Peptidomimetics derived from the bioactive hexapeptide angiotensin IV (Ang IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6) have been designed and synthesized. These peptidomimetics are aimed at inhibiting the insulin-regulated amino peptidase (IRAP), also known as the AT4 receptor. This membrane-bound zinc-metallopeptidase is currently under investigation regarding its potential as a target for cognitive enhancers. The work presented herein was based on stepwise replacement of the amino acid residues in Ang IV by natural and unnatural amino acids, non-peptidic building blocks, and also on the introduction of conformational constraints. Initially, we focused on the introduction of secondary structure mimetics and backbone mimetics. The C-terminal tripeptide His-Pro-Phe was successfully replaced by a γ-turn mimetic scaffold, 2-(aminomethyl)phenylacetic acid (AMPA), which was coupled via an amide bond to the carboxyl terminus of Val-Tyr-Ile. Substitution of Val-Tyr-Ile, Val-Tyr, Tyr-Ile and Tyr, respectively, by 4-hydroxydiphenylmethane scaffolds comprising a 1,3,5-substituted benzene ring as a central moiety unfortunately rendered peptidomimetics that were less potent than Ang IV. The subsequent approach involved the introduction of conformational constraints into Val-Tyr-Ile-AMPA by replacing Val and Ile by amino acid residues appropriate for disulfide cyclization or ring-closing metathesis. Chemically diverse structures encompassing an N-terminal 13- or 14-membered macrocyclic tripeptide and a C-terminal non-peptidic moiety were developed. Tyr2 and AMPA were modified to acquire further knowledge about the structure-activity relationships and, in addition, to improve the metabolic stability and reduce the polarity. Several of the compounds displayed a high capacity to inhibit IRAP and exhibited Ki values in the low nanomolar range. Hence, the new compounds were more than ten times more potent than the parent peptide Ang IV. Enhanced selectivity over the closely related aminopeptidase N (AP-N) was achieved, as well as improved stability against proteolysis by metallopeptidases present in the assays. However, additional investigations are required to elucidate the bioactive conformation(s) of the relatively flexible N-terminal macrocycles. The compounds presented in this thesis have provided important information on structure-activity relationships regarding the interaction of Ang IV-related pseudopeptides and peptidomimetics with IRAP. The best compounds in the series constitute important starting points for further discovery of Ang IV peptidomimetics suitable as tools in the investigation of IRAP and other potential targets for Ang IV. The literature presents strong support for the hypothesis that drug-like IRAP inhibitors would serve as a new type of future cognitive enhancers with potential use in the treatment of cognitive disorders, e.g. Alzheimer’s disease.

This dissertation comprises an introduction, background and current research progress in the area of classical antifolates as the targeted anticancer therapies.<br>In this study, twelve series of classical 5-, 6- and 7-substituted pyrrolo[2,3-d]pyrimidines were designed and synthesized. Extensive structure modifications of the pyrrolo[2,3-d] pyrimidine scaffold were investigated to determine selective transport via FR or/and PCFT and tumor targeted antifolates with GARFTase or multiple folate metabolizing enzyme inhibition.<br>The design strategies employed include: variation of the side chain substitution position (5-,6- and 7-substituted); variation of the side chain length (n=1-6); isosteric replacement of the 1,4-disubstituted phenyl ring with 1,2- and 1,3- disubstituted phenyl ring and 2,5- disubstituted thiophenyl ring; replacement the L-glutamate with variation at the á and ã carboxylic acids.<br>As a part of this study, a total of one hundred and fifty six new compounds (including new intermediates) were synthesized and separated. Of these, twelve series consisting of forty two classical antifolate final compounds were submitted for biological evaluation. In addition, bulk synthesis of some potent final compounds (2, 2.0 g; 161, 500 mg; 175, 1.0 g; 166, 500 mg; 194, 500 mg) was carried out to facilitate in vivo evaluation.<br>More importantly, a new Heck coupling of the thiophene iodide 301 and allyl alcohols to synthesize aldehydes in one step was discovered. Due to its potential use in analog synthesis of clinically used antifolates such as RTX and PMX, this mild conditioned and easy to handle Heck coupling reaction is highly attractive.<br>During this study, the SAR of folate transporters (RFC, FR and PCFT) and GARFTase inhibitors were extensively explored. The 6-substituted straight chain compound 166 (n=7) was extremely potent against KB tumor cells (IC50=1.3 nM, about 80-fold more potent than clinically used PMX) without any RFC activity. The 5- substituted phenyl compound 175 (n=4) showed AICARFTase as the primary target with potent KB tumor cell inhibition (IC50=7.9 nM, about 8-fold more potent than PMX) and also indirectly inhibited the mTOR pathway leading to tumor cell apoptosis. Due to their potent antitumor activities, these two compounds serve as leads for future structural optimization. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

