Pharmacology, Toxicology and Environmental Health Commons^™

Exogenous Surfactant As A Delivery Vehicle For Intrapulmonary Therapeutics, Brandon J. Baer

Electronic Thesis and Dissertation Repository

As an organ system, the lung has unique advantages and disadvantages for direct drug delivery. Its contact with the external environment allows for the airways to be easily accessible to intrapulmonary delivery. However, its complex structure, which divides into more narrow airways with each branch, can make direct delivery to the remote alveoli challenging. The objective of this thesis was to overcome this issue by using exogenous surfactant, a lipoprotein complex used to treat neonatal respiratory distress syndrome, as a carrier for pulmonary therapeutics. It was hypothesized that therapeutics administered with a surfactant vehicle would display enhanced delivery to the …

Effect Of Carbon Monoxide Releasing Molecule 3 (Corm - 3) On Platelet Adhesion To Human Brain Microvascular Endothelial Cells, Najat S. El-Farra

Undergraduate Student Research Internships Conference

Sepsis is characterized by the widespread inflammation of the body. Systemic inflammation activates and recruits inflammatory cells (e.g., leukocytes) and platelets to the affected organs.

During these inflammatory conditions, human brain microvascular endothelial cells (hBMEC) and platelets both upregulate adhesive molecules rendering platelets to adhere to hBMEC.

Although carbon monoxide is thought of as a toxic molecule to many, previous work shows its anti-inflammatory properties. Evidence has shown carbon monoxide-releasing molecules (e.g., CORM-3; that release small, non-toxic amounts of CO) can combat the effects of severe inflammation in several in vivo animal model.

In this current study, we are looking …

Characterizing The Structural, Biophysical And Functional Effects Of S-Glutathionylation On Stim1 Ca2+ Sensing, Christian Michael Sirko

Electronic Thesis and Dissertation Repository

Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca²⁺) sensing protein that initiates cytoplasmic Ca²⁺ influx via store-operated calcium entry (SOCE). STIM1, in conjunction with Orai, a plasma membrane (PM) protein, function as mediators of SOCE through the formation of calcium-release activated calcium (CRAC) channels. S-Glutathionylation of STIM1 at Cys56 has been shown to evoke constitutive Ca²⁺ entry in DT40 cells, however no studies have carefully investigated the biophysical and structural effects of this covalent modification to the luminal domain, which are critical for understanding the molecular mechanism underlying the regulation of …

Excess No Stabilizes The Luminal Domain Of Stim2 In A Cys-Specific Manner Thereby Regulating Basal Calcium Homeostasis And Store-Operated Calcium Entry, Matthew Novello

Electronic Thesis and Dissertation Repository

Stromal-interaction molecule 2 (STIM2) is an endoplasmic reticulum (ER) membrane-inserted Ca²⁺-sensing protein which, together with the plasma membrane Ca²⁺ channel Orai1, regulates basal Ca²⁺ homeostasis and store-operated Ca²⁺ entry (SOCE). Recent evidence suggests that S-nitrosylation, which is the covalent attachment of a nitric oxide (NO) moiety to a cysteine thiol, can attenuate the function of the paralog STIM1 protein. Compared to STIM1, STIM2 also functions as a basal Ca²⁺ homeostatic feedback regulator. Therefore, the objective of my study was to evaluate the susceptibility of STIM2 to S-nitrosylation and the effects that this …

The Wet Bridge Transfer System: An Novel In Vitro Tool For Assessing Exogenous Surfactant As A Pulmonary Drug Delivery Vehicle, Brandon J. Baer

Western Research Forum

Background:

Due to its complex branching structure, direct drug delivery to the remote areas of the lung is a major challenge. Consequently, most therapies, such as those treating pulmonary infection and inflammation, must utilize large systemic dosing, with the potential for adverse side effects. A novel alternative strategy is to use exogenous surfactant, a material capable of distributing throughout the lung, as a pulmonary drug delivery vehicle.

Objective:

Utilize an in vitro transferring system to assess exogenous surfactant (BLES) as a pulmonary delivery vehicle for different therapeutics.

Methods:

An in vitro technique was developed to simultaneously study surfactant delivery and …

Developing Novel Therapeutics For Bacterial Lung Infections, Brandon J. Baer, Ruud Veldhuizen, Cory Yamashita

Western Research Forum

Background: Bacterial lung infections are leading causes of death worldwide. Unfortunately, increasing resistance to antibiotics and the inflammation often accompanying these infections are leading to poor outcomes despite antibiotic intervention. Complicating treatment further, the tree-like branching structure of the lung makes drug delivery to distal sites of infection difficult. Our research aims to address these challenges by developing new therapeutics and new tools to improve and assess drug delivery, bacterial killing and inflammation. Our therapy combines host defense peptides, which have been shown to kill antibiotic-resistant bacteria and down regulate inflammation, with a pulmonary vehicle, exogenous surfactant, that can improve …

Pulmonary Surfactant Fortified With Cath-2 As A Novel Therapy For Bacterial Pneumonia, Brandon J. Baer

Western Research Forum

Background: Bacterial pneumonia is a leading cause of death worldwide, with high mortality rates persisting even after antibiotic treatment. Current treatments for pneumonia involve administration of antibiotics, however after the bacteria are killed they release toxic substances that induce inflammation and lung dysfunction. Host defense peptides represent a potential solution to this problem through their ability to down regulate inflammation. However, effective delivery to the lung is difficult because of the complex branching structure of the airways. My study addresses this delivery problem by using exogenous surfactant, a pulmonary delivery vehicle capable of improving spreading of these peptides throughout the …

The Effects Of Acetylenic Tricyclic Bis-(Cyano Enone) On Cell Migration, Eddie Chan

