Recent evidence suggests that leflunomide, a dihydroorotate dehydrogenase (DHODH) inhibitor, disrupts neural crest development and melanoma pathogenesis via inhibiting transcription elongation. DHODH is an enzyme in the pyrimidine biosynthetic pathway and inhibition of this enzyme by leflunomide triggers a low nucleotide state. Leflunomide effectively ablates the neural crest lineage in embryonic zebrafish, preventing the formation of mature melanocytes, among other neural crest lineages. This drug also effectively suppresses melanoma and is in a clinical trial, administered in combination with the BRAF inhibitor vemurafenib, for metastatic melanoma. Despite knowing that leflunomide targets transcription elongation, the mechanism by which low nucleotides directly regulates transcription is unknown.

<p> The studies described in this thesis explore the physiology of upper respiratory tract colonization by S. pneumoniae and S. pyogenes. We have shown that colonization is associated with biofilm formation and examined the effects of co-colonization on genetic exchange. Additional studies have investigated the virulence and inflammatory potential of biofilm bacteria, and identified factors influencing cellular egress from biofilm communities and the transition to acute disease. The remaining chapters explore the mechanism of action of the human milk protein lipid complex HAMLET and its potential for antimicrobial adjutancy.</p>

<p> Patients diagnosed with cancer often require interventions for the accompanying mental health distress of their diagnosis; patients&rsquo; mental distress can lead to hopelessness and noncompliance. Improvements for assessment and interventions are needed. This project provides recommendations for improving oncological nursing knowledge by implementing competencies for nurses through educational modules, focusing on nursing approach, confidence, and interventions necessary for understanding methods of treatment and the measurement of distress in oncology and oncological treatment. Using established standards and competencies will improve knowledge and skill in inpatient settings. Current established nursing standards from the American Nursing Association, Institute Of Medicine/National Comprehensive Cancer Network, C-Change, and Public Mental Health Essentials were explored in order to identify gaps and create a list of recommended competencies for oncology nursing. Six associated adaptable educational modules were developed based on the adult education framework of Knowles, and participant training entailed proper use and comprehension of the Distress Thermometer for measurement of distress. The C-Change observation displayed participant (<i>n</i> = 102) results of approximately 119% improvement, which was observed in knowledge, communication, and confidence. Participants used the resources to reduce distress levels by initiating the selected established interventions for management, all of which was made evident in patient self-reported outcomes, using resources from published, established, standardized competencies. Having such training will allow for improved care for patients with cancer, thus having an influence on positive social change. </p>

<p> The origins of pairs of monozygotic twins and higher order multiples, i.e. triplets, quadruplets, etc., have been extensively studied but still little is understood. To gain insight into this event, certain possible cell labeling schemes that model an organism&rsquo;s development are analyzed. The phenomenon of quadruplet twins is exposed during the process. We predict that monozygotic quadruplets are not really quadruplets but instead are two pairs of monozygotic twins where the pairs slightly differ. From the considered models, the probability of monozygotic twins is found to be (1/2)<i>^K</i>, and we discover from our analysis that the probability of monozygotic quadruplets, or triplets in the case of the death of an embryo, is (1/8)<i>^K</i>, where <i> K</i> is a species-specific integer representing the number of pairs of homologous chromosomes. This investigation into twinning provides a foundation for understanding the process of cell development through which the cell development mechanism is established. The failure of the internal cellular clock from this mechanism may play an important role in cancerogenesis. The parameter K may determine cancerization with a probability threshold that is approximately inversely proportional to the Hayflick limit, so exposure to small levels of ionizing radiation and chemical pollution may not produce cancer.</p>

