<p>Two experimentswere  conducted  using  pigs  at different  life stages to  determine  the  effects  of dietary  tryptophan  and  water  delivered  oregano  essential  oil  on growth performance, rectal temperature, water use,intestinal integrity and gene expression of biomarkers in the face heat or transport stress. In the first experiment, 192 grow-finish pigs were used to investigate the effects of  water  supplementation  of  oregano  essential  oil  (OEO)  on  growth  performance,  water  intake, rectal temperature, intestinal integrity, and expression of genetic biomarkers during an acute heat challenge. Pigs were randomly allotted to 2 X 2 factorial arrangement of treatments with pigs being heat  stressed  or  not  and  being  supplemented  with  OEO  or  not with 8  replicate  pens  of  each treatment  with  6  pigs/pen  (4  barrows,  2  gilts  per  pen).  Water  treatments  were administered immediately, with  dosing at  47 μL/L of OEO. One-half  of  the  pigs  on  each  water  treatment remained under thermoneutral conditions (TN; 21.1C), while the other half was subjected to a 3 d diurnal, acute heat stress (HS) with 12 hours at 33.3 oC (7AM-7PM) and 12 hours at 26.7oC (7PM-7AM). Three days post-HS, temperatures were reduced back to TN for the rest of the study, and pigs remained on their water treatments. Rectal temperatures were collected in the morning and evening  of  the  heat  stress  period  on  one  barrow  and  one  gilt  in  each  pen. Jejunal  tissue was collected for subsequent histological examination and determination of gene expression. All data were  analyzed  using  the  GLM  procedure  of  SAS  (ver.  9.4).  Pigs  subjected  to  heat  stress  had reduced ADG (P < 0.003) and G:F (P < 0.008) during the 3d heat stress compared to pigs reared under thermoneutral conditions. However, post-heatstress, heat stressed pigs had compensatory gain resulting in increased ADG (P < 0.001) and G:F (P < 0.001) compared to thermoneutral reared pigs.   Overall,  there  was an  interaction  (P  <  0.006)  observed  between  water  and  heat  treatment with  OEO  increasing  ADG  in  thermoneutral  pigs  but  not  in  heat  stressed  pigs.  Similarly, interactions  between  water  and  heat  treatment  were  observed  for  ADFI  during  heat  stress  (P  < 0.004),  post  heat  stress  (P  <  0.01),  and  overall  (P  <  0.004)  from  increasing  OEO  intake  in thermoneutral pigs but not in heat stressed pigs. Rectal temperatures were higher (P < 0.001) for heat stressed pigs at the end of d 1 and 2 of the acute heat challenge compared to TN housed pigs. Pigs exposed to HS also used more water than pigs housed in a thermoneutral environment (P < 0.002). There were no differences between villi height, crypt depth or VH:CD between treatment groups  (P  >0.05).  There  was  also  no  difference  in  TP53  and  CDKNA1  gene  expression  among treatments (P > 0.10). In the second experiment, 36 barrows were used in an 18d experiment to investigate the effects of pre-weaning tryptophan supplementation on performance and intestinal integrity following  weaning  with  or  without  transport  stress  at  weaning.  Pigs  were  randomly allotted to 2 X 2 factorial arrangement of treatments of pre-weaning tryptophan supplementation or not and weaning transport or not. Pigs on the tryptophan treatment received 0.35, 0.45, and 0.55 g Trp/d in 5 day intervals, beginning 15 d prior to weaning.Tryptophan was dissolved in chocolate milk and administered by oral gavage with control pigs receiving milk only. At weaning, 4 pigs from each pre-weaning treatmentwere euthanized for collection of jejunal tissue.  Of the remaining pigs, half the pigs oneach treatment were transported for 12 h, and half were moved into individual pens  with  no  transport.  Following  transport,  all  pigs  were  individually  housed  and  provided  ad libitum  access  towater  andfeed  from  a  common  diet.  On  d  3  post-weaning,  all  pigswere euthanized for collection of jejunal tissue. Jejunal tissue was used for histological examination and for determination of gene expression. All data were analyzed using the GLM procedure of SAS (9.4).  No  effects  of  Trp  supplementation  were  observed  on  pre-weaning  (P  >  0.10)  growth. Pig BW and ADFI were unaffected (P > 0.10) by Trp supplementation and transport at weaning. Post-weaning, there was a tendency (P < 0.06) for an effect of transport on ADG as transported pigs lost weight in the 3 d post-weaning period while non-transported pigs gained slightly. Gain:Feed post-weaning  was  lower  (P  <  0.04)  for  transported  pigs  compared  to  non-transported  pigs.  No differences  were  observed  for villus base  and  mid  width,  villus  height,  crypt  depth  or  villus height:crypt   depth.   There   was   a   tendency   for   an   interaction   of   transportation   and   Trp supplementation  (P  <  0.06)  on  villi  base  width  driven  by  an  increased  villus  width  in  non-transported pigs given supplemental Trp but a decrease in villus width in transported pigs given supplemental Trp.These results conclude that these alleviating agents had minimal effects when pigs  were  stressed, however TN grow-finish pigs  benefitted  from  OEO  water  supplementation among growth performance.</p>

