Approximately 10% of pregnant women take antidepressants. Prenatal exposure to selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, has been shown to alter serotonergic signaling in the brain. However, the effects of SSRIs on peripheral serotonin (5HT) synthesis and/or signaling have largely been ignored. Serotonin in the gut is critical for intestinal function and dysregulation of this pathway is associated with intestinal disease. Therefore, the goal of this study was to determine the effects of perinatal exposure to the SSRI fluoxetine (Prozac®) on intestinal health in the offspring. Dams were given vehicle or fluoxetine hydrochloride (FLX 10 mg/kg/d; N=15) for 2 weeks prior to mating until weaning. We assessed markers of serotonergic signaling, inflammation, and composition of the gut microbiota in the offspring. Male offspring of fluoxetine-treated dams had significantly elevated serum levels of 5-HT and decreased expression of the 5HT2A receptor and MAO. In female offspring there was no effect of SSRI exposure to alter any components of serotonergic signaling. Although we did not find any evidence of increased inflammation following fluoxetine exposure, there were significant alterations in the composition of the gut microbiota in the exposed offspring. Male offspring of SSRIs-exposed mothers had changes in key components of the gut serotonergic system in association with elevated levels of serum 5-HT and alterations in the gut microbiota in adulthood. The impact of these changes on intestinal health and the reasons for the sex specific effects remain to be determined. / Thesis / Master of Science in Medical Sciences (MSMS)

Dialkylsilanediols are a novel class of non-hydrolyzable analogues of the tetrahedral intermediate of amide hydrolysis, shown to be good inhibitors of HIV-1 protease, angiotensin converting enzyme (ACE), and thermolysin. An impediment to utilization of these silanediol structures, however, has been the methods for their assembly. This research describes the reductive lithiation of hydridosilanes and alkoxysilanes, and the use of the resulting silyl anions to develop efficient methods to synthesize silanediol precursors. In the first part of research, lithiation of hydridosilanes was studied. As part of this study, a simple 1H NMR method was developed for monitoring and analyzing the progress of lithiation. In addition, this method was converted to a titration for silyllithium reagents using BHT as an internal standard. Silanediols 107 and 177 are analogues of a potent chymase inhibitor, NK-3201 (82). In the second part, diphenylsilanes 108 and 170, precursors to silanediols 107 and 177, were synthesized using addition of silyllithium to sulfinimine 113 as a key step. In the third part, lithiation of alkoxysilanes was studied. (Si,O)-Dianions, generated from lithiation of silane alcohol 175 or 2,2-diphenyl-1-oxa-2-silacyclopentane (225), were reacted with a wide variety of electrophiles to give potentially useful silicon-containing building blocks. Addition of the (Si,O)-dianion 284 to sulfinimines gave silanediol inhibitor precursors with full control of stereochemistry. In the last part, a new method featuring 1,1-diphenyl-2-azaallyllithium chemistry were utilized to synthesize a series of protected α-amino silanes 323, 329 - 331. / Chemistry

Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA. We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3. The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.

