Acrylfentanyl is a synthetic opioid that has been widely used in the last year. To help in the fight against synthetic drugs two potential metabolites of acrylfentanyl, one monohydroxy and one dihydroxy were synthesized. These metabolites will hopefully later be implemented in the analytical methods for metabolites of acrylfentanyl in urine by the Swedish National Board of Forensic Medicine. To have metabolites for analysis are very important as they are the main target in drug testing. The method used to synthesize the metabolites is a five-step synthesis with an additional 6th step for the dihydroxy metabolite. The methods used in the synthesis includes protection of amine with tert-butyloxycarbonyl, reductive amination with sodium triaceto boronhydride, alkylation and demethylation with boron tribromide. The methods used produced good results with high yields in nearly all steps.

Fusarium graminearum is responsible for causing Fusarium head blight in cereals and maize imposing a significant impact in Canadian agriculture. While a handful of secondary metabolites produced by F. graminearum are recognized as contributors to disease virulence, the functions of numerous molecular products arising from biosynthetic gene clusters expressed during infection remain undiscovered. Presented here are the results of CRISPR-Cas9 mediated gene-deletion experiments disrupting core biosynthetic genes from four biosynthetic gene clusters with reported in-planta transcription: C08, C16, C13 and C70. Both wheat head infection assays and coleoptile infection assays were used to evaluate the pathology phenotypes of transformant strains illustrating potential links between C16 and pathogenicity. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products and attempt to identify unknown secondary metabolites of the biosynthetic gene clusters. While C08 secondary metabolite remained elusive, confirmation of C16 secondary metabolites led to hypotheses regarding their potential connections to the inhibition of plant immune response and untargeted secondary metabolite profiling of the C13/C70 transformant strains suggests that this BGC may have significant implications for global secondary metabolite production.

The compatible solute glycine betaine accumulates in many plants including spinach (Spinacea oleracea) under conditions of water deficit stress. The precursor to glycine betaine is choline, a ubiquitous metabolite in plants as a component of phosphotidylcholine. In spinach choline is synthesized from phosphocholine, a product of three sequential N-methylations of phosphoethanolamine catalysed by the cytosolic enzyme S-adenosyl-L-methionine: phosphoethanolamine-N-methyltransferase (PEAMT). PEAMT activity shows diurnal changes with peak activity at the end of the photoperiod and a decrease overnight. The activity of this enzyme is up-regulated 2 to 3-fold in salt-stressed plants relative to unstressed plants. The objective of this thesis is to determine how PEAMT activity is regulated in vivo. Thus, PEAMT activity, protein and transcript levels were quantified in spinach leaves from plants subjected to different light and salinity conditions. A spinach PEAMT eDNA sequence was used to over-express recombinant PEAMT in the protein expression vector pET30a (+). The presence of a polyhistidine-tag on the overexpressed protein allowed for purification by a cobalt metal affinity column. The affinity purified protein was used to produce polyclonal antibodies for immunoblot hybridization analysis. For these studies, PEAMT protein was first immunoaffinity purified from soluble extracts prepared from leaves and then the protein subjected to electrophoresis by SDS-p AGE. Enzyme assays and immunoblot analysis show PEAMT activity and protein levels increase and become relatively constant in leaves of plants exposed to continuous light. In continuous darkness, PEAMT activity and protein levels decrease and remain low and constant. Thus the pattern of changes in PEAMT activity levels are associated with changes in PEAMT protein levels. In contrast, Northern blot hybridizations show that under conditions of constant light, peamt transcript levels undergo cyclical changes with peak levels at 20 and 40 h and troughs at 28 and 52 h after the continuous light treatment was imposed. These peaks coincide with the dark and light cycles of the normal photoperiod. The same cyclical changes in peamt transcript levels was seen for plants transferred from a normal photoperiod to continuous darkness. Since these changes persist in the absence of a day/night cue we conclude that peamt transcript levels are circadian-regulated. The peamt transcript levels of control unstressed and salt-stressed plants also show circadian rhythms, however the levels found in salt-stressed plants were 0.5 to 2-fold higher than the controls. Therefore, while salinization of plants increases peamt transcript abundance, it does not alter the circadian rhythm that transcripts of this gene display. Changes in PEAMT activity and protein levels are likely controlled by other as yet unknown post-translational mechanisms, processes that override and obscure operation of a circadian rhythm in regulating the level of peamt transcripts. / Thesis / Master of Science (MS)

