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<p>Cytochrome P450s (CYPs) are enzymes that are found throughout the three domains of life. They function in the metabolism of endogenous and exogenous compounds. CYPs are extensively studied in mammalian systems due to their importance in drug metabolism and are highly expressed in detoxification organs like the liver and intestine. Fish CYP3s are not well understood. CYP3s have diversified in fish and subfamilies A, B, C and D constitute the CYP3 clade in fish. In this study, CYP3C1, CYP3C2, CYP3C3 and CYP3C4 in zebrafish (<em>Danio rerio</em>) and CYP3B4, CYP3B5 and CYP3B6 in medaka (<em>Orzyias latipes</em>) were quantified in hepatic and extrahepatic organs. CYP3C genes were quantified throughout development. All CYP3B and 3C isoforms were detected in all organs except CYP3B4 in male organs and in female brain. CYP3C1-C3 were maternally acquired and expressed in all embryonic stages. Higher expression of some of the isoforms occurred in the liver and intestine of zebrafish and medaka. This is indicative of a possible role in xenobiotic metabolism. Differences in expression between males and females gonad was observed, suggesting a possible role for estrogen in gene regulation. Further research will contribute to characterizing the upstream response elements in order to understand whether estrogens or other compounds are responsible for CYP3 regulation in fish. This knowledge will contribute to understanding the potential function these unique families of CYPs serve for fish.</p> / Master of Science (MSc)

In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes. Due to the lack of transcription in the fully-grown oocyte, critical oocyte processes including cell cycle progression, chromosome segregation, formation and maintenance of the meiotic metaphase spindles, maternal mRNA recruitment and degradation, fertilization and egg activation are all under post-transcriptional, translational, or post-translational control. Despite advances in understanding mechanisms regulating the translational control of cytoplasmic maternal mRNAs, it is unknown whether localized maternal mRNAs exist in mouse oocytes and what mechanisms are responsible for their control. Maternal mRNAs were isolated from metaphase II (MII) mouse oocytes, microsurgically-removed MII spindle-chromosome complexes, and enucleated MII oocytes and analyzed by cDNA microarray analysis. The analysis identified enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocyte metaphase II (MII) spindle. Maternal mRNAs involved in cellular compartments and processes related to the cytoskeleton, chromatin/nucleus, and cellular signaling were enriched on the MII spindle. Using immunofluorescence and confocal microscopy, MIS18A, a protein encoded by a spindle-localized maternal mRNA, was confirmed to be associated with the MII spindle along with components of the ribosome translational machinery. The key translational regulator, EIF4EBP1, was observed to undergo a dynamic and complex spatially regulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. These phosphorylation variants appeared at different positions along the spindle at different stages of meiosis. Overexpression of EIF4EBP1 mutants had a profound effect on the maintenance of MII arrest. Approximately 24% of oocytes expressing a phosphodeficient (Threonine 69 to Alanine) EIF4EBP1 mutant underwent spontaneous activation, suggesting EIF4EBP1 phosphorylation is important for translation of maternal mRNAs and maintenance of MII arrest. These results indicate that dynamic spatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation, maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindle formation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates and couples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understanding mechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs at the spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1 control of localized mRNA translation is likely key to correct segregation of genetic material across cell types. / Molecular Biology and Genetics

Retinoic acid receptor (RAR), a member of the steroid/thyroid hormone nuclear receptor superfamily, functions as a RA-dependent transcription activator bound to the RA response element (RARE) within the promoter or enhancer region of target genes. The transcriptional activity of RAR is modulated by a large number of coregulators including coactivators and corepressors. Acinus is a nuclear protein with three isoforms (Acinus-L, Acinus-S and Acinus-S'). Acinus-S' interacts with the A/B domain of RAR and represses RAR-regulated genes expression. Acinus (without isoform definition) has been identified as a component of nuclear speckles, the spliceosome and the exon junction complex (EJC), suggesting its localization in nuclear speckles and involvement in RNA processing. Acinus-S has been shown to localize in nuclear speckles. However, it is unclear whether the other two isoforms also localize in nuclear speckles. In addition, the role