This research utilizes Electrical Impedance Spectroscopy, a technique classically used for electrochemical analysis and material characterization, as the basis for a non-destructive, label-free assay platform for three dimensional (3D) cellular spheroids. In this work, a linear array of microelectrodes is optimized to rapidly respond to changes located within a 3D multicellular model. In addition, this technique is coupled with an on chip micro-pH sensor for monitoring the environment around the cells. Finally, the responses of both impedance and pH are correlated with physical changes within the cellular model. The impedance analysis system realized through this work provides a foundation for the development of high-throughput drug screening systems that utilize multiple parallel sensing modalities including pH and impedance sensing in order to quickly assess the efficacy of specific drug candidates. The slow development of new drugs is mainly attributed to poor predictability of current chemosensitivity and resistivity assays, as well as genetic differences between the animal models used for tests and humans. In addition, monolayer cultures used in early experimentation are fundamentally different from the complex structure of organs in vivo. This requires the study of smaller 3D models (spheroids) that more efficiently replicate the conditions within the body. The main objective of this research was to develop a microfluidic system on a chip that is capable of deducing viability and morphology of 3D tumor spheroids by monitoring both the impedance of the cellular model and the pH of their local environment. This would provide a fast and reliable method for screening pharmaceutical compounds in a high-throughput system.