Electronic Thesis and Dissertation Repository

Although cancer survival rates have significantly improved over the past few decades, the improvements are primarily due to early diagnosis and inhibiting cancer growth. Limited progress has been made in the treatment of cancer metastasis, which contributes to 90% of cancer related deaths, and therapeutic agents targeting the various aspects of metastasis are lacking. One potential approach is to utilize small pharmacological compounds to inhibit tumour cell motility, as a strategy against tumour cell migration, invasion, and metastasis. The acetylenic tricyclic bis-(cyano enone), TBE-31, has been shown to be a promising chemopreventative compound. However, its effects on cell migration are …

Characterization Of The Catalytic Ck2 Subunits With Substitutions At Residues Involved In Inhibitor Binding, Paul Desormeaux

Electronic Thesis and Dissertation Repository

CK2 is a constitutively active, ubiquitously expressed and pleiotropic serine/threonine protein kinase that is implicated in many cellular functions including tumorigenesis. CK2 has two catalytic subunits, CK2a and CK2a’, that carry out its function in the cell. Previous studies have indicated that inhibitor-refractory mutants have been effective in recovering residual CK2 activity, in the presence of inhibitors, when compared to wild type CK2. Based on these observations, inhibitor-refractory mutants were created for both CK2a and CK2a’ and tested with various concentrations with two CK2-specific inhibitors, CX-4945 and inhibitor VIII. The CK2a triple mutant (V66A/I174A/H160D) was tested in inducible U2OS Flp-In …

Sirna Targeting Of Thymidylate Synthase, Thymidine Kinase 1 And Thymidine Kinase 2 As An Anticancer Therapy: A Combinatorial Rnai Approach, Christine Di Cresce

Electronic Thesis and Dissertation Repository

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumour milieu, and may modulate resistance to TS-targeting drugs. We have previously shown that TS antisense molecules (oligodeoxynucleotides, ODNs, and small interfering siRNA, siRNA) sensitize tumour cells, both in vitro and in vivo, to TS targeting drugs. As both TS and TKs contribute to cellular …

Molecular Mechanisms Underlying The Early Life Programming Of The Liver, Gurjeev Sohi

Electronic Thesis and Dissertation Repository

Clinical studies have demonstrated that intrauterine growth restriction (IUGR) offspring, faced with a nutritional mismatch postpartum, have an increased risk of developing the metabolic syndrome. The maternal protein restriction (MPR) rat model has been extensively studied to investigate the adverse effects of a nutritional mismatch in postnatal life of IUGR offspring. Previous studies have demonstrated that MPR leads to impaired function of the liver, an important metabolic organ. However the underlying mechanisms which predispose these offspring to the metabolic syndrome remain elusive. In the following studies, low protein diet during pregnancy and lactation led to IUGR offspring with decreased liver …

Cadmium Accumulation And Distribution In Lettuce And Barley, Fardausi Akhter

Electronic Thesis and Dissertation Repository

Cadmium (Cd) is a non-essential trace element and its environmental concentrations are increasing due to human activities. Edible plants can accumulate high concentrations of Cd, which could be toxic to humans. Understanding how and where Cd is stored in plants is important for ensuring lower concentration of Cd in the food. In this thesis, the accumulation and distribution of Cd in three agricultural plants, namely lettuce (Lactuca sativa L.), barley (Hordeum vulgare L.) and radish (Raphanus sativus L.), were investigated with a focus on the potential mechanisms involved in the localization of Cd in the root. The …

Human Equilibrative Nucleoside Transporter Subtype 1: Structure-Function Analysis Using Cysteine Mutagenesis And Thiol Modifying Techniques, Jamie Park

Electronic Thesis and Dissertation Repository

Human equilibrative nucleoside transporter 1 is the main mediator of bi-directional nucleoside flux and is found ubiquitously. Inhibitor and substrate interactions with ENT1 are known to be affected by cysteine-modifying reagents. Our aim was to investigate the importance of cysteine residues in hENT1 function and identify which residues were sensitive to thiol modification for further application of cysteine scanning mutagenesis on extracellular loop 5. Transporter function was assessed by the binding of [³H]NBMPR and the cellular uptake of [³H]2-chloroadenosine. Treatment of hENT1 with the neutral sulfhydryl-modifier methyl methanethiosulfonate (MMTS) enhanced [³H]NBMPR binding but decreased …

Regulation Of G Protein Signaling By Goloco Motif Containing Proteins, Peishen Zhao

Electronic Thesis and Dissertation Repository

Signal transduction via heterotrimeric G proteins in response to transmembrane G protein-coupled receptors plays a central aspect in how cells integrate extracellular stimuli and produce biological responses. In addition to receptor-mediated activation of heterotrimeric G proteins, during the last few decades, accessory proteins have been found to regulate G protein activity via different mechanisms. Several proteins have been identified that contain multiple G protein regulatory domains. Using various molecular and biochemical approaches, we have characterized the effects of two such proteins, G18 and RGS14, on G protein activity. Both proteins contain a second G protein binding domain in addition to …

Structural Insights Into Dna Replication And Lesion Bypass By Y Family Dna Polymerases, Kevin N. Kirouac

Electronic Thesis and Dissertation Repository

Y family DNA polymerases are specialized enzymes for replication through sites of DNA damage in the genome. Although the DNA damage bypass activity of these enzymes is important for genome maintenance and integrity, it is also responsible for DNA mutagenesis due to the error-prone nature of the Y family. Understanding how these enzymes select incoming nucleotides during DNA replication will give insight into their role in cancer formation, aging, and evolution. This work attempts to mechanistically explain, primarily through X-ray crystallography and enzymatic activity assays, how Y family polymerases select incoming nucleotides in various DNA replication contexts. Initially, we sought …