A cDNA clone encoding a turkey intestinal peptide transporter, tPepT1, was isolated from a turkey small intestinal cDNA library by screening with our chicken PepT1 (cPepT1) cDNA probe. The tPepT1 cDNA is 2,921-bp long and encodes a 79.4 kDa protein of 714 amino acids (AA) with 12 predicted transmembrane domains. The isoelectric point (pI) of tPepT1 is 5.9, which is much lower than that of PepT1 cloned from chicken (pI = 7.5) and other species. The AA sequence of tPepT1 is 94.3% identical to cPepT1 and ~ 60% identical to PepT1 from rat, sheep, rabbit, and human. Using a two-electrode voltage-clamp technique in Xenopus oocytes expressing tPepT1, Gly-Sar transport was pH dependent, but independent of Na+ and K+. For the dipeptides Gly-Sar and Met-Met, the evoked inward currents indicated that the transporter was saturable and had a high affinity for these substrates. However, transport of the tetrapeptide, Met-Gly-Met-Met, exhibited a possible substrate inhibition. To study developmental regulation of PepT1 in broiler and turkey embryos, 12 Nicholas turkey or Cobb x Cobb broiler embryos (six males and six females) were sampled daily from 5 d before hatch to the day of hatch (d 0). The abundance of PepT1 mRNA in the small intestine was quantified densitometrically from northern blots after hybridization with full-length cPepT1 and tPepT1 cDNA as probes. There was a quadratic increase (P < 0.001) in PepT1 mRNA abundance with age in turkey and broiler embryos. The relative increase in abundance of PepT1 mRNA in intestinal tissue from 5 d before hatch to d 0 was much less in the turkey than in the broiler (3.2-fold vs 14-fold). The dramatic increase in PepT1 mRNA abundance indicates a developmental regulation of the PepT1 gene and that there may be a crucial role for PepT1 in the neonatal chick and poult. / Master of Science

The objective of this study was to evaluate developmental regulation of the expression of nutrient transporter and brushborder hydrolase genes in the small intestine of piglets. Seventy piglets from seven sows were killed at birth (d 0), during suckling (d 1, 3, 7, 14, 21) and postweaning (d 22, 24, 28, 35), and intestinal segments (duodenum, jejunum and ileum) were collected. The mRNA abundance was determined by Northern blot using specific cDNA probes for three disaccharidases (lactase-phlorizin hydrolase, LPH, sucrase-isomaltase, SI, and maltase-glucoamylase, MGA), three peptide hydrolases (aminopeptidase A, APA, aminopeptidase N, APN, and dipeptidyl peptidase IV, DPP IV), two sugar transporters (Na+-dependent glucose transporter 1, SGLT1, and facilitated glucose transporter 5, GLUT5), a peptide transporter (H+-dependent peptide transporter 1, PepT1), four amino acid transporters (excitatory amino acid carrier 1, EAAC1, Na+-dependent neutral amino acid transporter, ATB0, the light chain of a heterodimeric transport system b0,+ involved in the heteroexchange of cationic and neutral amino acids, b0,+AT, and Na+-independent large branched and aromatic neutral amino acid transporter 2, LAT2), and two iron transporters (divalent metal ion transporter 1, DMT1, and iron-regulated transporter 1, IREG1). Protein expression was quantified by Western blot using specific antibodies for LPH, SI, SGLT1, and PepT1. During suckling, the abundance of LPH, APA, APN, DPP IV, b0,+AT mRNA increased quadratically (P < 0.001) with age from birth to d 7 or 14 then remained unchanged or slightly declined with age to d 21. The mRNA abundance of SI increased and LAT2 decreased linearly (P < 0.001) with age, and the abundance of MGA and GLUT5 mRNA remained unchanged with age. There was an age x intestinal segment interaction (P < 0.001) for the abundance of EAAC1 and ATB0 mRNA. The abundance of EAAC1 mRNA increased from d 0 through 14 and remained stable to d 21 in the ileum, and it was low and slightly increased with age through d 21 in the duodenum and jejunum. The abundance of ATB0 mRNA generally increased from d 0 to 21 in the duodenum and ileum, and increased from d 0 to 7 and then decreased to d 21 in the jejunum. The abundance of SGLT1 and PepT1 mRNA was substantial at birth and transiently declined to d 1. The abundance of SGLT1 mRNA generally increased from d 1 to 21, and PepT1 mRNA abundance increased to d 3 and then plateaued through d 21. Postweaning, the mRNA abundance of all of these carbohydrate and protein assimilation related genes increased during the first day (3 d for ATB0) after weaning then declined to the levels at