The brain has the capacity to modify itself structurally and functionally, to adapt to novel circumstances. Adaptive changes in neural circuitry that become intransigent, such as continued hypervigilance after resolution of a threat situation, become maladaptive and may facilitate development of psychiatric disorders such as Major Depressive Disorder (MDD). Although MDD pathogenesis is unclear, hypothalamic-pituitary-adrenal axis dysregulation may facilitate the neuroplastic changes observed in MDD. Whether these neuroplastic changes facilitate the development of MDD or develop due to MDD remains unclear. The characterization of neuroplastic changes in MDD has resulted in sometimes contradictory findings. There are gaps in understanding the timing of neuroplastic changes in MDD, and how and when they are affected by antidepressant treatment. Characterization of neuroplasticity in MDD may uncover different phenotypes and aid in the discovery of a predictive biomarker of antidepressant treatment response. This dissertation presents the results of a series of neuroimaging studies. Chapter 1 provides an introduction to neuroplasticity and MDD. In Chapter 2 results of a study examining hippocampal memory function in treatment naïve patients with MDD are presented. Chapter 3 exhibits findings from a study examining effects of an acute tryptophan depletion paradigm in midlife women receiving estrogen-based treatment on an emotional conflict task. Chapter 4 discusses results from an examination of unmedicated patients with MDD and healthy control participants on an emotional conflict task. Chapter 5 presents longitudinal data of the sample from Chapter 4, and the effect of 8 weeks of treatment with antidepressant escitalopram on performance on an emotional conflict task. In Chapter 6 a case study is presented of a patient with long-standing overt ventriculomegaly, whose chief complaint was of mood and cognitive impairments. Chapter 7 summarizes the findings and contributions of this body of research and discusses clinical implications and future directions. / Dissertation / Doctor of Philosophy (PhD) / The characterization of brain changes in Major Depressive Disorder (MDD) has resulted in contradictory findings, and gaps in understanding how the brain changes in response to antidepressant treatment. This dissertation aims to characterize brain changes in MDD through a series of neuroimaging studies. Chapter 1 provides an introduction to MDD and brain changes in MDD. Chapter 2 presents an examination of memory in treatment naïve patients with MDD. Chapter 3 presents a study of acute tryptophan depletion in midlife women receiving estrogen-based treatment on an emotional conflict task. Chapter 4 examines unmedicated patients with MDD and healthy control participants on an emotional conflict task. Chapter 5 examines the effects of antidepressant treatment on performance on an emotional conflict task. Chapter 6 presents a case study of a patient with ventriculomegaly with mood and cognitive impairments. Chapter 7 summarizes the contributions of this research and discusses implications and future directions.

Background Previous studies have suggested that individuals recovered from anorexia nervosa (AN) are characterized by increased serotonergic (5-HT) activity that might be related to elevated levels of anxiety. Assuming these traits to be also present in individuals at risk for AN, it was further hypothesized that restricting food intake might be a means to temporarily alleviate dysphoric affective states by reducing central nervous availability of tryptophan (TRP), the sole precursor of 5-HT. One study that supported this hypothesis found anxiolytic effects in individuals with a history of AN during an experimentally induced short-term depletion of TRP supply to the brain. Methods In this placebo-controlled, double-blind cross-over study, 22 patients weight-recovered from AN (recAN) and 25 healthy control participants (HC) completed questionnaires assessing anxiety and momentary mood during acute tryptophan depletion (ATD), a dietary intervention that lowers central 5-HT synthesis. Results The ATD procedure effectively reduced the ratio of TRP to competing for large neutral amino acids in the peripheral blood, indicating decreased TRP supply to the brain. Effects of ATD on anxiety and mood did not differ between recAN and HC. Bayesian null hypothesis testing confirmed these initial results. Discussion Our results do not support the hypothesis that short-term depletion of TRP and its impact on the brain 5-HT reduces anxiety or improves mood in AN. As the evidence for the role of 5-HT dysfunction on affective processes in patients with AN is limited, further studies are needed to assess its relevance in the pathophysiology of AN.