Underground injection of acid gas has been studied for several decades for oil field applications, such as enhanced oil recovery (EOR), but is now being studied as a solution to climate change. This research aims to simulate underground conditions at injection sites, such as the pilot scale injection site located near the site of a coal fired power facility in the Black Warrior Basin of Alabama. This proposed carbon capture and sequestration (CCS) location would involve injection of liquid CO2 into a carbonaceous saline aquifer. The objective of this study was to investigate carbonate surface treatments that alter the kinetics and mechanism of mineral dissolution resulting from the injection of an acid gas (CO2) into a geologic formation. A variety of mineral coatings were tested in an attempt to preserve mineral integrity under acidic conditions. Surface active chemicals were first tested, including scale inhibitors, followed by a novel acid induced surface treatment that precipitates an inorganic layer on the calcite to preserve the acid soluble mineral. These experiments are the first to investigate the use of scale inhibitors for mineral preservation, although were found ultimately to have little impact on dissolution kinetics. However, anions of moderate to strong acids induced surface coatings that were determined to effectively inhibit dissolution. Additionally, a novel, high pressure flow-through experimental apparatus was developed to simulate pressure and temperature conditions relevant to injection sites. Similar mineralogical studies in the literature have used pressurized, unstirred, batch systems to simulate mineral interactions. Solids with an acid induced surface coating were tested in the high pressure column and no calcium was found to leave the column.

Underground injection of acid gas has been studied for several decades for oil field applications, such as enhanced oil recovery (EOR), but is now being studied as a solution to climate change. This research aims to simulate underground conditions at injection sites, such as the pilot scale injection site located near the site of a coal fired power facility in the Black Warrior Basin of Alabama. This proposed carbon capture and sequestration (CCS) location would involve injection of liquid CO2 into a carbonaceous saline aquifer. The objective of this study was to investigate carbonate surface treatments that alter the kinetics and mechanism of mineral dissolution resulting from the injection of an acid gas (CO2) into a geologic formation. A variety of mineral coatings were tested in an attempt to preserve mineral integrity under acidic conditions. Surface active chemicals were first tested, including scale inhibitors, followed by a novel acid induced surface treatment that precipitates an inorganic layer on the calcite to preserve the acid soluble mineral. These experiments are the first to investigate the use of scale inhibitors for mineral preservation, although were found ultimately to have little impact on dissolution kinetics. However, anions of moderate to strong acids induced surface coatings that were determined to effectively inhibit dissolution. Additionally, a novel, high pressure flow-through experimental apparatus was developed to simulate pressure and temperature conditions relevant to injection sites. Similar mineralogical studies in the literature have used pressurized, unstirred, batch systems to simulate mineral interactions. Solids with an acid induced surface coating were tested in the high pressure column and no calcium was found to leave the column.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

