Glioblastoma multiforme (GBM) is an incurable brain tumor, with patients only expecting to live 15 to 16 months post-diagnosis with the most current treatments options including surgery, chemotherapy, and radiation. How GBM resist treatment is still not very well known. However, cancer stem cells (CSCs), a subset of cells in GBM tissue considered responsible for therapeutic resistance and the poor patient prognosis. NANOG, a homeobox gene, is responsible for maintaining pluripotency of embryonic stem cells and is observed in CSCs. NANOGP8, a retrogene in the NANOG family is expressed in CSCs and provides cells with stem-like characteristics previously observed in stem cells. Thus, we hypothesize that NANOGP8 is not only useful as diagnostic and/or prognostic marker, but a target to improve the efficacy of current GBM treatments since it regulates signaling pathways responsible for cell proliferation. I will investigate the mechanisms in which NANOGP8 expression starts in CSCs and increase TMZ efficacy by silencing embryonic stem cell genes in Glioblastoma. As a retrogene, NANOGP8 derive from reverse transcription of the parent gene NANOG and lacks the promoters for expression in cells. We expect to detect some modifications to the upstream sequence of NANOGP8 that may serve as initiators of expression, such as an insertion of a promotor. Although NANOGP8 has similar function to original NANOG parent gene, NANOGP8 may also have additional oncogenic functions making CSCs more resistant to therapy.

Melanoma is one of the most aggressive and inherently resistant cancers and the most dangerous skin cancer. While it accounts for fewer than 5% of skin cancer cases, 80% of skin cancer related deaths are attributed to melanoma. While resection remains the gold standard for melanoma treatment, surgery is only effective in providing local control of the disease if the cancer is detected in the early stages. Once melanoma enters the later stages, and particularly in the metastatic phase, recurrence is probable, and no adequate treatment exists. Previous work in this group has shown that photodynamic therapy (PDT) presents an opportunity to induce cell death in melanoma cells through the production of ROS and singlet oxygen at doses high enough to overwhelm the resistance mechanisms of the cancer. In this study, we investigated the use of recombinant SNAP-tag-based antibody fusion proteins as a means of delivering phototoxic molecules directly to cancer cells expressing the CSPG4 and PD-L1 cell surface receptors. SNAP-tag is an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase. It reacts autocatalytically and in a strictly 1:1 coupling chemistry with substrates that have been modified with benzylguanine (BG). Through genetic fusion of this self-labelling protein with a tumour targeting antibody, we developed a recombinant immunoconjugate able to carry BG-modified photosensitizers to selectively target and eliminate malignant melanoma cells. Conjugation of the SNAP-tag fusion protein with the fluorescent dye Alex Fluor 488 showed that anti-CSPG4-SNAP binds specifically to melanoma cells expressing the CSPG4 surface antigen. Binding was tested across a range of cell lines presenting melanoma in its radial and vertical growth phases, in the metastatic growth phase, in its chemoresistant form, and in both its pigmented and unpigmented forms. This binding data thus confirms CSPG4 as a suitable targeted for this treatment strategy. Conjugation of the fusion protein with the BGmodified photosensitizer IRDye 700DX (IR700) has produced no phototoxicity as of yet. In light of the convincing binding analysis, it is concluded that inefficient solubilization of the lyophilized product resulted in inadequate conjugation of BG-IR700 with SNAP-tag. Nonetheless, steps have been planned to resolve the problem in future ongoing work on this project, and we remain confident in the applicability of this technology. The results for the PD-L1 fusion protein were inconclusive. In summary, SNAP-tag technology offers a simple and efficient method for immunofluorescent detection of cancerous cells. These fusions proteins are versatile as they 1) can contain any antibody targeting a tumour-associated or tumour-specific antigen of choice and 2) can be endowed with a wide variety of substrates, as long as the latter contains the BG moiety.