weaning in the jejunum and ileum, followed by a subsequent change pattern that varied among genes. During suckling, the mRNA abundance of LPH, SGLT1, and APA was greater in the duodenum and jejunum than the ileum (P < 0.001). The PepT1 and APN mRNA was evenly distributed among intestinal segments, and the expression of MGA, DPP IV, EAAC1, b0,+AT, ATB0, and LAT2 mRNA was generally greater in the jejunum and ileum than the duodenum or greatest in the ileum. Postweaning, the mRNA abundance of all of these carbohydrate and protein assimilation related genes examined was generally greater in the jejunum and ileum than the duodenum or highest in the ileum. From d 0 through 35, DMT1 and IREG1 mRNA was predominantly (P < 0.05) distributed in the duodenum, where the abundance of DMT1 and IREG1 mRNA increased with age during suckling, and then rapidly decreased after weaning. The protein expression of LPH and SI exhibited a similar developmental pattern as that for the mRNA abundance. Unlike the developmental regulation of their respective mRNA abundance, the protein expression of SGLT1 exhibited a general decline from suckling to postweaning. The protein expression of PepT1 gradually decreased with age from birth to d 35 in the duodenum, and initially declined from birth to the lowest value then slightly increased with age through d 21, followed by an increase to d 35 in the jejunum and ileum. In conclusion, the gene expression of these brushborder hydrolases and nutrient transporters was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages of life. These differences in ontogenetic regulation and the distribution may be related to the luminal substrate concentration as well as the nutrient categories, and the developmental regulation of these genes may occur not only at the transcriptional level but also at the posttranscriptional level. / Ph. D.

<p> Asphyxia (hypoxia, hypercapnia, and acidosis) sensing by neonatal adrenal chromaffin cells (AMC) plays a key role in the adaptation of the neonate to extrauterine environment. During birth, exposure of the neonate to episodes of acute asphyxia results in secretion of catecholamines from AMC, independent of neural inputs. This catecholamine (CAT) secretion promotes re-absorption of fluid and surfactant production in the lung, improved cardiac conductance, and assists in neonatal arousal. The asphyxia-sensing responses in AMC are lost postnatally during a time course that parallels splanchnic innervation. </p> </p> <p> The primary goal of this thesis was to uncover the mechanisms of asphyxia sensing in neonatal rat AMC, using primary and immortalized chromaffin cells, and to elucidate the potential role of innervation in the postnatal loss of these mechanisms. The experimental approaches relied on patch clamp techniques to record ionic currents and membrane potential, carbon fiber amperometry to monitor CAT secretion, chemiluminescence to measure reactive oxygen species (ROS) production and ATP secretion, and molecular techniques to examine protein and gene expression. </p> <p> In primary neonatal, but not juvenile AMC, hypoxia (PO₂ = 15 mmHg) caused a suppression of outward K⁺ current, membrane depolarization, and increased secretion. These effects were associated with a decrease in mitochondrial ROS production, were reversed by exogenous H₂O₂, and where mimicked by antioxidants. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only rotenone, a complex I blocker, mimicked and occulded the effects of hypoxia. The immortalized chromaffin cell line (MAH cells) behaved similarly, and became hypoxia-insensitive when depleted of functional mitochondria (ρ0 MAH). Both neonatal AMC and immortalized MAH cells also responded to increased CO₂ (hypercapnia) with K⁺ current inhibition, membrane depolarized, increased intracellular Ca²⁺, and CAT secretion. However, these responses were independent of functional mitochondria and were associated with the expression of carbonic anhydrase II (CAII). </p> <p> Since the splanchnic nerve supplies both cholinergic and opioid peptidergic innervation to AMC, I tested the hypothesis that postsynaptic activation of the corresponding receptors might underlie the postnatal loss of asphyxia-sensing. First, to determine whether nicotinic acetylcholine receptor stimulation was involved, pregnant rats were exposed to nicotine (or saline) during gestation and AMC were examined in the offspring, soon after birth. Control AMC isolated from pups born to saline-treated