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A senescence-like growth arrest succeeded by rapid recovery of proliferative capacity is observed in MCF-7 breast tumor cells exposed to fractionated radiation (5 x 2Gy) alone. Exposure to the vitamin D3 analog EB 1089 (100nM) prior to irradiation converts the initial growth arrest response to cell death in part through the inhibition of radiation-induced senescence and promotion of both apoptotic and autophagic cell death. More importantly, EB 1089 was shown to profoundly reduce the rate of recovery following fractionated irradiation. The effect of EB 1089 on the temporal response to radiation is also observed in MCF-7 cells expressing caspase 3, but not in cells where p53 function is abrogated. EB 1089 does not increase radiation-induced DNA damage or inhibit DNA repair, as measured by both the alkaline unwinding assay and 53BPl fociformation. EB 1089 inhibits radiation-induced down-regulation of myc; however, doxycyclin-induction of myc does not mimic the radiosensitizing effects of EB 1089. EB1089 increases radiation-induced reactive oxygen species (ROS) generation; however, both free radical scavengers, N-acetyl cysteine (NAC)and reduced glutathione (GSH), fail to attenuate the radio-sensitizing effects of EB 1089. These studies rule out increased radiation-induced DNA damage, inhibition of DNA repair, inhibition of myc suppression,or increased ROS generation as the mechanism(s) responsible for the radiosensitizingeffects of EB 1089. However, EB 1089 does cause an increase in radiation-induced ceramide generation in the cell, while the ceramide synthase inhibitor, fumonisin B1, inhibits apoptosis and increases cell viability following treatment with EB 1089 plus radiation. Signaling pathways that promote ceramide generation and autophagic cell death may provide insights as to the mechanisms underlying the interaction(s) between EB 1089 or vitamin D3 and radiation in breast tumor cells.

The goal of this escalating single-dose phase I research study was to determine the safety, tolerability, pharmacokinetics, pharmacodynamics as well as in vitro metabolism and plasma protein binding of MS-275, a novel histone deacetylase inhibitor, in patients with solid tumors and lymphomas. A validated LC/MS assay was developed to quantitate MS-275 in plasma, human liver microsomes and urine. The pharmacokinetic (PK) evaluation was done using a non-compartmental approach. In-vitro plasma protein binding profile of MS-275 was characterized by a validated micro-equilibrium dialysis method. In vitro phase I and phase II hepatic metabolism of MS-275 were evaluated using human liver microsomes. A correlative covariate analysis was performed in an effort to explain the wide inter-individual variability among patients.Results from the study demonstrate that the validated LC-MS assay is specific, accurate, precise and sensitive. MS-275 demonstrates a substantial inter-individual PK variability in systemic exposure and clearance; exposures increase in near-proportion, while peak concentrations increase more than-proportionally with an increase in dose. Mean apparent oral clearance (CL/F) is independent of dose and exhibits apparent dose-independent PK behavior over the studied dose range. Oral absorption is highly variable. MS-275 has a 50-fold longer half-life in humans compared to pre-clinical species. PK/PD analysis showed significant correlation between occurrence of DLT and higher systemic exposures. Although there was an increase in the acetylation of histone H3 and H4 over time, preliminary analysis showed no significant correlation between PK parameters and change in % histone acetylation after 24 hours. MS-275 is moderately bound to plasma proteins. Hepatic phase I and II metabolic pathways are only minor routes of elimination, and MS-275 is neither a substrate for liver-specific organic anion transporting proteins, OATP1B1 and OATP1B3, nor a substrate for gastrointestinal efflux transporters ABCB1 (P-gp) or ABCG2. No significant correlation was found between CL/F and demographic, body measures and other clinical covariates, and inter-patient variability in CL/F remained similar in magnitude even after correcting dose for body surface area (BSA) or other body measures. BSA is not a significant predictor of MS-275 PK, and flat-fixed dosing can be used in the future.