of Acinus in regulating pre-mRNA splicing is unclear. The goal of these studies was to examine the nuclear localization of Acinus-L and Acinus-S' and to determine the role of Acinus isoforms in RAR-dependent splicing. The sub-nuclear localization of Acinus-L and Acinus-S' was determined using fluorescence microscopy. Acinus-S' colocalizes with SC35 in nuclear speckles while Acinus-L localizes diffusely throughout the nucleoplasm. RA treatment has little effect on the sub-nuclear localization of Acinus-L and Acinus-S'. The domains/regions necessary for the distinct sub-nuclear localization of Acinus-L and Acinus-S' were identified. The speckled sub-nuclear localization of Acinus-S' is dependent on its C-terminal RS- and RD/E-rich region but is independent of the phosphorylation status of Ser-453 and Ser-604 within this region. The unique N-terminal SAP-motif of Acinus-L is responsible for its diffuse localization in the nucleus. Moreover, the sub-nuclear localization of Acinus isoforms is affected by each other, which is determined by the combinatorial effect of the more potent SAP motif of Acinus-L and the C-terminal RS- and RD/E-rich region in all Acinus isoforms. The C-terminal RS- and RD/E-rich region of Acinus mediates the colocalization of Acinus isoforms as well as with its interacting protein RNPS1. The role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S', with the activity of Acinus-L higher than that of Acinus-S', increase the splicing of a RA-responsive minigene containing a weak 5' splice site but not a RA-responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing activity of Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus on the splicing of pre-mRNAs containing the weak 5' splice site occurs to varying degrees using minigene constructs containing several different promoters while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by the RARE-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The ligand-dependent splicing activity of Acinus was further shown to be promoter-specific, depending on the ligand-dependent transcription activator. The RRM domain was identified to be necessary for the RA-dependent splicing activity of Acinus. The RA-independent splicing activity of Acinus is repressed by RNPS1. Unexpectedly, the C-terminal RS- and RD/E rich region is dispensable for the splicing activity of Acinus in regulating the minigene containing a weak 5' splice site. Importantly, measurement of the splicing of endogenous human RARâ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA-dependent manner for RARâ and in a RA-independent manner for Bcl-x. Taken together, these studies demonstrate the distinct sub-nuclear localization of Acinus-L and Acinus-S', and identified the domains that are responsible for their sub-nuclear localization, which shed light on possible distinct functions between Acinus isoforms. In addition, both Acinus-L and Acinus-S' have been shown to be splicing cofactors (with the activity of Acinus-L higher than that of Acinus-S') that facilitate constitutive splicing of pre-mRNAs containing a weak 5' splice site and regulate alternative splicing in favor of the isoform generated from the weaker alternative 5' splice site. Both Acinus-L and Acinus-S' have a RA-dependent splicing activity specific for RA-responsive genes, which suggests that Acinus functions in RAR-dependent splicing. / Biochemistry

Indiana University-Purdue University Indianapolis (IUPUI) / Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (Dhps) to post- translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of Dhps in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein Eif5a is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.

Grafenoxid (GO) har potential att användas i läkemedelsleveransapplikationer. Dess stora specifika yta gör det intressant som en effektiv bärare och skyddare av olika aktiva substanser för genterapi, såsom DNA och mRNA. Denna studie har fokuserat på att undersöka förhållandena för att ladda negativt laddat mRNA på GO. Kitosan (CS) och linjär polyetylenimin (PEI) har preadsorberats på GO för att underlätta mRNA-adsorption. Studien undersökte vid vilka förhållanden zeta-potentialen av GO/polyelektrolyt för det negativt laddade GO blir positivt. Dessa komplexerades sedan med mRNA vid olika N/P-förhållanden. Dessutom bedömde studien mRNA-frisättningskapaciteten genom att reducera pH.  GO/CS-komplexet vid förhållandet 1:2 visade positiv zeta-potential med N/P-förhållandena som sträcker sig från 1:1 till 10:1 visade att all mRNA och polyA har adsorberat till komplexet. N/P-förhållandet 10:1 var den enda som uppnådde en neutral zeta-potential, vilket tyder på tillräckligt mRNA för mättnad. Genom att öka koncentrationen av CS, kunde zeta-potentialen skifta till positivt vilket potentiellt förbättrar transfektionseffektiviteten. Visade en förbättring i signalen av det fria mRNA ökade när GO/CS/mRNA-komplexet utsattes för ett mer surt pH. Detta tyder på en potentiell frisättning när vektorn transfekteras in i cellen, eftersom den transporteras till lysosomerna som kännetecknas av sin sura miljö. GO/PEI-komplex visade endast negativ zeta-potential vid GO:PEI-förhållanden som når upp till 1:10, och därmed kommer det negativt laddade mRNA inte att adsorbera på dessa GO/PEI-komplex.  