Myelodysplastic syndromes (MDS) include a spectrum of stem cell malignancies characterized by ineffective hematopoiesis and predisposition to acute myeloid leukemia (AML) transformation. Patients are predominantly older (greater than 60 years old), with progressive cytopenias resulting from ineffective and cytologically dysplastic hematopoiesis. MDS subtypes are classified by morphologic features and bone marrow blast percentage, as well as cytogenetic pattern, as is the case for deletion 5q MDS. Interstitial deletion of the long arm of chromosome 5, del(5q), is the most common chromosomal abnormality in patients with MDS, and the 5q- syndrome, represents a distinct subset of del(5q) MDS characterized by an isolated deletion, megakaryocyte dysplasia, hypoplastic anemia, and an indolent natural history. MDS risk stratification is most commonly based on the International Prognostic Scoring System (IPSS) with survival outcomes ranging from a few months to many years based on risk factors. There are several therapeutic options for MDS including hematopoietic growth factors, immunosuppressive therapy, azanucleosides, and allogeneic stem cell transplant, however, there is still a need for more effective treatment options, particularly targeted therapeutics. One of the most effective treatments for MDS is selective for del(5q) MDS, and is the second generation immunomodulatory agent, lenalidomide (LEN). LEN is an analog of the known teratogen, thalidomide, and has broad biological effects including selective cytotoxicity to del(5q) clones, activation of T-cells, and expansion of erythroid precursors. In patients with del(5q) MDS, LEN is effective in up to 75% of patients, however, 50% of patients will become resistant within 2-3 years of treatment response. Studies in normal hematopoietic progenitors have shown that LEN induces expansion of the primitive erythroid precursors, which our laboratory has shown is accompanied by sensitization of progenitors to ligand induced erythropoietin receptor (EpoR) signaling. This sensitization is evidenced by increased and prolonged activation of the Signal Transducer and Activator of Transcription 5 (STAT5), compared to Epo stimulation alone. Although EpoR signaling is augmented by LEN, the exact mechanisms by which this is mediated to result in erythroid expansion are not fully characterized. In del(5q) MDS, we have shown that LEN selectively suppresses del(5q) clones via inhibition of the haploinsufficient phosphatases Cdc25c and PP2a, as well as stabilizing the human homolog of the murine double minute-2 protein (MDM2) to decrease expression of the tumor suppressor, p53, however, the mechanisms of action of LEN in non-del(5q) MDS remains elusive. Although most anemic MDS patients have normal or elevated endogenous levels of Epo, as well as comparable levels of progenitor EpoR density relative to healthy individuals, the biologic pathology underlying the impaired EpoR signaling in MDS is poorly defined. Recent reports have shown that membrane microdomains are important for T-cell, c-kit, and integrin signaling, however, there have been no reports on EpoR membrane localization. Lipid rafts are discrete membrane entities that provide platforms by which receptors aggregate and initiate downstream signaling. Furthermore, reports have indicated that there is a decrease in lipid raft density in GM-CSF primed MDS neutrophils, that consequently impaired production of reactive oxygen species (ROS) after fMLP stimulation, suggesting a role of rafts in MDS disease biology. Based on the role of rafts in signaling, and potential role in MDS pathogenesis, we sought to determine whether there was specific membrane localization of EpoR to the raft fractions, and whether disruption of rafts in MDS erythroids could impair EpoR signaling. To address this, we first examined the membrane localization of EpoR on the cell surface. We show here that EpoR translocates to lipid rafts in both erythroid progenitor cell lines as well as primary progenitor cells after stimulation by Epo. Furthermore, we found that Epo stimulation increases the assembly of lipid rafts, as well as the aggregation of rafts on the cell surface. Epo stimulation not only promoted the recruitment of EpoR into the raft fractions, but also downstream signaling intermediates such as Janus kinase 2 (Jak2), STAT5, and Lyn kinase. Moreover, a negative regulator of EpoR signaling, the CD45 tyrosine phosphatase, was redistributed outside of raft fractions after Epo stimulation, potentially enhancing receptor signal competence. Furthermore, disruption of lipid rafts by depletion of membrane cholesterol with MâCD (methyl-β-cyclodextrin) inhibited EpoR signaling in both cell lines and primary bone marrow progenitor cells. Additionally, we found that inhibition of Rho-associated, coiled-coil containing protein kinase (ROCK) and/or Ras-related C3 botulinium toxin substrate 1 (Rac1), blocked the recruitment of the receptor into the raft fractions indicating a critical role of these GTPases, and associated proteins, in the transport and localization of EpoR into raft microdomains. We next asked whether LEN could alter lipid raft assembly in erythroid precursors in the absence of Epo. LEN not only induced raft formation and aggregation but also increased F-actin polymerization. Similar to Epo stimulation, LEN alone was able to induce the recruitment of EpoR, Jak2, and STAT5 into raft fractions. Additionally, CD45 was redistributed outside of raft fractions after LEN treatment. Similarly, inhibition of ROCK blocked LEN induced raft formation and F-actin polymerization, indicating that LEN utilized effectors shared by Epo. Furthermore, LEN was able to increase raft density in raft deficient primary MDS erythroid progenitors. These data demonstrate that LEN may enhance erythroid expansion via induction of EpoR signaling competent raft platforms, to enhance survival and differentiation transcriptional response. Recently, ribosomal protein (RP), S-14, gene (RPS14) haplodeficiency was found to be a key determinant of the hypoplastic anemia in del(5q) MDS. Allelic loss of RPS14 compromises ribosome assembly, thereby causing nucleolar stress and release of free RPs that bind to and promote the degradation of MDM2, the principal negative regulator of p53. As a result, the accumulation of RPs causes lineage restricted stabilization of p53 in erythroid precursors. Our laboratory and colleagues confirmed that cellular p53 expression levels were elevated in del(5q) erythroid precursors, and that LEN decreased expression in responding patients. However, at the time of LEN treatment failure, p53 expression was again elevated at levels exceeding those at baseline. These results suggest that LEN is initially able to reverse p53 accumulation levels and that this action may be a mechanism by which LEN is selectively cytotoxic to del(5q) clones. Subsequent studies showed that LEN inhibits the cereblon E3 ubiquitin ligase complex, the newly discovered target of LEN. Cereblon has been reported to be the principal protein involved in thalidomide induced teratogenicity. Furthermore, the cytotoxic activity of LEN in multiple myeloma is dependent on cereblon. Our laboratory found that LEN inhibits the auto-ubiquitination of MDM2, thereby stabilizing the protein, and promoting ubiquitination of and ultimately the degradation of p53. Additionally, we found that LEN blocked the binding of free ribosomal proteins to MDM2, which are liberated from the nucleosome by ribosomal stress from RPS14 haploinsufficiency, consequently stabilizing the E3-ubiquitin ligase and fostering p53 degradation. In non-del(5q) MDS there is no cytotoxicity of MDS clones by LEN, suggesting an alternative method of erythropoiesis rescue. Although we know that LEN promotes the formation of signaling platforms, and recruitment of EpoR, we wished to determine whether there was an effect of LEN on EpoR expression, as EpoR expression is controlled through ubiquitination and proteasomal degradation. Treatment of erythroid progenitor cell lines and primary erythroid precursors with LEN increased cellular expression of Jak2-associated EpoR in a concentration dependent manner. There was no change in mRNA expression, supporting a post transcriptional mechanism. We then investigated whether receptor up-regulation was limited to EpoR, or included other cytokine receptors. We found that LEN induced expression of another Jak2 associated Type I receptor, IL3-R, but did not alter cellular expression of c-kit, a Type II cytokine receptor. Because Type I cytokine receptor turnover is regulated by a shared E3-ubiquitin ligase, and LEN inhibited both MDM2 and cereblon, we evaluated the effects of LEN on the E3-ubiquitin ligase, Ring Finger Protein-41 (RNF41), which regulates steady state or ligand independent, Jak2 associated Type I receptor internalization. We found that LEN inhibited the ubiquitination activity of RNF41, ultimately stabilizing EpoR membrane residence and increasing expression. In summary, MDS patients display ineffective hematopoiesis likely in part to decreased lipid raft assembly. Stimulation by Epo, or treatment by LEN, not only induced raft formation, but also induced the recruitment of both growth factor receptor, and downstream signaling intermediates into raft fractions to enhance EpoR signal fidelity. We have shown here two methods by which LEN may augment EpoR signaling. First, LEN increases lipid rafts and promotes recruitment of signaling effectors. Second, LEN increases and stabilizes the expression of EpoR through inhibition of the E3 ubiquitin ligase, RNF41. Therefore, we suggest here that LEN may have broad E3 ubiquitin ligase inhibitory effects. These data also indicate that lipid raft upregulation by LEN is mediated through GTPases, suggesting that GTPase activation may also occur via inhibition of specific E3 ubiquitin ligases, a question to be addressed in future studies.