Yes / Background: This study aims at characterizing the in vitro metabolism of cryptolepine using human and rat hepatocytes, identifying metabolites in rat plasma and urine after a single cryptolepine dose, and evaluating the single-dose oral and intravenous pharmacokinetics of cryptolepine in male Sprague Dawley (SD) rats. Methods: The in vitro metabolic profiles of cryptolepine were determined by LC-MS/MS following incubation with rat and human hepatocytes. The in vivo metabolic profile of cryptolepine was determined in plasma and urine samples from Sprague Dawley rats following single-dose oral administration of cryptolepine. Pharmacokinetic parameters of cryptolepine were determined in plasma and urine from Sprague Dawley rats after single-dose intravenous and oral administration. Results: Nine metabolites were identified in human and rat hepatocytes, resulting from metabolic pathways involving oxidation (M2-M9) and glucuronidation (M1, M2, M4, M8, M9). All human metabolites were found in rat hepatocyte incubations except glucuronide M1. Several metabolites (M2, M6, M9) were also identified in the urine and plasma of rats following oral administration of cryptolepine. Unchanged cryptolepine detected in urine was negligible. The Pharmacokinetic profile of cryptolepine showed a very high plasma clearance and volume of distribution (Vss) resulting in a moderate average plasma half-life of 4.5 h. Oral absorption was fast and plasma exposure and oral bioavailability were low. Conclusions: Cryptolepine metabolism is similar in rat and human in vitro with the exception of direct glucuronidation in human. Clearance in rat and human is likely to include a significant metabolic contribution, with proposed primary human metabolism pathways hydroxylation, dihydrodiol formation and glucuronidation. Cryptolepine showed extensive distribution with a moderate half-life. / Funded by Novartis Pharma under the Next Generation Scientist Program.

Genetic and molecular factors associated with chronic lung and kidney disease susceptibility, leading causes of domestic and global mortality, remain largely unexplained. Until recently, much of the research has focused on nucleotide differences in the human genome. However, the scope of gene studies has expanded beyond nucleotide variability, such as single nucleotide polymorphisms (SNPs). Current research utilizes multiple “omics” that consider not just inherited susceptibility factors, but also the macromolecules coded by the genome and gene-environment interactions. We conducted three studies to answer scientific questions, gain valuable insight into chronic lung and kidney disease processes, and provided meaningful guidance in prioritizing future research. This dissertation is particularly relevant to research in under-represented populations and when sample sizes are limited due to the nature of the disease or difficulty in obtaining biological specimens. We began with a common lung disease that affects millions annually, a potential risk factor for which little is known, and for which study is complicated because the diseased lung tissue is difficult to obtain for cases and more so for controls. In this study, we used easier-to-obtain nasal brushings and transcriptomics to measure counts of non-coding genetic elements. We then shifted to a kidney disease that has been best described in South America and South Asia, where genetic components of the disease might be very different from other more typical forms of kidney disease in North American and European populations. We used genetic variants explaining urinary metabolites to help prioritize future genetic research for the kidney disease, and we explored variants that explain blood concentration of urate to better understand metabolic processes in the at-risk population. In our first study, we examined the associations of Human Endogenous Retrovirus (HERV) transcription with lung function (FEV1/FVC) and Chronic Obstructive Pulmonary Disease (COPD) among current and former smokers. We sought evidence that greater transcription