The aim of this thesis was to establish the therapeutic value of the IFITM1 monoclonal antibodies and to design and develop therapeutically valuable recombinant monoclonal antibodies so as to study the implication of these novel antibodies in cancer therapy. Cancer metastasis is one of the main interests that has given rise to the design and development of innovative strategies for cancer therapeutics. The Interferon Induced Transmembrane Protein 1(IFITM1), a notable member of the IFITM family of proteins has been identified as one of the most up-regulated trans-membrane proteins in metastatic breast cancer and cervical adenocarcinoma. This interferon-regulated protein is also involved in cell migration, invasion in glioma and squamous cancers. This PhD aimed to study IFITM1 as a pro-invasive cancer target by the use of IFITM1 monoclonal antibodies that were raised against the extracellular domain of the human IFITM1 gene. The epitope mapping of IFITM1 revealed the binding activity of the IFITM1 monoclonal antibody. This gave the opportunity to design and generate to new IFITM1-specific molecular tools, in the form of recombinant IFITM1 targeted murine scFv antibody, IFITM1-CPG2 yeast fusion protein antibody for potential application in ADEPT as well as a Mouse-Human Chimeric IFITM1 antibody secreting mammalian cell line. The immunohistochemical staining of IFITM1 in tissue micro array from breast, colon and oeosphegal cancer has revealed that the majority of these cancers produce this protein. However, IFITM1 is over produced in cervical cancer indicating it’s selective over expression in cervical cells. This PhD endeavored to investigate the expression of IFITM1 at a translational and transcriptional level and to study the clinical significance of IFITM1 in cervical cancer. The antibody dependent cell mediated cytotoxic activity of the chimeric IFITM1 antibody was found to be cytotoxic to SiHa cells in vitro. In the future these molecular tools could be used to regulate and further characterize the activity of this transmembrane protein antibody. In an effort to better understand the mechanisms that regulate the activity and the over production of the IFITM1 gene and its interacting proteins, a proteomic screen of cervical cancer cells was carried out using data-independent SWATH-MS on an AB SCIEX TripleTOF™ mass spectrometer. This Mass Spec analysis provided us with a host of IFITM1 biomarkers and revealed that the IFITM1 gene and its binding proteins also cross link with the IRF1 pathway. The data presented in this thesis, demonstrates that the IFITM1 gene can be targeted to either stimulate or inhibit IFITIM1 signaling to engage IFITM1 as a potential pro-invasive extracellular receptor as a target in antibody cancer therapy. In summary, this thesis aimed to confirm the activity and the binding specificity of the IFITM1 antibody. Additionally, this thesis demonstrated a promising application of the recombinant antibody in the ADEPT technology. Characterization of IFITM1 mAb effector functions indicated that the antibody was cytotoxic to cervical cancer cells. This highlights an important element in the immune suppressive tumour microenvironment. And finally, this thesis also provides the basis for the production of recombinant mouse human chimeric antibodies that are a part of a new group of immunotherapeutic molecules paving the way for cancer therapeutics.

Pancreatic ductal adenocarcinomas (PDACs) are highly aggressive malignancies, associated with poor clinical prognosis and limited therapeutic options. The KRAS oncogene is mutated in over 90% of PDACs and plays a pivotal role in tumor progression. Global gene expression profiling of PDAC reveals 3-4 major molecular subtypes with distinct phenotypic traits and pharmacological vulnerabilities, including variations in oncogenic KRAS pathway dependencies. PDAC cell lines of the aberrantly differentiated endocrine exocrine (ADEX) subtype are robustly KRAS-dependent for survival. The KRAS gene is located on chromosome 12p11-12p12, a region amplified in 5-10% of primary PDACs. Within this amplicon, we identified co-amplification of KRAS with the STK38L gene in a subset of primary human PDACs and PDAC cell lines. This provided rationale to determine whether PDAC cell lines are dependent on STK38L expression for proliferation and viability. STK38L (also known as NDR2) encodes a nuclear Dbf2-related (NDR) serine/threonine kinase, which shares homology with Hippo pathway LATS1/2 kinases. We show that STK38L expression levels are elevated in a subset of primary PDACs and PDAC cell lines that display ADEX subtype characteristics, including overexpression of mutant KRAS. RNAi-mediated depletion of STK38L in a subset of ADEX subtype cell lines results in decreased cellular proliferation and increased apoptotic cell death. Concomitant with cytostatic and cytotoxic effects, STK38L depletion causes increased expression of the LATS2 kinase and the cell cycle regulator p21. LATS2 depletion partially rescues the cell proliferation and viability effects of STK38L depletion. Lastly, high STK38L mRNA expression is associated with worse patient prognosis compared to low STK38L expression in PDACs. Taken together, our study uncovers STK38L as a candidate, targetable vulnerability in a subset of molecularly defined PDACs. / 2019-11-01T00:00:00Z