dams displayed typical responses to hypoxia and hypercapnia, including inhibition of outward K⁺ current, membrane depolarization, increased cytosolic calcium, and CAT secretion. In contrast, AMC from pups born to nicotine-treated dams showed a dramatic loss of hypoxic sensitivity, though hypercapnic sensitivity and the expression of CO₂ markers (i.e. carbonic anhydrase I and II) appeared normal. This effect of chronic nicotine could be reproduced in cultured chromaffin cells in vitro and was mediated via activation of α7-nicotinic acetylcholine receptors (nAChR), leading to upregulation of ATP-dependent (K_ATP) K⁺ channels, which open during hypoxia and cause membrane hyperpolarization. The upregulation of K_ATP channels involved expression ofthe hypoxia inducible transcription factor (HIF 2α). Second, to determine whether opioid receptor stimulation was also involved, cultured chromaffin cells were chronically exposed to mu, delta, or kappa opioid agonists in vitro. Treatment with both mu and delta, but not kappa, agonists resulted in the loss of both hypoxia and hypercapnia sensing and a decrease in CAII expression. </p> <p> Taken together, these studies suggest that neonatal chromaffin cells sense hypoxia via a reduction in ROS generation located at or upstream of mitochondrial complex I. Chronic stimulation of nicotinic α7 ACh receptors leads to a selective loss of hypoxia sensing and this appears to be mediated via a HIF 2α-dependent upregulation of K_ATP channels. These channels open during hypoxia causing membrane hyperpolarization and reduced excitability. The result of this upregulation of K_ATP channels results in cells deficient in the ability to respond to hypoxia. </p> / Thesis / Doctor of Philosophy (PhD)

Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (-64 to -1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the -64 to -23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from -23 to -10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (-64 to -10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.

To better understand the digestive and absorptive capacities of the chick intestine so that we may feed diets that better meet the nutritional needs of the chick, it is important to understand how expression of nutrient transporter genes changes in response to various factors. A series of feeding trials were conducted to evaluate the dietary and developmental regulation of nutrient transporter mRNA abundance in the small intestine of two lines of broilers selected on corn-based (Line A) or wheat-based (Line B) diets. Abundance of mRNA was quantified in all experiments using real time PCR and the absolute quantification method. The objective of the first study was to investigate intestinal nutrient transporter and enzyme mRNA in Line A and B broilers at embryo day 18 and 20, day of hatch, and d 1, 3, 7, and 14 posthatch. Genes evaluated included the peptide transporter, PepT1, 10 AA transporters (rBAT, bo,+AT, ATBo,+, CAT1, CAT2, LAT1, y+LAT1, y+LAT2, BoAT and EAAT3), four sugar transporters (SGLT1, SGLT5, GLUT5, and GLUT2), and a digestive enzyme, APN. For PepT1, Line B had greater quantities of mRNA compared with Line A (P = 0.001), suggesting a greater capacity for absorption of AA as peptides. Levels of PepT1 mRNA were greatest in the duodenum (P < 0.05), whereas the abundances of SGLT1, GLUT5 and GLUT2 mRNA were greatest in the jejunum (P < 0.05). Abundances of EAAT3, bo,+AT, rBAT, BoAT, LAT1, CAT2, SGLT5 and APN mRNA were greatest in the ileum (P < 0.05). Quantities of PepT1, EAAT3, BoAT, SGLT1, GLUT5, and GLUT2 mRNA increased linearly (P < 0.01), while CAT1, CAT2, y+LAT1, and LAT1 mRNA decreased linearly (P < 0.05) with age. The objective of the second study was to evaluate the effect of dietary protein quality on intestinal peptide, AA, and glucose transporter, and digestive enzyme mRNA abundance in Line A and B broilers. At day of hatch (doh), chicks from both lines were randomly assigned to corn-based diets containing 24% crude protein (CP) with either soybean meal (SBM) or corn gluten meal (CGM) as the supplemental protein source, ad libitum. Groups of chicks from both lines were also assigned to the SBM diet at a quantity restricted to that consumed by the CGM group (SBM-RT). Abundance of PepT1, EAAT3, and GLUT2 mRNA was greater in Line B (P < 0.03), while APN and SGLT1 were greater in Line A (P < 0.04). When feed intake was equal (CGM vs restricted SBM), a greater abundance of PepT1 and bo,+AT