Gliomas are the most common and aggressive primary brain tumors. The most common treatment protocols for these brain tumors are combinations of surgery, chemotherapy and radiotherapy. However, even with the most aggressive combination of surgery and radiotherapy and/or chemotherapy schedules, gliomas almost always recur resulting in a median survival time for patients of not more than 12 months. This highly diffusive and invasive nature of brain tumors makes it very important to study the effects of these combined therapeutic strategies in an effort to improve the survival time of patients. It is also important to study the tumor microenvironment, since the complex nature of the cerebral vasculature, including the blood brain barrier and several other tumor-induced conditions such as hypoxia, high interstitial pressure, and cerebral edema affect drug delivery as well as the effectiveness of radiotherapy. Recently, a novel strategy using antiangiogenic therapy has been studied for the treatment of brain tumors. Antiangiogenic therapy interferes with the development of tumor vasculature and indirectly helps in the control of tumor growth. Recent clinical trials suggest that anti-angiogenic therapy is usually more effective when given in combination with other therapeutic strategies. In an effort to study the effects of the aforementioned therapeutic strategies, a spatio-temporal model is considered here that incorporates the tumor cell growth and the effects of radiotherapy and chemotherapy. The effects of different schedules of radiation therapy is then studied using a generalized linear quadratic model and compared against the published clinical data. The model is then extended to include the interactions of tumor vasculature and oxygen concentration, to explain tumor hypoxia and to study various methods of hypoxia characterizations including biomarker estimates and needle electrode measurements. The model predicted hypoxia is also used to analyze the effects of tumor oxygenation status on radiation response as it is known that tumor hypoxia negatively influences the radiotherapy outcome. This thesis also presents a detailed analysis of the effects of heterogenous tumor vasculature on tumor interstitial fluid pressure and interstitial fluid velocity. A mathematical modeling approach is then used to analyze the changes in interstitial fluid pressure with or without antiangiogenic therapy.

Gliomas are the most common and aggressive primary brain tumors. The most common treatment protocols for these brain tumors are combinations of surgery, chemotherapy and radiotherapy. However, even with the most aggressive combination of surgery and radiotherapy and/or chemotherapy schedules, gliomas almost always recur resulting in a median survival time for patients of not more than 12 months. This highly diffusive and invasive nature of brain tumors makes it very important to study the effects of these combined therapeutic strategies in an effort to improve the survival time of patients. It is also important to study the tumor microenvironment, since the complex nature of the cerebral vasculature, including the blood brain barrier and several other tumor-induced conditions such as hypoxia, high interstitial pressure, and cerebral edema affect drug delivery as well as the effectiveness of radiotherapy. Recently, a novel strategy using antiangiogenic therapy has been studied for the treatment of brain tumors. Antiangiogenic therapy interferes with the development of tumor vasculature and indirectly helps in the control of tumor growth. Recent clinical trials suggest that anti-angiogenic therapy is usually more effective when given in combination with other therapeutic strategies. In an effort to study the effects of the aforementioned therapeutic strategies, a spatio-temporal model is considered here that incorporates the tumor cell growth and the effects of radiotherapy and chemotherapy. The effects of different schedules of radiation therapy is then studied using a generalized linear quadratic model and compared against the published clinical data. The model is then extended to include the interactions of tumor vasculature and oxygen concentration, to explain tumor hypoxia and to study various methods of hypoxia characterizations including biomarker estimates and needle electrode measurements. The model predicted hypoxia is also used to analyze the effects of tumor oxygenation status on radiation response as it is known that tumor hypoxia negatively influences the radiotherapy outcome. This thesis also presents a detailed analysis of the effects of heterogenous tumor vasculature on tumor interstitial fluid pressure and interstitial fluid velocity. A mathematical modeling approach is then used to analyze the changes in interstitial fluid pressure with or without antiangiogenic therapy.

<p>This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation.</p><p>The interstitial fluid pressure (P_IF) is of importance for control of tissue fluid balance. A lowering of P_IF <i>in vivo</i> will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling P_IF.</p><p>Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of P_IF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of P_IF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored P_IF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered P_IF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered P_IF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from <i>Streptococcus equi</i> modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction <i>in vitro</i> and control of P_IF <i>in vivo</i>, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence P_IF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.</p>

This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation. The interstitial fluid pressure (PIF) is of importance for control of tissue fluid balance. A lowering of PIF in vivo will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling PIF. Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of PIF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of PIF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored PIF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered PIF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered PIF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from Streptococcus equi modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction in vitro and control of PIF in vivo, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence PIF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.

