Non-small cell lung cancer (NSCLC) patients respond poorly to generic chemotherapeutics. Despite recent advancements in the treatment of NSCLC, the overall five-year survival rate of NSCLC remains low at 14.6%. We have identified Immunoglobulin-containing and Cysteine-rich Receptor (IGCR-1) as a putative cell surface protein, which is expressed in lung epithelial cells. The main objectives of this study were to evaluate expression of IGCR-1 in normal lung epithelium and lung carcinoma. Our analysis of publicly available ATCG data via bioGPS revealed that IGCR-1 is highly expressed in human lung. Moreover, our immunohistochemical studies further confirmed that IGCR-1 is expressed in bronchial epithelial cells. Additionally, IGCR-1 is expressed in endothelial cells of blood vessels and localized on the cell surface of human embryonic kidney cells. Its cell surface localization suggests that IGCR-1 could be a cellular adhesion molecule (CAM) with roles in the tumor microenvironment. Of note, the analysis of a panel of human lung carcinoma cell lines via western blot analysis demonstrated that IGCR-1 is expressed at variable levels in these cell lines. Given the heterogeneity of NSCLSs and the corresponding differential expression of IGCR-1 in different lung cancer cell lines, IGCR-1 may play an important role in NSCLCs. / 2020-06-17T00:00:00Z

Osteoporosis (OP) is a chronic and progressive disease characterized by a decrease in bone mineral density (BMD), loss of bone tissue microstructure, and increased incidence of fracture. OP currently affects more than 10 million people in the United States, and its prevalence is expected to increase in the coming decades. This rising trend will add to the already $17 billion dollar per year healthcare industry which treats and rehabilitates those suffering from fragility related fractures. The causes of OP are multifactorial, with genetic and sex-dependent traits related to BMD and osteogenic cells having the largest effect on outcome. This study explored effective methods for culturing and inducing osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (MSCs). MSCs are multipotent progenitors cells, and of particular importance to the study of OP because of their contribution to bone and cartilage repair and regeneration throughout life. Specifically, our goals were to examine the relationships between osteoinductive medium containing dexamethasone (Dex) and collagen culture plate pretreatment on human bone marrow-derived MSC growth, differentiation, and mineralization. Bone marrow was obtained from the acetabular reamings of 10 patients (age range 51-68; sex: 6 female and 4 male) undergoing total hip arthroplasty. After isolation, cells were cultured in a basal medium of Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum and 10% penicillin/streptomycin. Plates were further designated as ±collagen pretreatment, and +collagen plates were treated with a thin layer of collagen prior to cell seeding. On day 6 post seeding, the basal medium was replaced with an osteoinductive medium of α-Modified Eagle Medium, 10% fetal bovine serum, 1% penicillin/streptomycin, 12.5µg/mL ascorbic acid and 8mM β-Glycerophosphate. Osteogenic conditions were designated as ±10-8M Dex. After a 21 day culture in osteoinductive medium, cells were fixed and biochemical assays were assessed for DNA, alkaline phosphatase (ALP) and mineralization content. Plain photographic and phase-contrast microscopy examinations of cell cultures showed an increased tendency of +Dex cultures to promote earlier proliferation, and greater osteogenic nodule formation as quantified by alizarin red staining. There were no apparent qualitative differences in culture plates pretreated with collagen. Biochemical assays showed no significant difference in DNA content between either the -Dex or collagen conditions compared to the control (p>0.05). However, there was a significant difference in DNA content between patients (p<0.01). The addition of Dex had a significant effect on the DNA normalized ALP content of cell cultures (p<0.05). There was no significant difference in ALP content between groups with collagen pretreatment (p>0.05), or across patients for either the -Dex group or collagen group as compared to the control (p>0.05). Neither Dex as an osteoinductive component nor collagen coating tissue culture plates had a significant effect on mineralization as measured by DNA normalized alizarin red stain content (p>0.05). There was also no significant difference in mineralization between patients for either the -Dex or collagen group as compared to the control (p>0.05). Our findings demonstrate the potential of Dex to induce in vitro MSC proliferation and differentiation, while cell culture plate pretreatment with collagen did not appear to have significant effects. Further study and larger sample sizes are needed to produce more statistically significant results and allow for examination of underlying patient genetics and co-morbidities.