The PI3K/mTOR signalling pathway plays a major role in biology and disease. Therefore, effective inhibitors that target proteins of this pathway have been developed. However, acquired resistance of cancer cells is a prevalent phenomenon that limits the durable response of these compounds. It is becoming apparent that experimental approaches for comprehensive biochemical analysis contribute to understand the complex mechanisms that confer drug resistance, and advances in largescale technologies including genomic sequencing and proteomics allow unprecedented molecular coverage without being biased for specific genes/cellular pathways. Initially, the phenotypic response of sensitive and resistant cells to the absence or presence of a PI3K inhibitor (PI3Ki), as well as other kinases, was examined. This study revealed that PI3Ki-resistant cells experience extensive phenotypic changes upon withdrawal of the PI3Ki from the culture media. The regulation of the proteome and phosphoproteome of sensitive and PI3Ki-resistant cells grown with or without the PI3Ki was analysed by shotgun mass spectrometry-based label-free quantitative technology. This analysis demonstrated that the proteomes and phosphoproteomes of drug-resistant cells are remodelled conditional to the presence of PI3Ki, and that the levels of enzymes with metabolic roles are modulated in resistant cells. Functional analysis of the metabolism of cells capable to survive in absence of PI3K/mTOR activity demonstrated that the bioenergetic activity of these cells is contingent on the presence of the selection drug. The complete set of protein-coding regions of the genome (exome) of sensitive and PI3Ki-resistant cells was then sequenced. This study unveiled common alterations in exome regions across PI3Kiresistant cell lines, as well as a degree of genomic heterogeneity between them. Lastly, the impact of lactic acid, a metabolic product, on a defined signalling network of the MCF7 breast cancer cell line was analysed. This study described the capacity of this metabolite to change the activity of signalling network branches.

Our lab recently described a role for JAK-STAT signaling in the maintenance of quiescence during the murine hair cycle. Research into signaling pathways and cytokines/growth factors involved in the mammalian hair cycle has not focused extensively on the JAK-STAT pathway. In this thesis, I investigated the upstream effector(s) and downstream mechanisms of JAK-STAT signaling in the HFSC during telogen, using a variety of methods, including murine conditional mutants of the JAK-STAT pathway, pharmacological and immunological techniques. The mechanism through which OSM exerts this effect is via JAK-STAT5 signaling downstream of the OSM receptor, which is antagonized by pharmacological JAK inhibition. Conditional epidermal ablation of OSMR or STAT5 during early- and mid-telogen (P42 – P60) shortens the telogen phase significantly, and inhibition of macrophages by way of neutralizing antibodies, small molecule inhibitors, and genetic ablation (with Csf1r-CreER::R26-iDTR mice) during telogen also promotes hair growth. Single-cell RNA sequencing of dermal immune cells across murine telogen identified a distinct subset of TREM2+ macrophages that are enriched for OSM, and gene-set analysis suggests these “trichophages” are similar to the microglia of the central nervous system. I show that this distinct subset of TREM2+ macrophages predominate during early- and mid-telogen, where they produce Oncostatin M (OSM), which is sufficient to maintain quiescence of hair follicle stem cells (HFSCs). Proliferation of HFSCs and hair growth is associated with depletion of this subset of TREM2+ macrophages. Interestingly, macrophage markers and OSM were found to be upregulated in the balding scalp of males with androgenetic alopecia, suggesting that this mechanism is physiologically relevant in the control of human hair cycling.