Los receptores GABA_A son canales iónicos activados por ligandos y funcionan como los principales mediadores de la inhibición en el sistema nervioso central de mamíferos. Están formados por cinco subunidades formando un poro central conductor de iones. Cada combinación de subunidades presenta una función y localización determinada, de las cuales el subtipo α1ß2γ2 es el más abundante en el ser humano. Los receptores GABA_A intervienen en una miríada de procesos neurológicos y su desregulación genera las denominadas canalopatías. Por lo cual, el estudio de estos sistemas es indispensable para el desarrollo de fármacos y de tratamientos para mejorar la calidad de vida. En este trabajo de tesis se propone el estudio in silico del receptor GABA_A α1ß2γ2 mediante el empleo de técnicas de bioinformática y biofísica computacional, que incluyen simulaciones de docking molecular, dinámica molecular y técnicas de muestreo avanzado. Se desarrolló un modelo por homología del receptor empleando el receptor GABA_A homopent mero de subunidades ß3. El modelo fue validado a través de un cuidadoso análisis de su estereoquímica y su estabilidad mediante simulaciones de dinámica molecular. A continuación, se realizó un exhaustivo análisis de la unión de compuestos a dos sitios de unión en el dominio extracelular del modelo: el sitio ortostérico (donde se unen los ligandos que actúan directamente sobre la activación del canal) y el sitio de unión de gran afinidad de las benzodiazepinas (moduladores alostéricos). Los modos de unión encontrados fueron contrastados con información experimental disponible y se halló muy buena concordancia. El trabajo finalizó con el primer estudio computacional sobre la interacción putativa entre este receptor y la proteína DBI y fragmentos peptídicos derivados de su digestión. Este análisis permitió elaborar, por primera vez, una hipótesis respecto a los residuos involucrados en la interacción. / GABA_A receptors are pentameric ligand-gated ion channels which act as the main mediators of inhibitory signalling in the central nervous system of mammals. They are formed by five subunits arranged around a central ion-conducting pore. Each combination of subunits has a specific function and localization, the α1ß2γ2 subtype being the most abundant in homo sapiens. These receptors intervene in a myriad of neurological processes and their disregulation cause several channelopathies. Although they are very complex systems, their study is fundamental for the development of new drugs and therapies aimed at improving life quality. In this thesis we performed an in silico study of the α1ß2γ2 GABA_A receptor through the use of bioinformatics and computational biophysics tools, which include molecular docking, molecular dynamics and enhanced sampling techniques. A homology model was developed using the structure of the GABAA_A ß3 homopentamer. The model was validated through a thorough analysis of its stereochemistry and its stability was evaluated from molecular dynamics simulations. Moreover, an exhaustive evaluation of the binding modes of ligands to two extracellular sites was performed: the orthosteric site (ligands which act directly on the activation of the channel) and the high affinity binding site for benzodiazepines (allosteric modulators). The comparison of the binding modes to available experimental information showed great agreement. Finally, a computational study was carried out for the first time regarding the putative interaction of this receptor with DBI and its peptide fragments. This study allowed the formulation of the first hypotheses regarding the aminoacids involved in the interaction.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24555号 / 薬科博第172号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 小野 正博, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

In humans, mammals, and perhaps all vertebrates, sphingolipids exist as a family of cellular signaling molecules and have been shown to be involved in a wide range of biological processes ranging from proliferation to apoptosis. As such, sphingolipid signaling has garnered the attention of numerous researchers as an attractive candidate for pharmacological manipulation. The synthetic pathway of one prominent sphingolipid, sphingosine 1-phosphate (S1P), has been implicated in a variety of disease states such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Formation of S1P is facilitated from the ATP dependent phosphorylation of sphingosine (Sph) through its generative enzyme's sphingosine kinase 1 and 2 (SphK1 and SphK2). Inhibition of SphK1 and SphK2 results in the manipulation of S1P levels, which has been shown to be therapeutic in various animal models of disease. While there are multiple examples of potent SphK1-selective and dual SphK1/2 inhibitors, SphK2-selective inhibitors are scarce. Herein, we describe the design, synthesis and biological testing of SphK2-selective inhibitors. We first describe the discovery that introducing a trifluoromethyl group onto the internal aryl ring of our inhibitor scaffold led to superior selectivity and potency towards SphK2. We demonstrate that the trifluoromethyl moiety is interacting with a previously unknown side cavity in the substrate binding site of SphK2 that is unique and could be exploited in the