Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice

Previous studies on the effect of glycosylation on the elimination rate of antibodies have produced conflicting results. Here, we performed pharmacokinetic studies in mice with two preparations of a monoclonal IgG1 antibody enriched for complex type or high mannose type oligosaccharides at the Fc glycosylation site. No significant difference in the serum half-life was found between the two antibody glycoforms, nor was any difference observed in the serum half-lives of different complex type glycoforms. To evaluate the influence of glycosylation within the variable domain, a second monoclonal antibody, glycosylated in both the Fc and Fv domains, was separated into fractions containing different amounts of Fv-associated sialic acid and administered to mice. Again, no significant difference was found in the clearance rates of variants carrying different amounts of Fv-associated sialic acid or lacking Fv-glycosylation. These results suggest that glycosylation has little or no impact on the pharmacokinetic behavior of these two monoclonal antibodies in mice.

Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. Conclusion: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.

Time-domain models for power system stability and unbalance

It is an important and difficult challenge to protect modern interconnected power system from blackouts. Applying advanced power system protection techniques and increasing power system stability are ways to improve the reliability and security of power systems. Phasor-domain software packages such as Power System Simulator for Engineers (PSS/E) can be used to study large power systems but cannot be used for transient analysis. In order to observe both power system stability and transient behavior of the system during disturbances, modeling has to be done in the time-domain. This work focuses on modeling of power systems and various control systems in the Alternative Transients Program (ATP). ATP is a time-domain power system modeling software in which all the power system components can be modeled in detail. Models are implemented with attention to component representation and parameters. The synchronous machine model includes the saturation characteristics and control interface. Transient Analysis Control System is used to model the excitation control system, power system stabilizer and the turbine governor system of the synchronous machine. Several base cases of a single machine system are modeled and benchmarked against PSS/E. A two area system is modeled and inter-area and intra-area oscillations are observed. The two area system is reduced to a two machine system using reduced dynamic equivalencing. The original and the reduced systems are benchmarked against PSS/E. This work also includes the simulation of single-pole tripping using one of the base case models. Advantages of single-pole tripping and comparison of system behavior against three-pole tripping are studied. Results indicate that the built-in control system models in PSS/E can be effectively reproduced in ATP. The benchmarked models correctly simulate the power system dynamics. The successful implementation of a dynamically reduced system in ATP shows promise for studying a small sub-system of a large system without losing the dynamic behaviors. Other aspects such as relaying can be investigated using the benchmarked models. It is expected that this work will provide guidance in modeling different control systems for the synchronous machine and in representing dynamic equivalents of large power systems.

Time-domain modeling of high-frequency electromagnetic wave propagation, overhead wires, and earth

Prediction of radiated fields from transmission lines has not previously been studied from a panoptical power system perspective. The application of BPL technologies to overhead transmission lines would benefit greatly from an ability to simulate real power system environments, not limited to the transmission lines themselves. Presently circuitbased transmission line models used by EMTP-type programs utilize Carson’s formula for a waveguide parallel to an interface. This formula is not valid for calculations at high frequencies, considering effects of earth return currents. This thesis explains the challenges of developing such improved models, explores an approach to combining circuit-based and electromagnetics modeling to predict radiated fields from transmission lines, exposes inadequacies of simulation tools, and suggests methods of extending the validity of transmission line models into very high frequency ranges. Electromagnetics programs are commonly used to study radiated fields from transmission lines. However, an approach is proposed here which is also able to incorporate the components of a power system through the combined use of EMTP-type models. Carson’s formulas address the series impedance of electrical conductors above and parallel to the earth. These equations have been analyzed to show their inherent assumptions and what the implications are. Additionally, the lack of validity into higher frequencies has been demonstrated, showing the need to replace Carson’s formulas for these types of studies. This body of work leads to several conclusions about the relatively new study of BPL. Foremost, there is a gap in modeling capabilities which has been bridged through integration of circuit-based and electromagnetics modeling, allowing more realistic prediction of BPL performance and radiated fields. The proposed approach is limited in its scope of validity due to the formulas used by EMTP-type software. To extend the range of validity, a new set of equations must be identified and implemented in the approach. Several potential methods of implementation have been explored. Though an appropriate set of equations has not yet been identified, further research in this area will benefit from a clear depiction of the next important steps and how they can be accomplished. Prediction of radiated fields from transmission lines has not previously been studied from a panoptical power system perspective. The application of BPL technologies to overhead transmission lines would benefit greatly from an ability to simulate real power system environments, not limited to the transmission lines themselves. Presently circuitbased transmission line models used by EMTP-type programs utilize Carson’s formula for a waveguide parallel to an interface. This formula is not valid for calculations at high frequencies, considering effects of earth return currents. This thesis explains the challenges of developing such improved models, explores an approach to combining circuit-based and electromagnetics modeling to predict radiated fields from transmission lines, exposes inadequacies of simulation tools, and suggests methods of extending the validity of transmission line models into very high frequency ranges. Electromagnetics programs are commonly used to study radiated fields from transmission lines. However, an approach is proposed here which is also able to incorporate the components of a power system through the combined use of EMTP-type models. Carson’s formulas address the series impedance of electrical conductors above and parallel to the earth. These equations have been analyzed to show their inherent assumptions and what the implications are. Additionally, the lack of validity into higher frequencies has been demonstrated, showing the need to replace Carson’s formulas for these types of studies. This body of work leads to several conclusions about the relatively new study of BPL. Foremost, there is a gap in modeling capabilities which has been bridged through integration of circuit-based and electromagnetics modeling, allowing more realistic prediction of BPL performance and radiated fields. The proposed approach is limited in its scope of validity due to the formulas used by EMTP-type software. To extend the range of validity, a new set of equations must be identified and implemented in the approach. Several potential methods of implementation have been explored. Though an appropriate set of equations has not yet been identified, further research in this area will benefit from a clear depiction of the next important steps and how they can be accomplished.