Protooncogene Crk was found to be upregulated in tumours with aggressive and invasive potential. The adaptor protein Crk has an important role in cell signaling: it integrates signals from activated integrins and growth factors receptors via its SH2 domain and transmits the signal to its SH3 domain binding partners that activate the small GTPases Rac1, Rap1 and Ras. This leads to regulation of cell migration, proliferation and survival. The aim of this thesis project was to inhibit the Crk dependent signaling by a competitive inhibition of the Crk SH3 domain, using a high affinity CrkSH3 binding peptoid. Binding of the inhibitor to the Crk SH3 domain prevents binding of cellular Crk SH3 interaction partners and the corresponding signal transmission is impaired. In this thesis project the effect of the Crk SH3 inhibition on the invasiveness of cancer cells was analyzed. The observed inhibitory effect on cell invasion as well as on anchorage independent growth provides a proof of therapeutical relevance of targeting CrkSH3N domain by peptoide-based inhibitors. Powered by TCPDF (www.tcpdf.org)

Master of Science / Department of Anatomy & Physiology / Deryl L. Troyer / Breast cancer ranks second as a cause of cancer death in women in the USA. Detection of early tumors and tumor-targeted treatments could decrease the problems associated with breast cancer management. Photodynamic therapy (PDT) is a cancer treatment that uses a photosensitizer and a specific wavelength of light and is currently in clinical trials for breast cancer. When tumor cells which have absorbed photosensitizer are exposed to the correct wavelength of light, reactive oxygen species are generated, resulting in tumor cell death. Poor tissue penetration of light is a major limitation in PDT, restricting its use to treatment of localized tumors. Light generation at the tumor area might increase the effectiveness of PDT. Polymorphonuclear neutrophils (PMNs) are known to often infiltrate breast adenocarcinoma, and their activatation in tumor stroma produces luminescence in the presence of luminol. Here, we hypothesized that luminol can be used as a theranostic agent for luminescence-based early tumor detection (diagnosis) and in situ PDT (treatment). BALB/c mice were transplanted with 4T1 mammary adenocarcinoma cells to establish a breast adenocarcinoma model. The early tumor detection objective was tested by daily intraperitoneal injection of luminol and in vivo luminescence imaging. To test the PDT treatment objective,the photosensitizer 5-aminolevulinic acid (ALA) and luminol were administered to mice through intraperitoneal and intravenous routes, respectively. This treatment regimen was repeated six times and ALA alone/luminol alone/saline treated tumor-bearing mice were used as controls. Results demonstrated that luminol allowed detection of activated PMNs only two days after 4T1 cell transplantation, even though tumors were not yet palpable. Relative differences in the increase of tumor volume and final tumor weights were analyzed to test the in situ PDT. Analysis of the data showed luminol treatments resulted in breast adenocarcinoma tumor growth attenuation. In conclusion this study provides evidence that luminol can be a theranostic agent for breast adenocarcinoma.

Usefulness of a particular clinical assay is directly correlated with its ability to extract highest possible signal from available data. This is particularly relevant for personalized radiation therapy since early plan modifications confer greater benefits to treatment outcome. Recent studies have demonstrated capability of single-cell Raman microscopy to detect cellular radiation response at clinical (below 10Gy) doses, but only in certain strongly responding cell lines and after at least two day incubation. One possible cause is rather unoptimized signal processing used. This work investigates application of several advanced multivariate methods - weighted principal component analysis (WPCA), robust PCA, probabilistic PCA, and nonlinear PCA to increase radiation response signal. Representative datasets from strongly (H460 - human lung) and weakly (LNCaP - human prostate) responding cell lines were analysed in 0-50Gy and 0-10Gy dose ranges and results quantified to determine relative and absolute algorithm performance. It was found that with careful tuning, significant improvements in sensitivity and better signal separation could be achieved as compared to conventional PCA. / Graduate

<p>Implantable biomedical devices have been widely used to treat a variety of diseases for many decades. If allowed by the size and form factor, batteries have been the power source of choice in implantable devices (e.g., cardiac pacemakers). Batteries are, however, still big and come in shapes that are not ideal for minimally invasive deployment. Inductive powering is another commonly used energy source in which two well-aligned coils allow a transmitter to power the implanted receiver (e.g., cochlear implants). Once the receiver coil becomes small (mm-scale), the inductive powering link becomes very inefficient and sensitive to slight misalignment between the coils. Hence, it becomes increasingly difficult to power small devices implanted deep (>5 cm) within the tissue using inductive powering. Ultrasonic powering is an attractive alternative for powering miniature devices since it can penetrate deep into the tissue, it has greater efficiency at mm-scale receiver size, it can be omni-directional, and it is more amenable to miniaturization.</p> <p>In this dissertation, I describe the use of ultrasonic waves to power and control mm-scale implantable devices. After a detailed look at ultrasonic transmission link, I will discuss factors affecting the power transfer efficiency. These include the effect of receiver aspect ratio and size on the resonant frequency and factors related to acoustic and electrical matching. A 3D printed acoustic matching layer in then described. I will discuss two applications using ultrasound to power and control implantable devices. The first is a low-power on-off acoustic control scheme to reduce the standby power consumption in implantable devices. The second is an ultrasonically powered electrolytic ablator with an on-board micro-light-source for the treatment of cancer.</p>