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The biochemical pathway for the conversion of pentoses to hexoses is readily demonstrated in erythrocytes because of the abundance of the enzymes transaldolase. The pathway of pentose-phosphate synthesis from nucleosides is of interest because it is a means of providing erythrocytes with hexose- and triosephosphates without the participation of adenosine triphosphate. In contrast to glycolysis, the major metabolic chemical sequence in the erythrocyte, the pentose pathway, can be considered to be an energy conserving one [TRUNCATED] / 2031-01-01

The unique chemical, optical and physical properties of inorganic nanocrystals have generated tremendous interest to develop a variety of applications, most importantly as novel probes in biology. Recent developments have advertised them as promising platforms for sensing, drug delivery, imaging cells and tissue, and as diagnostic tools. However, one impediment to achieve these goals has remained the lack of effective means to deliver them into the cytosol of live cells. A variety of techniques have been explored to achieve this goal including receptor mediated internalization, lipid-based transfection, electroporation and viral peptide-mediated delivery. Nevertheless, most if not all those approaches tend to sequester the nanoparticles within endocytic vesicles. This prevents them from reaching intracellular targets, thus limiting their use in cellular studies. Despite several strategies and efforts there is still a need for an easy and reproducible approach to deliver exogenous nanomaterials to cells directly via membrane translocation. In this dissertation, we summarize different approaches to overcome this issue by employing two distinct peptides to deliver nanoparticles into the cell cytoplasm through the plasma membrane. We also describe the use of gold nanoparticles to develop a potential platform for thiol sensing. In Chapter 1, we introduce the optical and physical properties of inorganic nanomaterials including, quantum dots (QDs), gold nanoparticles (AuNPs) and gold nanorods (AuNRs). We further briefly describe their syntheses approach followed by an overview of surface functionalization and bio-conjugation strategies employed to assemble colloidally stable and biocompatible nanoparticle-biomolecule conjugates. This followed by a brief discussion about various recent applications of nanoparticles in biology and biomedicine focused on imaging, biosensing, drug delivery and photothermal therapy. Finally, we conclude by presenting several approaches that have been applied for intracellular delivery of nanoparticles into the cell cytoplasm. In Chapter 2, we characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-imidazole coordination, where parameters such as NP size and number of attached proteins were varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ≈ 95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further describe the use of this system to develop a solution phase sensing platform targeting thiolate compounds. This is based on the use of Energy Transfer (ET) as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. We then demonstrate that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction and type of thiol derivatives used and also provide a measure for the equilibrium dissociation constant (Kd-1) for these compounds. In Chapter 3, we describe a new quantum dot (QD)-conjugate prepared with a lytic peptide, derived from a non-enveloped virus capsid protein, capable of bypassing the endocytotic pathways and delivering large amounts of QDs to living cells. The polypeptide, derived from the Nudaurelia capensis Omega virus, was fused onto the C-terminus of maltose binding protein that contained a hexa-HIS tag at its N-terminus, allowing spontaneous self-assembly of controlled numbers of the fusion protein per QD via metal-HIS interactions. We illustrate how the efficacy of QD-peptide conjugate uptake by several mammalian cell lines was substantial even for small concentrations (10-100 nM). Upon internalization the QDs were primarily distributed