The coupling efficiency of carbon nanotubes with near infrared laser radiation at 940nm wavelength was investigated. Nanotubes treated with different post processing methods were irradiated at different laser power intensities as dry samples and suspensions in water or ethanol. The interaction with the laser beam was measured and quantified based on the temperature increase in the samples as well as the amount of energy transmitted through them. Parallel experiments using carbon black revealed better performance of carbon nanotubes in terms of coupling efficiency and heat dissipation to their surroundings. It was found that most of the incident radiation on an individual carbon nanotube is absorbed, resulting in extreme local temperature increases proportional to the laser intensity, which can lead to instant tube oxidation in air. Such high heats are efficiently transferred to the material in immediate contact with the nanotubes, increasing its temperature very rapidly. The most intriguing results were obtained in the presence of water where the observations suggested, disintegration of carbon nanotubes with each laser pulse. It is shown that extremely high local temperatures vaporise the water in the immediate vicinity of a carbon nanotube and result in a water-gas reaction. It is further postulated that such effects can be achieved with laser beams at power intensities near the skin tissue's safe exposure thresholds, and therefore can potentially be used as a method of removing nanotubes from living tissue. This has advantages in providing an exit route for nanotubes whether introduced on purpose for reasons of medicine or therapy, or possibly, as a result of inadvertent exposure. Further studies on laser heating and transmission through different dry samples, highlighted that more crystalline structures such as that of a heat-treated nanotube, are more effective in causing extinction of the laser beam and a reduction in the transmitted beam intensity, however the tubes with more defects or with a length comparable to the radiation wavelength are very effective in converting the absorbed laser energy to heat. This effect is exacerbated when the laser beam is polarised parallel to the long axis of the carbon nanotubes. These heating effects were exploited to create welds in high density polyethylene using through transmission laser welding. The resultant welds showed better than or equal mechanical performance to welds made using industrial absorbers such as carbon black or Clearweld®.

