Indiana University-Purdue University Indianapolis (IUPUI) / Taxane-induced musculoskeletal pain (TIMP) is musculoskeletal pain that includes myalgia (i.e., diffuse muscle pain, usually accompanied by malaise) and/or arthralgia (i.e., joint pain) that occurs following treatment with taxane-based chemotherapy. TIMP is a symptom that is clinically reported as negatively affecting most cancer survivors receiving taxane-based chemotherapy; however, TIMP is not comprehensively understood. The purpose of this dissertation was to conduct a cross sectional, descriptive, correlational pilot study to describe TIMP in women with ovarian cancer who were being or had been treated with paclitaxel-containing regimens. Specific aims were to: (1) describe the TIMP symptom experience (intensity, distress, duration, location, quality, temporal pattern, aggravating and alleviating factors, and pain management); (2) describe the associations between TIMP (intensity, distress) and co occurring symptoms (pain [general], peripheral neuropathy, impaired sleep, fatigue, emotional distress, and/or hot flashes); and (3) identify associations between TIMP (intensity, distress) and patient-reported outcomes (interference with daily activities, physical functioning, and health-related quality of life). Primary data collection was performed on a convenience sample of 15 women with ovarian cancer. Participants were recruited from an outpatient cancer clinic, local cancer support communities, and a national cancer survivors’ research registry. Descriptive statistics and Spearman’s correlations were used. Findings showed TIMP is moderate to severe in intensity on average, constant, affecting a large area of the body, and aggravated by everyday walking. Greater TIMP intensity or distress was associated with greater intensity and interference of most co-occurring symptoms and was associated with greater interference with daily activities, worse physical functioning, and worse health-related quality of life. Nurses are encouraged to comprehensively assess TIMP using structured, validated tools for pain to better intervene on aggravating and alleviating factors and pain management regimens. Prospective, longitudinal studies with larger sample sizes are needed to further understand TIMP and its impact on cancer survivors.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第21041号 / 人健博第57号 / 新制||人健||4(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 市橋 則明, 教授 田村 恵子, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small(„5% deformation) and large strains(>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules

Recent advancements in cancer research has led to the synthesis of a new drug known as docetaxel. Meant to replace paclitaxel, its more natural counterpart whose ingredients are difficult to obtain, the drug is known to effectively treat a wide array of cancers, including breast cancer, ovarian cancer, and prostate cancer. The establishment of a synthetic alternative to paclitaxel has increased its bioavailability, thereby lowering the cost needed to utilize the drug. Still, the limiting factor in minimizing costs is the method in which the drug is processed. Current methods in drug processing have their limitations, which include the introduction of impurities and a low effective yield due to poor powder geometry. Thus, the goal of this study looks to explore a new way to process the drug in a more efficient manner. In this study, a new method for processing docetaxel is explored on in great detail. A more direct method of using electrospray deposition is utilized for the creation of monodisperse nanoparticles, with the main intention of increasing the efficiency at which the drug is processed and prepared for drug delivery to the patient by means of injection. A key feature in electrospray deposition is its ability to produce droplets that are sized homogenously. These droplets eventually evaporate at homogenous rates. These two concepts have been exploited to consistently produce nanoparticles of the cancer drug, which is made possible by the fact that the minimal variation in droplet sizes has easily translated to minimal variation in dry particle sizes. Compared to other methods of drug processing, one other benefit that electrospray deposition conveys is that through evaporation, virtually all impurities and unwanted foreign material are eliminated.

Cancer remains an elusive killer due, in part, to the suppression of normal immunologic antitumor responses. Normal host (NH) macrophage (Mϕ) populations have tumoricidal effects such as tumor antigen phagocytosis and presentation, and cytokine production. Tumor-infiltrating Mϕs may evade these activities by dysregulating production of immunostimulatory cytokines (including Interleukin [IL]-1β, IL-18, and tumor necrosis factor-α [TNF-α]), by production of antagonistic factors. The restoration of IL-1β, IL-18, and TNF-α production by Mϕs could re-establish antitumor host immune responses. Previous work in our laboratory suggests that tumor distal (TD) Mϕs produce more IL-1β than NH Mϕs when stimulated with IFN-γ and lipopolysaccharide (LPS). We hypothesize that the presence of immunomodulatory factors like IL-10 and TGF-β dysregulate IL-1β production in tumor proximal (TP) Mϕs. Indeed, IL-1β production was downregulated among in situ TP Mϕs. We have proposed that IL-18, a structural homologue to IL-1β was similarly dysregulated in TD and TP Mϕs. IL-18 was enhanced in both distal and proximal Mϕs. Differences in the functions of these cytokines could account for this dissimilarity. TNF-α, another proinflammatory cytokine, followed the dysregulation pattern of IL-1β in our tumor-burdened hosts (TBH), likely because of the similar functions of these cytokines. Because it is a potential vehicle for immunotherapeutic treatment, paclitaxel's action on the immune response (TAXOL™) was investigated. Paclitaxel is a potent Mϕ activator that upregulates a variety of cytokines in an LPS-like manner. Paclitaxel enhanced TD Mϕ production of IL-1β, IL-18, and TNF-α in an LPS-like manner. Production of IL-1β and TNF-α was reduced in TP Mϕs when treated with paclitaxel; however, IL-18 production was enhanced. This difference could be due to the different functions of IL-1β and IL-18. To determine whether production of these cytokines translates into downstream expression of transcription products, IL-12 and nitric oxide (NO) were assayed. NO was enhanced distally, but paclitaxel treatment failed to enhance NO production. When treated with paclitaxel, IL-12 was produced by NH and TD Mϕs. Collectively, these studies suggest that tumor-induced cytokine imbalances compromise antitumor immunity and paclitaxel may reverse this activity. / Master of Science