Resultaten tyder på en lovande utgångspunkt för pre-formuleringen av GO/CS-komplexet för vidare forskning. Detta arbete ger ett bidrag för framtida studier inom detta område. / Graphene oxide (GO) has a potential to be used in drug delivery applications. The large surface-to-mass ratio makes it interesting as efficient carrier and protector of various substances aimed for therapy, including DNA and mRNA. This study has focused on determining the ideal conditions for loading negatively charged mRNA onto GO using chitosan (CS) and linear polyethyleneimine (PEI) to facilitate mRNA adhesion. This was achieved by examining at what ratios of GO/polyelectrolyte the zeta potential of the negatively charged GO becomes positive, which were then subjected to mRNA complexation at different N/P (nitrogen/phosphate) ratios. Moreover, the study assessed the mRNA release capability by altering the pH.  The GO/CS complex at ratio 1:2 showed positive zeta potential with the N/P ratios ranging from 1:1 to 10:1 presented 100% loading efficiency of the added nucleic acids. With the N/P ratio 10:1 standing out as it achieved a neutral zeta potential, suggesting enough mRNA for saturation. By increasing the concentration of CS, the zeta potential could shift to positive potentially enhancing transfection efficiency. During the release assessment, the GO/CS/mRNA complex displayed increased amount of unbound mRNA when subjected to a more acidic pH. This suggests potential release when transfected into the cell, as the vector is transported to the lysosomes characterized by their acidic environment. GO/PEI complexes demonstrated only negative zeta potential at GO:PEI ratios reaching to 1:10, and thus the negatively mRNA will not adsorb on these GO/PEI complexes.  The findings suggest a promising starting point for the pre-formulation of the GO/CS complex for further research. This work provides a solid foundation for future studies in this area.

Immediately post-hatch, broiler chicks are exposed to microbes that begin colonizing the gut, including environmental pathogens. One of the costliest enteric diseases in broiler production is necrotic enteritis (NE), caused by the ubiquitous opportunistic bacteria Clostridium perfringens (CP). With the worldwide reduction in antibiotic growth-promoters (AGPs), there is increased interest in natural alternatives to reduce disease and improve broiler health. The overall objective of the studies described herein was to apply probiotics or synbiotics to birds by in ovo application or orally before they leave the hatchery, then evaluate bird performance and various intestinal responses. Data were analyzed in JMP with LS Means to separate means with significance assigned at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. The first 21-day (D) study used 480 male Cobb 500 broilers randomly divided into one of four treatments using a 2x2 factorial design: a no-additive control (CTRL), a one-time oral application of synbiotic at the hatchery fed a basal diet (HS), an oral application of water at the hatchery with dietary synbiotics (DS), and a hatchery synbiotic plus dietary synbiotic (HSDS). Performance was measured on day-of-hatch (DOH), D3, D7, D14, and D21. mRNA abundance of various intestinal markers was measured at D7 and D21, including tight junction proteins ZO-1, ZO-2, and CLD-1; nutrient transporters SGLT1 and PepT1; and immune response markers TLR2, TLR4, and IL-10. HS lowered feed intake (FI) and feed conversion ratio (FCR) without lowering body weight (BW) from D14-21. There was greater abundance of PepT1 mRNA (P ≤ 0.1) and IL-10 mRNA (P ≤ 0.05) on D21 in HSDS. Second, a 21-day pilot study with 480 male and female Cobb 500 broilers was conducted to determine the optimum in ovo dosage level of a probiotic or synbiotic (PROB or SYNB) applied at embryonic day 18 (E18) with subsequent NE challenge using seven treatments: in ovo application of sterile water (CTRL), low (PROB-L or SYNB-L: 1x105 CFU), medium (PROB-M or SYNB-M: 1x106 CFU), or high (PROB-H or SYNB-H: 1x107 CFU) probiotic or synbiotic doses dissolved in sterile water. Performance measurements were taken on DOH, D4, D8, D14 and D21. On D8, NE lesion scores were not impacted by treatment. D8 ileal samples were taken for mRNA abundance of TLR4, IL-10, IL-1β, AvBD8, AvBD10, and AvBD13. SYNB-H had higher abundance of AvBD10 mRNA compared to CTRL (P ≤ 0.1), and higher IL-1β mRNA compared to SYNB-L (P ≤ 0.05). PROB-H and SYNB-H had better performance than the low and medium doses, but were not better than the CTRL. The high doses were chosen for subsequent studies. Third, a longer 42-day study using 1,630 Ross 708 male and female broilers was conducted consisting of the following six treatments. A negative control (NC): sterile water in ovo fed basal corn/soybean meal mash diet