<p><i>S. sclerotiorum</i> has been studied for over 100 years. Despite this, a definite resistance mechanism to this plant pathogen remains to be identified. Researchers continue to examine the <i>S. sclerotiorum</i> life cycle to identify stages where effective disease management strategies can be applied. The development of molecular tools has allowed for a better understanding of the pathogen and created new opportunities for research on plant-pathogen interactions.</p> <p>Most of the past research on pathogenicity factors produced by this pathogen, such as hydrolytic enzymes, studied them in isolation. This thesis examines how <i>S. sclerotiorum</i> pathogenicity factors, including cutinases, polygalacturonases and necrosis-inducing peptides, work in concert during the infection. The first study explored processes for cuticle penetration leading to the identification of the gene encoding S. sclerotiorum cutinase A and the characterization of the factors that govern its expression during the infection. The second study investigated how the pathogen penetrates the cell wall and proliferates within the host. In this regard, the mechanism with which expression of <i>S. sclerotiorum</i> polygalacturonase genes is regulated was elucidated. The interplay with host polygalacturonase inhibitor proteins was also demonstrated and related to the mechanisms of host resistance. The third study examined factors involved in tissue necrosis and two necrosis-inducing proteins were characterized. This study also unraveled part of the signaling mechanisms that allow for the pathogen to regulate pathogenicity gene expression during the infection. The signaling mechanisms were found to involve calcium, cAMP and at least one <i>S. sclerotiorum</i> mitogen activated protein kinase (SMK3) working in concert to coordinate the infection process. SMK3 was found to play a major role in a variety of vital functions, such as mycelial branching, infection cushion formation and sclerotia production. Genetic transformation of <i>S. sclerotiorum</i> was required to enable certain aspects of this study. My approach to this led to the development of a highly efficient method to isolate homokaryotic lines of filamentous fungi. In conclusion, this thesis has advanced the understanding of <i>S. sclerotiorum</i>-host interactions and identified a number of factors involved in pathogenesis.</p>

This research examined the impacts of water quality, phosphate-based corrosion inhibitors and pipe wall exposure on free chlorine (HOCl)/chloramine (NH2Cl) degradation and haloacetic acid (HAA)/N-nitrosodimethylamine (NDMA) formation in simulated distribution system water mains and household plumbing at bench-scale and pilot scale. In bench-scale bottle tests, the reactivity of fresh/pre-corroded pipe materials with HOCl/NH2Cl in decreasing order was: ductile iron, copper, lead. The addition of phosphate-based corrosion inhibitors generally increased HOCl/NH2Cl degradation for fresh iron coupons, but decreased HOCl/NH2Cl decay only for fresh copper coupons. Generally, these corrosion inhibitors did not impact HAA formation. Copper corrosion products, including Cu(II), Cu2O, CuO and Cu2(OH)2CO3, catalyzed HAA and NDMA formation. For HAAs, copper catalysis increased with increasing pH from 6.6 to 8.6 and/or increasing concentrations of these copper corrosion products. Interactions of copper with natural organic matter (NOM), likely by complexation, and the subsequent increase in the reactivity of NOM were proposed to be the primary reason for the increased HAA formation.NDMA formation increased with increasing Cu(II) concentrations, DMA concentrations, alkalinity and hardness but was inhibited by the presence of NOM. The transformation of NH2Cl to dichloramine (NHCl2) and complexation of copper with DMA were proposed to be involved in elevating the formation of NDMA at pH 7.0. Finally, in pilot-scale modified pipe loop tests, copper catalysis of NDMA formation was confirmed, especially under laminar flow conditions, and iron was shown to possibly catalyze NDMA formation under turbulent conditions. Orthophosphate increased the catalytic effects of iron but decreased copper catalysis on NDMA formation by either modifying the properties of the iron-associated suspended particles or reducing the dissolved metal concentrations. Orthophosphate increased chloramine decay when in contact with iron, likely by promoting nitrite formation, but orthophosphate decreased chloramine decay for copper and lead by reducing the availability of metal corrosion products.

It is well known boronic acids have its unique chemistry and related applications in organic synthesis. The boronic acid functionally group also plays very important roles in medicinal chemistry and chemical biology. For example, boronic acids have been developed as potential therapeutic agents, chemical biology tools. All these applications are directly related to the unique electronic and chemical properties of the boronic acid group. Herein, several application of boronic acids have been studied: 1) several groups of compounds were found as bacterial quorum sensing inhibitors; 2) a boronate compound was developed as a probe for detecting reactive oxygen species (ROS); and 3) boronic acid-modified aptamers can be used for glycoprotein recognition.