Taxol (generic name paclitaxel), a plant‐derived antineoplastic agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. Obtaining taxol from this source requires destruction of trees. It has been used alone or in combination with other chemotherapeutic agents for the treatment of breast, ovarian as well as many other types of cancer, including non‐small cell lung carcinoma, prostate, head and neck cancer, and lymphoma, as well as AIDSrelated Kaposi’s sarcoma. The mode of action of taxol against a number of human cancer cells is by preventing the depolymerization of tubulin during cell division. This molecule increases microtubule stability in the cell and induces apoptosis. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the dry weight. The commercial isolation of 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000‐3000 well‐grown trees. The limited supply of the drug has prompted efforts to find alternative sources of taxol. Alternative methods for taxol production, such as chemical synthesis, tissue and cell cultures of the Taxus species are expensive and give low yields. A fermentation process involving any microorganism would be the most desirable means to lower the cost and increase availability. The first report on the isolation of taxol‐producing fungi from Taxus brevifolia appeared in 1993 (Stierle, et al., 1993). Several taxol‐producing fungi have been identified since, such as Taxomyces andreanae, Taxodium disticum, Tubercularia sp., Pestalotiopsis microspora, Alternaria sp., Fusarium maire and Periconia sp (Li, et al., 1996, Strobel, et al., 1996a, Strobel, et al., 1996b, Li, et al., 1998b, Ji, et al., 2006, Xu, et al., 2006). This thesis investigates the isolation of an endophytic fungus, isolated from the stem cuttings of Taxus celebica, which produces taxol and related taxanes. We observed morphological and cultural characteristics and analyzed the sequences of rDNA ITS from the strain. The isolated fungus grew on potato carrot agar (PCA) medium at 25 °C and the colonies were white to off‐white, floccose, with irregular margins. The reverse side of the culture was cream in color. The morphology was examined microscopically following staining with cotton blue in lactophenol. Cultures produced macroconidia on slender, 85 μm long phialides. The macroconidia were 25‐40 X 3.75 μm. Cultures also produced round or oval microconidia. Analysis of the ITS and D1/D2 26S rDNA sequence revealed 99 % identity with Fusarium solani voucher NJM 0271. Based on its morphological, cultural characteristics and 26S rDNA sequence, the fungus was identified as F. solani. This fungus is different from the previously reported endophytic taxol‐producing species of Fusarium. Taxol and baccatin III, produced by this fungus, were identified by chromatographic and spectroscopic comparison with standard compounds. The amount of taxol produced by F. solani in potato dextrose liquid medium is low (1.6 μg l‐1) (Chakravarthi, et al., 2008). We further investigated different growth media and various factors of cultivation to select the medium and conditions that maximize production of taxol and other taxanes by this fungus. F. solani was grown in five well‐defined culture media under stationary and shake conditions separately for various time intervals and the amounts of taxol, baccatin III and other taxanes produced were estimated by competitive immunoassay. The modified flask basal medium (MFBM) was shown to yield the highest production of taxol (128 μg l‐1) which is 80 times more than when grown in potato dextrose liquid medium, baccatin III (136 μg l‐1) and total taxanes (350 μg l‐1) under shake conditions. From our results the highest taxol production of F. solani was achieved when cultured in MFBM. The production in MFBM was 80 times higher than that cultured in the potato dextrose liquid medium. In conclusion, it was shown that the culture medium plays a major role in taxol and other taxanes production and fungal growth. MFBM is the best medium, among the media studied, to produce taxol and other taxanes. The higher concentrations of NH4NO3, MgSO4, KH2PO4 and FeCl3 in the FBM medium seem important for production of taxol and other taxanes. These results can be considered as starting‐point for the research directed to improve taxol and baccatin III production by F. solani via different approaches including fermentations, strain improvement and genetic engineering techniques. Finally, in order to get more insights into the mode of action of this fungal taxol and baccatin III (for the first time), their apoptotic activity on different cancer cell lines was determined. We elucidated the biochemical pathways leading to apoptotic cell death after fungal taxol‐ and baccatin III‐ treatment in different cancer cell lines. Experiments are done on various cancer cell lines namely JR4 Jurkat (T‐cell leukemia), J16 Bcl‐2 Jurkat T cells, HepG2 (hepatoma), caspase‐8‐deficient Jurkat T cells, HeLa (human cervical carcinoma), Ovcar3 (human ovarian carcinoma) and T47D (human breast carcinoma) cells. We were able to demonstrate that both fungal taxol and baccatin III can induce apoptosis in all the cell lines tested, by flow cytometric analysis. Hallmarks of apoptosis following the signaling pathway to far more upstream‐located events were investigated using biochemical and cell biological methods. It has shown that during fungal taxol‐ and baccatin III‐induced apoptosis, DNA is degraded resulting in a increased number of hypodiploid cells reaching up to 65‐70% after 48 h. Disruption of mitochondrial membrane potential was examined by flow cytometric analysis using mitochondrial membrane potential sensitive dye JC‐1 and JR4‐Jurkat cells were shown to undergo significant loss of mitochondrial membrane potential loss of mitochondrial membrane potential reaching up to 70% in 6 nM fungal taxol and 65 % in 3.5 μM baccatin III after 36 h. These results were similar to those observed with standard taxol and baccatin III. We further investigated the role of caspases in fungal taxol‐ and baccatin III‐induced apoptosis, caspase‐8‐deficient Jurkat cells, Bcl‐2‐over‐expressed J16‐Jurkat cells and caspase inhibitors were used. Results derived from caspase‐8‐deficient Jurkat cells show that caspase‐8 is not involved in fungal taxol‐ and baccatin IIIinduced apoptosis of Jurkat cells. Using the pan‐caspase inhibitor (Z‐VAD‐FMK), caspase‐9 inhibitor (Z‐LEHD‐FMK), caspase‐3‐inhibitor (Z‐DEVD‐FMK), caspase‐2‐ inhibitor (Z‐VDVAD‐FMK) and caspase 10‐inhibitor (Z‐AEVD‐FMK), it was shown that caspase‐10 is involved in fungal taxol‐ and baccatin III‐ induced apoptosis in JR4‐Jurkat cells. It was also shown that inhibitors of caspases‐9, ‐2 or ‐3 partially inhibited fungal taxol‐ and baccatin III‐ induced apoptosis, whereas the caspase‐ 10 inhibitor totally abrogated this process. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in fungal taxol‐ and baccatin III‐treated JR4‐Jurkat and HeLa cells. DNA fragmentations were shown by agarose gel electrophoresis method. Our work showed that treatment of JR4‐ Jurkat and HepG2 cells with fungal taxol and baccatin III induces apoptosis as shown by DNA ladder formation. Herein it was demonstrated that fungal taxol and baccatin III have a similar mechanism of action, but the efficacy of fungal taxol to induce apoptosis is higher. In summary, fungal baccatin III is found to be effective in inducing apoptosis similar to taxol but at higher concentration and both fungal taxol and baccatin III induce apoptosis via caspase‐10 and mitochondrial pathway in Jurkat cells. In conclusion, the present study describes isolation of a taxol‐producing endophyte F. solani IISc.CJB‐1. The growth requirements of this fungus for production of taxol, baccatin III and other taxanes were studied. The apoptotic activity of taxol and baccatin III (for the first time) was observed. In addition, our results show that the culture medium plays a major role in taxol and other taxanes production and fungal growth. Among the media studied, modified flask basal medium (MFBM) is the best to produce taxol and other taxanes. It is evident from this data that this fungal strain can be promising candidate for large‐scale production of taxol and related taxanes.