of HERVs was associated with lower lung function and higher risk of COPD. HERVs associated with FEV1/FVC were located within or near COPD genes, and HERV transcription was correlated with gene-regulation involved in FEV1 decline. RNA-seq data from two Detection of Early lung Cancer Among Military Personnel (DECAMP) cohorts were used to identify transcribed HERVs and estimate locus-specific HERV counts. A weight-of-evidence framework was then used to prioritize HERVs and was compared to a p-value based approach. We found 55 HERV loci with evidence that transcription was associated with higher or lower FEV1/FVC. Consistent with the prevailing theory that transcription and expression of HERVs is suppressed in adults, we observed HERV loci with lower mean counts and increased transcription was associated with worse lung function. However, we found some HERV loci with higher mean counts, and increased transcription was associated with better lung function. Most of the lung function-associated HERVs were intergenic and none were near COPD genes. Higher counts of HUERSP1 (15q21) were correlated with higher expression of down-regulated FEV1 decline genes (rho= 0.82; 95%CI: 0.77, 0.85; p= 1.1 x10-65). We also found, albeit with limited evidence, that overall higher transcriptional levels of some HERVs were associated with differential COPD risk. Contrary to our expectation, we observed lower odds of COPD among those with the highest transcription across all loci of HERLEQUIN (beta= 0.45; 95%CI: 0.21, 0.96), HERV-P71A (beta= 0.44; 95%CI: 0.21, 0.91) and HML2 (beta= 0.45; 95%CI: 0.22, 0.93) compared to those with the lowest transcriptional levels. Use of both weight-of-evidence and p-values to prioritize HERVs was better than exclusive use of either framework. We were unable to rule out the possibility that, for some HERVs, transcription might exhibit Goldilocks-like effects on pulmonary function and COPD risk. For the second study, we explored the role of genetic variants in metabolism as measured by urinary metabolite concentrations. Our sample population was Mesoamericans residing in Nicaragua who were at risk for chronic kidney disease of non-traditional origins (CKDnt). High serum urate, also indicative of altered metabolism, is more common than expected in this genetically distinct population. We suspected a potential genetic component that might explain both CKDnt risk and differences in urinary metabolites. However, our limited sample size for genome-wide association studies (GWAS) motivated the design of three approaches to test our hypotheses: (1) for our main hypothesis, we began by investigating associations between nucleotide variability of all variants and concentration of all urine metabolites, and we identified statistically significant associations after accounting for multiple testing; (2) we tested a more limited hypothesis that analyzed all variants but selected metabolites that differed in concentration from similar European workers; (3) for our most specific hypothesis we analyzed associations between a selection of variants that affect protein structure and all metabolites. We included 313 non-cases of CKDnt from our case-control genetics study and with available metabolite data in our analyses. Metabolites were identified, and their concentrations measured, using nuclear magnetic resonance. The effect of variants on metabolite concentration and CKDnt case odds ratios were estimated using generalized linear mixed model association tests. We accounted for population structure, using the top ten genetically determined principal components, and cryptic relatedness using a genetic relatedness matrix. We found that Mesoamericans in our study population carried genetic variants that predispose them to altered energy metabolism and renal transport of solutes, but the allele frequencies did not differ significantly between cases and non-cases of CKDnt. A non-synonymous AGXT2 variant (rs37370) significantly explained 3-Aminoisobutyric acid (beta= -0.68; 95%CI: -0.73, -0.47, p= 9.55 x10 -20) and Dihydrothymine (beta= -1.05; 95%CI: -1.23, -0.88; p= 1.83 x10 -31). Our other approaches identified variants in several other genes including TTC23L, ACADS, HAO2, TCIRG1, NDUFA10, and SLC26A10. While we failed to find a shared genetic link between metabolism and CKDnt, we observed genetic variability in biologically plausible genes such as AGXT2 and HAO2. We concluded that Mesoamericans at risk of CKDnt, due to heat and dehydration, may also carry gene variants that predispose them to higher body heat production. Our third study was a GWAS to further understand genetic factors related to serum uric acid (sUA) metabolism among Nicaraguans at risk of CKDnt. Prior longitudinal studies have found that sugarcane workers experience post-shift elevation in sUA, and abnormally high concentration (hyperuricemia) is a known risk factor for CKD. After onset of CKD, higher sUA is associated with a worse prognosis. Also, previous sUA GWAS have identified a set of genetic variants explaining urate concentration across ethnic populations and found SNPs where the effect on concentration depends on renal function. However, it was unclear if known sUA associated variants explain concentration among populations at risk of CKDnt. We also suspected that genetic variability, unique to Mesoamericans, might explain serum concentration more than known sUA associated SNPs. Therefore, we examined SNP variability and associations with sUA among Nicaraguans. We chose to separately analyze our cases and non-cases of CKDnt because sUA in these groups had different possible biological etiologies. Our aims were to: (1) determine if previously published variants, with established association to sUA among global populations, explained concentration among those diagnosed with CKDnt and those at-risk of the disease; (2) discover novel sUA SNPs among our sample of Mesoamericans; (3) boost our discovery of variants by exploiting kidney function as an effect modifier; (4) identify SNPs where association depends on renal function; and (5) identify SNPs explaining sUA among both CKDnt cases and non-cases. We drew two analytical samples from the Mesoamerican Nephropathy Case-Control Genetic Study: case-only (n= 609) and control-only (non-cases= 385). Renal function was measured as serum creatinine (sCr). In our case-only GWAS, we used two models: generalized linear mixed model (GLMM) that included top 10 principal components (PCs) and use of sUA-lowering medication (allopurinol), and also included an additional gene*sCr interaction term. Only among cases, we tested for joint effects of variant and the interaction with renal function. SNPs, which explained sUA in case-only GWAS or joint test, were examined for modification by renal function using the gene*sCr coefficient. Control-only GWAS was based on GLMM that only included top 10 PCs. In this pilot study, we observed a high prevalence of hyperuricemia (CKDnt cases= 78.8% and non-cases= 23.6%). Despite our limited power, we successfully replicated prior findings for the ABCG2 variant, rs74904971, which was among the top three SNPs explaining sUA in global populations. Among CKDnt cases, rs74904971 significantly explained sUA concentration (beta = 1.1; 95%CI: 0.8, 1.4; p= 9.2 x10 -12), but not among controls (beta = 0.41; 95%CI: 0.16, 0.66; p= 1.2 x10 -3). We found no evidence that renal function modified the genetic effects of rs74904971 (betaG*sCr = 0.44, pG*sCr = 0.17). Most other established urate-SNPs were not significant even at 0.05 significance-level. We found five novel SNPs associated with sUA in our control-only and case-only GWAS, but these variants all had low minor allele frequency (MAF < 5%). The best SNP from control-only GWAS, a GALNTL6 intron variant (rs17057585), was significantly associated with a 1.9 mg/dL increase in sUA (95%CI: 1.3, 2.6, p= 3.6 x10 -9). In our case-only GWAS, we found participants with more alternative alleles of rs61156970, located 89kbp downstream from MCTP2, had suggestively lower sUA (beta= -1.9; 95%CI: -2.6, -1.2; p= 1.8 x10 -7). The joint test allowed us to identify an additional 20 variants, of which 11 SNPs had MAF ≥ 10%, and we found 16 SNPs with significantly greater effects among cases with worse renal function compared to cases with better functioning kidneys. We used The Human Protein Atlas, an online database, to access the biological plausibility for the most significant SNPs from the joint test. We found limited support for the top intronic SNPs from the joint test: rs9499393 (within ERCC3) and rs89572695 (within TMEM9B) have MAF < 10% and are highly expressed genes that are non-specific to renal cells. The effect of indel rs11404676 (within MTHFD1L), which was significantly greater among CKDnt cases with worse renal function than cases with better function (betaG*sCr= 1.4, pG*sCr = 1.4 x10 -6), is more likely to be biologically relevant to purine biosynthesis and urate concentration. We have demonstrated that we can identify known sUA associated SNPs with large effects, and that other Mesoamerican-specific variants better explain serum urate than known ABCG2 SNPs. Renal function among non-cases should be addressed in future GWAS from this population and caution is advised when extrapolating multi-ethnic GWAS results to Mesoamericans.