M. S.

Plant aromatic amino acid decarboxylases (AAADs) are a group of economically important enzymes categorically joined through their pyridoxal 5'-phosphate (PLP) dependence and sequence homology. Extensive evolutionary divergence of this enzyme family has resulted in a selection of enzymes with stringent aromatic amino acid substrate specificities. Variations in substrate specificities enable individual enzymes to catalyze key reactions in a diverse set of pathways impacting the synthesis of monoterpenoid indole alkaloids (including the pharmacologically active vinblastine and quinine), benzylisoquinoline alkaloids (including the pharmacologically active papaverine, codeine, morphine, and sanguinarine), and antioxidant and chemotherapeutic amides. Recent studies of plant AAAD proteins demonstrated that in addition to the typical decarboxylation enzymes, some annotated plant AAAD proteins are actually aromatic acetaldehyde synthases (AASs). These AASs catalyze a decarboxylation-oxidative deamination process of aromatic amino acids, leading to the production of aromatic acetaldehydes rather than the AAAD derived arylalkylamines. Research has implicated that plant AAS enzymes are involved in the production of volatile flower scents, floral attractants, and defensive phenolic acetaldehyde secondary metabolites. Historically, the structural elements responsible for differentiating plant AAAD substrate specificity and activity have been difficult to identify due to strong AAAD and AAS inter-enzyme homology. Through extensive bioinformatic analysis and experimental verification of plant AAADs, we have determined some structural elements unique to given types of AAADs. This document highlights structural components apparently responsible for the differentiation of activity and substrate specificity. In addition to producing primary sequence identifiers capable of AAAD activity and substrate specificity differentiation, this work has also demonstrated applications of AAAD enzyme engineering and novel activity identification. / Ph. D.

M. S.

The effects of intragastrically administered tryptophan and tyrosine, which are direct precursors of brain neurotransmitters affecting food intake, were investigated. Differences in sensitivities and/or responses in relatively fast-growing and slow-growing strains of chickens were also tested. The amino acids were intubated intragastrically in a series of four experiments. Food consumption following tryptophan intubation decreased in both the slow and fast-growing strains. Increased food intake was observed in the slow-growing birds as a result of tyrosine treatment suggesting that food consumption can be altered by manipulation of dietary amino acids. Tyrosine treatment did not stimulate food intake in the fast-growing strain, implying that chickens in this population were in a state of maximal or near maximal stimulation, and thus relatively incapable of increasing food intake. To determine whether the changes observed in feeding behavior were perhaps due to some general inhibition or stimulation of the central nervous system rather than the result of mediation of feeding mechanisms in the brain, the effects of intragastrically administered tyrosine and tryptophan on tonic immobility (TI) were also tested. Duration and susceptibility of TI were unaffected by tyrosine and tryptophan, suggesting that these amino acids acted upon mechanisms specifically involved in the regulation of food intake. / Master of Science

No

Das Enzym Tryptophan Synthase ist ein ausgezeichnetes Beispiel einer molekularen Fabrik auf der Nanoskala mit zwei katalytischen Zentren. Der katalytische Zyklus des Moleküls beruht zudem auf zahlreichen allosterischen Wechselwirkungen sowie der Übertragung des Intermediats Indol durch einen intramolekularen Tunnel. In dieser Arbeit wird das erste kinetische Modell eines einzelnen Tryptophan Synthase Moleküls konstruiert und analysiert. Simulationen zeigen starke Korrelationen zwischen den Zuständen der Katalysezentren sowie die Ausbildung von Synchronisation. Mit stochastischer Thermodynamik wird die experimentell unzugängliche Reaktionskonstante für die Rückübertragung des Indols aus Messdaten rekonstuiert. Methoden, die den Informationsaustausch in bipartiten Markovnetzwerken charakterisieren, werden auf beliebige Markovnetzwerke verallgemeinert und auf das Modell angewendet. Der abschließende Teil befasst sich mit chemischen Reaktionsnetzwerken von Metaboliten und Enzymen. Es werden algebraische Modelle (Halbgruppen) konstruiert, welche aufeinanderfolgende und simultane katalytische Funktionen von Enzymen und von Unternetzwerken erfassen. Diese Funktionen werden genutzt, um eine natürliche Dynamikum sowie hinreichende und notwendige Bedingungen für seine Selbsterhaltung zu formulieren. Anschließend werden die algebraischen Modelle dazu genutzt, um eine Korrespondenz zwischen Halbgruppenkongruenzen und Skalenübergängen auf den Reaktionsnetzwerken herzustellen. Insbesondere wird eine Art von Kongruenzen erörtert, welche dem Ausspuren der globalen Struktur des Netzwerkes unter vollständiger Beibehaltung seiner lokalen Komponenten entspicht. Während klassische Techniken eine bestimmte lokale Komponente fixieren und sämtliche Informationen über ihre Umgebung ausspuren, sind bei dem algebraischen Verfahren alle lokalen Komponenten zugleich sichtbar und eine Verknüpfung von Funktionen aus verschiedenen Komponenten ist problemlos möglich. / The channeling enzyme tryptophan synthase provides a paradigmatic example of a chemical nanomachine with two distinct catalytic subunits. It catalyzes the biosynthesis of tryptophan, whereby the catalytic activity in a subunit is enhanced or inhibited depending on the state of the other subunit, gates control the accessibility of the reactive sites and the intermediate product indole is directly channeled within the protein. The first single-molecule kinetic model of the enzyme is constructed. Simulations reveal strong correlations in the states of the active centers and the emergent synchronization. Thermodynamic data is used to calculate the rate constant for the reverse indole channeling. Using the fully reversible single-molecule model, the stochastic thermodynamics of the enzyme is closely examined. The current methods describing information exchange in bipartite systems are extended to arbitrary Markov networks and applied to the kinetic model. They allow the characterization of the information exchange between the subunits resulting from allosteric cross-regulations and channeling. The final part of this work is focused on chemical reaction networks of metabolites and enzymes. Algebraic semigroup models are constructed based on a formalism that emphasizes the catalytic function of reactants within the network. A correspondence between coarse-graining procedures and semigroup congruences respecting the functional structure is established. A family of congruences that leads to a rather unusual coarse-graining is analyzed: The network is covered with local patches in a way that the local information on the network is fully retained, but the environment of each patch is not resolved. Whereas classical coarse-graining procedures would fix a particular patch and delete information about the environment, the algebraic approach keeps the structure of all local patches and allows the interaction of functions within distinct patches.

