-chain substrates, and have equal preference for FabC and RedQ ACPs. RedR would then catalyze four subsequent elongation steps with malonyl-RedQ, with appropriate 3-keto group processing after each step. The proposed role and substrate specificities of condensing enzymes RedP and FabH have not been investigated in S. coelicolor. The genes encoding FabG, FabA, and FabI have not been characterized in Streptomyces. Analysis of the S. coelicolor genome sequence has revealed the presence of one fabI gene (SCO1814, encoding an enoyl-ACP reductase), and three likely fabG genes (SCO1815, SCO1345, and SCO1346, encoding β-ketoacyl-ACP reductase). In the current study the substrates specificities of both RedP and FabH were determined from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC) (FabC is a dedicated ACP for fatty acid biosynthesis and RedQ for undecylprodiginine biosynthesis in S. coelicolor). For RedP, activity was only observed with a pairing of acetyl-CoA and malonyl-RedQ. No activity was observed with isobutyryl-CoA consistent with the proposed role for RedP and the observation that acetyl CoA-derived prodiginines predominate in S. coelicolor. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonyl-FabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyl-FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyryl-CoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines. The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-ketoacyl-RedQ substrates for undecylprodiginine biosynthesis. Similar to FabG, spectrophotometric and LC/MS assays were also carried out to elucidate the functional role and substrate specificity of S. coelicolor FabI. The kinetic analyses demonstrate that SCO1814 has NADH-dependent enoyl-ACP reductase activity. Spectrophotometric and LC/MS assays demonstrated that FabI does not differentiate between straight and branched-chain substrates, and has equal preference for FabC and RedQ ACPs. These observations provide experimental support for the hypothesis that these enzymes are shared and process the intermediates in the elongation cycle of both fatty acid and undecylprodiginine biosynthesis. In summary, these studies have demonstrated the activity of enzymes RedP, FabH, FabG and FabI (InhA) previously uncharacterized in S. coelicolor and clarified their role in fatty acid and undecylprodiginine biosynthesis.

A rapid method was developed for the quantitative analysis of N, N'hexamethylenebisacetamide (HMBA) and its deacetylated metabolites, N-acetyl-1, 6- diaminohexane (NADAH) and 1, 6-diaminohexane (DAH). The method used flowinjection liquid chromatography (LC)-atmospheric pressure chemical ionization (APCI)selected ion monitoring at low/high resolving power (LR/HR-SIM)-mass spectrometry (MS). Samples were extracted from murine erythroleukemia (MEL) cell culture and were analyzed without chemical derivatization. The limits of detection (LOD) for HMBA, NADAH and DAH measured by HR-SIM were 0.05, 0.1 and 0.2 picomole, respectively. Calibration curves were constructed by using tetradeuterated DAH as an internal standard for both SIM methods. The ratio of the analyte concentration in the extract of a MEL cell culture treated with HMBA for 120 hours was found to be 1. 7, 27 and 17 times that of the control for DAH, NADAH and HMBA, respectively. The HRSIM that used a lock-mass technique helped eliminate chemical interferences that had mlz values similar to the one of interest. Although this technique was developed using HMBA, NADAH and DAH, it is expected to work for natural polyamines and their acetylated metabolites. Qualitative analysis was performed by both positive electrospray (ES)- and APCIMS under full mass range scanning. The mass spectra of polyamine standards were characterized. HMBA exhibited an ability to form mono- and bi-molecular adducts with metal ions, itself and with other polyamine molecules in ES-MS. The LODs estimated by ES-MS were consistently higher than the APCI-SIM methods that is described here.

CHARLES UNIVERSITY PHARMACEUTICAL FACULTY IN HRADEC KRÁLOVÉ DEPARTMENT OF PHARMACEUTICAL BOTANY Title of the Diploma thesis: FENOLIC ACIDS IN PLANTS Candidate: Lucie Klížová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Diploma thesis 2020/2021, pp. 153 Phenolic acids belongs between secondary metabolites, which are part of fruits, vegetables, cereals and other plants of different families. For example, it can be plants of family Lamiaceae, Asteraceae and Ericaceae, which contain significant amounts of these substances. In plants phenolic acids play an important role and they have many human health-promoting effects including antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, anticancer activity and other. Plants containing phenolic acids are preferably also used as antidiabetics, expectorants, insecticides, hypolipidemics, cardioprotective and neurological compounds, etc. The content of these compounds in plants may be different according to monitored cultivar, the place of growth, the time of harvest and many other aspects. Presence, identification and subsequent quantification of individual phenolic acids are usually done through the HPLC and MS methods and total content of these compounds is determined through the Folin-Ciocalteu method. The diploma thesis is a literature...

