The American alligator (Alligator mississippiensis) is an aquatic diving reptile with a periodic breathing pattern. Previous work has identified pulmonary stretch receptors (PSR), both rapidly- and slowly-adapting, and intrapulmonary chemoreceptors (IPCs) that modulate breathing patterns in alligators. The purpose of the present study was to identify the effects of prolonged lung inflation and deflation (simulated dives) on PSR and/or IPC firing characteristics in the alligator. The effects of airway pressure, hypercapnia, and hypoxia on dynamic and static responses of pulmonary stretch receptors (PSR) were studied in juvenile alligators (mean mass = 246 g) at 24°C. Receptor activity appeared to be a mixture of slowly-adapting PSRs (SARs) and rapidly-adapting PSRs (RARs) with varying thresholds and degrees of adaptation, but no CO2 sensitivity. Dives were simulated in order to character receptor activity before, during, and after prolonged periods of lung inflation and deflation. Some stretch receptors showed a change in dynamic response, exhibiting inhibition for several breaths after 1 min of lung inflation, but were unaffected by prolonged deflation. For SAR, the post-dive inhibition was inhibited by CO2 and hypoxia alone. These airway stretch receptors may be involved in recovery of breathing patterns and lung volume during pre- and post-diving behavior and apneic periods in diving reptiles. These results suggest that inhibition of PSR firing following prolonged inflation may promote post-dive ventilation in alligators.

Prototoxin proteins have been identified as members of the Ly6/uPAR super family whose three-finger motif resembles that of α-bungarotoxin. Though they are known to modify the function of nAChRs, their specificity is still unclear. Our lab identified three prototoxin proteins in the chicken ciliary ganglion: Ch3ly, Ch5ly, and Ch6ly. Ch6ly was later identified as prostate stem cell antigen (PSCA), and specifically decreased the amount of calcium influx through the homomeric α7 nAChR subtype. I then identifiedCh3ly and Ch5ly as LY6E and LYPD6B, respectively. I focused my attention onLYPD6B because of its expression in the brain. This dissertation tests whether LYPD6Bis a prototoxin protein that specifically co-localizes with and modifies the function of the heteromeric α3β4* nAChRs (the other nAChR subtype expressed in the chicken ciliary ganglia). In the first part of my dissertation I performed intracellular two-electrode voltage clamp on Xenopus oocytes co-expressing human LYPD6B and different stoichiometries of the α3β4* nAChR, these included two (α3)2(β4)3 withβ4−α3−β4−β4−α3 and β4−α3−β4−α3−β4 stoichiometries, two (α3)3(β4)2 with stoichiometries β4−α3−α3−β4−α3 and β4−α3−β4−α3−α3, two (α3β4)2(α5D)β4−α3−α5D−β4−α3 and β4−α3−β4−α3−α5D, and (α3β4)2(α5N) with stoichiometries β4−α3−α5N−β4−α3 and β4−α3−β4−α3−α5N. Concatemeric constructs are designed to link nAChR subunits, thus when translated it is done so as a single polypeptide. LYPD6Bincreased the acetylcholine (ACh) potency and desensitization rate, but decreased the maximum current response (Imax) for the (α3)3(β4)2 nAChR subtype. Yet, LYPD6Bonly decreased the Imax for the (α3β4)2α5 D-variant and not the N-variant (associated with increase nicotine consumption). For the second part of my dissertation, I determined if the expression of LYPD6B correlated with nAChRs in an activity dependent manner. Though LYPD6B mRNA expression correlates with nAChR subunit mRNA expression levels, it seemed to be independent of nAChR activity. To determine if fluorescent colocalization occurs between LYPD6B and a specific nAChR subtype, I genetically engineered LYPD6B to express a human influenza hemagglutinin (HA) epitope tag and cloned into a chicken retrovirus. LYPD6B was shown to co-localize only with the α3β4*heteromeric and not the homomeric α7 nAChRs, in a nAChR activity dependent manner. This study adds to the complexity of a prototoxin’s function by suggesting that the specificity is dependent on nAChR type and stoichiometry. It is the first in identifying a prototoxin protein, LYPD6B, which specifically modulates the function of the(α3)3(β4)2 and (α3β4)2(α5 D-variant) heteromeric nAChR subtypes. For the (α3β4)2(α5D-variant) nAChR subtype LYPD6B decreased the Imax. Such observation may be telling of a novel mechanism involved with nicotine dependence. For the(α3)3(β4)2 nAChR subtype LYPD6B increases its ACh sensitivity, desensitization rate, while decreasing Imax. Additionally, the co-localization of LYPD6B and α3β4* nAChRsin the lack of nAChR activity highlights the relevance of the functional effects α3β4*nAChRs exhibit due to LYPD6B. Such relevance may be the utilization of limiting Ach amounts.