mRNA was associated with the higher quality SBM (P < 0.04), while a greater abundance of EAAT3 and GLUT2 mRNA was associated with the lower quality CGM (P < 0.01). When feed intake was restricted (SBM vs SBM-RT), a greater abundance of PepT1 mRNA was associated with the restricted intake (P < 0.04). The objective of the third study was to determine the effect of dietary protein composition on mRNA abundance of peptide and AA transporters, and a digestive enzyme. From day 8 to day 15 posthatch, Line A and B broilers were fed equal amounts of 1 of 3 diets (24% CP). Dietary protein sources included whey protein concentrate (whey), a partial whey hydrolysate (hydro), or a mixture of free amino acids (AA) similar to the composition of whey. Intestine was collected at days 8, 9, 11, 13, and 15. Expression of all genes except LAT1 was greater (P < 0.05) in Line B compared with A. Abundance of PepT1, EAAT3, y+LAT2, CAT1, bo,+AT, and APN mRNA varied little across diets in Line A but for CAT1 mRNA was greatest (P = 0.005) in Line A birds that consumed the AA diet. Expression of these genes was greatest (P < 0.006) in Line B birds consuming the hydro diet. A greater (P < 0.05) age response of bo,+AT, EAAT3, CAT1, and APN mRNA was observed in birds consuming the hydro or AA diets relative to the whey diet. Results from these studies collectively demonstrate that nutrient transporter gene expression is responsive to a variety of factors, including developmental stage, dietary manipulation, and genetic selection. Information from these studies can be used to improve dietary formulation so that nutrient utilization is enhanced, resulting in improved growth of the broiler. / Ph. D.

Konjac glucomannan (KGM), the main biologically active constituent of konjac flour extracted from corms of Amorphophallus konjac (konjac), can be used to prepare functional foods and may also have potential as a pharmaceutical product to combat obesity. The current study employed three experimental approaches to study the biology and chemistry of konjac, namely (1) glasshouse experiments to study the morphogenesis, growth and productivity of konjac plants, (2) a histological and immunocytochemical investigation of the localisation and developmental regulation of the deposition and metabolism of KGM in developing corm tissues, and (3) a comparative study of methodologies for the extraction and analysis of KGM. The current data demonstrated a morphological and functional separation between the ventral and dorsal regions of corms. The ventral region appeared to function as a source during the initial period of shoot development, while the dorsal region appeared to operate as a sink after the development of mature canopy. Once the corm reached maturity, both an inflorescence and a leaf were produced within a single season. It has also been demonstrated that the age of the ‘mother’ corm is an important factor affecting the quality of offsets produced. An anti-mannan antiserum detected a temporally regulated pattern of mannan epitope production within glucomannan idioblasts in developing corm tissues, with increased expression as the corm approached maturity/dormancy. The current observations also suggest that the mobilization of KGM initiates at the periphery of the corm and proceeds inwards towards the centre of the corm. Compositional analysis showed that the purified konjac flour (PKF) produced using a modified extraction procedure contained 92% glucomannan, with a weight average molecular weight (Mw), polydispersity index (PDI) and degree of acetylation (DA) of 9.5 ± 0.6 x 105 gmol-1, 1.2 and 2.8 wt. %. These data, plus Fourier-transform infrared spectral (FTIR) and zero shear viscosity analyses of the extract (PKF) were all consistent with the literature. Comparison of three existing methodologies for the quantitative analysis of the KGM content, namely 3,5-dinitrosalicylic acid (3,5-DNS), phenol-sulphuric acid and enzymatic colorimetric assays; indicated that the 3,5-DNS colorimetric assay was the most reproducible and accurate method, with a linear correlation coefficient of 0.997 and recoveries between 97% and 103% across three spiking levels of starch. In summary, this study has provided a better understanding of aspects of the biology and cultivation of A. konjac and has also produced methodologies which can be used as the basis for an improved good laboratory practice (GLP) for the commercial extraction and analysis of this multifunctional natural polymer.