This study introduces a method for multiparallel analysis of small organic compounds in the unicellular green alga Chlamydomonas reinhardtii, one of the premier model organisms in cell biology. The comprehensive study of the changes of metabolite composition, or metabolomics, in response to environmental, genetic or developmental signals is an important complement of other functional genomic techniques in the effort to develop an understanding of how genes, proteins and metabolites are all integrated into a seamless and dynamic network to sustain cellular functions. The sample preparation protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity and process large sample quantities. As a result of the rapid sampling, extraction and analysis by gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) more than 800 analytes from a single sample can be measured, of which over a 100 could be positively identified. As part of the analysis of GC-TOF raw data, aliquot ratio analysis to systematically remove artifact signals and tools for the use of principal component analysis (PCA) on metabolomic datasets are proposed. Cells subjected to nitrogen (N), phosphorus (P), sulfur (S) or iron (Fe) depleted growth conditions develop highly distinctive metabolite profiles with metabolites implicated in many different processes being affected in their concentration during adaptation to nutrient deprivation. Metabolite profiling allowed characterization of both specific and general responses to nutrient deprivation at the metabolite level. Modulation of the substrates for N-assimilation and the oxidative pentose phosphate pathway indicated a priority for maintaining the capability for immediate activation of N assimilation even under conditions of decreased metabolic activity and arrested growth, while the rise in 4-hydroxyproline in S deprived cells could be related to enhanced degradation of proteins of the cell wall. The adaptation to sulfur deficiency was analyzed with greater temporal resolution and responses of wild-type cells were compared with mutant cells deficient in SAC1, an important regulator of the sulfur deficiency response. Whereas concurrent metabolite depletion and accumulation occurs during adaptation to S deprivation in wild-type cells, the sac1 mutant strain is characterized by a massive incapability to sustain many processes that normally lead to transient or permanent accumulation of the levels of certain metabolites or recovery of metabolite levels after initial down-regulation. For most of the steps in arginine biosynthesis in Chlamydomonas mutants have been isolated that are deficient in the respective enzyme activities. Three strains deficient in the activities of N-acetylglutamate-5-phosphate reductase (arg1), N2 acetylornithine-aminotransferase (arg9), and argininosuccinate lyase (arg2), respectively, were analyzed with regard to activation of endogenous arginine biosynthesis after withdrawal of externally supplied arginine. Enzymatic blocks in the arginine biosynthetic pathway could be characterized by precursor accumulation, like the amassment of argininosuccinate in arg2 cells, and depletion of intermediates occurring downstream of the enzymatic block, e.g. N2-acetylornithine, ornithine, and argininosuccinate depletion in arg9 cells. The unexpected finding of substantial levels of the arginine pathway intermediates N-acetylornithine, citrulline, and argininosuccinate downstream the enzymatic block in arg1 cells provided an explanation for the residual growth capacity of these cells in the absence of external arginine sources. The presence of these compounds, together