design of SphK2-selective inhibitors. The synthesis of 21 derivatives with various substituents spanning off the internal aryl ring was completed, therefore characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site. This work led to the development of the most potent SphK2-selective inhibitor known at the time. We then describe the transformation of our SphK2-selective inhibitors into an orally bioavailable drug. We explain how the guanidine functionality on our inhibitor scaffold hinders our compounds from being orally bioavailable. Consequently, a library of 24 derivatives with various modifications to the guanidine functionality was synthesized and evaluated for improved orally bioavailability. Highlighted in this work is the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832), which displays a hSphK2 Ki of 82 nM and 122-fold selectivity for SphK2. Chemical modification and in vivo assessment of 3.14 (SLS1081832) prodrugs was explored. / Doctor of Philosophy / In humans, sphingosine 1-phosphate (S1P) is a signaling molecule that is generated through an ATP dependent reaction of sphingosine (Sph) via sphingosine kinase 1 and 2 (SphK1 and SphK2). Furthermore, S1P has been shown to be implicated in various diseases such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Inhibition of SphK1 and SphK2 has been shown to be therapeutic towards the symptoms of these diseases. Therefore, in order to alleviate these disorders, the concentrations of S1P must be controlled through pharmacological inhibition of SphK1 and SphK2. There are multiple reported examples of potent SphK1-selective and dual SphK1/2 inhibitors; however, SphK2-selective inhibitors are scarce. This work describes the synthesis and biological assessment of 21 compounds for their effectiveness in selectively targeting and inhibiting SphK2. The work led to the discovery of a previously unrecognized side cavity in the binding pocket of SphK2 that enhances inhibitor potency and selectivity towards SphK2. Furthermore, studies characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site led to the development of the most potent SphK2- selective inhibitor known at the time. Building on this work, we next focused on the transformation of our SphK2-selective inhibitors into a drug that could be administered orally. We describe the synthesis of 24 compounds with various modifications to one portion of our scaffold and their effect on improved orally bioavailability. This work led to the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832).

Soybean is an important source of protein in animal feed, and growing demand for meat consumption worldwide has led to increased soybean production. Over 120 million metric tons of soybean were harvested in the United States in 2018, approximately one-third of the world production. In the Mid-Atlantic region, soybean is one of the most valuable field crops. Major foliar diseases that reduce soybean yield in the Mid-Atlantic region are frogeye leaf spot (FLS) and Cercospora leaf blight. In addition to crop rotation and host resistance, foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage these soybean foliar diseases. Yield benefits of foliar fungicides have been inconsistent and this may be the result of low disease pressure, unfavorable environmental conditions for disease development, or the presence of fungal pathogen populations that have developed resistance to fungicides. The objectives of this research were 1) to develop a pyrosequencing-based assay to rapidly quantify QoI resistance frequencies in Cercospora sojina, the causal agent of FLS, 2) to examine the effects of fungicide application timings, disease pressure, and environmental factors on soybean yield, and 3) to develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. using a threshold decision rule. A pyrosequencing assay targeting the G143A mutation was designed, and a Virginia survey of C. sojina populations indicated that the G143A mutation conferring QoI resistance is widespread. In small plot fungicide application timing experiments, five weekly fungicide applications starting at beginning pod (R3) resulted in the greatest yield, but for single fungicide applications, R3 or 1 week after R3 resulted in the greatest yields. There was positive relationship between the cumulative number of disease favorable days (mean daily temperature 20-30°C and ≥ 10 hours of relative humidity >90%) from planting to R3 and disease severity at the full pod stage (r = 0.97, P = <0.01). Higher disease severity was associated with greater yield loss (r2 =0.53, P = 0.10) suggesting foliar fungicide applications are more likely to have yield benefits as the number of disease favorable days prior to R3 increase. A disease favorable-days threshold (FDT) using the environmental parameters indicated above was evaluated in on-farm experiments throughout Virginia, Maryland, and Delaware. Based on decision rules, FDT = 8 three weeks prior to R3 was the best predictor of a yield benefit with an R3 fungicide application. The decision aid was also able to correctly predict when a fungicide application would not be profitable ≥90% of the time. This weather-based decision aid along with monitoring of fungicide resistance development within the region will provide soybean growers