The advent of effective oral, molecular-targeted drugs in oncology has changed many incurable malignancies such as chronic myeloid leukemia into chronic diseases similar to coronary artery disease and diabetes mellitus. Oral agents including monoclonal antibodies, kinase inhibitors and hormone receptor blockers offer cancer patients incremental improvements in both overall survival and quality of life. As it is imperative to recognize and manage side effects of platelet inhibitors, beta blockers, statins, HIV drugs, and fluoroquinolones by all healthcare providers, the same holds true for these newer targeted therapies, patients may present to their generalist or other subspecialist with drug-related symptoms. Cardiovascular adverse events are among the most frequent, and potentially serious, health issues in outpatient clinics, and among the most frequent side effects of targeted chemotherapy. Data support improved patient outcomes and satisfaction when primary care and other providers are cognizant of chemotherapy side effects, allowing for earlier intervention and reduction in morbidity and health care costs. With the implementation of accountable care and pay-for-performance, improved communication between generalists and subspecialists is essential to deliver cost-effective patient care.

Imaging techniques provide a method for non-invasive longitudinal monitoring of cancer therapies, but common metrics such as tumour size are late markers and do not indicate heterogeneity of response. Apoptotic cell death is an earlier marker of tumour response and produces molecular and cellular-level changes (macromolecular breakdown, membrane changes and cell shrinkage) that may be detectable by magnetic resonance imaging (MRI). Previous studies using conventional MRI methods have shown little sensitivity to apoptosis. In this thesis it is hypothesized that, using an extracellular contrast agent to affect the MRI property of relaxation for extracellular water preferentially, parameters related to water in the intracellular and extracellular environments and the exchange between them can be obtained and will be sensitive to apoptosis. It is also hypothesized that membrane changes and macromolecular breakdown are detectable by the technique of magnetization transfer. Measurements of relaxation in the presence of contrast agent in vitro demonstrated a decrease in extracellular water fraction and an increase in the rate of water exchange across the plasma membrane during apoptosis. In vivo, this method was complicated by the difficulty of delivering contrast agent to the tumour, but regions with good delivery showed correlation between high water exchange rates from MRI and apoptosis in histology. Magnetization transfer studies indicated only small changes in vitro during apoptosis and these were largely related to changes in the free water, so this method was not investigated further. Further work is required to determine the tumour lines where the water exchange methods may be applied reliably. Nevertheless, the method of measuring water exchange presented in this thesis can be performed in a clinically-feasible amount of time (~20 minutes). It therefore has potential in detecting apoptosis and predicting therapy response. It also emphasizes the role of water exchange in conventional MRI relaxation experiments.

Imaging techniques provide a method for non-invasive longitudinal monitoring of cancer therapies, but common metrics such as tumour size are late markers and do not indicate heterogeneity of response. Apoptotic cell death is an earlier marker of tumour response and produces molecular and cellular-level changes (macromolecular breakdown, membrane changes and cell shrinkage) that may be detectable by magnetic resonance imaging (MRI). Previous studies using conventional MRI methods have shown little sensitivity to apoptosis. In this thesis it is hypothesized that, using an extracellular contrast agent to affect the MRI property of relaxation for extracellular water preferentially, parameters related to water in the intracellular and extracellular environments and the exchange between them can be obtained and will be sensitive to apoptosis. It is also hypothesized that membrane changes and macromolecular breakdown are detectable by the technique of magnetization transfer. Measurements of relaxation in the presence of contrast agent in vitro demonstrated a decrease in extracellular water fraction and an increase in the rate of water exchange across the plasma membrane during apoptosis. In vivo, this method was complicated by the difficulty of delivering contrast agent to the tumour, but regions with good delivery showed correlation between high water exchange rates from MRI and apoptosis in histology. Magnetization transfer studies indicated only small changes in vitro during apoptosis and these were largely related to changes in the free water, so this method was not investigated further. Further work is required to determine the tumour lines where the water exchange methods may be applied reliably. Nevertheless, the method of measuring water exchange presented in this thesis can be performed in a clinically-feasible amount of time (~20 minutes). It therefore has potential in detecting apoptosis and predicting therapy response. It also emphasizes the role of water exchange in conventional MRI relaxation experiments.