The native polyamines (putrescine, spermidine, and spermine) are low molecular weight amines that exist as polycations at physiological pH. These polycations interact with DNA, RNA, and influence many cellular processes. Intracellular polyamine levels are maintained via a balance of polyamine biosynthesis, catabolism, and transport. Ornithine, an amino acid obtained from L-arginine, is decarboxylated by ornithine decarboxylase (ODC) to form putrescine. The biosynthesis of the higher polyamines, spermidine and spermine, requires the addition of an amino-propyl group donated by decarboxylated S-adenosylmethionine (dc-SAM), which itself is derived from the amino acid L-methionine (Met). Tumor cells are heavily reliant on methionine because it is required for the translation of proteins, methylation of biomolecules and anabolic processes like polyamine biosynthesis. Pancreatic ductal adenocarcinoma cells (PDAC) are heavily reliant upon polyamines, and one way to affect the growth of PDAC cells is to reduce their intracellular methionine pools. LAT1 is the principal transporter of methionine into human cells and this thesis describes the discovery of a new methionine efflux agonist, which uses LAT1 to export methionine into the extracellular space. As expected, this agonist reduced both intracellular methionine and polyamines levels and inhibited cell growth of L3.6pl human pancreatic cancer cells. The agonist contains two chiral centers. Utilizing organic synthesis, the four possible stereoisomers (RR, SS, SR, and RS) were synthesized and. evaluated for their ability to inhibit L3.6pl cell growth, deplete intracellular methionine levels, and reduce intracellular polyamine pools. A success here may lead to new drugs which target pancreatic cancers via methionine starvation.

Transplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate regeneration following myocardial infarction (MI), however, the role of exosomes derived from ES cells (ES-Exo) in activating resident cardiac stem cells (CSCs) to undergo cardiac differentiation is not established. In the present study, Stem cell antigen 1 positive (Sca-1+ve) CSCs were isolated, incubated with exosomes, and evaluated for differentiation into the major heart cell types in vitro. Observations of in vitro cardiac differentiation were further established in an in vivo model of MI. Ligation of the coronary artery, or a sham surgery was performed in C57BL/6 mice 8-12 weeks of age. Mice were split among four study groups: sham, MI, MI + H9c2-Exo (a cell line control), & MI + ES-Exo. ES-Exo were transplanted via intramyocardial (IM) injection immediately following coronary artery ligation. At day 14 (D14), chocardiography was used to evaluate cardiac function. Differentiation into the major heart cells was determined by sarcomeric α-actin (cardiomyocytes) and smooth muscle α-actin (vascular smooth muscle cells) immunostaining. Hematoxylin and Eosin and Masson’s Trichrome staining assessed cardiomyocyte hypertrophy and fibrosis, respectively. Immunostaining for major heart cellular markers revealed significant activation of resident Sca-1+ve CSCs to undergo cardiac differentiation after ES-Exo treatment. Cardiomyocyte hypertrophy and myocardial fibrosis were significantly increased following coronary artery ligation. Results from histological staining revealed significantly decreased levels of hypertrophy and fibrosis in hearts transplanted with ESExo following coronary ligation. In summary, our findings advocate ES-Exo as a viable treatment option to repopulate the myocardium with viable heart cells, attenuate cardiac remodeling, and rescue cardiac function.

Translational frameshifting is a process that leads to the production of multiple different proteins from a single mRNA. Programmed translational frameshifting is typically utilized by retroviruses to increase genome coding capacity and to control the ratio between proteins necessary for different infection stages. In E. coli, translational frameshifting regulates the production of several essential proteins, including the replication/clamp loader subunits tau (τ) and gamma(γ), which are products of the gene dnaX. In E. coli, τ is an essential auxiliary factor of DNA polymerase III holoenzyme. τ can dimerize Pol III and interact with the DnaB helicase, which allows synthesis of leading and lagging DNA strands. The role of γ is unknown since it does not share similar properties with τ apart for clamp loading; but, γ is implicated in DNA repair and likely has key role in polymerase switching. In this study, the effects of ribosomal availability in the cell on the dnaX frameshift frequencies (regulation of τ/γ) were investigated. It was found that lower ribosomal availability in the cell resulted in lower frameshift frequencies on reporters containing the dnaX frameshift motif. Also, E. coli strains with tagged genomic dnaX and disrupted ompT were generated. Although, it is generally accepted that τ/γ are produced at equimolar concentrations, it was discovered that endogenous γ was produced at substantially higher concentrations than τ when cells were grown in defined media (EZ MOPS). This finding shows that that the ratio of τ/γ can be regulated. These findings suggest there are yet uncharacterized roles of γ, such as polymerase switching, that may be the reason for its production in many bacteria.