No treatment that would completely stop the progression of Alzheimer's disease has not been found yet. Recently used tacrine showed good results in the treatment of Alzheimer's disease, however long-term use led to chronic hepatotoxicity due to its metabolites. This master thesis deals with the compound 7-phenoxytacrine, one of the promising tacrine derivatives, which is one of the candidates for potential use in the therapy of Alzheimer's disease. Due to the formation of hepatotoxic metabolites of tacrine after the biotransformation in human liver, it appears necessary to identify the emerging metabolites of 7-phenoxytacrine molecule. Within this master's thesis in vitro biotransformation study of 7-phenoxytacrine using human liver microsomes was performed. High performance liquid chromatography with tandem mass spectrometry was used to determine the parent substance and the seventeen 7-phenoxytacrine metabolites. The analytical method showed the formation of six monohydroxylated and eleven dehydroxylated metabolites of 7-phenoxytacrine. Thus, we concluded that hydroxylation is the major metabolic reaction after in vitro microsomal biotransformation. In addition to the identification of metabolites, a quantification and microsomal stability study, including the determination of the amount of...

In recent years, there has been a rise in the use of New Psychoactive Substances (NPS) that mimic the effects of controlled drugs and licensed medicines, which have become a significant public health concern globally. Synthetic cannabinoids, a rapidly expanding category of NPS, with much higher potency and binding affinity to the cannabinoid receptors than ∆9-tetrahydrocannabinol (THC), have led to serious psychiatric complications and other adverse effects. This project aims to synthesize and analyze four potential metabolites of a synthetic cannabinoid, ADB-5´Br-BUTINACA, to determine if the synthesized metabolites correspond to those produced in human hepatocytes. The metabolites were synthesized by alkylation, amide coupling, and hydrolysis/TFA treatment and analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR). The reaction strategy combined all three steps into a single process, making it significantly easier to carry out and not requiring much expertise. The results suggest that the synthetic approach used in this project was successful in generating the desired potential metabolites, with overall yields varying from 27.5 % to 57.6 % and high purities ranging from 95.6 % to 99.3 %. However, the overall yields were lower than expected due to product loss during the transfer of the solution mixture, the possibility of side reactions, and incomplete conversion. Further optimization of reaction conditions may be necessary to improve the yield of the synthesized metabolites.

Secondary bacterial superinfections have been a significant source of debilitating morbidity and mortality outcomes during both seasonal influenza outbreaks and historical pandemics. As many as 40% of those infected with influenza that develop a secondary bacterial infection such as S. aureus or S. pneumoniae will succumb to the infection. The complex relationships between the immune system, the pathogens, and host response makes this facet of biomedical research a topic of continual discoveries. During superinfection, there is a novel hypothesis that the failure of resolution causes a cascade of uncontrolled inflammatory responses leading to excessive morbidity or death. Here, we investigate a specific metabolite receptor, PPARα, and how its induction during superinfection affects the host immune response and the ability to resolve the infection. During superinfections, previous studies from our lab has found that the CYP450 metabolites are produced in abundance compared to their respective levels during singular viral or bacterial infections. These metabolites furthermore induced PPARα, which has been found to enhance necroptosis during superinfection conditions. Understanding programming of cell death provides insights on the resulting inflammation during an active infection. We further examined PPARα properties by inducing it without the presence of pathogens and found PPAR’s mechanisms to be context dependent. When PPARα is stimulated solely with a chemical agonist WY14643, the induction drives macrophages to apoptosis. When we started examining PPARα induction while inducing programmed cell death profiles, we found that the effect of PPARα to be uninfluential to apoptosis, but highly influential in necroptotic cell death. Latest studies associate very long chain fatty acid accumulation in cells undergoing necroptosis, and fatty acid isolation and analysis was completed. Fatty acid isolation showed an accumulation of ceramides and a significant decrease of both vital eicosanoid precursors and cell membrane associated phospholipids. In these studies, we next examined how induction of PPARα affects macrophage immune response and ultimately hinders resolution of infection. In vivo animal studies showed that macrophages were polarizing toward anti-inflammatory M2 phenotypes during the superinfection. When flow cytometry studies were performed to examine if metabolites stimulating PPARα were responsible for this change, we found that macrophages were polarizing to the M2b phenotype. This finding has been highly intriguing to our studies, as M2b macrophages are most abundantly present when resolution occurs in influenza infection and mice have successfully produced anti-influenza antibodies. Further examination was done to see how macrophage immune response was affected. Using Nanostring and examining PPARα activated macrophages via microscopy, several cytokines and chemokines for immune response were dampened. Microscopy specifically showed the nuclear localization of NFκB is effectively diminished during PPAR activation, demonstrating that the immune response is impaired. Finally, macrophage function was considered and analyzed by CFU assays and live cell microscopy. Both indicated that phagocytosis was impaired in macrophages, but microscopy elucidated that there was a lack of bacterial killing due to PPAR activation. Understanding the mechanisms of superinfection and how to effectively ameliorate them has potential to not only reduce the amount of morbidity and mortality around influenza each year, but advance the understandings of how these different systems come together in other immune contexts. The understanding of programmed cell-death, inflammatory gene networks, immune response and function all have changed by lipid profiling and how these metabolites can influence traditionally well studied systems. Having a greater appreciation for how metabolites induce immune, transcriptional, or cellular changes, as well as how metabolites and lipid profiling can be altered would be groundbreaking for inflammation research of several diseases and conditions that are not fully understood. / Biomedical Sciences

