This study was designed to determine the effects of stage of lactation and subsequent energy status on metabolic and endocrine measures, follicular development, and the quality of oocytes obtained from Holstein cows. Holstein cows were selected prior to calving and assigned to the early lactation (EL) group (n=8) while, cows at d 90 postpartum were selected for the mid-lactation (ML) group (n=7). Blood samples were taken twice weekly from 4 wk prior to the start of follicular aspirations and then on through the aspiration periods for metabolite and hormone determination. Ultrasound-guided transvaginal follicular aspiration (TVFA) was conducted twice weekly for a 10-wk period on all cows. Follicular fluid samples were obtained from the largest follicle, > 10 mm in diameter, for hormone determination. All data were analyzed by ANOVA, using the general linear model procedures. Mean energy balance was positive for (2.43 ± 0.32 Mcal/kg) for ML cows and negative (-1.55 ± 0.33 Mcal/kg) for EL cows. In ML cows serum progesterone (P4) decreased rapidly from 2.7 ± 0.1 ng/ml at the first aspiration session to a nadir of 0.33 ± 0.1 ng/ml at wk 8, while follicular fluid P4 increased from 0.9 ± 0.5 to 5.6 ± 0.5 ng/ml. In the EL cows serum and follicular fluid P4 remained relatively constant over the course of aspirations. There was a linear increase in follicular fluid insulin-like growth factor I (IGF-I) for EL and ML cows, however the increase was more rapid for ML cows (159 ± 36 to 200 ± 36 ng/ml) than for EL cows (145 ± 36 to 164 ± 36 ng/ml). Over the aspiration period nonesterified fatty acids (NEFA) declined rapidly for the EL cows (0.32 ± 0.2 to 0.22 ± 0.2 mEq/L), while serum NEFA for the ML cows were relatively stable (0.19 ± 0.2 to 0.22 ± 0.2 mEq/L). The number of follicles observed during the aspiration sessions increased linearly for both EL and ML cows (P < 0.05) over the 10-wk period. However, the increase was larger for the ML cows than for the EL cows, going from 14.2 ± 0.5 to 18.1 ± 0.5 and 14.9 ± 0.3 to 15.7 ± 0.5, respectively. These results show that cows in early lactation are physiologically under more production stress than cows in mid lactation. Furthermore, increasing levels of serum and follicular fluid IGF-I in mid lactation may reflect differences in follicle and oocyte measures. / Master of Science

Mémoire numérisé par la Direction des bibliothèques de l’Université de Montréal. / Methodology was developed for synthesizing chemical libraries for discovering new peptide honnone receptor ligands. In particular, a combinatorial strategy was used for library synthesis of neruokinin and somatostatin receptor ligand candidates. This strategy was based on the design and synthesis of indolizidin-2-one amino acid scaffolds and their subsequent incorporation into a series of amido amides (88-140) using a novel solid-phase approach. Dipeptide mimic 7-benzylindolizidinone amino acid 48 was synthesized on a gram scale by a literature procedure and then introduced into a library of candidates (88-140) to discover new somatostatin and nemokinin receptor ligands. A combinatorial strategy was employed for their synthesis which featured oxime resin as solid support. 7-Benzylindolizidinone AL(BOC) amino acid 48 was coupled to oxime resin. The library members were then obtained by a sequence featuring deprotection of the BOC group, free basing and coupling to the amine with 5 different carboxylic acids, followed by displacement with 9 different primary amines to give amido amide targets. (J A second scaffold was conceived and synthesized in the second part of the thesis. Enantiopure 7^-(BOC)amino-7-[3-azidopropyl]mdolizidm-2-one acid 141 has been synthesized by displacement of the methanesulfonate of its 7- hydroxypropyl counterpart 151 with sodium azide and subsequent ester hydrolysis. ^V-(BOC)Amino-7-[3-hydroxypropyl]indolizidin-2-one ester 151 was obtained from a sequence commencing with the alkylation of (2S,SS)-di-tertbutyl 5-oxo-2,8-di-[^-(PhF)amino]azelate 75 (PhF = 9-(9-phenylfluorenyl)). Stereoselective allylation of 75, regioselective olefin hydroboration, selective primary alcohol protection as a silyl ether and oxidation of the secondary alcohol gave (2S,4R,SS)-di-tert-buty\ 4-[3-tër/-butyldimethylsiloxypropyl]-5-oxo-2,8-din [AL(PhF)amino]azelate 149 as a pure diastereomer in 33% overall yield. Linear ketone 149 was then converted into the indolizidinone heterocycle by a route featuring reductive amination, lactam cyclization and isolation by way of a silyl ether which provided the (6>S',7^?)-isomer of 151. Enantiopure 7V-(BOC)amino-7- (3-azidopropyl)indolizidin-2-one acid 141 was also achieved by converted 151 to its respective methanesufonate followed by displacement with sodium azide. In summary, this thesis provides first an introduction into the importance of the peptide hormones Substance P and somatostatin. The combinatorial strategy is then illustrated by the introduction of 7-benzylindolizidin-2-one 48 into a focused library. Finally, methodology is presented for preparing alternative 7-alkylindolizidin-2-one amino acid scaffolds. / Une méthodologie a été développée pour la synthèse d'une librairie de molécules dans le but de mettre au point de nouveaux ligands pour les récepteurs d'hormones peptidiques. En particulier, une stratégie combinatoire a été utilisée dans la synthèse d'une librairie de ligands potentiels