All blood vessels in the microvasculature are embedded in loose connective tissue, which regulates the transport of fluid to and from tissues. The intersti-tial fluid pressure (IFP) is one of the forces that control this transport. A lowering of IFP in vivo results in an increased transport of fluid from the circulation into the underhydrated connective tissues, resulting in edema formation. During homeostasis, contractile connective tissue cells exert a tension on the connective tissue fibrous network by binding with β1 in-tegrins, thereby actively controlling IFP. During inflammation, the IFP is lowered but platelet-derived growth factor (PDGF)-BB induces an IFP nor-malization dependent on integrin αVβ3. We demonstrate that extracellular proteins from Streptococcus equi subspecies equi modulated cell-mediated and integrin αVβ3-directed collagen gel contraction in vitro. One of these proteins, the collagen- and fibronectin binding FNE, stimulated contraction by a process dependent on fibronectin synthesis. This study identified a pos-sible novel virulence mechanism for bacteria based on the ability of bacteria to modulate the edema response. Another protein, the collagen-binding pro-tein CNE, inhibited contraction and this led to the identification of sites in collagen monomers that potentially are involved in connecting αVβ3 to the collagen network. PDGF-BB and prostaglandin E1 (PGE1) stimulate and inhibit collagen gel contraction in vitro and normalize and lower IFP, respec-tively. We showed that these agents affected both similar and different sets of actin-binding proteins. PDGF-BB stimulated actin cytoskeleton dynamics whereas PGE1 inhibited processes dependent on cytoskeletal motor and adhesive functions, suggesting that these different activities may partly ex-plain the contrasting effects of PGE1 and PDGF-BB on contraction and IFP. Mutation of the phosphatidylinositol 3’-kinase (PI3K), but not phospholipase C (PLC)γ activation site, rendered cells unable to respond to PDGF-BB in contraction and in activation of the actin binding and severing protein cofilin. Ability to activate cofilin after PDGF-BB stimulation correlated with ability to respond to PDGF-BB in contraction, suggesting a role for cofilin in this process downstream of PDGF receptor-activated PI3K. Many proteins can modulate contraction either by affecting the extracellular matrix and cell adhesions or by altering cytoskeletal dynamics. Knowledge on how these proteins might influence IFP is likely to be of clinical importance for treat-ment of inflammatory conditions including anaphylaxis, septic shock and also carcinoma growth.

A brief introduction to cancer biology and treatment is presented with a focus on current clinical advances in the delivery of chemotherapy and antiangiogenic therapies. Mathematical oncology is then surveyed with summaries of various models of tumor growth, tumor angiogenesis and other relevant biological entities such as angiogenic growth factors. Both strictly time-dependent ordinary differential equation (ODE)-based and spatial partial differential equation (PDE)-based models are considered. These biological models are first developed into an ODE model where various treatment options can be compared including different combinations of drugs and dosage schedules. This model gives way to a PDE model that includes the spatially heterogeneous blood vessel distribution found in tumors, as well as angiogenic growth factor imbalances. This model is similarly analyzed and implications are summarized. Finally, including the effects of interstitial fluid pressure into an angiogenic activity model is performed. This model displays the importance of factor convection on the angiogenic behaviour of tumours.

A brief introduction to cancer biology and treatment is presented with a focus on current clinical advances in the delivery of chemotherapy and antiangiogenic therapies. Mathematical oncology is then surveyed with summaries of various models of tumor growth, tumor angiogenesis and other relevant biological entities such as angiogenic growth factors. Both strictly time-dependent ordinary differential equation (ODE)-based and spatial partial differential equation (PDE)-based models are considered. These biological models are first developed into an ODE model where various treatment options can be compared including different combinations of drugs and dosage schedules. This model gives way to a PDE model that includes the spatially heterogeneous blood vessel distribution found in tumors, as well as angiogenic growth factor imbalances. This model is similarly analyzed and implications are summarized. Finally, including the effects of interstitial fluid pressure into an angiogenic activity model is performed. This model displays the importance of factor convection on the angiogenic behaviour of tumours.