These projects were conducted to determine the digestibility of forage diets with differing CP levels in llamas and alpacas. The Utah study was designed to compare llama and alpaca nutritional parameters to determine if nutritional recommendations for llamas can be directly extrapolated to alpacas. The first study evaluated the effects of forage quality on blood metabolites and nitrogen balance in mature, intact male llamas (n = 4, 36 ± 4.4 months, 87 ± 17 kg) at high altitude in Letanias, Bolivia (4,267 m = aprox.14,000 ft above sea level). A second experiment was conducted with eight adult gelded camelids (n = 8; 4 llamas, 24-36 months, 90 ± 10.7 kg; 4 alpacas, 24-36 months, 50 ± 4 kg) at Brigham Young University, Provo, UT (altitude 1370 m). Animals were randomly fed barley hay (B) and 80% barley/20% alfalfa hay (BA). A fresh cut grass pasture (P) was included as the third forage for Bolivian llamas. Animals were housed in metabolism crates and diets were fed for a 7 d adjustment period followed by a 5 d collection period. Feed, feed refusal, feces and urine were collected, dried and N content determined by combustion analysis. Venous blood samples were collected on d 12 at 30 min intervals over a 6 h period. Plasma was harvested and analyzed for electrolytes (Na, K, Cl, Ca, Ca++, P, Mg) and metabolites (glucose, non- esterified fatty acids (NEFAs), urea N, creatinine, albumin, total protein (TPP), osmolality (Osm)). Llamas and alpacas demonstrated differences with respect to nitrogen metabolism when consuming forage diets with differing protein concentration. Llamas showed a N maintenance requirement of 0.75 g crude N/ W0.75. Using the standard CP to digestible protein (DP) conversion factor of 0.8, llamas required 0.60 digestible N/W0.75. When consuming the same high protein barley alfalfa diet, llamas had a much greater increase in N retention than alpacas. These species differences indicate that alpacas have a higher N requirement to meet metabolic needs, and extrapolations with respect to nitrogen requirements and balance are not valid between llamas and alpacas. In the Bolivian llama trial, locally grown and harvested hycrested and Siberian wheat grass pasture (P), barley (B), and barley80%/alfalfa20% (BA) hays were fed. The Bolivian llamas were in negative N balance when fed the B and P diets. Dry matter digestibility was greater with the B and BA than P forage, and N digestibility was significantly higher with BA than either the B or P forages. Nitrogen maintenance requirement for Bolivian llamas at 4,267 m was 0.58 compared to 0.75 g crude N/W0.75 for Utah llamas, an increased digestive efficiency and a lower N maintenance requirement at higher altitude.

The human gut microbiota plays a crucial role in human health and therefore imbalances in gut microbiota functioning can lead to the development of metabolic disorders. Short-chain fatty acids (SCFA), tricarboxylic acid (TCA) cycle metabolites, bile acids (BA) and other metabolites are the essential compounds for the metabolome. Per- and Polyfluoroalkyl substances (PFAS) are a group of chemicals that can be absorbed onto the lumen by food. Since many PFAS has shown to possess a long list of adverse effects on human health, the short term impact of PFAS on gut microbiota functioning using an in vitro model that mimics fermentation in the human colon was explored. Samples obtained from in vitro fecal fermentation were then analysed through high-performance liquid chromatography coupled to time-of-flight mass spectrometry (LC-qToF-MS) using targeted and non-targeted approaches. Fecal samples were donated by four donors which were put through a fermentation over 24 hours and treated with no PFAS mixture (control), low concentration mixture of PFAS (PFAS-L) and high concentration mixture of PFAS (PFAS-H). The PFAS mixture contained perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA). For the fermentation, samples were collected after 0, 2, 4, 6, 8 and 24 hours. The samples were spun down and the supernatant was collected. Two approaches were applied to explore the metabolism in the fermentation extracts: The first one was a “targeted SCFA and TCA cycle metabolites analysis’’ and the second one was “untargeted analysis of polar and nonpolar metabolites’’. Results show that all TCA cycle metabolites displayed different trends with the compound and little to no variation between the treatments. However, although there was no significant difference, 4 core compounds of the TCA metabolism were lower after treatment with PFAS-L or PFAS-H. For the untargeted method, 78 of 2855 compounds were identified. When comparing control samples, PFAS-H and PFAS-L, 200 features showed statistically significant differences. Most of these had higher concentrations for PFAS-treated samples. When PFAS-H was compared only with PFAS-L, 30 features were found to be statistically significant, indicating that distinct concentrations of PFAS differentially affect gut microbiota metabolism. For future identification, further analysis must be done, preferably with MS/MS, to obtain more structural information for these significant metabolites, since the masses of the fraction ions are needed to narrow down the search in databases used to obtain the identity of an ion. PFAS and the concentration seem to have an impact on the gut microbiota, the study should be done with additional donors to archive trends applicable to a whole population.