aux récepteurs de la neurokinine et de la somatostatine. Cette stratégie est basée sur le design et la synthèse des acides aminés indolizidin-2-one comme squelettes et leur incorporation dans une série d'amido amides utilisant une nouvelle approche sur support solide. Le peptidomimétique 7-benzylindolizidinone 48 a été synthétisé sur une échelle multigramme selon un protocole de la littérature et a ensuite été incorporé dans une librairie de ligands potentiels aux récepteurs de la somatostatine et de la neurokinine. L'approche combinatoire utilisée pour la synthèse fait appel à la résine d'oxime comme support solide. L'acide aminé 7- benzylindolizidinone N-(BOC) 48 a été couplé à la résine. La librairie moléculaire a ensuite été obtenue par la déprotection du groupe BOC, l'obtention de la base libre et le couplage de l'amine avec cinq différents acides carboxyliques, suivi du déplacement avec neuf aminés primaires différentes pour donner les amido amides ciblés. Un deuxième squelette a été conçu et synthétisé dans la deuxième partie du projet. L'acide ^V-(BOC)amino-7-[3-azidopropyl]mdolizidm-2-one énantiopur 141 a été synthétisé par le déplacement du méthanesulfonate du 7-hydroxypropyl 151 avec l'azoture de sodium suivi de l'hydrolyse de l'ester. L'ester N- (BOC)amino-7-[3-hydroxypropyl]indolizidin-2-one 151 fût obtenu par une séquence de réactions commençant par l'alkylation du (25', SS)-di-tert-butyï-5- oxo-2,8-di-[^-(PhF)amino]azélate 75 (PhF = 9-(9-phénylfluorényl)). L'allylation stéréosélective de 75 , suivi de l'hydroboration régiosélective de l'oléfme, de la IV protection sélective de l'alcool primaire sous forme d'ether silylé puis de l'oxidation de l'alcool secondaire a donné le (25', 4R, SS)-di-tert-bvtyl-4-[3-tertbutyldiméthylsiloxypropyl]- 5-oxo-2,8-di-[AL(PhF)amino]azélate 149 comme seul diastéréoisomère dans un rendement global de 33%. La transformation de la cétone 149 en hétérocycle indolizidinone a par la suite été réalisée par une amination reductive, une cyclisation à la lactame et l'isolation sous forme d'ether silylé pour donner l'isomère (65, 77?) de 151. L'acide 7^-(BOC)amino-7-[3- azidopropyl]indolizidm-2-one 141 énantiopure a aussi été synthétisé par la conversion de 151 au méthanesulfonate correspondant suivi du déplacement avec l'azoture de sodiuni. En résumé, ce mémoire donne premièrement une introduction à l'importance des hormones peptidiques que sont la substance P et la somatostatine. La stratégie utilisée est ensuite illustrée par l'introduction du 7- beiizylindolizidin-2-one 48 dans une librairie de molécules. Finalement, la méthodologie pour préparer différents acides aminés 7-alkylindolizidin-2-one est présentée.

Advanced healthcare requires novel technologies capable of real-time sensing to long-term health monitoring. One example includes biomarker detection for disease diagnosis and deciding treatment options. But several limitations exist with current technologies; however, the COVID pandemic brought these limitations to a global presence as the authorities struggled to quickly authorize a facile test for the early detection of SARS-CoV-2. An important next step is to research alternate strategies, utilize the current infrastructure available, and build sensors that meet standards the current technologies fail to. One of the strategies involves identifying novel sensing parts. To this end, we turned our attention to bacteria as they provide a plethora of novel sensing parts. Bacteria respond to stimuli using a wide range of biomolecules that include enzymes and transcription factors. Our group reported an optical progesterone biosensor based on a novel progesterone responsive allosteric transcription factor (aTF). Firstly, the electrochemical transduction of the binding affinity between this aTF and its cognate DNA sequence is discussed. The binding and unbinding of aTF-DNA results in an impedance change and is directly proportional to progesterone concentration. The limit of detection is comparable to the optical progesterone sensor and relevant to the physiological ranges of progesterone present in bodily fluids. Secondly, to convert the sensor into a point of care system, the expression of the aTF-enzyme fusion protein that undergoes the binding-unbinding event is discussed. The enzyme in presence of its excess substrate acts as a signal amplifier to track the binding changes. The signal depends on the proximity of the fusion protein to the electrode surface and correlates to the progesterone concentration. As we recover from the deadly COVID pandemic, we realize that early diagnosis is a key pillar of disease containment, in addition to other approaches such as contact tracing, distancing, and personal protective equipment. A truly transformative technology in the fight against future viruses is a rapid and quantitative point-of-care (POC) test with a low limit of detection and a high specificity. To that end, an inverted glucometer technology for the detection of infectious diseases is presented. As a model system, SARS-CoV-2 antigens – nucleocapsid protein, antibodies against it, and an inflammatory biomarker are detected. Antigen of interest is sandwiched between capture and detection reagents with biotin and glucose oxidase tags respectively. Glucose oxidase, a widely used enzyme in glucometers, amplifies the output signal in presence of excess glucose. The following chapters encompass designs, different immobilization techniques, characterization, and optimization methods to develop biosensors that meet requirement standards. This research serves as a platform for development of state of the art technologies for diagnostics applications. / 2025-01-16T00:00:00Z