This thesis reports a study of the chemical stability and coordination chemistry of several antitumour metallocenes Cp2MCl2 (Cp = h5-C5H5; M = Ti 1, V 2, Nb 3, Mo 4), as well as derivatives of Cp2TiCl2 1, with nucleic acids, nucleic acid constituents and proteins. These studies were carried out in order to identify the biologically active species and more fully understand the molecular level mechanism of action of the antitumour metallocenes, in particular Cp2TiCl2 1, which is currently undergoing phase II clinical trials. The interactions of Cp2MoCl2 4 with four oligonucleotides were studied by 1H and 31P NMR spectroscopy. In 50 mM salt solutions of Cp2MoCl2 4, hydrolysis of the halide ligands occurred to give a solution with pD -2, containing a species in which both Cp rings remain metal bound for 24 h. At pD -7, partial hydrolysis of the Cp rings (-30percent) occurred after 24 h. Addition of an aqueous solution of Cp2MoCl2 4 in 50 mM salt to the self-complementary sequence d(CGCATATGCG)2, maintaining the pD at 6.0-7.0, showed no evidence for the formation of a metallocene-oligonucleotide complex and only peaks arising from hydrolysis of Cp2MoCl2 4 were detected. A similar result was obtained in titration experiments with the single stranded sequence d(ATGGTA) at pD 6.5-7.0. However, at pD 3.0, new signals assigned to a molybdocene-oligonucleotide complex(es), which was stable for hours at pD 3.0, were detected; while at pD -7 the complex is destabilised and only peaks arising from hydrolysis of Cp2MoCl2 4 were detected. Titration experiments at low pD with Cp2MoCl2 4 and the dinucleotide dCG were consistent with formation of a complex arising due to coordination of molybdenum to guanine N7 and/or cytosine N3. The results obtained showed that stable oligonucleotide adducts were not formed in 50 mM salt at pD -7 and hence it is highly unlikely that formation of molybdocene-DNA adducts in vivo is the primary action that is responsible for the antitumour properties of Cp2MoCl2 4. The rate of hydrolysis of the aromatic rings of Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) and the dimethylsubstituted derivatives (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41), in aqueous solutions at pD 2-8 was studied by 1H NMR spectroscopy. Rapid hydrolysis of both the halide/glycine and Cp ligands in Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) occurred and predominantly gave a precipitate at pD -7. In contrast, under the same experimental conditions, the predominant species present in aqueous solutions of (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) at pH 2-8 contained both MeCp rings metal bound. At pD < 5, Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) and (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) formed similar complex(es) with purine nucleotides. However, at pD >5, stable adducts between nucleotides and Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) were not formed. In contrast, (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) formed complex(es) with 5'-dAMP or 5'-dGMP, which were stable for 24 h. These results suggest that formation of stable chelates between (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) and nucleic acid constituents in vivo is possible. However, the methyl substituted derivatives 34 and 41 did not show any antitumour activity against EAT in mice when administered in either 10percentDMSO/90percentsaline or in water at pH 6.2-6.4, which suggests that the labile Cp-Ti bond present in Cp2TiCl2 1 is required for antitumour activity. The synthesis of a range of Cp substituted titanocene derivatives was investigated in an attempt to prepare derivatives with modified Cp stability in comparison to the methyl substituted derivatives. The synthesis of derivatives (CpCH2Y)2TiCl2 where Y equals ?CHO 43, ?CONMe2 44, ?NO2 45, (RCp)2TiCl2 where R equals ?COMe 46, ?COOMe 47 or ?CONMe2 48, (CpNMe2)2TiCl2 62 and (Cp(CH2)2NMe2)2TiCl2 63 was unsuccessful, due to the presence of coordinating substituents on the Cp rings and poor stability in polar, protic solvents. Hence, these derivatives were excluded from further studies. The rate of hydrolysis of the Cp rings of Cp2TiX2 (X equals Cl 1, OCOCCl3 22 and OCOCH2NH3Cl 27) in aqueous solutions, 10percentDMSO/90percentD2O and 100percent DMSO was monitored by 1H NMR spectroscopy. Rapid hydrolysis of both the carboxylate and Cp ligands of Cp2TiX2 (OCOCCl3 22 and OCOCH2NH3Cl 27) occurred in DMSO to give biologically inactive species. The rate of these reactions were concentration dependent as dilution of these samples with saline or water to give the therapeutic conditions of 10percentDMSO/90percentD2O slowed the hydrolysis chemistry. In contrast, samples of Cp2TiX2 (X equals Cl 1 and OCOCH2NH3Cl 27) dissolved in water, gave solutions containing the presumed antitumour active species in which the halide or glycine ligands have been hydrolysed but the Cp rings remain metal bound. Thus, charged X ligands may be incorporated into Cp2TiX2 and will give comparable activity to Cp2TiCl2 1 provided the samples are administered in water. The antitumour metallocenes Cp2MCl2 (M equals Ti 1, V 2, Nb 3, Mo 4) and the inactive derivative (MeCp)2TiCl2 34 were found to inhibit the relaxation of supercoiled plasmid DNA pBR322 by human topoisomerase II in vitro. These results implicated the inhibition of topoisomerase II in the mechanism of antitumour activity although there was no direct correlation between the in vitro results with biological activity against EAT in vivo. UV spectroscopy confirmed that the metallocenes Cp2MCl2 (M equals Ti 1, Mo 4) became associated with and were stabilised to hydrolysis by calf thymus DNA but not with human serum albumin. ICP-AES was used to measure the amount of metal associated with either DNA or human serum albumin after incubation with Cp2MCl2 (M equals Ti 1, Nb 3, Mo 4) and dialysis of these solution. The results confirmed that DNA stabilises or becomes associated with the metallocenes. However, errors associated with the ICP-AES measurements did not allow these results to be quantified. 1H NMR spectroscopy was used to show that the antitumour metallocene Cp2MoCl2 4 formed an adduct with glutathione 72 in the pH range 3-7 through the sulfur donor group. In comparison, the antitumour metallocenes Cp2MCl2 (M equals Ti 1, Nb 3) showed limited adduct formation with glutathione 72 at pH -3 and no adducts were detected at pH > 5.5.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Degree awarded (2004): PhDBmS, Neuroscience Program, George Washington University / Glutamate is critically important as an excitatory neurotransmitter in the central nervous system. Increasing evidence suggests additional signaling roles for glutamate in cell proliferation and migration in normal and oncogenic states. Recently, glutamate release from glioma cells has been shown to increase tumor growth in vivo. To investigate the mechanism of glutamate enhancement of tumor growth, we investigated the effect of glutamate on tumor cell proliferation, invasion, and glioma-induced cell death. Here we demonstrate that glutamate enhances tumor growth via increasing tumor cell proliferation and inducing excitotoxic death of cells surrounding the solid tumor mass, thereby facilitating tumor expansion. The evidence that glutamate enhances tumor growth suggests that regulating extracellular levels of glutamate may restrict tumor growth. In the normal brain, extracellular glutamate levels are maintained by a family of glutamate transporters. To investigate the therapeutic potential of regulating extracellular glutamate concentrations on tumor growth, we utilized a transgenic mouse model of EAAT2 glutamate transporter overexpression. In this report, we demonstrate that increased glutamate transport limits tumor growth in vivo and provides protection against glioma-associated neuronal cell death. In addition, seizure activity, often associated with the presence of a CNS tumor, is attenuated in transgenic mice overexpressing the glutamate transporter, EAAT2. These findings suggest that glutamate transporters may provide a new therapeutic target for limiting tumor expansion and secondary epileptogenesis. / Advisory Committee: Dr. Margaret Sutherland (Chair), Dr. Steven Patierno (Chair), Dr. Tim Hales, Dr. Vincent Chiappinelli, Dr. Linda Werling, Dr. Frances Noonan