with the unusual accumulation of N-Acetylglutamate, the first intermediate that commits the glutamate backbone to ornithine and arginine biosynthesis, in arg1 cells suggests that alternative pathways, possibly involving the activity of ornithine aminotransferase, may be active when the default reaction sequence to produce ornithine via acetylation of glutamate is disabled. / Entwicklung und Anwendung von Methoden zur multiparallelen Analyse von Metaboliten in der einzelligen Grünalge Chlamydomonas reinhardtii, einem der wichtigsten Modellorganismen der Zellbiologie, sind Gegenstand dieser Arbeit. Metabolomanalyse, die umfassende Analyse von Veränderungen der Konzentrationen von Stoffwechselprodukten durch Umweltreize oder genetische und entwicklungsbedingte Signale, ist ein wichtiges Komplement anderer Genomanalysemethoden, um die Integration von Genen, Proteinen und Metaboliten in ein nahtloses und dynamisches Netzwerk zur Aufrechterhaltung der Lebensfunktionen eines Organismus zu verstehen. Die Methode wurde im Hinblick auf schnelle Inaktivierung enzymatischer Aktivität, Maximierung der Extraktionskapazität und Behandlung großer Probenmengen optimiert. Im Ergebnis der Probenaufarbeitung, Extraktion und Analyse mittels Gaschromatographie und Time-Of-Flight-Massenspektrometrie konnten mehr als 800 analytische Signale in Einzelproben dargestellt werden, von denen über 100 identifiziert werden konnten. Die Arbeit stellt methodische Innovationen zur systematischen Erkennung von Artefakten in GC-MS Chromatogrammen und Werkzeuge zur Anwendung der Hauptkomponentenanalyse auf Metabolom-Daten vor. Zellen unter Stickstoff- (N), Phosphor- (P), Schwefel- (S), oder Eisen- (Fe) Mangel zeigen deutliche Unterschiede in ihrer Metabolitenausstattung. Die Anpassung an die einzelnen Nährstoffmangelsituationen ist durch spezifische Änderungen einer Reihe von Metaboliten zentraler Prozesse des Primärstoffwechsels gekennzeichnet. Die Konzentrationsänderungen von Substraten für die Stickstoffassimilation und den oxidativen Pentosephosphatweg deuten darauf hin, dass die Fähigkeit zur schnellen Aktivierung der N-Assimilation auch unter Bedingungen herabgesetzter Stoffwechsel- und Wachstumsaktivität aufrechterhalten wird. Die Akkumulation von 4-Hydroxyprolin unter Schwefelmangel könnte im Zusammenhang stehen mit der Degradation von Proteinen der Chlamydomonas-Zellwand, deren wesentlicher Bestandteil hydroxyprolinreiche Glykoproteine sind und die unter Schwefelmangel aktiv umgebaut wird. Die Anpassung an Schwefelmangel wurde mit größerer zeitlicher Auflösung in Wildtyp-Zellen und Zellen des sac1-Stammes untersucht. SAC1 ist ein zentraler Regulator der Schwefelmangelantwort in Chlamydomonas. Zeitgleiche Ab- und Zunahme von Metaboliten ist ein charakteristisches Element der Anpassung an Schwefelmangel in Wildtypzellen. Die Reaktion von SAC1-Mutanten auf Schwefelmangel ist durch weit reichenden Verlust zur Steuerung von Prozessen gekennzeichnet, die normalerweise zur vorübergehenden oder dauerhaften Anreicherung bestimmter Metabolite führen. Die Verfügbarkeit von Chlamydomonas-Stämmen mit fehlender Enzymaktivität für fast jeden der Schritte der Argininbiosynthese eröffnet die Möglichkeit, das Potential der Metabolitenanalyse zur Untersuchung der Regulation der Aminosäurebiosynthese in photosynthetischen Eukaryoten zur Anwendung zu bringen. Drei Stämme, mit fehlender Aktivität für N-Acetylglutamat-5-phosphat Reduktase (arg1), N2 Acetylornithin-Aminotransferase (arg9) beziehungsweise Argininosuccinat Lyase (arg2) wurden in Bezug auf die Aktivierung ihrer endogenen Argininbiosynthese nach Entzug externer