Oncolytic viruses (OVs) are novel biological agents that selectively infect and kill malignant cells. OVs can also generate anti-cancer immunity. Our lab exploited this phenomenon and developed an in vitro vaccine with infected leukemia cells with oncolytic virus vaccine – and named immunotherapy by leukemia-oncolytic virus (iLOV) – that provided in vivo protection in a murine model for acute lymphoblastic leukemia. This work further characterizes iLOV biology and the interaction of its immune responses. An in vitro immune response assay was optimized to detect and quantify the in vivo anti-leukemia immunity generated by iLOV. Anti-viral immunity is an obstacle for OV therapy. Although iLOV created anti-viral antibodies towards itself, these neutralizing antibodies did not hinder the vaccine’s ability to initiate complement or dendritic cell activation. We envision personalized versions of iLOV for leukemia patients in remission to prevent the possibility of relapse. This work highlights new advantages for infected cell vaccines and supports the progress of iLOV toward clinical testing.

Oncolytic viruses are novel candidates for cancer therapy and their efficacy relies on their capacity to overcome the host’s anti-viral barriers. In mammalian cells, the anti-viral response involves a protein-signaling cascade known as the interferon pathway, which alerts the immune system and limits the propagation of infection. Given that most cancer cells have defects in this pathway, they are susceptible to viral infection and responsive to oncolytic virotherapy. For reasons that remain unknown, many cancers are still refractory to oncolytic viruses, which suggests the existence of additional antiviral mechanisms. In this study, we investigate the potential involvement of an alternative antiviral pathway in cancer cells. Given that insects and plants rely on the RNA silencing pathway for their anti-viral protection, we investigated the presence of a similar mechanism in cancer cells. We found viral genome-derived small RNAs in various cancer cell lines upon infection, which is indicative of an RNA-mediated antiviral response. Also, various viruses encode suppressors of the RNA interference pathway. To determine if an oncolytic virus could benefit from such a factor, we engineered an oncolytic virus variant to encode the Nodamura virus B2 protein, a known inhibitor of RNA silencing-mediated immune responses. Using this virus, we observed enhanced cytotoxicity in 33 out of the 38 human cancer cell lines tested. Furthermore, our results show inhibition of viral genome cleavage and altered microRNA processing by our B2-expressing oncolytic virus. Taken together, our data suggests the blockade of RNA silencing antiviral pathways and/or antiviral microRNA processing improves the efficacy of our B2-encoding virus in a cell-line specific manner. Overall, our results establish the improved potential of our novel virus therapy and demonstrate for the first time the involvement of RNA pathways in the antiviral defense of cancer cells.

