Thesis (PH.D.) -- Syracuse University, 2006 / "Publication number AAT 3241858."

DEF6 is a Rho-Guanine Nucleotide Exchange Factor (GEF) with a poorly characterised role in T-cell receptor signalling and unknown structure1-3. Its importance in T-cell signalling has been highlighted by observations that DEF6-deficient T cells display defective actin polymerisation and polarisation, have a decreased susceptibility to CD3-induced apoptosis4, and exhibit aberrant expression of the inflammatory cytokine IL-172. DEF6 is a GEF for Rho-family GTPases including CDC42, RhoA and Rac11, which unlike other Rho-GEFs exhibits a reversal of the N-C terminal orientation of the DH and PH domains only found in DEF6 and its homolog in B-cells, SWAP70. Through this role DEF6 has been shown to be involved in the spatiotemporal organisation of T cell signalling through the GTPase CDC425. DEF6-deficient mice have been shown to develop autoimmune diseases and have also been shown to be resistant to the development of other autoimmune diseases, indicating a loss of control of the immune response. The molecular mechanism behind these phenotypes remains to be identified. Exogenously expressed DEF6 has been shown to form cytoplasmic granules under conditions of arrested translation, similar to processing bodies and stress granules which are involved in translational regulation. Data presented here furthers this work and demonstrates endogenous DEF6 forms granules in activated T-cells. Exogenously expressed DEF6 shows partial co-localisation with markers of P-bodies, and demonstrates slow recovery from FRAP indicating a structural role within granules. The granules move within the cell, and analysis of this movement suggests the granules move via active transport, suggesting a link to the microtubule network. Examination of the individual domains of DEF6 demonstrates that the N-terminus is likely to have a role in the co-localisation of DEF6 with P-bodies, whilst the C-terminal DHL domain drives aggregation of DEF6 into granules. Data here confirms the predicted formation of a coiled-coil structure within the DHL domain.

Cell-based metabolomics using LC-MS systemizes the study of the uniqueness of small-molecule metabolite (metabolomes) profiles in cellular processes. Cell-based metabolomics can potentially be used in many applications for the study of biological perturbation from stimulants in cellular pathways. The advantages of cell-based metabolomics include ease of control and interpretation when compared to the study of human subjects and animal models. Furthermore, this method can decrease some highly challenging problems that occur in genomics, transcriptomics and proteomics. Nowadays, cell culture in metabolomics studies has been used in many applications. These include cell culture and bioreactor optimisation, phenotype classification, stimulant testing effect, target and toxicity analysis, metabolic networks determination and modelling, and biomarker and drug target discovery. In this study, the reverse phase-liquid chromatography-mass spectrometry and hydrophilic interaction chromatography-mass spectrometry for comprehensive metabolic profiling well suited to the untargeted analysis of non-polar and polar metabolites in mammalian cells were developed, optimized and validated. These methods can separate and detect most of hydrophobic and polar metabolites that are normally found in mammalian cell lines. After that the LC-MS methods were applied to assess the effects of drugs with known and unknown cellular metabolic effects on three mammalian cell lines, namely HMVECs for antipsychotics experiment, MCF-7 cells for cordycepin experiment and MIN6 cells for fluoxetine experiment by using untargeted metabolic profiling. The global effects of antipsychotics at high therapeutic dosage in HMVECs were investigated. The results support for the toxicity hypothesis with measurements that confirm previous findings and reveal the exact biological pathways of antipsychotic-altered BBB functions. It was found that antipsychotics may affect the bioenergetics pathway due to mitochondrial dysfunction resulted in ketoacidosis and inducing oxide stress by reactive oxygen species generation. In the MCF- cell experiment, the results of the untargeted metabolite profiling demonstrated the clear anti-breast cancer effects of cordycepin and pentostatin. By investigating the metabolite profiles, clear synergistic effects of cordycepin and pentostatin combined in comparison to cordycepin activity alone in MCF-7 cells was observed. Furthermore, the pathway analysis indicated that anti-breast cancer activity was mainly responsible for alterations in purine and pyrimidine metabolism and bioenergetics. Additionally, cordycepin may be involved in the inhibition of cell proliferation and differentiation, and the activation of cell apoptosis. The last experiment on MIN6 cells, the developed and optimized HILIC-MS approach in order to determine the biological pathways which are impaired by fluoxetine on glucose-stimulated insulin secretion on MIN6 cell lines was performed. It is found that fluoxetine may impair glycolysis, TCA and fatty acid metabolism on MIN6 cell lines. Moreover, it is also reveal that the alteration of biological pathways on MIN6 cells by known ETC inhibitors (rotenone (Complex I inhibitor) antimycin (Complex III inhibitor)) and azide (a complex IV inhibitor). From comparison with these ETC inhibitors, it is found that fluoxetine may have the same effect pattern with azide.