outside the endosomes/lysosomes. We further provide evidence indicating an entry mechanism that does not involve endocytosis, but rather the perforation of the cell membrane by the lytic peptide on the QD surfaces. In Chapter 4, we propose the use of a chemically-synthesized anticancer peptide, SVS-1, as an efficient vehicle to promote the rapid delivery of ligated quantum dots across the cell membrane and directly into the cytoplasm of live cells. We describe the assembly of QD-SVS-1 bioconjugates by functionalizing the QD surface with maleimide groups, which were then subsequently reacted with the N-terminal thiol of a cysteine containing SVS-1 analogue (CGG-SVS-1) to form a stable thioether linkage. We provide epi-fluorescence, confocal microscopy, and flow cytometry data, combined with specific endocytosis inhibition measurements to demonstrate that conjugates stain the cytoplasm, without interactions with endosomes or the nuclei. We have also provided QD-conjugate internalization data collected by live cell imaging as supplemental files. Finally, in Chapter 5, we demonstrate the use of SVS-1 to promote non-endocytic uptake of both small size gold nanoparticles (AuNPs) and larger size gold nanorods (AuNRs) into mammalian cells. We describe the preparation of colloidally stable AuNPs and AuNRs with an amine-functionalized polymer, His-PIMA-PEG-OCH3/NH2, as their capping ligand. Subsequently, the amine groups were utilized for covalent attachment of cysteine terminated SVS-1 (via a thioether linkage) and NHS-ester-Texas-Red dye onto the nanocrystal surface. We further demonstrate nanocrystal staining throughout the cytoplasmic volume of the cells incubated with these conjugates via fluorescence microscopy. We further provide additional endocytosis inhibition experiment results to confirm that physical translocation of these conjugates takes place through the cell membrane independent of endocytosis. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / May 9, 2018. / Includes bibliographical references. / Hedi Mattoussi, Professor Directing Dissertation; Samuel C. Grant, University Representative; Brian Miller, Committee Member; Hong Li, Committee Member; Joseph B. Schlenoff, Committee Member.

Using a bioinformatics approach to explore the oxidation chemistry of peroxidases in microorganisms, I generated a sequence similarity network (SSN) of proteins that fall under the umbrella of diheme peroxidases. Bacterial cytochrome c peroxidases (bCCPs) are responsible for the electrochemical conversion of H2O2 to water by use of two c-type heme prosthetic groups. Despite structural similarity to bCCPs, the diheme enzyme MauG from P. denitrificans does not catalytically convert H2O2 to water. MauG, part of the mau operon, instead utilizes H2O2 to generate a highly unusual bis-Fe(IV) intermediate for the biosynthesis of tryptophan trpytophanyl quinone (TTQ), the cofactor required for methylamine dehydrogenase (MADH). The results within this thesis provide evidence for unreported diheme proteins conserved in all strains of Burkholderia, a gram-negative bacterium with implications in cystic fibrosis. Sharing sequence similarity to MauG, the Burkholderia orthologs separate into two classes referred to as Class A and Class B, but genomic analysis reveals a lack of a mau operon for all species. Instead these diheme enzymes are highly conserved downstream of a putative phosphatase protein partner. The work presented in this thesis expands our knowledge of the peroxidatic chemistry of heme containing proteins in microbes. The results for the biochemical and biophysical characterization of Class A enzyme BthA and Class B diheme enzyme BthB from non-pathogenic B. thailandensis prove BthA and BthB are dual functioning peroxidases, capable of both H2O2 reduction and formation of the same bis-Fe(IV) intermediate proposed for MauG. Mutational analysis of the six-coordinate heme ligand of BthA, Tyr463, addresses the electrochemical and spectroscopic properties important in the dual functionality, as well as provides evidence for a role of BthA in O2 reactivity. Furthermore, the biochemical characterization of PhosA, the putative protein partner of BthA based on genomic analysis, confirms presence of a dimetal Fe-Zn center reminiscent of purple acid phosphatases, and reactivity toward nucleotide diphosphates. While studies to understand the biological role of BthA and PhosA are still underway, results from the biophysical characterization of both proteins supports a role of the Class A system in unprecedented chemistry. / 2020-11-27T00:00:00Z