in the Mid-Atlantic U.S. with tools to maximize yields and profitability. / Doctor of Philosophy / Soybean is the third most valuable field crop in the world, ranked only behind rice and wheat in value. Over 98% of the soybean crop is used for animal feed due to its high protein content. The United States is the largest soybean producer in the world, responsible for one-third of global production. Soybean is the top cash crop in the Mid-Atlantic region. Foliar fungal diseases can reduce the soybean yield by causing lesions on the leaves that reduce photosynthesis and cause premature defoliation. Frogeye leaf spot (FLS) caused by Cercospora sojina is a major yield reducing soybean foliar diseases in the Mid-Atlantic region. Foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage the disease. However, fungicide efficacy has been inconsistent. Inconsistencies may be due to low disease pressure, improper application timing, or fungicide resistance. The purpose of this research was to investigate the fungicide efficacy inconsistencies and to develop management tools to improve yield and maximize profitability. Our objectives were to 1) develop a molecular assay to quantify frequencies of the mutation conferring fungicide resistance in Virginia populations of C. sojina, 2) examine the effects of fungicide application timings, disease severity, and weather on soybean yield, and 3) develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. The C. sojina fungicide resistance mutation was widespread in Virginia, but overall frequencies were relatively low compared to findings from Midwest and Southern states. In fungicide timing experiments, beginning pod (R3) applications resulted in the most consistent yield benefits, and disease severity and yield loss increased as the number of weather-based disease favorable days prior to R3 increased. We used data from on-farm experiments in Virginia, Maryland, and Delaware to develop a weather-based disease favorable-days threshold that increased the probability that a fungicide application at R3 would have a yield benefit in soybean. The results of our research have led improved fungal disease management recommendations for soybean in the Mid-Atlantic that will maximize yields and profitability.

Apple scab (causal organism: Venturia inaequalis) is an economically devastating disease of apples that is predominantly controlled with fungicides. Of the chemical classes currently available, the sterol-inhibiting (SI) and strobilurin (QoI) fungicides are the most commonly used. Recent observations indicate that V. inaequalis populations in Virginia have developed resistance to myclobutanil and other SIs. However, little is known about the frequency and distribution of SI and QoI resistance in Virginia's scab populations. The first objective of this research was to evaluate V. inaequalis populations in Virginia for SI and QoI resistance. Fungal isolates were collected from experimental orchards at the Alson H. Smith Jr., Agricultural Research and Extension Center (AHS AREC) and from commercial orchards in Virginia and Maryland. Sensitivities were determined by assessing colony growth at 19°C on potato dextrose agar (PDA) amended with 0 or 1.0 µg ml-1 of myclobutanil (SI) (N=87) or trifloxystrobin (QoI) (N=25) at 28 days. A range of fungicide sensitivity was observed for both chemical classes. The second objective of this research was to monitor the temporal dynamics of SI resistance over five sequential field seasons. To monitor shoot growth, neon rubber bands were placed over actively growing shoot tips following myclobutanil application or sample collection. Fungal isolates were collected from the same trees from 2007 through 2010 (N=176) and compared with isolates collected from wild apple seedlings (N=3). A continuum of SI resistance was observed for each year, and the V. inaequalis population exhibited a baseline shifted toward reduced sensitivity. The third objective of this research was to examine the spatial distribution of SI fungicide resistance within the tree canopy in a lower-density orchard (less than 150 trees A-1). Leaves collected from larger trees (>8m) in a lower-density orchard at the AHS AREC were analyzed for manganese deposition, pre- and post-mancozeb application. Fungal isolates (N=105) were collected from several locations within the canopy in replicated trees in the same orchard. Weather sensors also monitored the microclimates within those tree canopies. Spray deposition, microclimate and SI resistance were influenced by canopy location. The fourth objective of this research was to investigate potential SI resistance mechanisms. Previously classified isolates were screened for point mutations within the CYP51A1 gene (Appendix C), differences in polymorphic bands (alleles) (Appendix D), and differences in metabolism of myclobutanil (Appendix E). The consensus sequences for the CYP51A1 gene were identical for all isolates tested (N=9), and results from amplified fragment length polymorphism experiment (N=82) were inconclusive. There were, however, significant differences among incubation time and myclobutanil concentration in the bioassay (N=11). Our results indicate that myclobutanil is still an effective compound for control of apple scab in many areas of Virginia. / Ph. D.