Tvelve-species of the alqa class Eustigmatophyceae were studied by means of light and electron microscopy, with particular reference to structural aspects of the vegetative cells and the flagellar apparatus of the zoospores. The Vegetative cells are shown to have microfibrils (probably cellulose)' in the cell wall (Vischeria stellata), lamellate vesicles in the cytoplasm of all the species observed and a clear connection between the cfiloroplast endoplasmic reticulum and the nuclear envelope only in representatives of the Monodopsidaceae. Microfibrils (probably cellulose) were also found in the cell wall of the tribophycean species Ophiocytium malus. The most significant results on uni- and biflagellate zoospores include the observation of a Golgi body for the first time in a eustigmatophycean zoospore (Vischeria helvetica) and the first reconstruction of the system of flagellar roots in the Eustigmatophyceae (V. stellata). This consists of a rhizoplast and four microtubular roots: roots R1 (3 MTs) and R2 (2 MTs) originate-at basal body B1 and run anteriorly around the flagellar swelling; root R3 (5 MTs) arises between the basal bodies and runs to the posterior end of the cell; root R4 (2 MTs) originates at basal body B2 and curves around the eyespot. For comparison, zoospores of the tribophycean species Heterococcus marietanii and H. protonematoldes were also studied. A system of flagellar roots consisting of a small rhizoplast and three microtubular roots, two directed anteriorly and one posteriorly was confirmed. A double helix was shown to be typical of the transition region of the flagella in-this genus. The few observations on settling cells shoved the withdrawal of the complete flagellar apparatus including the swelling-and the possibility of reformation of the pyrenold in Vischeria from material stored in the spiral vesicles during the motile stage. In preliminary observations on mitosis and cytokinesis it was found that, at early stages, basal bodies appear near the nuclear surface and the chloroplast and the pyrenoid divide. Cytokinesis seems to occur by a cleavage furrow. My reconstruction of the flagellar root system in Eustigmatophyceae shows sufficient similarities with the flagellar, roots of other heterokont algal and fungal classes to justify its, inclusion with them in a single division,the Heterokontophyta. On the basis of this observation and the main ultrastructural features known for these classes,a phylogeny is, constructed for the whole group and the probable characteristics of the, common ancestor are proposed.

>Magister Scientiae - MSc / Life is an emergent property of a complex network of interacting cellular-machines. Three-dimensional (3D), cellular structure captured at supra-atomic resolution has the potential to revolutionise our understanding of the interactions, dynamics and structure of these machines: proteins, organelles and other cellular constituents, in their normal functional states. Techniques, capable of acquiring 3D cellular structure at sufficient resolution to enable identification and interpretation of individual macromolecules in the cellular milieu, have the potential to provide this data. Advances in cryo-preservation, preparation and metal-coating techniques allow images of the surfaces of in situ macromolecules to be obtained in a life-like state by field emission scanning – and transmission electron microscopy (FE/SEM, FE/TEM) at a resolution of 2-4 nm. A large body of macromolecular structural information has been obtained using these techniques, but while the images produced provide a qualitative impression of three-dimensionality, computational methods are required to extract quantitative 3D structure. In order to test the feasibility of applying various photogrammetric and tomographic algorithms to micrographs of well-preserved metal-coated biological surfaces, several algorithms were attempted on a variety of FE/SEM and TEM micrographs. A stereoscopic algorithm was implemented and applied to FESEM stereo images of the nuclear pore basket, resulting in a high quality digital elevation map. A SEM rotation series of an object of complicated topology (ant) was reconstructed volumetrically by silhouette-intersection. Finally, the iterative helical real-space reconstruction technique as applied to cryo-TEM micrographs of unidirectionally heavy-metal shadowed. These preliminary results confirm that 3D information obtained from multiple TEM or SEM surface images could be applied to the problem of 3D macromolecular imaging in the cellular context. However, each of the various methods described here comes with peculiar topological, resolution and geometrical limitations, some of which are inherent shortcomings of the methodologies described; others might be overcome with improved algorithms. Combined with carefully designed surface experiments, some of the methods investigated here could provide novel insights and extend current surface-imaging studies. Docking of atomic resolution structures into low-resolution maps derived from surface imaging experiments is a particularly exciting prospect.

No description available.