Thesis advisor: Jianmin Gao / Thesis advisor: Eranthie Weerapana / Nature has developed a generous number of peptides carrying out various essential functions in all living organisms. Human body produces peptides as signaling molecules, such as hormones, to transmit messages from cell to cell and regulate metabolic homeostasis. Microbes synthesize peptides as antibiotics to inhibit the growth of other microorganisms. These peptides display an exceeding diversity of amino acid composition, peptide sequence, secondary structure and post-translational modification. Inspired by nature, researchers have developed peptides as a unique modality of therapeutics, combining the best attributes of small-molecule drugs and protein-based biopharmaceuticals. This work has sought to explore the potential of peptides as synthetic receptors, binders of protein and probes for bacteria detection. The research started from a foldable cyclic peptide scaffold, prolinomycin, a proline-rich analogue of valinomycin. The peptide can chelate a potassium ion folding into a drum like structure, which provides a platform to display and preoganize functional side chains for target binding. We first investigated its folding behavior under physiological conditions. We demonstrate that the metal-assisted folding of the prolinomycin scaffold tolerates various side chain mutations. The stability of the structure can be improved by introducing crosslinking moieties. Based on this scaffold, we rationally designed synthetic receptors of various amines by utilizing iminoboronate chemistry with acetylphenyl boronic acid (APBA). Furthermore, I pursued phage display, a powerful technique to develop high affinity peptide binders of protein targets. Proteins are the most appealing targets for drug development and disease biomarkers discovery. We chose sortase A (SrtA) as a model target protein to screen for potent peptide binders. A peptide inhibitor of sortase A with single-digit micromolar affinity was identified from a cyclic peptide library displayed by phage. In addition, from the chemically modified phage display peptide library presenting APBA motifs, peptide binders with specificity and micromolar affinity towards SrtA were discovered. Instead of binding to the active site, the peptide could recognize the surface of the protein. Additionally, to further expand the chemical space of phage display, I constructed a phage display peptide library presenting N-terminal cysteine (NCys) which can undergo site-specific chemical modifications. Two pieces of chemistry were applied, including thiazolidino boronate (Tzb) mediated acylation reaction of NCys and 2-cyanobenzothiazole (CBT)-NCys condensation. The site-specific dual modifications on NCys and internal Cys of phage-encoded peptides were achieved. Furthermore, a strategy to N, S-doubly label NCys via an alternative pathway of CBT condensation was reported, which presents a significant addition to the toolbox for site-specific protein modifications. Finally, by functionalizing graphene field effect transistors (G-FET) with peptide probes, we developed the first selective, electrical detection of the pathogenic bacterial species Staphylococcus aureus and antibiotic resistant Acinetobacter baumannii on a single platform. Overall, peptides provide enormous opportunities for therapeutics development. Research herein demonstrated principles of peptide design for specific molecular recognition. Novel chemistry strategies have been developed to expand the molecular diversity of peptide libraries. We believed that the advances in peptide design and screening would promote peptide-based drug discovery. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Blood flow to the previously active skeletal muscle remains elevated for several hours following an acute bout of aerobic exercise and is dependent on activation of H1 and H2 histamine receptors. Many questions remain unanswered in humans regarding the mechanisms mediating this sustained post-exercise vasodilation and what benefits come of this physiological phenomenon. The studies detailed in this dissertation were designed to examine the upstream mechanisms and explore a potential benefit associated with sustained post-exercise vasodilation. In chapter IV, we examined if oxidative stress is the upstream exercise-related factor mediating sustained post-exercise vasodilation. Intravenously infusing the antioxidant ascorbate blunted sustained post-exercise vasodilation, and this reduction was similar in magnitude to that observed with H1/H2 blockade. However, ascorbate may directly degrade histamine and may also inhibit its formation. Therefore, we conducted a follow-up study to verify the findings in study 1. In this study, we intravenously infused n-acetylcysteine, a potent antioxidant with no known histaminergic interactions. We found that n-acetylcysteine had no effect on sustained post-exercise vasodilation, indicating that exercise-induced oxidative stress is not the exercise related factor mediating sustained post-exercise vasodilation. In chapter V, we attempted to measure interstitial histamine in an effort to demonstrate that exercise induces the local formation of histamine in previously active skeletal muscle. We found that histamine is increased in the interstitial fluid within skeletal muscle during and after exercise. Additionally, we determined that de novo synthesis via histidine decarboxylase contributes to the rise in histamine during and following exercise. We also demonstrated a possible role of mast cells as an additional mechanism augmenting histamine in skeletal muscle. Collectively, these studies demonstrate that histamine is the ligand activating histamine receptors and activation is due to the induction of histidine decarboxylase and mast cell activation. In chapter VI, we attempted to determine if histamine receptor activation contributes to the expression of pro- and anti-angiogenic growth factors during the recovery from exercise. Our preliminary findings indicate that activation of histamine receptors may play a role in the expression of pro-angiogenic growth factors during the recovery from acute aerobic exercise.