Plant cell culture is an attractive platform technology for production and supply of important plant derived medicinals. A unique characteristic of plant cells is the ability to grow as multicellular aggregates in suspension. The presence of these non-uniform aggregates results in creation of distinct microenvironments, which can induce variations in cellular metabolism (e.g., growth, oxygen consumption and secondary metabolite synthesis). This heterogeneity can lead to unpredictable and suboptimal performance in large scale bioprocesses. One example is the Taxus cell culture system, which produces a widely used chemotherapeutic drug - paclitaxel (Taxol ®). Despite extensive process engineering efforts which have led to increased yields of paclitaxel, Taxus cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces the accumulation of paclitaxel, but to varying extents in culture. A significant negative correlation was observed between paclitaxel level and mean aggregate size of the culture, demonstrating the relevance of measuring, and potentially controlling aggregate size during long term subculture. Understanding the regulation of gene expression can provide rational engineering strategies to control variability and optimize performance of Taxus cell cultures. Biosynthetic pathway gene analyses revealed upregulation of genes upon elicitation with MeJA; results also suggested additional molecular regulatory points outside of the biosynthetic pathway. In order to fully understand Taxus molecular regulation and the relationship to paclitaxel production variability, a transcriptome-wide analysis using next generation sequencing (454 and Illumina) methods was performed. Several pathways outside of paclitaxel biosynthesis were found active upon MeJA elicitation. Global comparison of gene expression amongst cultures accumulating different levels of paclitaxel is being performed to completely understand the interactions amongst the paclitaxel biosynthetic pathway and other complimentary and competing pathways to suggest effective targets for metabolic engineering. This work collectively represents the first molecular studies to understand metabolic regulation in Taxus cell cultures. Apart from inducing paclitaxel biosynthesis, MeJA decreases cell growth in Taxus cell cultures. The MeJA-mediated repression of cell growth was shown to correlate with inhibition of cell cycle progression as evident both at the culture level through flow cytometric analyses and at the transcriptional level by repression of key cell cycle-associated genes. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.

Plant derived natural products represent some of the most effective anti-cancer and anti-infectious disease pharmaceuticals available today. However, uncertainty regarding the feasibility of commercial supply due to the limited availability of many plants in nature has resulted in a dramatic reduction in the use of natural products as leads in modern drug discovery. Plant cell suspension culture, consisting of dedifferentiated plant cells grown in vitro and amenable to large scale industrial biotechnology processes, is a production alternative which promises renewable and economical supply of these important drugs. The widespread application of this technology is limited by low product yields, slow growth rates, challenges in scale-up, and above all, variability in these properties, which is poorly understood. Plant cells grow as aggregates in suspension cultures ranging from two to thousands of cells (less than 100 micron to well over 2 mm). Aggregates have long been identified as an important feature of plant cell culture systems, as they create microenvironments for individual cells with respect to nutrient limitations, cell-cell signaling, and applied shear in the in vitro environment. Despite its purported significance, a rigorous engineering analysis of aggregation has remained elusive. In this thesis, aggregation was characterized, analyzed, and modeled in Taxus suspension cultures, which produce the anti-cancer drug paclitaxel. A technique was developed to reliably and routinely measure aggregate size using a Coulter counter. The analysis of aggregate size as a process variable was then used to evaluate the effect of aggregation on process performance, and the analysis of single cells isolated from different sized aggregates was used to understand the effect of aggregation on cellular metabolism and heterogeneity. Process characterization studies indicated that aggregate size changed over a batch cycle as well as from batch to batch, so a population balance equation model was developed to describe and predict these changes in the aggregate size distribution. This multi-scale engineering approach towards understanding plant cell aggregation serves as an important step in the development of rational strategies aimed at controlling the process variability which has heretofore limited the application of plant cell culture technology.

Dans le cadre d'un vaste programme de domestication de l'if du Canada (Taxus canadensis Marsh.) à des fins de production de taxanes, de jeunes plants en première, deuxième ou troisième années de culture en contenants ont été soumis à différentes sources d'azote (nitrate d'ammonium, nitrate de calcium, nitrate de sodium, sulfate d'ammonium et urée), appliquées à différentes doses, et à divers pH du substrat pour étudier leur influence sur la croissance des plants, sur la teneur en éléments minéraux de leurs tissus et sur leur teneur en taxanes. Tous les autres éléments essentiels ont été ajoutés en quantités et en proportions normalement favorables à la croissance des jeunes conifères forestiers. Les résultats indiquent que l'if du Canada est fortement affecté par le nitrate de sodium, mais répond favorablement aux autres sources azotées dont la concentration en azote se situe entre 100 et 400 ppm pour les plants en première et deuxième année de culture. Une concentration de 450 ppm de N est très favorable à la croissance de plants en troisième armée de culture. Tout traitement fertilisant favorable au bon développement des plants n'a eu aucune incidence sur leur teneur en taxanes. Une fertilisation adéquate permet donc d'augmenter la capacité de récolte de principe actif.

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