Ribosomes translate messenger RNA (mRNA) three nucleotides at a time until translation is terminated at a stop codon. However, during all translation, frameshifting can occur, leading to the formation of proteins with amino acid sequences that differ from the in- frame product. Spontaneous frameshifting can be harmful to an organism. For instance, antibiotics such as streptomycin inhibit bacterial growth by increasing misreading and frameshifting. However, programmed translational frameshifting (which can induce high levels of frameshifting) can be used in some instances to control the ratio of specific proteins (as seen with the dnaX gene) or to increase the density of genomic information. This study explored the effects of endogenous small molecules on the IS3-frameshift-motif that is found in the transposase genes of many mobile elements. Using a cell-free protein synthesis system and a luminescent frameshift reporter, it was discovered that the addition of a small molecule extract derived from E. coli significantly decreased frameshifting, suggesting that it contains molecules that can alter translational fidelity. These experiments also revealed that the addition of the translation inhibitor chloramphenicol to translation assays at sub-inhibitory concentrations, reduced frameshift efficiency. During the studies, the role of the stability of luminescent protein reporters on the reported frameshifting levels was also explored, which is omitted in many studies. Finally, a method was developed that allows for the isolation of molecules that weakly associate with ribosomes, which opens the door for more detailed investigations of chemicals that alter translational fidelity. In conclusion, these studies provide new insight on the potential modulation of translational frameshifting by endogenous small molecules, and they set the stage to reveal the important players in this important biochemical process.

Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections worldwide and is also responsible for trachoma, the leading cause of preventable blindness. Despite being susceptible to most antibiotics, Chlamydia trachomatis is an enormously successful pathogen capable of causing serious health issues. Chlamydia trachomatis harbors a type III secretion system which delivers chlamydial proteins (a.k.a. effectors) into the mammalian host cell during the initial stages of infection and throughout the subsequent intracellular developmental cycle. One of the early secreted effectors is called translocated early phosphoprotein (TepP). In order to gain a better understanding of TepP, we employed Drosophila melanogaster as a model organism to identify novel gene functions. Transgenic fruit flies engineered to express TepP in designated tissues were used to identify macroscopic and/or microscopic phenotypic changes to Drosophila development. The fruit fly model allowed us to explore potential roles for TepP in disease progression. Using this in vivo model, a border cell migration defect was observed in the Drosophila egg chambers in response to the Chlamydia trachomatis effector protein TepP. Through knockdown and overexpression studies as well as qPCR we identified a likely interaction in the PVF1/PVR pathway between TepP and Crk to cause this migration defect. Development of this in vivo model provides a platform for rapid and efficient investigation of bacterial effector proteins of unknown function and interrogation of the mechanisms by which these proteins interact with host pathways to contribute to disease. By developing a better understanding of how chlamydial effectors contribute to disease we are able to identify novel targets to explore for prevention of C. trachomatis infections.

In recent years, there is a growing interest in developing metal based antimicrobial nanomaterials suitable for agricultural and biomedical applications. For centuries, Copper (Cu) biocide has been used for protecting a wide variety of crops from devastating bacterial and fungal diseases. However, prolonged and aggressive use of Cu led to the development of resistance and accumulation in soil. The latter has been linked to aquatic toxicity due to soil Cu leaching. Furthermore, copper build up in soil causes phytotoxicity and reduces uptake of micronutrient Zn through the root system. In biomedical field, Cu has been historically used as an antimicrobial agent in wound dressing. In healthcare facilities, Cu based touch surfaces are shown to significantly reduce antimicrobial infection rates. Emerging biomedical applications include wound healing, sensing and even potential use of nano-Cu as cancer therapeutic. However, Cu cytotoxicity is a serious concern. There is a strong need for developing advanced Cu based composite material that will retain antimicrobial properties as reduced Cu load until a suitable alternative becomes available. In this thesis, the objective is to develop a Cu coated Iron Oxide nanocomposite. The idea is to distribute Cu over the high surface area of biocompatible Iron Oxide nanoparticle (IONP) to improve Cu bioavailability. In the design of Cu-IONP nanocomposite, we have introduced N-Acetyl Cysteine (NAC, an antioxidant biomolecule) that anchors IONP to Cu. The composite was synthesized using a co-precipitation technique. Characterization of the Cu/NAC-IONP nanocomposite was done using Atomic Absorption Spectroscopy (AAS), Dynamic Light Scattering, Fourier Transform Infrared Spectroscopy (FTIR), Scanning electron microscopy and X-Ray Photoelectron Spectroscopy. Characterization results support the formation of composite and NAC as a bidentate ligand conjugating Cu to IONP. Antimicrobial activity of Cu/NAC-IONP nanocomposite was studied using ASTM published protocol. The Cu/NAC-IONP nanocomposite shows higher Colony Forming Unit Percent reduction when compared with NAC-Iron Oxide and no treatment. This suggests that antimicrobial activity of Cu is retained in the nanocomposite. Additionally, AAS study revealed an interesting property of the nanocomposite that the Cu release is strongly dependent on incubation temperature. Cu ion release is increased with the increase in incubation temperature. This new finding may lead to design of a delivery system where Cu release can be controlled by tuning temperature.