Through oral contact, saliva can be transferred to a variety of objects commonly encountered in forensic casework, such as cigarette butts, condoms, drinkware, masks, clothing, and vaginal or penile swabs. As saliva does not require external stimulation for secretion, it is readily available for exposure to the external environment. For this reason, the detection and analysis of saliva are relevant to many types of crime, including but not limited to sexual assaults, kidnappings, homicides, and burglaries. Although the presence of saliva does not serve as a definitive indication of crime, it can link individuals to a crime scene or serve as evidence of physical contact. When sufficient quality and quantity of saliva is present at a crime scene or on an item of evidence, it typically serves as a source of DNA.1 Therefore, detection of saliva is also pertinent to the identification of potential victims and perpetrators. Currently, forensic identification of biological stains is limited to individual body fluids, such that analysis of a mixture containing multiple body fluids will require a separate test for each biological component. In addition, all routine forensic assays for saliva are of a preliminary nature. Due to the lack of confirmation techniques for saliva, surface-enhanced Raman spectroscopy (SERS) emerges as a potential technique for rapid, sensitive, and confirmatory identification of saliva stains. This study is designed to examine the efficacy of a SERS based methodology for the detection, characterization, and identification of human saliva. Using this technology, the major molecular component detected in saliva was salivary thiocyanate. A three-peak thiocyanate SERS signature was observed at 440 cm-1, 740 cm-1, and 2107 cm-1 and is recommended for use as a biomarker in the detection and identification of liquid and dried saliva. Assessment of validation parameters was also performed to determine the robustness, accuracy, and sensitivity of this methodology. Examination of donor variability among and within individuals demonstrates variability in salivary thiocyanate concentrations. As a result, the sensitivity of this technology is limited by the inherent thiocyanate concentrations of individuals. In the samples utilized for this study, SERS could readily detect salivary thiocyanate at concentrations as low as 1/100. Classification of spectral data from 12 donors indicates some donor variability arising from differences in protein content. However, deviations in matrix proteins do not hinder the identification of saliva because this methodology uses a salivary thiocyanate SERS signature for the basis of saliva detection. A preliminary sample aging study demonstrates that saliva can be rapidly identified in 20-month old samples aged at room temperature. This suggests that the salivary thiocyanate SERS signature is stable without the need for controlled environments and, therefore, highly suitable for use in forensic detection of human body fluids. At many crime scenes, biological fluids are present in a mixed sample consisting of human fluids such as saliva, semen, blood, and vaginal fluid. Salivary mixtures with blood and vaginal fluid were respectively analyzed to assess the efficiency of this SERS methodology in detecting and identifying saliva from the mixture. As thiocyanate is also an inherent component of blood, the ability to identify saliva from a salivary mixture with blood is limited to mixtures where saliva is a predominant component. For samples used in this study, saliva identification is limited to mixtures containing 75% saliva and 25% blood. As vaginal fluid does not contain salivary thiocyanate, this SERS methodology can readily identify salivary thiocyanate from mixtures containing 1% saliva and 99% vaginal fluid extract. This study examined two potential substrates and their corresponding efficiencies in body fluid detection for forensic casework. The thiocyanate signature is readily observed on both gold (Au) and silver (Ag) substrates. With respect to sensitivity, Ag substrates are more sensitive and capable of detecting thiocyanate in lower concentrations of saliva. Au substrates exhibit greater selectivity for other salivary components at low thiocyanate concentrations, which can be useful in characterizing the protein content. In view of all experimental results, this study demonstrates that SERS is a rapid and sensitive technique capable of detecting and identifying saliva among complex body mixtures for forensic science applications.