Die Entwicklung des grampositiven Bodenbakteriums Streptomyces ist in einem komplexen Lebenszyklus koordiniert, bestehend aus drei Stufen: vegetativem Hyphenwachstum, Luftmycelbildung und Sporulation. C-di-GMP kontrolliert die Enwicklung über zwei Effektorproteine: dem Masterregulator BldD und dem Anti-Sigmafaktor RsiG. In dieser Arbeit konnte gezeigt werden, dass das membranständige GGDEF-EAL Protein CdgC eine wichtige aktive Diguanylatzyklase (DGC) in S. venezuelae ist. Chromosomale Deletion von cdgC führte zu einer flachen, gräulichen Koloniemorphologie mit radialen Stegen und hydrophiler Oberfläche sowie zu frühzeitiger Sporulation ohne Lufthyphenbildung. Phänotypische Analysen zeigten, dass die DGC-Aktivität von CdgC essentiell ist für dessen biologische Rolle und deuten auf einen zusätzlichen Protein-spezifischen morphologischen Effekt von CdgC hin. CdgC-FLAG akkumuliert im Laufe des Lebenszyklus und scheint BldD-abhängig über eine c-di-GMP vermittelte Feedbackschleife reguliert zu werden. Frühere RNA-seq Daten, verifiziert für repräsentative Gene mittels qRT-PCR, deuten eine differentielle Expression der Bestandteile des hydrophoben Mantels als Ursache der Lufthyphendefizienz an. Konfokalmikroskopische Aufnahmen des bakteriellen Tubulin-Homologons FtsZ deuten einen c-di-GMP-sensitiven Einfluss von CdgC auf die Koordination der Zellteilung an. Zudem konnte nachgewiesen werden, dass CdgC mit sich selbst sowie drei potentiellen Membranproteinen interagiert. Demnach trägt CdgC zur Koordination von Zellteilungs- und hydrophoben Zelloberflächenproteinen bei und beeinflusst damit c-di-GMP-abhängig den Zeitpunkt der Sporenbildung. Insgesamt führt diese Studie CdgC als GGDEF-EAL-Tandemprotein mit spezifischem Knockout- Phänotyp ein, der von seiner DGC-Aktivität sowie seinem Membrananker bestimmt wird. Zudem ist CdgC, als Reaktion auf eine noch unbekannte Signalübertragungskaskade, an der Koordinierung von Zeitpunkt und Verlauf der Sporulation ausschlaggebend beteiligt. / The proliferation of Gram-positive soil bacteria Streptomyces is temporally and genetically coordinated with a complex developmental life cycle, including three main stages of differentiation: vegetative hyphal growth, formation of aerial mycelium and sporulation. The key factor of Streptomyces developmental control is c-di-GMP with to-date two identified effector proteins: the master regulator BldD and the anti-sigma factor RsiG. In this thesis, the membrane-associated GGDEF-EAL protein CdgC, was identified as a major active diguanylate cyclase (DGC) in S. venezuelae. Deletion of cdgC results in the unique flat gray colony morphology with radial wrinkles and a hydrophilic surface, that shows enhanced sporulation without forming aerial hyphae. Phenotypic analyses suggest, that the DGC activity is essential for its biological role, but hint to an additional protein specific role. The protein levels of CdgC-FLAG were found to accumulate during the life cycle of S. venezuelae. Further investigation of CdgC-FLAG in a strain carrying a DNA-binding deficient BldD_D116A allele indicated, that BldD represses the expression of CdgC in a regulatory feedback loop along with the DGCs CdgA, CdgB and CdgE. RNA‐sequencing data indicated that reduced expression levels of the major compounds of the hydrophobic sheath result in the initiation of sporulation out of the vegetative mycelium and were verified for representative examples via qRT-PCR. Confocal microscopic imaging of the bacterial tubulin homolog FtsZ indicated a contribution of CdgC via its DGC activity in coordination of the cell division. In addition, BTH screenings revealed self-interaction and identified three membrane associated interaction partners. In conclusion, this study introduces the GGDEF-EAL tandem protein CdgC, whose specific knockout phenotype is governed by its DGC activity and membrane association. CdgC seems to drive timing and mode of sporulation in response to an unknown signal to a major extend.