Nanomaterials have been widely investigated for improving the treatment of diseases acting as vectors for diverse therapies and as diagnostic tools. Iron-based nanowires possess promising potential for biomedical applications due to their outstanding properties. The combination of different therapeutic and diagnostic strategies into one single platform is an approach for more efficient and safer treatments. In this thesis, I investigate the application of iron-iron oxide core-shell nanowires as therapeutic agents for cancer treatment. In particular, a novel method for multimodal cancer cell destruction was developed combining the optical, magneto-mechanical and chemotherapeutic properties of functionalized nanowires. By functionalizing the nanowires with doxorubicin through a pH-sensitive linker, the first treatment modality was achieved by selective intracellular drug release. The second treatment modality utilizes the mechanical disturbance exerted by the nanowires upon the application of a low-power alternating magnetic field. The third treatment modality exploits the capability of the nanowires to transform optical energy, absorbed from near-infrared irradiation, into heat. The efficiency of the three treatment modalities both independently and combined were tested in breast cancer cells with near complete cell death (90%). The combination of the different strategies can potentially reduce side effects and treatment time. Additionally, I studied the potential of these iron-iron oxide core-shell nanowires as diagnostic tools, included in the Appendix of this dissertation. Specifically, I studied their capability to act as magnetic resonance imaging contrast agents for cell labeling, detection and tracking. Therein, a high performance as T2 contrast agents was confirmed evaluating the effect of oxidation and surface coatings on the T2 contrast in the tailored transverse relaxivities. The detection of nanowire-labeled cancer cells was demonstrated in T2-weighted images of cells implanted in tissue-mimicking phantoms and in mouse brain. Labeling the cells with nanowires enabled high-resolution cell detection after in vivo implantation (~10 cells) over a minimum of 40 days. The capability of these magnetic nanowires of being remotely controllable and detectable make them an attractive option in the treatment and diagnosis of cancer and in cell therapy. Future directions include preclinical studies for testing the nanowire-based photothermal therapy for tumor ablation.

This study aims to prepare theranostic nanoparticles (NPs) that are expected to increase cancer diagnostics and therapeutic efficacy. We prepared the NPs constituting carbon dots (CDs) as an imaging agent, folic acid as a targeting agent, doxorubicin (DOX), or gemcitabine (GEM) as chemotherapy agents. The NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. Through ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Fourier transform-infrared spectroscopy, the fabrication of these NPs was confirmed. It was discovered that the high drug loading efficiency is the noncovalent series while the high drug loading capacity is the covalent series The in-vitro pH-dependent drug release data indicate the NPs release more drugs at around pH 5.0 than at pH 7.4. The NPs sizes are between 2-5 nm. The Cell viability was investigated using the Alamar Blue assay and the three NPs complexes exhibited strong therapeutic efficacy against MDA-MB-468 breast cancer cells as compared with CDs-drug.

Cancer prevails as one of the most devastating diseases being at the top of death causes for adults despite continuous development and innovation in cancer therapy. Nanotechnology may be used to achieve therapeutic dosing, establish sustained-release drug profiles, and increase the half-life of drugs. In this context, magnetic nanowires (NWs) have shown a good biocompatibility and cellular internalization with a low cytotoxic effect. In this thesis, I induced cancer cell death by combining the chemotherapeutic effect of iron NWs functionalized with Doxorubicin (DOX) with mechanical disturbance under a low frequency alternating magnetic field. Two different agents, APTES and BSA, were separately used for coating NWs permitting further functionalization with DOX. Internalization was qualitatively and quantitatively assessed for both formulations by confocal reflection microscopy and inductively coupled plasma-mass spectrometry. From confocal reflection analysis, BSA formulations demonstrate to have a higher internalization degree and a broader distribution within the cells in comparison to APTES formulations. Both groups of functionalized NWs generated a comparable cytotoxic effect in MDA-MB-231 breast cancer cells in a DOX concentration-dependent manner, (~60% at the highest concentration tested) that was significantly different from the effect produced by the free DOX (~95% at the same concentration) and non-functionalized NWs formulations (~10% at the same NWs concentration). A synergistic cytotoxic effect is obtained when a low frequency magnetic field (1 mT, 10 Hz) is applied to cells treated with the two formulations that is again comparable (~70% at the highest concentration). Furthermore, the cytotoxic effect of both groups of coated NWs without the drug increased notoriously when the field is applied (~25% at the highest concentration tested). Here, a novel bimodal method for cancer cell destruction was developed by the conjugation of the magneto-mechanical properties of the iron NWs coupled with the chemotoxic effect of an anticancer drug. Moreover, it was demonstrated that iron nanowires possess an outstanding biocompatibility and showed high efficacy as drug delivery agents coupled to a high degree of cell internalization. Finally, the proposed method benefits from the low power fields applied during treatment. This poses much less safety risks and allows using cheaper and simpler equipment.