Macrophages are sentinels present in most tissues of the body, where they recognise and respond to biological dangers. Recognition and uptake of particles is mediated through phagocytic receptors which upon activation induce appropriate responses. These responses need to be tightly regulated in order to destroy pathogens but prevent uncontrolled inflammation. Phagocytosis is an evolutionarily conserved process required for host defence and homeostasis. During phagocytosis, particles are recognised by cell surface receptors that trigger rearrangement of the actin cytoskeleton and internalization of the bound particle into a de novo, membranous organelle known as the phagosome. Regulation of phagocytosis and phagosome maturation can be achieved through changes in transcription/translation and differential recruitment of proteins but also through their non-translational modifications. Here I explored the role of ubiquitylation in the phagosome biogenesis of Interferon-gamma (IFN-ɣ) activated macrophages. Ubiquitylation is a diverse, reversible post-translational modification which is not only involved in protein degradation but also in vesicle trafficking and immune signalling. My data shows that phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ. I applied a targeted AQUA peptide approach by which we quantified ubiquitin chain linkage peptides from phagosome samples by PRM. This data shows that all chain linkages apart from M1/linear chains are present on phagosomes. Furthermore, IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, I characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. I demonstrated that most ubiquitin chains are on the cytoplasmic site of the phagosome enabling an interaction of ubiquitin chains with cytosolic proteins such as Rab7. Rab7 a major regulator of vesicle trafficking could be shown to be ubiquitylated on phagosomes. I further showed that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation and RNF115 is responsible for most of the increase of K63 polyubiquitylation of phagosomal proteins. Knock-down of RNF115 promotes phagosome maturation and induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome and disruption of this pathway induces pro-inflammatory responses in macrophages. In conclusion, this is the first study showing unbiasedly that ubiquitylation plays an important role in vesicle trafficking to the lysosome.

No description available.

Clinical pathways have been studied to promote best practices in nursing and enhance patient satisfaction. For 10 years a pediatric orthopedic surgical team at a Midwest hospital has not had a clinical pathway to treat or standardize care for adolescents following posterior spinal fusion surgery. Pain scores and patients' length of stay were collected using a retrospective chart review. This information was used to revise preoperative education materials and develop a visual poster. The purpose of this project was to identify and develop a way to educate the pediatric nursing staff on the use of the developed educational materials, poster, and clinical pathway prior to its implementation. David A. Kolb's learning cycle and the experiential learning model was used as the theoretical foundation of this study. The quality improvement project for the nurses will be developed using a flipped classroom approach as the learning environment. Videos, scenarios, and small group activities will be created and used in an interactive learning environment. The study will use a pretest-posttest design of retrospective chart review data with the independent variable being the education provided to the nurses. Social implications related to this project are to provide information on the plan of care following surgery to the adolescent and caregiver. This project will promote positive social change for adolescents and caregivers who will be engaged in the postoperative care to increase their satisfaction and decrease their anxiety.

The outpatient care of the heart failure (HF) patient is fragmented due to the lack of evidence-based practice guidelines use. The primary goal of this project was to improve the care of the HF patient in the outpatient arena through use of clinical pathways using the logic model as the project framework. The intervention was carried out over a 4-week period on a convenience, random sample of patients (n = 80) attending a cardiology practice. The patients were recruited from 2 physicians' patient populations and were selected based on an adult diagnosis of HF, reduced ejection fraction of <40% at some point in time, and the New York Heart Association (NYHA) functional class II-V. Comparisons were made in the documentation of care between patients on or off the pathway. The intervention included documentation of patient education, care follow-up, medications, NYHA functional class, and symptom exacerbation, documented in the electronic medical record. The quality of care data were evaluated based on 3 of the Joint Commission core measures for outpatient care of the HF patient. Additional data were collected regarding use of the clinical pathway based on provider and week of implementation. Data were analyzed via a Chi-square test of independence comparing pathway use by provider and use of pathway as study progressed. The comparative results show statistically significant differences in use of the pathway by provider and a statistically significant increase in use during the project . The quality of care results varied in statistical significance. The pathway utilization increased over time and provided a method for standardizing documentation of care for the HF patient in this outpatient clinic, a benefit for HF patients and providers in this cardiology practice and beyond.