<p> In spite of the advances made in experimental and mutational studies, understanding the importance of remote interactions is crucial for refining the knowledge of enzyme catalysis. In this study, a model system for Glu97 was developed in <i>Homo sapiens</i> triosephosphate isomerase (<i> h</i>TIM) to investigate the energetic contribution and structural role of this fully conserved glutamate residue in the TIM-catalyzed isomerization reaction. Recombinant human triosephosphate isomerase (<i>h</i>TIM) was altered using site-directed mutagenesis, in which an aspartate, glutamine, or alanine residue was substituted for Glu97. In steady-state kinetics, the E97D variant exhibited the most significant catalytic activity while the E97Q enzyme was the least active. Observing both the forward and reverse directions of the TIM-catalyzed reaction, the results revealed that the enzymatic activity for E97D and E97A TIM was diminished by ~3-fold or less, and the rate was essentially unchanged for the E97D mutation. The E97Q mutant observed a greater rate effect, ~10-fold decrease in <i>k_cat</i> and ~20-fold decrease in catalytic efficiency (kcat/ K_M). To determine the conformational stability of the WT and mutant <i>h</i>TIM, unfolding of all four enzymes was monitored by circular dichroism, tryptophan and ANS fluorescence spectroscopy. The dimer stability was evaluated by gel-filtration analysis and the mutants showed similar chromatograms compared to that of the WT. The similar behavior observed for the WT and E97D suggests that the Asp mutation has little effect on catalysis, enzyme stability, and the unfolding pathway. On the contrary, the statistical significance observed in the E97Q and E97A mutants suggests that the Gln and Ala mutations affect the stability of the structure and may affect the unfolding pathway. Overall, these point-mutations support the model that remote interactions of Glu97 may have a modest role in catalysis. One explanation is that the direct role of Glu97 may have evolved in the human species and plays a less significant role compared to earlier species in evolution in which Glu97 mutations showed larger rate effects. Possibly, the network of unfavorable interactions is reduced and therefore, the mutational effect of Glu97 is less deleterious in <i>h</i>TIM. </p><p>

The term "diabesity" is a pandemic that is threatening populations worldwide and is the term is finding itself as a household name, fueling itself through the high-fat diet and sedentary lifestyle. As a result of these living choices, the population is suffering from lipotoxicity, the underlying cause of Type 2 Diabetes Mellitus (T2DM) and insulin resistance. Lipolytic control resides around the rate-limiting enzyme, adipose triglyceride lipase, which is normally downregulated in the insulin-stimulated state. This is the signal that is aberrant in patients who have T2DM and could be a significant factor in lipotoxicity. This state of lipotoxicity leads to many complications, increasing the risk of heart disease, heart attack, blindness, nephropathy, neuropathy, and stroke. Vasculature damage also can lead to poor perfusion of the lower extremities, increasing the risk of ulcer, gangrene, and amputation of the foot. An area of research delving into this issue lies within the mammalian target of rapamycin complex 1 (mTORC1), as it mediates the antilipolytic signal and decreases ATGL expression. This discovery was further clarified when the Kandror lab identified the transcription factor early growth factor 1 (Egr1) as the protein that binds to the promoter region of ATGL and downregulates its transcription. Egr1 has also been thought to be a factor in the development of insulin resistance in the hyperinsulemic state. To date, we have experimented upon murine 3T3-L1 and human adipocytes. We have developed a double transgenic mouse positive for reverse tetracycline transactivator (rtTA) and myc-tagged ras homolog enriched in brain (Rheb), which is a misnomer as it is located in adipose tissue (AT). With the mTORC1 axis hyperactive, we seek to show that Egr1 is a rich target in diabetes and lipotoxicity, regulating and inhibiting ATGL levels through mTORC1.