The cytochrome o and cytochrome d oxidase complexes provide twin termini for the branched respiratory chain of aerobically grown Escherichia coli. Combined use of mutant strains, modulated growth conditions and high resolution analytical techniques enabled cytochromes to be resolved, identified and partially characterized. The cytochrome complement of everted membrane vesicles and detergent extracts fractionated by liquid chromatography is more complex than previously recognised. Multiple type-b cytochromes were resolved by potentiometry and by high resolution spectrophotometry in membrane vesicles from mutant strains lacking the cytochrome d oxidase complex and grown under conditions minimising respiratory chain diversity. Cytochrome o was identified with Em = +235 mV (vs. NHE) as were low potential cytochromes associated with dehydrogenases. Spectrally distinct components of the cytochrome d complex yielded Em values of +125 mV (cytochrome 6595) and +187 mV (cytochrome d). The latter displayed atypical redox behaviour with extreme hysteresis during potentiometric titrations. Several cytochromes b₅₅₆ displaying single, symmetrical redox α-bands at 77 K were resolved from detergent extracts of vesicles. Mutant strains identified one with Mr = 52500 (gel filtration) and Em = +20 mV as the sdhC gene product, a component of succinate dehydrogenase. DL-lactate induced another while a hydroperoxidase, Mr = 386000 (gel filtration) with twin Em values of -2mV and -121 mV and a split Soret absorption band at 77 K (λ[symbol omitted]max= 426.0 nm + 434.0 nm) was produced under limited oxygen tension. The Triton-solubilized and purified cytochrome 0 complex exhibited Mr = 516000 (gel filtration) with five component peptides of Mr= 55000, 32000, 31000, 21000 and 16000 (SDS-PAGE). It displayed mid-point potentials of -58 mV, +127 mV and +260 mV and three a-absorption maxima at 77 K : 554.5 nm, 557.0 nm and 563.5 nm. These components were reduced equivalently during poised-potential low temperature spectrophotometric analyses. Carbon monoxide binding changed the complex's redox α-absorption spectrum minimally but shifted the high potential Em to approximately +420 mV. Quinone analogues inhibited both reduction and reoxidation of the complex. Cytochrome o complex prepared from cloned sources contained a significantly greater proportion of the component with mid range electrochemical potential absorbing at 554.0 nm. These results are discussed in relation to possible structures of the complex, its respiratory interactions and the identity of cytochrome o itself. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Hemopoiesis is thought to be regulated in part by specific, but as yet undefined, interactions between primitive hemopoietic cells and fixed, non-hemopoietic marrow elements collectively referred to as the stroma. Recently, a marrow culture system has been described that allows the maintenance of primitive human hemopoietic progenitor cells for many weeks in the absence of exogenously added hemopoietic growth factors. The formation of a heterogeneous adherent layer in which many stromal elements are found appears to be important to the maintenance of hemopoiesis in this system. As part of the overall goal of delineating the cellular and molecular interactions involved, my first objective was to develop an experimental system for assessing the hemopoiesis-sustaining function of the adherent layer of long-term human marrow cultures. This required the identification of a suitable procedure for separating the hemopoietic and non-hemopoietic regulatory components so that the former could be used to quantitate the function of the latter. This was achieved using irradiation to selectively inactivate residual hemopoietic cells in long-term culture adherent layers, and using a medium containing cis-4-hydroxy-L-proline to selectively inactivate stromal cells and their precursors present in suspensions of unseparated human marrow which were then added back in co-culture experiments. My second objective was to develop a strategy for obtaining purified populations of cells corresponding to the various mesenchymal cell types in long-term adherent layers. I therefore prepared a high titre SV-40 virus stock and used it to establish permanent, cloned lines from human marrow "fibroblast" colonies, long-term culture adherent layers, and umbilical cord endothelial cells. Characterization of the transformants generated showed that they were all positive for SV-40, and in general expressed the phenotypic characteristics of the cells originally infected. Functional studies showed that these transformants, like their normal counterparts, respond to activation by producing two types of hemopoietic growth factors. These studies suggest that marrow mesenchymal cells may regulate the growth and maintenance of primitive hemopoietic cells by producing hemopoietic growth factors in response to appropriate perturbation. The availability of permanent cloned lines of human marrow stromal cells should facilitate future analysis of these events at the molecular level. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The neuroendocrine system presents a diversity of patterns in hormone secretion across a large range of temporal scales. These patterns of secretion are crucial to many aspects of animal survival and behavior. This offers a wealth of important and interesting topics of study amenable to theoretical, experimental, and computational approaches. The pituitary gland plays a large role in neuroendocrine physiology by decoding signals from the brain, translating those signals and relaying them to the glands and tissues of the body via the circulatory system. The cells of the anterior pituitary gland use a diverse set of biochemical processes to accomplish this task. The first step is the activation of a hormone receptor, which convert an extracellular signal to an intracellular one. These receptors in turn trigger cascades of chemical reactions involving enzymes and intracellular messengers, as well as changes in membrane potential dynamics and intracellular Ca2+ ion concentrations, all of which are important in controlling the production and secretion of hormones at appropriate levels. The first portion of this dissertation studies how such biochemical systems respond to patterned inputs, such as pulsatile patterns that are characteristic of hormones in the reproductive system, growth hormone, and stress-responsive hormones. We examine how models of some common intracellular signaling components such as receptor binding and protein phosphorylation respond to pulsed inputs. We then study how a preference for a specific input pulse frequency may arise from the interactions between transcription factors, which are molecules regulating the expression of genes and in this case responsible for production of a hormone. The next portion of this work describes experiments performed to demonstrate the direct actions of a hormone, oxytocin, on three pituitary cell types. Intracellular Ca2+ ion concentration was measured in primary cultured female rat pituitary cells in vitro using video microscopy and a fluorescent \ca-sensitive dye. Oxytocin triggered increases in intracellular Ca2+ concentration as well as secretion of hormone from gonadotrophs, somatotrophs, and lactotrophs in a manner consistent with a direct action via the oxytocin receptor. Finally, computational tools are presented for harnessing the computational power of graphics processing units to rapidly compute numerical solutions to initial value problems such as those that arise in the study of pituitary cell electrophysiology. This provides tools for more rapid model exploration by computing the ensembles of parameter combinations required in parameter sweep and parameter space sampling computations in parallel. This allows rapid computations to be performed on inexpensive modern desktop or laptop computers. As a case study, we use a model of membrane potential dynamics of pituitary lactotroph cells, which is produces spiking and bursting patterns. We compare the actions of three K+ channel conductances known to be increased by the action of the hormone dopamine in lactotrophs. Paradoxically, low levels of dopamine stimulate Ca2+ increases despite only increasing these typically inhibitory conductances, a result previously hypothesized to be due to a transition from spiking to bursting. We compare the mechanisms by which one of the K+ channels is able to promote this stimulatory effect while the other two are not, and we find that this effect is robust in a population of model cells with randomized background parameters. / A Dissertation submitted to the Department of Mathematics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2015. / June 2, 2015. / Anterior pituitary, Electrophysiology modeling, Mathematical modeling, Mathematical neuroendocrinology, Parallel computing, Singaling pathways / Includes bibliographical references. / Richard Bertram, Professor Co-Directing Dissertation; Joel Tabak, Professor Co-Directing Dissertation; Wei Yang, University Representative; Nick Cogan, Committee Member; Michael Mascagni, Committee Member; Washington Mio, Committee Member.