Phosphonates represent an important class of organophosphorus compounds. Their use as reagents in organic synthesis is prevalent, and there is a plethora of examples of biologically active compounds possessing the phosphonate moiety. To further our exploration of phosphonates as both reagents and biologically active compounds we have developed a one-flask protocol for the direct synthesis of phosphonates from benzylic and allylic alcohols. This transformation is unprecedented and is applicable to a range of substrates. Both electron rich and electron deficient benzylic alcohols react under the conditions developed. Furthermore, good yields are achieved when converting allylic alcohols to the corresponding allylic phosphonates. In at least one case, the one-flask protocol allows for phosphonate formation that was not achievable under the standard conditions. Bisphosphonates represent a significant subclass of phosphonates. Several nitrogenous bisphosphonates have found use in the clinic as treatments for bone-related disease including osteoporosis, and there is speculation that bisphosphonates that are enzyme-specific inhibitors may be used as cancer therapies. To develop our understanding of isoprenoid metabolism, we have prepared a range of bisphosphonates as potential inhibitors of geranylgeranyl pyrophosphate synthase. After much experimentation, an α-amino analog of a potent inhibitor of GGDPS has been synthesized and biological data is forthcoming. Furthermore, a new class of aromatic bisphosphonates, analogs of digeranyl bisphosphonic acid, has been synthesized and assayed. The bioassay results indicate that this series of compounds retains its specificity for the GGDPS enzyme, and that the dialkyl analogues retain much of their potency in the assays in spite of the increased steric bulk of the aromatic substructure. We have also begun the design and synthesis of compounds as potential inhibitors of Rab geranylgeranyl transferase (RGGTase). The lead compound, 3-PEHPC, is documented to inhibit RGGTase selectively, albeit at less than desirable concentrations. Using 3-PEHPC as the model compound we have elected to probe the impact of modifications on the hydrophilic "head" portion of the molecule. Using the phosphonophosphinate functionality as a surrogate for the phosphonocarboxylate moiety we have successfully synthesized digeranyl phosphonophosphinate. Initial assay data indicates that this novel phosphonophosphinate does not act upon GGDPS as does the analogous bisphosphonate substructure. The bioassay data to probe this compound's impact on RGGTase is forthcoming. Given the worldwide impact of tuberculosis infection and the emergence of drug-resistant strains of tuberculosis-causing pathogens, new and potent treatments for tuberculosis are necessary. We have engaged in the synthesis of several compounds as inhibitors of Rv2361c, an enzyme key to cell wall biosynthesis in Mycobacterium tuberculosis, the principle causative agent of tuberculosis in humans. To probe the impact of modifications at the C-9-position of the most potent of our Rv2361c inhibitors, we have made several analogues having phenyl and indole substituents. The in vitro enzyme assay data for the set of compounds has clarified understanding of the essential components of the pharmacophore, and helped to establish the direction for future efforts.

Suicidal behavior represents the most severe, yet inherently preventable, outcome of psychiatric disease. Despite tremendous efforts to improve the awareness and treatment of psychiatric illness, suicidal behavior rates have been on the rise. The greatest challenge to confronting this crisis is the effective identification and treatment of those at risk for suicide. This challenge has been difficult to address due, in part, to the lack of a clear biological basis for suicidal behavior. Toward addressing this knowledge gap, evidence has been identified of a significant heritable component to suicidal behavior. Subsequent genetic research efforts have focused on the examination of common sites of genetic variation within candidate genes and throughout the genome. These efforts have identified many potentially important risk loci, but the majority of the risk expected to arise from genetic variation remains unexplained by current data. The primary objective of this dissertation was to examine the contribution of largely unexplored rare and potentially damaging genetic variation within suicidal behavior. To do this, targeted next-generation sequencing approaches were employed within a cohort of individuals diagnosed with bipolar disorder, a group particularly enriched for suicidal behavior. Sequence data was generated that examined essentially all protein-coding regions of the human genome (“exome”), with expanded sequencing around and within candidate genes hypothesized to play a role in suicidal behavior risk. The secondary objective of this dissertation focused on the assessment of rare variation within bipolar disorder through sequenced pedigrees and followup in a large collaborative bipolar disorder versus normal control sequencing dataset. These objectives were addressed through the thoughtful application of diverse and complimentary methods. These methods were selected to investigate individual variants, genes, and biological pathways. This approach offered examinations of the potential impact of rare genetic variation within focused regions and across complex biological process pathways that could be disrupted through damaging variation in many different genes. The presented efforts represent the largest examinations of rare functional variation with suicidal behavior and bipolar disorder performed, to date. No individual variant or gene survived correction for multiple testing for either phenotype. These results are consistent with other initial sequencing efforts in complex psychiatric phenotypes, offering conclusions that larger samples will likely be required to identify significant associations for single variants and genes. Within pathway analyses, however, we identified a significant enrichment of rare damaging variation that segregated within bipolar disorder pedigrees in genes that have been implicated in de novo studies of autism. This finding was further replicated within three large case/control sequencing samples, providing support to emerging evidence of a potential overlap of risk loci for autism and bipolar disorder. Many additional results approached significance that bear further consideration. These results offer potential candidate genes and pathways that could be utilized in future sequencing efforts for suicidal behavior and bipolar disorder. In addition, highly valuable resources in the form of datasets strongly enriched for novel rare loci were produced that can significantly contribute to ongoing efforts to investigate bipolar disorder and suicidal behavior. These data can be used in combination with other emerging datasets to generate more powerful meta- and mega-analyses to confidently identify risk loci for both phenotypes.