Argininquellen analysiert. Die einzelnen enzymatischen Blocks konnten durch Precursor-Anreicherung, wie die Anhäufung von Argininosuccinat in arg2-Zellen, und Erschöpfung von Intermediaten nachgelagerter Reaktionen, beispielsweise die deutliche Abnahme von N2-Acetylornithin, Ornithin und Argininosuccinat in arg9-Zellen charakterisiert werden. Das unerwartete Vorhandensein von zum Teil das Wildtyp-Niveau überschreitender Mengen von N2-Acetylornithin, Citrullin und Argininosuccinat, die Produkte bzw. Substrate dem enzymatischen Block nachgelagerter Reaktionen in arg1-Zellen sind, bot eine Erklärung für eine noch vorhandene Restkapazität zum Wachstum des arg1-Stamms auch ohne äußere Arginingabe. Der Nachweis dieser Verbindungen sowie die ungewöhnliche Anreicherung von N-Acetylglutamat, der ersten Verbindung, die das Glutamat-Gerüst für die Ornithin- und Argininsynthese bindet, in arg1-Zellen könnte auf alternative Reaktionen, möglicherweise unter Beteiligung von Ornithin-Aminotransferase, zur Synthese von Ornithin hindeuten, die in Erscheinung treten, wenn die Synthesekette nach Acetylierung von Glutamat blockiert ist.

Maize anthracnose, caused by the fungus Colletotrichum graminicola, is an economically important species contributing to major yield losses. C. graminicola is a hemibiotroph; initially it invades its host while it is alive, and then it switches to destructive necrotrophic growth and the host is killed. Establishment of compatible interactions by biotrophic pathogens is usually associated with suppression of host defenses and cell death, while necrotrophic pathogens typically secrete phytotoxic compounds and induce cell death. To understand the relationship of hemibiotrophy in C. graminicola to biotrophy and necrotrophy, I compared a compatible and an incompatible interaction, utilizing a non-pathogenic mutant strain that is very similar to the wild type in vitro. I developed an assay to visualize in detail living fungal and host cells during pathogenic and nonpathogenic interactions. My results provided evidence that C. graminicola produces diffusible substances during colonization that predispose nearby living host cells for fungal invasion. My observations further suggested that the mutant is nonpathogenic because it fails to produce these substances. To explore the possibility that the C. graminicola mutant is impaired in the production and/or secretion of one or more secondary metabolites (SM), I characterized the range of SM-associated genes in C. graminicola. C. graminicola has a large and diverse repetoire of these genes, indicating significant capacity for the production of SM. I then characterized the global expression of fungal genes during different developmental phases in both compatible and incompatible interactions. I found that SM-associated genes are expressed during early and late stages of maize infection. Secreted proteins and putative effectors were overrepresented among differentially regulated predicted gene products. There were relatively few differences in expression between the mutant and wild type, suggesting that differences between them may relate to post-transcriptional events. The transcriptional analysis indicated that the mutant was defective very early in biotrophy. This study indicates that biotrophy and necrotrophy coexist in this pathosystem in different cells, and that arrays of differentially regulated and locally expressed genes are involved in maintaining this balance. Understanding the nature of induced susceptibility may lead to new therapeutic targets for management of this damaging disease.

No description available.