The regulation of fluid (water) volume in the body is crucial for tissue homeostasis. The interstitial fluid, which comprises almost 20% of the body fluid, is stored in the loose connective tissue and its volume is actively regulated by components of this tissue. The loose connective tissue provides a path for fluid flow from capillaries to the tissue and lymphatics. This fluid is partially stored in the interstitium and the remainder is directed to the lymphatics. The fibroblasts in the loose connective tissue actively compact the fibrous extracellular matrix (ECM) through mechanotransduction via integrins. This in turn, maintains the interstitial fluid pressure and keeps the ground substance underhydrated. The interstitial fluid pressure is part of the forces that regulate the efflux of fluid from capillaries and keep the ground substance underhydrated. The underhydrated ground substance has a potential to take up fluid 3-fold the plasma volume. Therefore, the active contraction of the ECM via fibroblasts is crucial to prevent the risk of evacuation of fluid from capillaries. During pathologies, such as inflammation and carcinogenesis, the interstitial fluid pressure and hence the interstitial fluid volume is altered. The results presented in this thesis show that the signaling events downstream of αVβ3 integrin, collagen-binding β1 integrins, and platelet-derived growth factor receptor β, that induce cell-mediated matrix contraction, included paired function of PI3K and PLCγ, cofilin activation, actin turnover, and generation of actomyosin forces. Furthermore, the results highlight new potential roles for fibrin and αVβ3 integrins, for instance during clearance of edema. Notably, fibrin extravasation at inflammatory sites induced αVβ3 integrin-dependent matrix contraction, leading to normalization of the altered interstitial fluid volume. It also reprograms the expression of ECM-related genes and hence induces ECM turnover. Taken together, these results provide further insight into the regulatory mechanism through which the loose connective tissue actively regulates the interstitial fluid volume.

High interstitial fluid pressure (IFP) in tumours is recognized as a barrier to drug delivery resulting in reduced efficacy. High IFP impedes the normal process of convective fluid flow (CFF) from blood vessels into the interstitium. The aim of this study was to develop an in vitro model that could be used to measure CFF and to study its effects on drug delivery. The model consists of a transwell cell culture insert which supports the growth of multicell layers (MCL) on collagen coated membranes. A graduated tube is inserted into the transwell and a pressure gradient is applied across the membrane by raising the volume of medium in the tube above that of the bottom chamber. CFF is determined by measuring the weight of medium in the bottom chamber as a function of time. CFF was inversely proportional to MCL thickness and 41.1±3.6µm thick MCL has completely stopped CFF. Using a physiologically relevant hydrostatic pressure of 28mmHg, a CFF of 21µL/min was recorded using a DLD-1 MCL that was 12.21±3.2µm thick. Under these conditions, the rates of penetration of doxorubicin, imatinib and gefitinib were respectively 42, 26 and 13 folds greater than when no CFF exists. Reversing the CFF so that it opposed the drug diffusion gradient significantly impairs drug penetration. In conclusion, a novel in vitro model for assessing the impact of CFF on drug delivery has been developed. This model could be used to evaluate strategies designed to increase drug delivery to solid tumours by modifying the CFF.

<p>Molecular mechanisms in the development and progression of thyroid carcinomas are still not fully understood. In the present thesis the highly malignant anaplastic thyroid carcinoma (ATC) was used to study regulation of apoptosis and tumor interstitial fluid pressure (IFP).</p><p>Addition of a natural estrogen metabolite, 2-Methoxyestradiol (2-ME), induced a G2/M cell cycle arrest and apoptosis in five out of six human ATC cell lines. Treatment with 2-ME induced DNA-fragmentation as well as activation of caspase-3. Inhibitors of JNK and p38 MAPKs activity decreased the effect of 2-ME suggesting involvement in the induction of apoptosis.</p><p>Solid tumors have an elevated IFP. High IFP forms or reflects a barrier for exchange of molecules between microvessels and surrounding tissue. The mechanisms for the generation of the high IFP were investigated using a specific TGF-β inhibitor in an ATC model in athymic mice. Tumor IFP was lowered in TGF-β inhibitor-treated compared to control mice. Affymetrix microarray analysis showed a decreased expression of macrophage-associated genes in treated tumors. Furthermore, the number and activity of tumor-associated macrophages was reduced after TGF-β inhibition. A decreased protein leakage together with an increased coverage of α-smooth-muscle actin (SMA)-expressing cells indicated vessel normalization. An adjuvant treatment with the TGF-β inhibitor resulted in an increased treatment efficacy of doxorubicin. Thus, TGF-β inhibitor-treatment suggests improved microvessel function which results in a lowering of tumor IFP and increased tumor drug uptake.</p><p>To create a model for specific inactivation of genes in the thyroid, a transgenic mouse with a thyrocyte-specific expression of Cre recombinase was generated. The thyroglobulin promoter together with an inducible Cre recombinase (<i>creER</i><i>T2</i>) was used. Two transgenic founder lines were identified expressing cre mRNA solely in the thyroid. Functional activity of the CreER^T2 protein was demonstrated by using a ROSA26-LacZ reporter mouse.</p>