<p>  </p> <p>In commercial settings, piglet weaning and transportation occur concurrently due to the expansive application of multi-site production systems across the United States and the combination of these events can be defined as an early life stressor. Early life stress is known to reduce pig performance, efficiency, and immune resilience contributing to reduced welfare and increased production losses. To combat the deleterious effects of stress on pigs, the essential amino acid tryptophan (Trp) supplemented above current National Research Council (NRC 2012) recommended levels, improves neuroendocrine responses to stress, lowers plasma cortisol and norepinephrine concentrations, and improves hypothalamic-pituitary-adrenal axis recovery time following stress. However, there are discrepancies concerning the Trp requirement for nursery pigs, suggesting the 2012 recommendations for Trp may be inaccurate for optimizing growth performance and health in modern pigs. Three experiments were conducted to determine the effects of supplemented standard ileal digestible Trp above NRC (2012) recommended levels on performance, feed efficiency, immune vigor, and stress tolerance. The objective of experiment 1 was to eliminate or reduce short- and long-term, transport-induced reductions in piglet feed efficiency and growth by supplementing Trp above NRC (2012) recommendations pre-weaning and/or during the nursery phase. An oral gavage of Trp or a control milk carrier was provided to pre-allotted piglets beginning at day 5 of lactation and continuing to weaning. At weaning all pigs were blocked by sex, weaning weight and pre-wean treatment and randomly assigned to transport and post-wean treatments. Pigs were fed in four nursery phases with diets containing 1X or 2X NRC recommended concentrations of SID Trp and a common grower diet fed in 6 phases during the grow-finish period. At market, loin characteristics were measured via ultrasound and carcass data was collected from the packing plant. Pre-weaning Trp supplementation had no effect on pre-weaning growth performance; however, post-wean Trp increased overall body weight and average daily gain in nursery pigs when Trp was supplemented pre-wean. The objective for experiment 2 was to mitigate the adverse effects of early life transport stress on subsequent immune challenges by providing supplemental Trp during the nursery period. At weaning, pigs were transported for 8 hours and assigned to treatments of vaccine-induced immune challenge and dietary treatment. Pigs were fed standard nursery diets, in four phases, over 35 days with pigs receiving 1X or 2X the NRC (2012) recommended Trp concentration. Half the pigs on each dietary treatment were subjected to a 3-wk vaccine challenge consisting of circovirus, mycoplasma, and influenza vaccines administered in wk 2, 3, and 4 post-wean, respectively. At market, loin characteristics were measured via ultrasound and carcass data was collected.  At the culmination of the nursery period, unchallenged pigs supplemented with Trp were heavier compared to control pigs. This resulted from an overall improvement in average daily gain for 2X Trp fed pigs. However, increasing Trp did not affect market weight, loin eye area, or lean percent in market pigs, although 2X Trp increased back fat. The objective of experiment 3 was to determine what ratio of SID tryptophan, relative to lysine, maximizes growth performance and feed efficiency in weaned pigs during the nursery period. Pigs were blocked by sex and weaning weight and randomly allotted to one of five dietary treatments with Trp increasing stepwise by 0.33%-units, respectively. Pigs were fed standard nursery diets, in four phases, over 35 days with pigs receiving 1X, 1.33X, 1.66X, 2X, or 2.33X the NRC (2012) recommended Trp concentration.  Overall, there were no differences in performance or efficiency across dietary treatments during a 35-day nursery period in unstressed healthy pigs.  In conclusion, increasing Trp in swine diets prior to and/or following stress events can mitigate stress-related perturbations in performance and efficiency.      </p>