The amphibian retina is commonly used as a model system for studying function and mechanism of the visual system in electrophysiology, since the neural structure and synaptic mechanism of the amphibian retina are similar to higher vertebrate retinas. I determined the specific subtypes of receptors and channels that are involved in chemical and electrical synapses in the amphibian retina. My study indicates that glycine receptor subunits of GlyRº1, 3 and 4 and glutamate receptor subunit of GluR4 are present in bipolar and amacrine dendrites and axons to conduct chemical synapses in the retinal circuit. I also found that the gap junction channel, pannexin 1a (panx1a), is present in cone-dominated On-bipolar cells and rod-dominated amacrine processes possibly to connect rod-and cone-pathway in the inner retina. In addition, panx1a may form hemi-channels that pass ATP and Ca2+ signals. The findings of my study fill the gap of our knowledge about the subtypes of neurotransmitter receptors and gap junction channels conducting visual information in particular cell types and synaptic areas. / by Yufei Liu. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

In the tissue distribution study, the expression of GHR2 is significantly higher than GHR1 in many tissues of the seabream including the gonad, kidney, muscle, pituitary and spleen. In vivo hormone treatment data indicated that cortisol and testosterone have differential expression regulation between GHR1 and GHR2. On the other hand, hepatic expression of both GHR1 and GHR2 in seabream was decreased by estradiol treatment. In primary cultures of seabream hepatocytes, the expression patterns after treatment by the various concentrations of hormones were consistent with the in vivo results. / To study the actions of environmental estrogens on the somatotropic axis, a transgenic yeast system was developed for estrogenicity screening. The fish estrogen receptor (gfER) and a reporter vector containing the estrogen responsive element (ERE) were expressed in yeast cells as a means to identify potential estrogens. Using this system, more than fifty chemicals including pesticides, herbicides, industrial chemicals and phytoestrogens were screened. Ten compounds including dibutyl phthalate (DBP) and bisphenol A (BPA) were demonstrated to exhibit estrogenic activities. And a compound (malachite green, MG) with novel anti-estrogenenic activities was identified. Then BPA and MG were focused to explore the disrupting effects of environmental estrogens on the two GHRs. Through the method of real-time PCR, both compounds could attenuate the gene expression level of GHRs in seabream hepatocytes. Using the method of luciferase assay, the signal transduction of the two GHRs was found to be desensitized by both BPA and MG. / Two genomic contigs of putative growth hormone receptor (GHR) were identified in fugu and zebrafish genomes by in silico analysis, suggesting the presence of two GHR subtypes in a single teleost species. This hypothesis was tested by cloning the full-length cDNA sequence of a second GHR subtype from the black seabream in which the first GHR subtype has been previously reported. Phylogenetic analysis of known GHR sequences from various vertebrates revealed that fish GHRs cluster into two distinct clades, viz. GHR1 and GHR2. The biological activities of both GHR subtypes from seabream had been examined using the reporter transcription assays in cultured eukaryotic cells. It was demonstrated that both of them have differential signal transduction upon Spi 2.1, beta-casein and c-fos promoter activities. / by Jiao, Baowei. / "December 2006." / Adviser: Christopher H. K. Cheng. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5662. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 150-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Kan Ka-wing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. [161]-[184]). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Karl Wah-keung Tsim. / Bibliography: leaves 109-119 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

David Chi-cheong Wan. / Bibliography: leaves 75-85 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