<div>Under the guidance of cancer treatment data, this thesis emphasizes the development of radiation-responsive nanomaterials for the effective implementation of localized, multimodal therapy for solid tumors. Evidence from decades of treatment outcomes underscores the benefits provided by employing multiple therapeutic agents in concert to improve prognoses for cancer patients. As a pillar of standard care in oncology, radiation therapy (RT) is a particularly appropriate choice as a component of combination therapies, acting as a localized tool for achieving long-term tumor control. By combing primary RT with radio-sensitizing, polymer-encapsulated formulations of crystalline calcium tungstate nanoparticles (CWO NPs), this work has shown significant improvements in efficacy in in vitro and murine xenograft models of primary human head and neck tumors as well as in spontaneous sarcoma in a clinical case study. Under X-ray radiation, CWO NPs emit long-wavelength ultraviolet (UV-A) and visible light, a property referred to as radioluminescence. This work focuses on utilizing these properties in combination with encapsulant functionalization strategies to further improve therapeutic outcomes through specific mechanistic enhancements.</div><div><br></div><div>Ordinarily used primarily to improve biocompatibility and colloidal stability, the polymeric materials used to encapsulate the CWO NPs were tailored to serve distinct functions in the overall combination therapy scheme. Approaches explored in this work include surface functionalization of these polymers with a cancer-specific ligand, folic acid, and the incorporation of photo-responsive/sensitizing bilirubin-polymer conjugates as an encapsulant. The predicted outcomes of surface functionalization and photo-active encapsulation were confirmed to significantly enhance radiotherapy efficacy. Finally, exploration of intratumoral NP distribution after dose administration was conducted to preliminarily evaluate strategies for dose homogeneity improvement. Mechanical agitation of the injection site somewhat improves distribution of NPs in tumor xenografts but requires future exploration for improved understanding and implementation.</div>

The main aim of this study is to synthesize theranostic nanoparticles (NPs) that will drastically increase the diagnostics and therapeutic efficacy for cancer. In this research, we had prepared the NPs which constitute carbon dots (CDs), the imaging agent, Folic acid, the targeting agent, and Doxorubicin (DOX) or Gemcitabine (GEM) as the chemotherapy agents. The prepared NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. The spectroscopy, ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy, and Fourier transform-infrared spectroscopy (FT-IR), were used to confirm the successful fabrication of these complexes. Through UV-vis analysis, the drug loading capacity (DLC) and drug loading efficiency (DLE) of the complexes were determined. The noncovalent series had a higher DLE of about 83% while the covalent series showed higher DLC, 70% on average indicating high drug content. The in-vitro pH-dependent drug release shows that the noncovalent FA-CDs-DOX and the covalent FA-CDs-GEM series release more drugs into the cancer cells (pH of 5.0) than into healthy normal (pH of 7.4). The sizes of NPs were measure around 2-5 nm with Dynamic light Scattering (DLS). The toxicity of CDs, CDs-drug, and FA-CDs-drug on MDA-MB468 breast cancer cell was tested through the methylthiazolytetrazolium (MTT) assay and found that the FA bonded NPs exhibited strong therapeutic efficacy. More pharmaceutical data towards the cancer cells are investigated by our research collaborators – the pharmaceutical department at ETSU and Xavier University at Louisiana.

Sphingolipids are a class of lipid molecules that function both as structural membrane components and as bioactive signaling molecules. Sphingolipids can be produced de novo or salvaged and recycled. Despite the established roles of sphingolipids such as sphingosine 1-phosphate and ceramides in regulating signaling involved in pro- and anti-tumorigenic cellular processes, the role of the de novo sphingolipid biosynthesis pathway in cancer is unclear. The main objective of this thesis study was to determine whether there is an essential role for this pathway in cancer and whether its disruption can be a cancer-specific metabolic vulnerability. Here, we find that de novo sphingolipid synthesis through the rate-limiting enzyme serine palmitoyltransferase (SPT) is not required in cancer cells due to their salvage capacity. However, upregulation of SPT in cancer cells creates a requirement to detoxify its product, 3-ketodihydrosphingosine (3KDS), via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). We demonstrate that KDSR is essential in cancer cells both in vitro and in vivo to restrain the levels of its substrate 3KDS, the accumulation of which can disrupt ER structure and function, resulting in proteotoxic stress and cell death. Our findings also reveal that KDSR is essential specifically in cancer cells and not normal cells and that upregulation of SPT in cancer may act as a biomarker for sensitivity to targeting KDSR. Altogether, this thesis study provides new insights into the role of KDSR in the de novo sphingolipid biosynthesis pathway in both cancer and ER homeostasis and demonstrates the potential to exploit this for therapeutic purposes in a cancer-specific manner.