Computational fluids domain reduction to a simplified fluid network

The primary goal of this project is to demonstrate the practical use of data mining algorithms to cluster a solved steady-state computational fluids simulation (CFD) flow domain into a simplified lumped-parameter network. A commercial-quality code, “cfdMine” was created using a volume-weighted k-means clustering that that can accomplish the clustering of a 20 million cell CFD domain on a single CPU in several hours or less. Additionally agglomeration and k-means Mahalanobis were added as optional post-processing steps to further enhance the separation of the clusters. The resultant nodal network is considered a reduced-order model and can be solved transiently at a very minimal computational cost. The reduced order network is then instantiated in the commercial thermal solver MuSES to perform transient conjugate heat transfer using convection predicted using a lumped network (based on steady-state CFD). When inserting the lumped nodal network into a MuSES model, the potential for developing a “localized heat transfer coefficient” is shown to be an improvement over existing techniques. Also, it was found that the use of the clustering created a new flow visualization technique. Finally, fixing clusters near equipment newly demonstrates a capability to track temperatures near specific objects (such as equipment in vehicles).

PHYLOGENETIC ANALYSES, GENE DOSAGE EFFECT AND SELECTION IN MEMBERS OF THE LATERAL BOUNDARY DOMAIN (LBD) GENES TRANSCRIPTION FACTOR FAMILY

Wood formation is an economically and environmentally important process and has played a significant role in the evolution of terrestrial plants. Despite its significance, the molecular underpinnings of the process are still poorly understood. We have previously shown that four Lateral Boundary Domain (LBD) transcription factors have important roles in the regulation of wood formation with two (LBD1 and LBD4) involved in secondary phloem and ray cell development and two (LBD15 and LBD18) in secondary xylem formation. Here, we used comparative phylogenetic analyses to test potential roles of the four LBD genes in the evolution of woodiness. We studied the copy number and variation in DNA and amino acid sequences of the four LBDs in a wide range of woody and herbaceous plant taxa with fully sequenced and annotated genomes. LBD1 showed the highest gene copy number across the studied species, and LBD1 gene copy number was strongly and significantly correlated with the level of ray seriation. The lianas, cucumber and grape, with multiseriate ray cells showed the highest gene copy number (12 and 11, respectively). Because lianas’ growth habit requires significant twisting and bending, the less lignified ray parenchyma cells likely facilitate stem flexibility and maintenance of xylem conductivity. We further demonstrate conservation of amino acids in the LBD18 protein sequences that are specific to woody taxa. Neutrality tests showed evidence for strong purifying selection on these gene regions across various orders, indicating adaptive convergent evolution of LBD18. Structural modeling demonstrates that the conserved amino acids have a significant impact on the tertiary protein structure and thus are likely of significant functional importance.