Nucleosome distributions are critically important in regulating access to the eukaryotic genome. Cells with different physiologies have strikingly similar nucleosome distributions. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. Factors regulating the maintenance of the basal state as well as changes in nucleosome distribution following a response must be investigated. In our first set of experiments we used the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) as a model system for stimulus-induced nucleosome distribution changes. We measured nucleosome distribution at high temporal resolution in human cells at the 2 kb flanking the transcription start sites (TSSs) of hundreds immunity-related loci, using microarray technology, during the reactivation of KSHV. We show that nucleosome redistribution peaks at 24 hours post KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 hours post KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans. We next wanted to affirm and extend our previous observations regarding the widespread and transient nature of nucleosome redistributions during viral reactivation. We tested if this widespread nucleosome remodeling was a genome wide event or limited solely to the hundreds of immunity-related loci measured by microarray. We measured nucleosome distributions at high temporal resolution following KSHV reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the TSS of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the sophisticated underlying DNA sequence. We demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes have allowed us to propose a new hierarchical model for chromatin-based regulation of genome response. Given that we discovered that nucleosome distributions are widespread and transient, it was important for us to understand the forces maintaining the basal state. We It would be interesting to understand the forces and factors that maintain nucleosome architecture in a basal state and regenerate it following a response, such as KSHV reactivation. An appealing group of candidates that might maintain and regenerate the nucleosome architecture in its basal state is the transcriptional machinery. We identified RNA polymerase II (RNA Pol II) as a likely candidate as it is found throughout the genome and not always associated with transcription. We next were interested in the role RNA Pol II plays in the maintenance of chromatin structure. We measured nucleosome distributions in response to RNA Pol II inhibition by δ-amanitin treatment. Nucleosome distribution changes, following RNA Pol II inhibition, were widespread and the TSSs with nucleosome distribution changes were enriched for RNA Pol II independent of it's role it plays in active transcription. This work gives new insight into understanding the role of chromatin structure regulates and maintains the human genome. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2015. / April 9, 2015. / Chromatin, KSHV, microarray, next generation sequencing, nucleosome, RNA Pol II / Includes bibliographical references. / Jonathan H. Dennis, Professor Directing Dissertation; Michael G. Roper, University Representative; Hank W. Bass, Committee Member; P. Bryant Chase, Committee Member; Debra A. Fadool, Committee Member.