A seleção de animais para consumo alimentar residual (RFI) está intrinsecamente associada com a diminuição do consumo matéria seca e é independente do ganho de peso corporal, selecionando animais de eficiência produtiva e econômica, além também de diminuir a emissão de gases de efeito estufa provinda do gado. Neste estudo, amostras de soro de 16 animais selecionados divergentemente para eficiência de alimentação foram coletadas antes do confinamento (dia -21) e avaliadas em uma abordagem metabolômica global, com o objetivo de usar análise diferencial, análise de co-expressão e enriquecimento funcional, identificando marcadores para eficiência de alimentação antes do confinamento. Um analito foi diferencialmente presente entre os animais de baixo e alto RFI. A análise WGCNA identificou 22 e 25 módulos no modo positivo e negativo, respectivamente e, 1 módulo de cada modo foi fortemente associado a RFI (r = 0,53, p-valor &lt;0,05 e r = 0,52, p-valor &lt;0,1 nos modos negativo e positivo, respectivamente). A análise de enriquecimento funcional predize 13 processos biológicos associados à eficiência alimentar, incluindo alterações no metabolismo de vitaminas lipossolúveis, inflamação, estresse oxidativo, metabolismo de aminoácidos e metabolismo de ácidos graxos. Esse trabalho evidencia a possibilidade de se identificar um biomarcador para eficiência alimentar e também sugerem que as diferenças nas respostas ao estresse oxidativo e nos processos inflamatórios já influenciam na variação da eficiência alimentar previamente ao confinamento. / Animal selection for residual feed intake (RFI) is intrinsically associated with decreased consumption of dry matter independent of body weight gain, selecting yielding increased production and economic efficiency but also decreasing the greenhouse gas emission of livestock. In this study, serum samples of 16 animals selected for divergent feed efficiency were collected prior to feedlot (day -21) and evaluated in an untargeted metabolomics approach, with the goal of using differential analysis, co-expression analysis and functional enrichment to identifier markers for feed efficiency prior to the feedlot. One feature was differentially accumulated between low and high RFI. WGCNA analysis identified 22 and 25 modules in positive and negative mode, respectively, of 1 module of each mode was strongly associated with RFI (r= 0.53, p-value &lt;0.05 and r=0.52, p-value &lt; 0.1 to negative and positive mode, respectively). Pathway enrichment analysis yielded 13 biological processes associated with feed efficiency including alterations in vitamins liposoluble metabolism, inflammation, oxidative stress, amino acid metabolism and fatty acid metabolism. Our findings suggest the possibility to identify a biomarker for feed efficiency and also discuss that differences in oxidative stress responses and inflammatory processes could explain the feed efficiency variation prior to feedlot.

Mammals and fish rely on hormones to regulate blood glucose levels. The two major glucose regulating hormones are insulin and glucagon. Literature on mammalian insulin and glucagon is quite extensive, however, there is limited information on how these hormones regulate blood glucose levels in fish. The material available for fish mostly pertains to changes in glucose concentration and gene expression of enzymes, but there is no information on the direct influence they have on glucose kinetics. Therefore, the main goal of my thesis is to measure the change in hepatic glucose production and glucose disposal when rainbow trout are administered insulin or glucagon. The beginning of my research focused on insulin. I hypothesized that rainbow trout respond to insulin by decreasing hepatic glucose production and increase glucose disposal, just like mammals. To test this, I infused insulin for 4 hours at 1.5 g insulin kg 1 min-1. I measured glucose disposal (Rd glucose), hepatic glucose production (Ra glucose), and blood glucose concentration. Following insulin administration the glucose fluxes decreased steadily (Rd glucose -37% and Ra glucose -43%). The decline in blood glucose levels follows the difference between Rd and Ra. These results explain why rainbow trout are unable to clear a glucose load to the same degree as mammals. The second major glucose hormone (glucagon) is what interested me for the second part of the research. The limited information on fish glucagon is even less than that of fish insulin. I speculated that trout respond to glucagon the same way mammals do (increase hepatic glucose production and show no affect on glucose disposal). To study the effects of glucagon on glucose fluxes, I tracked the changes in Ra and Rd glucose. The results showed glucose fluxes showed no siginificant difference from baseline in the first few hours, then steadily decreasing until the final time point reached values below baseline. Therefore, these experiments revealed that glucagon follows a similar pattern of effects in trout as mammals. However, the strength of the response to glucagon is different between trout and mammals. This thesis is the first to investigate the effects of insulin and glucagon on glucose kinetics in rainbow trout. I have concluded that rainbow trout have different responses to insulin and glucagon when compared to mammals. Furthermore, fish showing limited glucoregulatory capacity can be partially explained by their responses to insulin and glucagon.