CONVERSION OF DOMAIN TYPE ENFORCEMENT LANGUAGE TO THE JAVA SECURITY MANAGER

With today's prevalence of Internet-connected systems storing sensitive data and the omnipresent threat of technically skilled malicious users, computer security remains a critically important field. Because of today's multitude of vulnerable systems and security threats, it is vital that computer science students be taught techniques for programming secure systems, especially since many of them will work on systems with sensitive data after graduation. Teaching computer science students proper design, implementation, and maintenance of secure systems is a challenging task that calls for the use of novel pedagogical tools. This report describes the implementation of a compiler that converts mandatory access control specification Domain-Type Enforcement Language to the Java Security Manager, primarily for pedagogical purposes. The implementation of the Java Security Manager was explored in depth, and various techniques to work around its inherent limitations were explored and partially implemented, although some of these workarounds do not appear in the current version of the compiler because they would have compromised cross-platform compatibility. The current version of the compiler and implementation details of the Java Security Manager are discussed in depth.

A novel FIP1L1-PDGFRA mutant destabilizing the inactive conformation of the kinase domain in chronic eosinophilic leukemia/hypereosinophilic syndrome

BACKGROUND: The Fip1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) gene fusion is a common cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES), and patients suffering from this particular subgroup of CEL/HES respond to low-dose imatinib therapy. However, some patients may develop imatinib resistance because of an acquired T674I mutation, which is believed to prevent drug binding through steric hindrance. METHODS: In an imatinib resistant FIP1L1-PDGFRA positive patient, we analyzed the molecular structure of the fusion gene and analyzed the effect of several kinase inhibitors on FIP1L1-PDGFRA-mediated proliferative responses in vitro. RESULTS: Sequencing of the FIP1L1-PDGFRA fusion gene revealed the occurrence of a S601P mutation, which is located within the nucleotide binding loop. In agreement with the clinical observations, imatinib did not inhibit the proliferation of S601P mutant FIP1L1-PDGFRA-transduced Ba/F3 cells. Moreover, sorafenib, which has been described to inhibit T674I mutant FIP1L1-PDGFRA, failed to block S601P mutant FIP1L1-PDGFRA. Structural modeling revealed that the newly identified S601P mutated form of PDGFRA destabilizes the inactive conformation of the kinase domain that is necessary to bind imatinib as well as sorafenib. CONCLUSIONS: We identified a novel mutation in FIP1L1-PDGFRA resulting in both imatinib and sorafenib resistance. The identification of novel drug-resistant FIP1L1-PDGFRA variants may help to develop the next generation of target-directed compounds for CEL/HES and other leukemias.

Intracellular Ca(2+) operates a switch between repair and lysis of streptolysin O-perforated cells

Pore-forming (poly)peptides originating from invading pathogens cause plasma membrane damage in target cells, with consequences as diverse as proliferation or cell death. However, the factors that define the outcome remain unknown. We show that in cells maintaining an intracellular Ca(2+) concentration [Ca(2+)](i) below a critical threshold of 10 microM, repair mechanisms seal off 'hot spots' of Ca(2+) entry and shed them in the form of microparticles, leading to [Ca(2+)](i) reduction and cell recovery. Cells that are capable of preventing an elevation of [Ca(2+)](i) above the critical concentration, yet are unable to complete plasma membrane repair, enter a prolonged phase of [Ca(2+)](i) oscillations, accompanied by a continuous shedding of microparticles. When [Ca(2+)](i) exceeds the critical concentration, an irreversible formation of ceramide platforms within the plasma membrane and their internalisation drives the dying cells beyond the 'point of no return'. These findings show that the extent of [Ca(2+)](i) elevation determines the fate of targeted cells and establishes how different Ca(2+)-dependent mechanisms facilitate either cell survival or death.

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

Electrical neuroimaging in the time domain

A publication entitled “A default mode of brain function” initiated a new way of looking at functional imaging data. In this PET study the authors discussed the often-observed consistent decrease of brain activation in a variety of tasks as compared with the baseline. They suggested that this deactivation is due to a task-induced suspension of a default mode of brain function that is active during rest, i.e. that there exists intrinsic well-organized brain activity during rest in several distinct brain regions. This suggestion led to a large number of imaging studies on the resting state of the brain and to the conclusion that the study of this intrinsic activity is crucial for understanding how the brain works. The fact that the brain is active during rest has been well known from a variety of EEG recordings for a very long time. Different states of the brain in the sleep–wake continuum are characterized by typical patterns of spontaneous oscillations in different frequency ranges and in different brain regions. Best studied are the evolving states during the different sleep stages, but characteristic EEG oscillation patterns have also been well described during awake periods (see Chapter 1 for details). A highly recommended comprehensive review on the brain's default state defined by oscillatory electrical brain activities is provided in the recent book by György Buzsaki, showing how these states can be measured by electrophysiological procedures at the global brain level as well as at the local cellular level.