<p>Microwave imaging is an efficient diagnostic modality for non-invasively visualizing dielectric contrasts of non-metallic bodies. An increasing interest of this field has been observed during the last decades. Many application areas in biomedicine have been issued, recently the breast tumor detection application using microwave imaging.</p><p>Many groups are working in the field at the moment for several reasons. Breast cancer is a major health problem globally for women, while it is the second most common cancer form for women causing 0.3 % of the yearly female death in Sweden. Medical imaging is considered as the most effective way of diagnostic breast tumors, where X-ray mammography is the dominating technique. However, this imaging modality still suffers from some limitations. Many women, mostly young ones, have radiographically dense breasts, which means that the breast tissues containing high rates of fibroglandular tissues. In this case the density is very similar to the breast tumor and the diagnosis is very difficult. In this case alternative modalities like Magnetic Resonance Imaging (MRI) with contrast enhancement and Ultrasound imaging are used, however those are not suitable for large scale screening program.Another limitation is the false-negative and false-positive rate using mammography, in general 5–15 % of the tumors are not detected and many cases have to go though a breast biopsy to verify a tumor diagnosis. At last the mammography using breast compression sometimes painful, and utilizing ionizing X-rays. The big potential in microwave imaging is the reported high contrast of complex permittivity between fibroglandular tissues and tumor tissues in breasts and that it is a non-ionizing method which probably will be rather inexpensive.</p><p>The goal with this work is to develop a microwave imaging system able to reconstruct quantitative images of a female breast. In the frame of this goal this Licentiate thesis contains a brief review of the ongoing research in the field of microwave imaging of biological tissues, with the major focus on the breast tumor application. Both imaging algorithms and experimental setups are included. A feasibility study is performed to analyze what response levels could be expected, in signal properties, in a breast tumor detection application. Also, the usability of a 3D microwave propagation simulator, (QW3D), in the setup development is investigated. This is done by using a simple antenna setup with a breast phantom with different tumor positions. From those results it is clear that strong responses are obtained by a tumor presence and the diffracted responses gives strong information about inhomogeneities inside the breast. The second part of this Licentiate thesis is done in collaboration between Mälardalen University and Supélec. Using the existing planar 2.45 GHz microwave camera and the iterative non-linear Newton Kantorovich code, developed at Département de Recherches en Electromagnétisme (DRE) at Supélec, as a starting point, a new platform for both real-time qualitative imaging and quantitative images of inhomogeneous objects are investigated. The focusing is related to breast tumor detection. For the moment the tomographic performance of the planar camera is verified in simulations through a comparison with other setups. Good calibration is observed, but still experimental work concerning phantom development etc. is needed before experimental results on breast tumor detection may be obtained.</p>

Multiple myeloma is a malignant bone marrow plasma cell tumor which is responsible for approximately 12,000 deaths per year in the United States and two percent of all cancer deaths. It is recognized clinically by the presence of more than ten percent bone marrow plasma cells, the detection of a monoclonal protein (M-protein), anemia, hypercalcemia, renal insufficiency, and lytic bone lesions. The disease is usually preceded by a premalignant tumor called monoclonal gammopathy of undetermined significance (MGUS), which is present in one percent of adults over the age of fifty, three percent over the age of seventy and ten percent of those in the tenth decade. MGUS is also recognized by the detection of M-protein, but with less than ten percent bone marrow plasma cells and without the other features exhibited by myeloma. The majority of MGUS patients remain stable for long periods without ever developing myeloma. Only a small percentage of patients with MGUS eventually develop multiple myeloma. However, the reason for this is not yet known. Once the myeloma stage is reached, a sequence of well-understood mutational evets eventually lead to the escape of the tumor from the control of the immune system. We propose a mathematical model of tumor-immune system interactions at the onset of the disease in an effort to better understand the early events that take place and their influence on the outcome of the disease. The model is calibrated with parameter values obtained from available data and we study the resulting dynamics. Next, we study how the behavior of the system is affected as parameters are varied. Finally, we interpret the results and draw some conclusions.

A treatment strategy for cancer is the suppression of tumor growth by directing an immune response to the tumor vessels, which will destroy the tissue. In this thesis we describe the development of a vaccine that targets antigens expressed around angiogenic vasculature in most solid tumors. These antigens are alternative spliced extra domains of glycoproteins present in the extracellular matrix; e.g. the extra domain-B (ED-B) and extra domain-A (ED-A) of fibronectin and the C-domain of tenascin-C (TNCC). We show that it is possible to break self-tolerance and induce a strong antibody response against ED-B by vaccination. Furthermore, tumor growth was inhibited and the changes observed in the tumor tissue were consistent with an attack of the tumor vasculature by the immune system. For clinical development of therapeutic vaccines, targeting self-molecules like ED-B, a potent but non-toxic biodegradable adjuvant is required. The squalene-based Montanide ISA 720 (M720) in combination with CpG DNA fulfilled these requirements and induced an equally strong anti-self immune response as the preclinical golden standard Freund’s adjuvant. We have further characterized the immune response against ED-B generated with the adjuvant M720/GpG.  The ED-B vaccine also inhibited tumor growth in a therapeutic setting in a transgenic mouse model of pancreatic insulinoma in which tumorigenesis was already initiated. Furthermore, antibodies against ED-A and TNCC could be induced in mice and rabbits. We analyzed the expression of ED-A in breast tumors of transgenic MMTV-PyMT mice, a metastatic breast cancer model, with the aim to use this model to study the effect of an ED-A vaccine on metastasis. We also detected ED-B in canine mammary tumor tissue. Therefore vascular antigens might also represent potential therapeutic targets in dogs.  All together our preclinical data demonstrate that a vaccine targeting tumor blood vessels is a promising new approach for cancer treatment.