by Chi-shing Sum. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 84-96). / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / LIST OF ABBREVIATIONS --- p.iv / TABLE OF CONTENTS --- p.v / Chapter CHAPTER 1 --- p.1 / Chapter 1.1 --- Biochemistry of Renin-Angiotensin System --- p.1 / Chapter 1.2 --- Physiological Roles of Angiotensin --- p.5 / Chapter 1.3 --- Biochemistry of Angiotensin Receptors --- p.6 / Chapter 1.4 --- Tissue Renin-Angiotensin System --- p.13 / Kidney --- p.13 / Blood vessels --- p.14 / Heart --- p.15 / Brain --- p.16 / Testes --- p.17 / Chapter 1.5 --- Structure and Function of Vas Deferens --- p.18 / Chapter 1.6 --- Aim of Study --- p.21 / Chapter CHAPTER 2 --- p.22 / Chapter 2.1 --- introduction --- p.22 / Chapter 2.2 --- Materials --- p.23 / Chapter 2.3 --- methods --- p.23 / Chapter 2.3.1 --- Preparation of isolated epididymal rat vas deferens --- p.23 / Chapter 2.3.2 --- Concentration-responses to angiotensins --- p.24 / Chapter 2.3.3 --- Effects of angiotensin II in the presence of protease inhibitors --- p.24 / Chapter 2.3.4 --- Effect of losartan and CGP 42112 --- p.24 / Chapter 2.3.5 --- Schild analysis --- p.25 / Chapter 2.3.6 --- Interaction of angiotensin II with exogenous noradrenaline --- p.25 / Chapter 2.3.7 --- Statistical analysis --- p.25 / Chapter 2.4 --- results --- p.25 / Chapter 2.4.1 --- Effect of angiotensin on epididymal rat vas deferens --- p.25 / Chapter 2.4.2 --- Concentration-responses to angiotensins in epididymal rat vas deferens --- p.27 / Chapter 2.4.3 --- Effect of angiotensin II in the presence of protease inhibitors --- p.27 / Chapter 2.4.4 --- Effect of losartan ami CGP 42112 --- p.27 / Chapter 2.4.5 --- Schild analysis --- p.36 / Chapter 2.4.6 --- Interaction of angiotensin II with exogenous noradrenaline --- p.36 / Chapter 2.5 --- Discussion --- p.36 / Chapter CHAPTER 3 --- p.39 / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.2 --- Materials --- p.39 / Chapter 3.3 --- Methods --- p.40 / Chapter 3.3.1 --- Preparation of isolated prostatic rat vas deferens --- p.40 / Chapter 3.3.2 --- Concentration-responses to angiotensins --- p.40 / Chapter 3.3.3 --- Effects of angiotensin II in the presence of protease inhibitors --- p.41 / Chapter 3.3.4 --- Effect oflosartan and CGP 42112 --- p.41 / Chapter 3.3.5 --- Schild analysis --- p.41 / Chapter 3.3.6 --- Interaction of angiotensin II with exogenous noradrenaline --- p.42 / Chapter 3.3.7 --- Concentration-response to angiotensin II after reserpine treatment --- p.42 / Chapter 3.3.8 --- Concentration-response to angiotensin II after desensitization of P2-purinoceptors --- p.42 / Chapter 3.3.9 --- Statistical analysis --- p.42 / Chapter 3.4 --- Results --- p.43 / Chapter 3.4.1 --- Effect of angiotensin on prostatic rat vas deferens --- p.43 / Chapter 3.4.2 --- Concentration-responses to angiotensins in prostatic rat vas deferens --- p.43 / Chapter 3.4.3 --- Effect of angiotensin II in the presence of protease inhibitors --- p.43 / Chapter 3.4.4 --- Effect of losartan and CGP 42112 --- p.49 / Chapter 3.4.5 --- Schild analysis --- p.49 / Chapter 3.4.6 --- Interaction of angiotensin II with exogenous noradrenaline --- p.49 / Chapter 3.4.7 --- Concentration-response to angiotensin II afier reserpine treatment --- p.54 / Chapter 3.4.8 --- Concentration-response to angiotensin II after desensitization of P2-purinoceptors --- p.54 / Chapter 3.5 --- Discussion --- p.63 / Chapter CHAPTER 4 --- p.66 / Chapter 4.1 --- introduction --- p.66 / Chapter 4.2 --- Materials and Methods --- p.66 / Chapter 4.2.1 --- Preparation of polyclonal angiotensin II antiserum --- p.66 / Chapter 4.2.1.1 --- Preparation of peptide conjugate --- p.66 / Chapter 4.2.1.2 --- Protein determination --- p.67 / Chapter 4.2.1.3 --- Immunization of rabbit with peptide conjugate --- p.67 / Chapter 4.2.1.4 --- Collecting rabbit serum --- p.68 / Chapter 4.2.2 --- Characterization of BSA-Ang II and Thy-Ang II antisera --- p.68 / Chapter 4.2.2.1 --- Slot blotting --- p.68 / Chapter 4.2.2.2 --- Enzyme-linked immunosorbent assay (ELISA) --- p.69 / Chapter 4.3 --- RESULT --- p.69 / Chapter 4.3.1 --- Preparation of polyclonal angiotensin II antiserum --- p.69 / Chapter 4.3.2 --- Characterization of BSA-Ang II and Thy-Ang II antisera --- p.70 / Chapter 4.3.2.1 --- Slot blotting --- p.70 / Chapter 4.3.2.2 --- Enzyme-linked immunosorbent assay (ELISA) --- p.70 / Chapter 4.3 --- discussion --- p.76 / Chapter CHAPTER 5 --- p.78 / Chapter 5. 1 --- General Discussions --- p.78 / REFERENCES --- p.84 / APPENDIX --- p.97 / Published Abstract and Paper --- p.97