without NE challenge; antibiotic growth-promoter (AGP+): sterile water in ovo fed basal diet with virginiamycin (0.5 kg/MT) as an AGP with NE challenge; NC+: same as NC plus NE challenge; SI+: synbiotic in ovo fed the basal diet and NE challenged; SD+: sterile water in ovo fed basal diet supplemented with synbiotic (0.5 kg/MT feed) and NE challenged; and SID+: synbiotic in ovo fed basal diet with synbiotic (0.5 kg/MT feed) with NE challenge. Cumulatively, SID+ had lower FI and FCR than NC+, but no change in BW or BWG. The combination treatment (SID+) often had an additive effect compared to SD+ or SI+ alone on mRNA abundance and D7 cecal fatty acid profiles. SD+ and SID+ also had higher D42 lean:fat ratios compared to NC+. Last, a 42-day study was conducted using 1,630 male and female Ross 708 broilers and the in ovo application of probiotics and subsequent NE challenge with five treatments. NC: sterile water in ovo, fed basal corn/soybean meal mash diet without NE challenge; AGP+: sterile water in ovo, fed basal diet with virginiamycin (0.5 kg/MT of feed) as AGP with NE challenge; NC+: NC treatment, with NE challenge; PI+: probiotic in ovo, fed basal diet, with NE challenge; PD+: sterile water in ovo, fed basal diet supplemented with probiotic (1.3 kg/MT of feed), with NE challenge. The use of probiotics in this study had little effect on performance, lean:fat ratios, and cecal fatty acid profiles, but PD+ increased mRNA abundance of D14 TLR2, D14 TNF-α, and D42 LEAP2 in cecal tonsils compared to controls. PI+ increased mRNA abundance of D7 and D42 MUC2, D7 LEAP2, and D42 TNF-α in the ileum. PI+ increased mRNA abundance in the cecal tonsils of D7 TLR2 and D42 TNF-α. These studies yielded interesting results about probiotics and synbiotics during a NE challenge by evaluating performance, intestinal immune responses, and fatty acid profiles in the ceca of broilers. In conclusion, the probiotic in this study did not improve broiler health during a NE challenge, but synbiotic use in ovo and continuation in the feed showed improvement over in ovo or dietary application alone. Synbiotic improved FCR over a challenged control, and altered mRNA abundance in the small intestine. / Doctor of Philosophy / The poultry industry is one of the most popular animal protein sources worldwide. As with any livestock operation, industry goals include optimizing animal health and well-being, maximizing animal productivity, and producing quality products in the most cost effective manner. Improvements in genetics, nutrition, and management have increased productivity and cut costs. One important application was the low-level use of antibiotics in feed. These medications reduced the risk of disease outbreak in flocks, which led to healthier birds and improved growth rates. However, when global concern of antibiotic resistance in human medicine came to light, both the livestock industry and governing bodies implemented voluntary and mandatory reduction or elimination of antibiotics. Previously, these important antibiotics helped to control costly diseases. As they are removed, alternatives to antibiotics will be important in disease control and prevention. A major group of alternatives to antibiotics in poultry includes probiotics, prebiotics, and synbiotics. Probiotic bacteria are considered 'good bacteria' in the gut, and provide various health benefits to the host. Prebiotics are non-living substances that support the growth of healthy bacteria. A synbiotic is the combination of both probiotics and prebiotics in a single application method. The goal of this research project was to give probiotics or synbiotics to broiler chicks and evaluate their potential benefits and effects on bird performance and the immune response. Ideally, applying probiotic bacteria as early as possible might translate into early colonization of the gut with healthy bacteria. This included oral application of synbiotics at the hatchery, or by safely injecting them into part of the egg that is swallowed by the chick embryo before hatch. This egg application, or in ovo application, is a safe, effective, widely-practiced method of vaccinating chicks to jumpstart their defense against disease. By vaccinating them in ovo, they can start to prime the immune system before they even hatch. Applying probiotics in ovo may improve health after early gut colonization with beneficial microbes. Numerous studies on natural alternatives to antibiotics have been conducted, with varying results. Results of this research indicate that in ovo application of probiotics and synbiotics is safe. Birds that received probiotics in the feed often performed similar to those that received none. However, the in ovo use of synbiotics combined with the continued use in the feed after hatch improved efficiency in broilers during an intestinal disease challenge and improved various aspects of gut function. Overall, as antibiotics are phased out, using probiotics and synbiotics may improve poultry health, but continued research will help understand the optimum ways to use them.