Microwave imaging is an efficient diagnostic modality for non-invasively visualizing dielectric contrasts of non-metallic bodies. An increasing interest of this field has been observed during the last decades. Many application areas in biomedicine have been issued, recently the breast tumor detection application using microwave imaging. Many groups are working in the field at the moment for several reasons. Breast cancer is a major health problem globally for women, while it is the second most common cancer form for women causing 0.3 % of the yearly female death in Sweden. Medical imaging is considered as the most effective way of diagnostic breast tumors, where X-ray mammography is the dominating technique. However, this imaging modality still suffers from some limitations. Many women, mostly young ones, have radiographically dense breasts, which means that the breast tissues containing high rates of fibroglandular tissues. In this case the density is very similar to the breast tumor and the diagnosis is very difficult. In this case alternative modalities like Magnetic Resonance Imaging (MRI) with contrast enhancement and Ultrasound imaging are used, however those are not suitable for large scale screening program.Another limitation is the false-negative and false-positive rate using mammography, in general 5–15 % of the tumors are not detected and many cases have to go though a breast biopsy to verify a tumor diagnosis. At last the mammography using breast compression sometimes painful, and utilizing ionizing X-rays. The big potential in microwave imaging is the reported high contrast of complex permittivity between fibroglandular tissues and tumor tissues in breasts and that it is a non-ionizing method which probably will be rather inexpensive. The goal with this work is to develop a microwave imaging system able to reconstruct quantitative images of a female breast. In the frame of this goal this Licentiate thesis contains a brief review of the ongoing research in the field of microwave imaging of biological tissues, with the major focus on the breast tumor application. Both imaging algorithms and experimental setups are included. A feasibility study is performed to analyze what response levels could be expected, in signal properties, in a breast tumor detection application. Also, the usability of a 3D microwave propagation simulator, (QW3D), in the setup development is investigated. This is done by using a simple antenna setup with a breast phantom with different tumor positions. From those results it is clear that strong responses are obtained by a tumor presence and the diffracted responses gives strong information about inhomogeneities inside the breast. The second part of this Licentiate thesis is done in collaboration between Mälardalen University and Supélec. Using the existing planar 2.45 GHz microwave camera and the iterative non-linear Newton Kantorovich code, developed at Département de Recherches en Electromagnétisme (DRE) at Supélec, as a starting point, a new platform for both real-time qualitative imaging and quantitative images of inhomogeneous objects are investigated. The focusing is related to breast tumor detection. For the moment the tomographic performance of the planar camera is verified in simulations through a comparison with other setups. Good calibration is observed, but still experimental work concerning phantom development etc. is needed before experimental results on breast tumor detection may be obtained.

MicroRNAs are important biomolecules for regulating biological processes. Moreover, the secondary structure of microRNA is important for its activity and has been used previously as a mean for finding unknown microRNAs. A phylogenetic study of the microRNA secondary structure reveals more information than its primary sequence, because the primary sequence can undergo mutations that give rise to different phylogenetic relationships, whereas the secondary structure is more robust against mutations and therefore sometimes  more informative. Here we constructed a phylogenetic tree entirely based on microRNA secondary structures using tools PHYLIP (Felsenstein, 1995) and RNAforester (Matthias Höchsmann, 2003, Hochsmann et al., 2004), and compared the overall topology and clusters with the phylogenetic tree constructed using microRNA sequence. The purpose behind this comparison was to investigate the sequence and structure similarity in phylogenetic context and also to investigate if functionally similar microRNA genes are closer in their structure-derived phylogenetic tree. Our phylogenetic comparison shows that the sequence similarity has hardly any effect on the structure similarity in the phylogenetic tree. MicroRNAs that have similar function are closer in the phylogenetic tree based on secondary structure than its respective sequence phylogeny. Hence, this approach can be very useful in predicting the functions of the new microRNAs whose function is yet to be known, since the function of the miRNAs heavily relies on its secondary structure.

High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments. This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.