Serotonin is a normal constituent of the brain and has been claimed to act as a neurohormonal or regulatory agent in nerve transmission. Previous workers have only studied its whole brain or subcellular particulate distribution. Because the brain itself is divided into many distinct ganglia and nuclei, the distribution of serotonin in these sites is considered to be of greater importance than either the whole brain or subcellular distribution. This paper is a study of the distribution of serotonin in particular brain sites in rats with normal and elevated levels of the amine, with and without electroshock treatment. The results indicate: (1) a heterogenous distribution of serotonin and 5-hydroxytryptophan in the rat brain; (2) serotonin and 5-hydroxytryptophan are transported down the neuron axon; (3) serotonin and 5-hydroxytryptophan are not utilized by all neurons; (4) iproniazid and/or electroshock can alter the uptake, distribution and transportation of serotonin and 5-hydroxytryptophan in the different areas and fibers of the central nervous system.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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Background: Herpes simplex virus type 2 (HSV-2), the primary cause of genital herpes, is one of the most widespread, lifelong sexually transmitted infections (STIs). Incidence is disproportionately higher in women compared to men, so a better understanding of vaginal transmission, the primary mode for HSV-2 infection in women, is crucial for developing preventative strategies. Female sex hormone, estrogen (E2), has been shown to play a protective role against sexually transmitted viral infections and previous studies have shown that vaginal epithelial cells treated with E2 are protected against HSV-2 infection; however, the underlying mechanism of E2 protection remains unclear, so a transcriptome analysis followed by functional studies was performed. Method of Study: In this study, VK2/E6E7 (vaginal epithelial) cells were used to study HSV-2 entry, infection and replication. VK2s were grown in Air-Liquid-Interface (ALI) cultures, allowing for their proliferation and stratified layer formation in transwells; closely mimicking physiological conditions. Media was supplemented with no hormone (NH) or physiological concentrations of E2, P4 and MPA for 7 days. After 24 hours of HSV-2 infection in these cultures, VK2 cells were lysed and processed for RNA isolation. We performed a comprehensive genome-wide microarray to profile gene expression of VK2 cells pre-treated with and without E2, prior to, and following HSV-2 infection. For data analysis, “R” software was used to perform all pre-processing steps and normalization. Gene Set Enrichment Analysis (GSEA) was performed to identify potential cellular pathways regulated by E2 after infection using the Hallmark database, relative to NH conditions. Immunofluorescence staining was used for functional analysis to confirm transcriptomic data. After selecting a pathway for investigation, small-molecule inhibitors and activators of this pathway were used in combination with NH or E2. Vero plaque assay and HSV-2-GFP infection were used to identify examine the protective effects of E2 and the selected pathway. In addition, we also used siRNA to specifically knockdown proteins part of the pathway and investigate the specific effects on protection against HSV-2. Results: Microarray analysis indicated that exposure to HSV-2 in the presence of E2 resulted in differential transcriptional profile compared to NH and P4. GSEA assigned one of the highest enrichment scores to the p53 pathway compared to other pathways under the influence of E2 compared to NH, following HSV-2 infection. Studies to correlate bioinformatic results with functional analysis showed significant increase in p53 protein expression after E2 treatment compared to NH. Vero plaque assay demonstrated 10-fold decrease in viral replication following E2 treatment as well as by direct activation of p53 in absence of E2. In contrast, p53 inhibition even in the presence of E2 resulted in 100-fold increased viral replication compared to E2 alone, suggesting that the p53 is involved in E2-mediated protection. We deduced that E2 particularly affects HSV-2 replication and not entry into VK2 cells. We also found that BST2 is strongly regulated by E2-mediated p53 and also contributes to protection against HSV-2. Lastly, we demonstrated that E2 demonstrates anti-inflammatory effects that correlate with its increase in barrier integrity seen with VK2s. Conclusions: With bioinformatic and functional analysis, we found that E2 provides protection through the p53 pathway, as well as through downstream BST2. Our data provides the first comprehensive overview of host cellular responses to HSV-2 and female sex hormones at a transcriptional level and highlights the protective role of E2-mediated p53 pathway. This study is the first to deduce the antiviral mechanism of E2 against HSV-2 infection in human vaginal epithelial cells. / Thesis / Master of Science (MSc)

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