The UVB portion (280--320 nm) of the ultraviolet spectrum contributes to the development of non-melanoma skin cancer (NMSC) in humans. UVB irradiation causes epigenetic alterations in target keratinocytes, including the upregulation of Activator Protein-1, a transcription factor complex that alters normal cellular gene expression. c-Fos expression is induced in a manner that correlates with the UVB-induced activation of AP-1. This suggests that c-Fos functions as a major regulatory component in the UVB-induced transactivation of AP-1. The purpose of this dissertation is to characterize UVB-induced regulation of c-Fos expression. Transcriptional regulation of c-fos is investigated by evaluating the role of each of four cis elements within the c-fos promoter. While mutation of each of these four cis elements results in significantly lower levels of UVB-induced promoter activity, the CRE and FAP1 elements mediate most of the UVB transactivation of c-fos. In addition, UVB irradiation induces homodimers of phosphorylated CREB to bind to the CRE and FAP1 elements. To identify cellular signal transduction pathways that are induced by UVB-irradiation to regulate c-Fos expression, a UVB-inducible enzyme, phosphatidylinositol 3-kinase (PI 3-kinase), is studied. Inhibition of PI 3-kinase reduces c-Fos expression in UVB-irradiated cells. Akt and GSK-3beta, constituents of the PI 3-kinase signaling pathway, are also found to be part of this UVB-induced signaling pathway. To identify potential molecular targets for the development of skin cancer chemoprevention strategies, the polyphenolic compound nordihydroguaiaretic acid is tested and found to prevent UVB-induced c-Fos expression and AP-1 transactivation by inhibiting the PI 3-kinase signal transduction pathway. Thus, phospho-CREB binding to the CRE and FAP1 cis elements and PI 3-kinase signaling are both identified as molecular mechanisms and potential molecular targets that are involved in UVB-induced c-Fos expression and AP-1 transactivation.

Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, the human colon tumor cell line HT29 (wtAPC -/-) was stably transfected with a zinc-inducible wild-type APC gene. Addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein, and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the beta-galactosidase (betaGal) gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. To examine the role of APC-dependent regulation of ODC, the two sets of E-boxes were analyzed. When the E-box domain in the 5' flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results indicate that APC expression can inhibit ODC via the 5' E-box. Using the cell model previously described, APC selectively represses the ODC A allele, apparently through selective binding of Mad1. These results demonstrate that wild-type APC suppresses c-myc and activates Mad1 expression in HT29 colon-derived cells. Treatment of Min mice with the ODC inhibitor, difluoromethylornithine (DFMO), suppresses intestinal polyamine contents and intestinal tumorigenesis. The data presented in this dissertation indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. Apoptosis is significantly reduced in both the small intestines and colons of Min (multiple intestinal neoplasia) mice when compared to normal littermates. Apoptotic indices can be restored by treating the mice with alpha-difluoromethylornithine (DFMO). DFMO is a specific, irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. These results indicate that APC induces apoptosis via the mitochondrial pathway rather than through the death receptor pathway. APC also affects a variety of other proteins involved in the regulation of apoptosis including transcription factors (i.e., ets2, FKHR, JunB, etc.) and bcl-2 (i.e., Bcl-xL) family members. The multiple levels at which APC functions suggest a variety of possible targets for the prevention and treatment of colon cancer. (Abstract shortened by UMI.)

Purpose. Explore how nurses and patients talk about cancer pain management during an oncology clinic visit. Describe the elements of these interactions and the patient-researcher discussions in order to evaluate the communication process used to report pain and to plan pain management. Design. Exploratory design of nurse-patient oncology clinic interactions and patient-researcher discussions. Methods. Nurse participants completed (1) a questionnaire about clinic time spent with patients and (2) Ward's Barrier Questionnaire (BQ) which concerns beliefs about the use of analgesics. Patient participants also completed a questionnaire about pain and Ward's BQ. Nurse-patient clinical interactions were audio-taped and analyzed by means of narrative analysis. Post-questionnaire patient-researcher discussions were analyzed also by narrative analysis. Quantitative data analysis was conducted on data from the questionnaires. Findings. Audio-taped nurse-patient interactions were divided by theme grouping into four summary examples: (1) Beginning to want to put it all together (56%), Communicating personal uniqueness (22%), (2) Active patient participation (13%), and (3) Learning about tests for future treatment (9%). Analysis revealed that while over 60% of the participants reported to be presently in pain, pain and pain management were rarely mentioned during the interactions. Patients did talk about pain extensively during the post-questionnaire discussions. Conclusions. Narrative analysis of nurse-patient interactions can provide health care professionals with examples of the quality and extent of information that cancer patients need regarding pain management. Not enough attention is given to patients' pain reports in the planning of pain management. Without systematic study of patients' pain reports and patients' comments on the effectiveness of analgesics, oncology clinic pain management will continue to remain inadequate.

