Computational and mathematical models of cellular processes promise great benets in important elds such as molecular biology and medicine. Increasingly, researchers are incorporating the fundamentally discrete and stochastic nature of biochemical processes into the mathematical models that are intended to represent them. This has led to the formulation of models for genetic networks as continuous-time, discrete state, Markov processes, giving rise to the so-called Chemical Master Equation (CME), which is a discrete, partial dierential equation, that governs the evolution of the associated probability distribution function (PDF). While promising many insights, the CME is computationally challenging, especially as the dimension of the model grows. In this thesis, novel methods are developed for computing the PDF of the Master Equation. The problems associated with the high-dimensional nature of the Chemical Master Equation are addressed by adapting Krylov methods, in combination with Finite State Projection methods, to derive algorithms well-suited to the Master Equation. Variations of the approach that incorporate the Strang splitting and a stochastic analogue of the total quasi-steady-state approximation are also derived for chemical systems with disparate rates. Monte Carlo approaches, such as the Stochastic Simulation Algorithm, that simulate trajectories of the process governed by the CME have been a very popular approach and we compare these with the PDF approaches developed in this thesis. The thesis concludes with a discussion of various implementation issues along with numerical results for important applications in systems biology, including the gene toggle, the Goldbeter-Koshland switch and the Mitogen-Activated Protein Kinase Cascade.

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Neurosciences. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 223-251). Also available online through Digital Dissertations.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22150号 / 医博第4541号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 稲垣 暢也, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein Na +/H+ exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response.

Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-α (PFT-α), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 μM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-α followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-α also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-α interferes with multiple steps of Dox-induced apoptosis.

Atherosclerosis propagates when innate immune cells, myeloid-derived macrophages, undergo unregulated uptake of cholesterol-rich modified low-density lipoproteins (LDL). Excess storage and retention of this cholesterol leads to development of lipid-laden macrophage foam cells, that accumulate within the intima of arteries as developing plaque. Formation of atherosclerotic lesions reduces blood flow and can further lead to more serious complications such as myocardial infarction, stroke, and cardiovascular disease. AMP-activated protein kinase (AMPK), a master regulator of cellular energetics, has been shown to participate in many anti-atherogenic pathways within myeloid cells such as (but not limited to) the inhibition of cholesterol synthesis and stimulation of reverse cholesterol transport. However, a recent report described a pro-atherogenic role for myeloid AMPK, showing it is expression required for myeloid cell recruitment and longevity within the atherosclerotic microenvironment. Despite this, multiple reports all corroborate describing a protective role for systemic pharmacological AMPK activation. We sought to determine the consequence of modified LDL variants in myeloid AMPK signaling and to further clarify the role of myeloid AMPK signaling within atherosclerosis. In cultured macrophages primed with modified LDL variants underwent AMPK activation, which was also associated with increased markers of autophagy. In an in vivo model of intermediate atherosclerosis, we observed that neither myeloid AMPK expression nor systemic AMPK-activating therapy influenced lesion myeloid content, necrosis, or autophagic markers. Furthermore, despite a suggestive trend, both myeloid AMPK and AMPK-therapy did not significantly influence lesion size in male or female mice. Interestingly, we found that in animals lacking AMPK signaling to only one substrate, HMGCR (the rate limiting enzyme in cholesterol synthesis), knock-in mice developed accelerated atherosclerosis when compared to their wild-type littermate. Furthermore, we determined that AMPK signaling to HMGCR in the hematopoietic compartment alone is enough to protect against atherogenesis. Taken together, these studies show the benefit of interrogating specific AMPK-regulated pathways in the context of atherosclerosis, and sheds light on the benefit of utilization of single point mutation knock-in models opposed to global or cell type-specific knockout models for investigations into AMPK within atherosclerosis.

Background. Esophageal cancer is ranked in the top ten of diagnosed tumors worldwide. Even though improvements in survival could be noticed over the last years, prognosis remains poor. ETS translocation variant 1 (ETV1) is a member of a family of transcription factors and is phosphorylated by mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2). Aim of this study was to evaluate the prognostic role of MK2 and ETV1 in esophageal cancer. Methods. Consecutive patients that underwent surgical resection at the department of surgery at the Medical University of Vienna between 1991 and 2012 were included into this study. After microscopic analysis, tissue micro arrays (TMAs) were created and immunohistochemistry was performed with antibodies against MK2 and ETV1. Results. 323 patients were included in this study. Clinical data was achieved from a prospective patient data base. Nuclear overexpression of MK2 was observed in 143 (44.3%) cases for nuclear staining and in 142 (44.0%) cases a cytoplasmic overexpression of MK2 was observed. Nuclear and cytoplasmic ETV1 overexpression was detected in 20 cases (6.2%) and 30 cases (9.3%), respectively. In univariate survival analysis, cMK2 and nETV1 were found to be significantly associated with patients' overall survival. Whereas overexpression of cMK2 was associated with shorter, nETV1 was associated with longer overall survival. In multivariate survival analysis, both cMK2 and nETV1 were found to be independent prognostic factors for the subgroup of EAC as well. Discussion. Expression of MK2 and ETV1 are prognostic factors in patients, with esophageal adenocarcinoma.

This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5’-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3’-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.