Polycyclic aromatic hydrocarbons (PAH) are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. Because many of them are highly suspect as etiological agents in human cancer, chemical analysis of PAH is of great environmental and toxicological importance. Current methodology for PAH follows the classical pattern of sample preparation and chromatographic analysis. Sample preparation preconcentrates PAH, simplifies matrix composition, and facilitates analytical resolution in the chromatographic column. Among the several approaches that exist to pre-concentrate PAH from water samples, the Environmental Protection Agency (EPA) recommends the use of solid-phase extraction (SPE). High-performance liquid chromatography (HPLC) and gas chromatographymass spectrometry (GC-MS) are the basis for standard PAH identification and determination. Ultraviolet (UV) absorption and room temperature fluorescence detection are both widely used in HPLC, but the specificity of these detectors is modest. Since PAH identification is solely based on retention times, unambiguous PAH identification requires complete chromatographic resolution of sample components. When HPLC is applied to "unfamiliar" samples, the EPA recommends that a supporting analytical technique such as GC-MS be applied to verify compound identification and to check peak-purity HPLC fractions. Independent of the volume of extracted water, the approximate time required to separate and determine the sixteen "priority pollutants" (EPA-PAH) via HPLC is approximately 60min. If additional GC-MS analysis is required for unambiguous PAH determination, the total analysis time will reach 2-3 hours per sample. If the concentrations of target species are found to lie outside the detector’s response range, the sample must be diluted and the process repeated. These are important considerations iv when routine analysis of numerous samples is contemplated. Parent PAH are relatively inert and need metabolic activation to express their carcinogenicity. By virtue of the rich heterogeneous distribution of metabolic products they produce, PAH provide a full spectrum of the complexity associated with understanding the initial phase of carcinogenesis. PAH metabolites include a variety of products such as expoxides, hydroxyl aromatics, quinines, dihydrodiols, dioepoxides, tetrols and water soluble conjugates. During the past decades tremendous efforts have been made to develop bio-analytical techniques that possess the selectivity and sensitivity for the problem at hand. Depending on the complexity of the sample and the relative concentrations of the targeted metabolites, a combination of sample preparation techniques is often necessary to reach the limits of detection of the instrumental method of analysis. The numerous preparation steps open ample opportunity to metabolite loss and collection of inaccurate data. Separation of metabolites has been accomplished via HPLC, capillary electrophoresis (CE) and GC-MS. Unfortunately, the existence of chemically related metabolic products with virtually identical fragmentation patterns often challenges the specificity of these techniques. This dissertation presents significant improvements in various fronts. Its first original component – which we have named solid-phase nano-extraction (SPNE) - deals with the use of gold nanoparticles (Au NPs) as extracting material for PAH. The advantages of SPNE are demonstrated for the analysis of PAH in water samples via both HPLC1 and Laser-Excited TimeResolved Shpol’skii Spectroscopy (LETRSS).2 The same concept is then extended to the analysis of monohydroxy-PAH in urine samples via SPE- HPLC3 and In-Capillary SPNE-CE.4 The second original component of this dissertation describes the application of Shpol’skii Spectroscopy to the analysis of polar PAH metabolites. The outstanding selectivity and v sensitivity for the direct analysis of PAH at trace concentration levels has made Shpol’skii spectroscopy a leading technique in environmental analysis.5 Unfortunately, the requirement of a specific guest-host combination - typically a non-polar PAH dissolved in an n-alkane - has hindered its widespread application to the field of analytical chemistry. This dissertation takes the first steps in removing this limitation demonstrating its feasibility for the analysis of polar benzo[a]pyrene metabolites in alcohol matrixes.6

Ministry of Higher Education; Sultanate of Oman / Digital full-text is unavailable. Submitted disc unusable.