Prostate cancer is the most common non-cutaneous malignancy in men and is the second leading cause of cancer death in American men, trailing only lung cancer. About 1 man in 7 will be diagnosed with prostate cancer and about 1 in 38 will die of prostate cancer. Prostate cancer does not usually present any symptoms until it has advanced or metastasized and thus screening for prostate cancer is an arduous task. Three of the most common techniques used to screen for prostate cancer includes digital rectal exam, transrectal ultrasound, and the use of biomarkers, specifically Prostate Specific Antigen (PSA), which has proven controversial. Due to the need for a more rapid, specific marker for the early detection of prostate cancer, this study aims to identify a new biomarker for prostate cancer. A novel strategy to identify a protein biomarker for prostate cancer was explored, a highly specific hybridoma against the novel biomarker was generated, the efficacy of the biomarker detection tools in prostate cancer was observed and an attempt to identify the biomarker protein sequence was made. Every time a prostate cancer specimen was tested, it was found that the clone 164 antibody that was generated was able to identify unique antigens in the prostate cancer tissue that were not evident in normal tissue. In addition, it was noticed that the clone 164 antibody could identify the marker protein in urine as well. It is believed that the clone 164 antibody is highly specific for early stage prostate cancer diagnosis. Finally, using mass spectrometry, four candidate protein biomarkers that clone 164 recognizes were isolated, with the closest match being Ig alpha-1 chain C region. It is believed that the antigens recognized by clone 164 promises great potential as a future biomarker for prostate cancer. Since the protein is only seen in the urine of patients with prostate cancer, it appears that the clone 164 antibody is suitable to include in a device that can be used in a urine-based, rapid diagnostics point of care kit. Future steps involve animal studies before proceeding to the next step of clinical trials. If the clone 164 antibody identified biomarker proves successful, the respective biomarker protein can be analyzed in detail. Once the expression profile of this biomarker is elucidated, it can be compared to the normal prostate DNA and may help in determining the location in the DNA, which may eventually lead to the idea of treating prostate cancer through gene therapy or the possibility of preventing or curing prostate cancer. Also, the specific antibody against this biomarker can be used as a preventive agent by humanizing this antibody and using it as a therapeutic vaccine.

<p> Dynamic Kinetic Selection (DKS) suggests that kinetic, rather than thermodynamic, stability will dictate the composition of a replicating population of biomolecules. Here, the results obtained from a series of five related reactions involving gradually increasing percentages of randomly-mutated substrate fragments to generate variants of full-length <i>Azoarcus</i> group I intron through an autocatalytic self-assembly reaction involving a series of recombination events, showed DKS as a driving factor in dictating the population composition of full-length product assembled from substrates that had fewer positions available to randomization. </p><p> In trying to elucidate a plausible scheme for the origins of complex biomolecules on the prebiotic Earth, the suggestion that networks comprised of interacting molecules were more likely to evolve into biomolecules capable of obtaining and sustaining characteristics attributed to living molecules has gained traction within the past few years. Of specific interest is the catalytic efficacy of ribozymes whose genotypes require that they interact with molecules of the same genotype (selfish systems) to be effective catalysts versus those that are more effective when accomplishing catalysis by cooperating with ribozymes of a different genotype (cooperative systems). Here, the <i> Azoarcus</i> I ribozyme was used to compare these two types of system. Both systems were shown to robustly produce full-length product. Two different methods of introducing random mutations into substrate fragments for the reactions described in this thesis were employed. The differences in the preparation methods for the substrates was not expected to have an impact on the nature of the full-length product. However, there was no correlation between the positions that tended to be more tolerant of accepting random mutations between the products arising from the two preparation methods. One preparation method yielded full-length ribozymes more consistent with the secondary structure of the wild-type ribozyme and followed substitution patterns found in <i> in vivo</i> nucleic acid substitutions, whereas the other method provided full-length ribozymes that tolerated mutations that would be expected to greatly affect the secondary structure of the ribozyme and those positions tended to mutate evenly to any of the three possible alternative nucleobases. </p><p> Point mutations introduced into ribozyme substrate fragments may have a deleterious, neutral, or beneficial effect, depending on their impact on the catalytic capability of the molecule vis-&aacute;-vis the effect, if any, the change has to the secondary and tertiary structure of the ribozyme. In this dissertation, the results of two series of point mutation reactions are addressed. The first set showed a point mutation to have a deleterious effect, whereas concerted mutations did not significantly affect activity of the ribozyme. The second series of reactions involved point mutations at a position that had previously been determined to be highly tolerant of random mutations. Results suggested that substitutions at this position had a minimal impact on ribozyme activity.</p><p>