In the first part of this dissertation work, Al bond pad corrosion behavior was investigated in the presence of common industrial contaminants such as chloride (Cl-) and fluoride (F-). Al corrosion while in direct contact with Cu displayed rapid hydrogen (H2) gas evolution and dendrite propagation. In contrast, Al without bimetallic contact showed only minor surface roughening. This observed difference in the corrosion mechanism between Cl- and F- is attributed to the solubility of the corrosion products (AlCl3 vs. AlF3) formed on the Al surface. Our subsequent work explored corrosion prevention inhibition of wire-bonded devices (WBD) in the Cl- environment. Our research shows that the Al bond pad was protected against corrosion by chemically modifying the surface of the Cu wires, thereby preventing the H2 evolution. The inhibitor was observed to be highly selective, thermally stable, hydrophobic, and cost-effective, making it viable for industrial application of this coating for Al bond pad corrosion prevention. In the second part of the dissertation work, we utilized a novel approach of using ultraviolet-visible spectroscopy (UV-Vis) as a chemical-sensitive monitoring tool of the chemical environment in Cu etch bath. The UV-Vis technique illuminates the roles of H+, Cl-, Cu+, and Cu2+ to the etch bath while also providing a means to monitor the Cl- in the broad UV peak at 250 nm. The UV-Vis probe successfully demonstrated the etch rate difference between the two etch bath solutions and help in the restoration of the etching bath. Additionally, the proof-of-concept experiments (POC) to investigate UV enhanced etching for achieving anisotropic etching in PCB fabrication showed promising preliminary results with the need to develop additional etching techniques.

Eukaryotic organisms require the ability to respond to their environments. They do so by utilizing signal transduction pathways that allow for signals to effect final biological responses. Many times, these final responses require new gene expression events that have been stimulated or repressed within the nucleus. Thus, much of the understanding of signal transduction pathways converges on the understanding of how signaling affects gene expression alterations (Kumar et al., 2004). The regulation of gene expression involves the modification of chromatin between condensed (closed, silent) and expanded (open, active) states. Histone modifications, such as acetylation, can determine the open versus closed status of chromatin. The PHD (Plant HomeoDomain) finger is a structural domain primarily found in nuclear proteins across eukaryotes. This domain specifically recognizes the epigenetic marks H3K4me2 and H3K4me3, which are di- and tri-methylated lysine 4 residues of Histone H3 (Loewith et al., 2000; Kuzmichev et al., 2002; Vieyra et al. 2002; Shiseki et al., 2003; Pedeux et al., 2005, Doyon et al., 2006). It is estimated that there are ~150 proteins that contain the PHD finger in humans (Solimon and Riabowol, 2007). The PHD finger is conserved in yeast and plants, however an analysis of this domain has only been performed done in Arabidopsis thaliana (Lee et al., 2009). The work presented in this report aims to extend the analysis of this domain in plants by identifying the PHD fingers of the crop species Oryza sativa (rice). In addition, a phylogenetic analysis of all PHD fingers in Arabidopsis and rice was undertaken. From these analyses, it was determined that there are 78 PHD fingers in Arabidopsis and 70 in rice. In addition, these domains can be categorized into classes and groups by defining features within the conserved motif. In a separate study, I investigated the function of two of the PHD finger proteins from Arabidopsis, ING1 (INhibitor of Growth1) and ING2. In humans, these proteins can be found in complexes associated with both open and closed chromatin. They facilitate chromatin remodeling by recruiting histone acetyltransferases and histone deacetylases to chromatin (Doyon et al., 2006, Pena et al., 2006). In addition, these proteins recognize H3K4me2/3 marks and are believed to be "interpreters" of the histone code (Pena et al., 2006, Shi et al., 2006). To understand the function of ING proteins in plants, I took a reverse genetics approach and characterized ing1 and ing2 mutants. My analysis revealed that these mutants are altered in time of flowering, as well as their response to nutrient and stress conditions. Lastly, I was able to show that ING2 protein interacts in vitro with SnRK1.1, a nutrient/stress sensor (Baena-Gonzalez et al., 2007). These results indicate a novel function for PHD proteins in plant growth, development and stress response. / Master of Science