Actin is an essential cytoskeletal protein that plays key roles in several cellular functions such as phagocytosis and cell motility with the help of actin binding proteins (ABPs). Gelsolin is a calcium regulated ABP that severs and caps actin filaments. Gelsolin helps control actin filament assembly dynamics that are required for cell survival. Cleavage products of gelsolin lead to Familial Amyloidosis, Finnish type, and conformational changes to gelsolin are implicated in disease progression. The majority of in vitro studies of gelsolin and actin have been performed in dilute buffer conditions which do not simulate the molecular interactions occurring in the intracellular environment. The intracellular space is packed with many macromolecules such as carbohydrates and other proteins. These macromolecules induce steric hindrance and excluded volume effects and have been shown to alter protein-protein interactions and protein conformations. We hypothesize that gelsolin and actin filaments present in crowded environments will produce greater gelsolin severing activity due to steric hinderance and induced conformational changes. To test this hypothesis, we have visualized actin filament severing by gelsolin in solution with macromolecular crowders utilizing total internal reflection fluorescence (TIRF) microscopy. Steady-state average filament lengths and filament length distributions were analyzed to determine the effect crowding has on gelsolin-mediated filament severing. Real-time filament severing assays visualized by TIRF allowed us to compare gelsolin's severing efficiency in the presence of crowders to those in dilute buffer conditions. Co-sedimentation assays were performed in order to determine the effect of crowding on gelsolin binding to actin filaments. Taken together, this study demonstrates that macromolecular crowding modulates gelsolin-mediated actin filament severing activities, offering insights into the interactions between actin and gelsolin inside the cell. These insights will deepen our understanding of in vivo cytoskeletal regulation which is linked to cell physiology and may aid researchers studying actin-related diseases.

Current U.S. National guidelines allow for the collection of sexual assault evidence up to 5 days after the incidence occurs. In these cases, the ability to obtain an autosomal STR (aSTR) profile of the male donor in these cases diminishes as the time interval increases. This inability to recover an aSTR profile from the semen donor is not due to a complete lack of sperm cells, as studies have frequently shown that sperm persists in the vaginal canal or cervix up to 10 days post coitus. Thus, the inability to recover an aSTR profile of the sperm donor is likely due to a low quantity of sperm cells and/or degradation of sperm cells, which pose significant problems to existing DNA extraction and typing methods. A typical DNA workflow for this type of evidence in a forensic casework laboratory includes the use of a differential extraction to separate sperm cells from non-sperm cells. These often harsh extraction methods can cause degraded and fragile sperm cells to be prematurely lysed into the non-sperm cell fraction. The significant amounts of vaginal epithelial cells in the sample can overwhelm the minute number of sperm cells present in this fraction, resulting in a complete masking of the male profile. For most sexual assault samples collected more than 48-72 hours after an incident, Y-STR analysis might be used instead of aSTR analysis, as it allows for an increased time frame of DNA recovery by detecting only the male donor Y-haplotype, circumventing the need for a differential extraction and avoiding potential competition during amplification. However, Y-STR loci are part of the non-recombining region of the Y-chromosome, and thus do not have the same discrimination power of aSTR loci. Therefore, enhanced methods for the recovery of sperm cells that allow researchers to bypass the limitations of a typical DNA workflow and obtain an aSTR profile need to be developed. This study aimed to do so by utilizing enhanced, non-standard methods to collect individual sperm cells via direct physical recovery from semen containing samples. Optimized direct lysis and micro-volume aSTR amplification were also used in order to obtain DNA profiles of the sperm donor. The quality of the obtained DNA profiles was evaluated using metrics such as allele recovery, stutter occurrence and percentage, and drop-in allele levels. Using these developed methods, the ability to analyze single sperm cells was demonstrated and the minimum number of sperm cells required in order to obtain probative and reliable DNA profiles was determined.