Choline metabolites are elevated in breast cancer, decrease in response to effective therapy and are detected non-invasively by magnetic resonance modalities. Decreases in choline metabolites occur early-on after initiation of treatment. There is potential for use of choline metabolites as non-invasive diagnostic and therapeutic response indicators. Choline metabolites are detected in vivo by magnetic resonance spectroscopy (MRS) in broad resonances which are composites of multiple compounds. Tumor extract studies have suggested that phosphocholine (PCho) is the component of these resonances with the greatest potential for use as a diagnostic marker or therapeutic response indicator. Since other compounds present in these broad resonances vary in concentration with cancer progression and in response to therapy, changes in these other resonances can potentially diminish the overall signal or dampen the detectable therapeutic response. The ability to resolve and quantify PCho in vivo increases the sensitivity of this detection method, and hence, increases its potential utility. Herein is reported the in vivo resolution and quantification of PCho in a human breast cancer xenograft model in mice. A significant PCho decrease is detected following treatment with the taxane docetaxel. This PCho decrease is correlated with the diffusion-weighted magnetic resonance imaging (DWMRI) measured increase in tumor water mobility, and with mitotic catastrophe, a non-apoptotic mode of cell death. By studying model system of human breast cancer cells, other metabolites in the choline pathway varying with cancer progression are determined, and the transcriptional expression of genes in the choline pathway is quantified. From these data and enzyme activity data reported by other groups, a model is proposed where a number of metabolic perturbations combine to elevate PCho in breast cancer. These perturbations include the elevation of choline transporter, choline kinase, and phospholipase activities, in combination with decreased CTP:PCho cytidylyltransferase (CCT) activity. By changes in metabolites and gene expression following therapy, it is proposed that increased CCT activity combined with decreased phospholipase and GPC phosphodiesterase activity lead to decreased PCho. In addition, expression of a putative choline transporter (CTL1 variant A) and a putative choline kinase (CHKL) is quantified in human breast cells.

The cellular infiltration of solid tumors is indicative of an immune response to cancerous growths. Unfortunately, most tumors grow progressively despite this infiltration. Therefore, the infiltrate from a regressing tumor is necessary in order to examine the requirements for tumor rejection. Due to the rarity of tumor rejection, elucidating the requirements is difficult without an animal model. The sponge model of concomitant tumor immunity allowed the examination of the components associated with tumor rejection. In the model of concomitant tumor immunity an animal is given a primary tumor followed by a secondary tumor challenge. Despite the progression of the primary tumor, the secondary tumor challenge is rejected. In this model the secondary tumor challenge is delivered into a preimplanted gelatin sponge matrix which can be retrieved in order to capture the components associated with tumor rejection. Retrieval of both the primary progressing tumor and the gelatin sponge allowed a direct comparison of the factors associated with tumor progression and rejection. Using this model, we have examined the progressing and rejected tumor sites for differences in T cell cytotoxicity, V beta T cell receptor usage, and the expression of cytokine genes and signal transducing proteins. The results from this study demonstrated that the T cells isolated from progressing tumor sites were not cytolytic, whereas the T cells from the rejection sites showed significant cytolysis towards the autologous tumor cells in vitro. Surprisingly, the T cell infiltration into the progressing and rejected tumor sites were similar with V beta 1 and V beta 8 T cell receptor bearing T cells predominating at both locations. The T cell response also showed clonal restriction upon examination of the complementarity determining region 3 (CDR3) of the T cell receptor. Significantly, the rejection site showed higher gene expression levels of IFN-γ, TNF-α, IL-2, IL-4, IL-10, and IL-12 and reduced TGF-β gene expression compared to the progressing tumor site. Finally, although the T cells from the progressing tumor site showed an altered pattern of tyrosine phosphorylation, the signaling molecules p59ᶠʸⁿ and CD3 ζ were expressed at comparable levels in the T cells from both sites. These data strongly suggest that the tumor microenvironment may play a major role in orchestrating an anti-tumor immune response.