Browsing by Author "Balaji Rao, Committee Member"

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Functional genomics analysis of carbohydrate conversion to biohydrogen by pure and mixed cultures of hyperthermophilic Thermotoga species.
(2009-09-03) Gray, Steven Randall; Jason Haugh, Committee Member; Robert Kelly, Committee Chair; Amy Grunden, Committee Member; Balaji Rao, Committee Member
The genus Thermotoga, is comprised fermentative anaerobes with optimal growth temperatures as high as 80Ã‚Â°C. To understand the genetic and physiological diversity within this genus, the genome sequences of five Thermotoga species (T. maritima, T. neapolitana, T. sp. RQ2, T. petrophila, T. lettingae) were compared using bioinformatics tools. Except for T. lettingae, the genomes exhibited high degrees of homology and shared organizational traits. The in silico comparison was supported by genomic DNA cross-hybridization to a T. maritima cDNA microarray, where 83-94% of the probes in the three other Thermotoga species were recognized. These results indicated that the four Thermotoga species share a core genome (~1470 ORFs); ORFs unique to particular species likely reflect the influence of specific environmental or evolutionary factors. The significant homology among the four Thermotoga species facilitated development of a multi-species cDNA microarray for use in pure and mixed culture transcriptional response studies. The Thermotoga multi-species cDNA microarray was used to examine pure and mixed culture transcriptomes for growth on glucose and on a polysaccharide mixture. The multi-species array was used to estimate species composition of the mixed culture; composition varied from 6:1.5:1:1 for glucose batch culture to 2.3:2:2:1 in glucose continuous culture for T. sp. RQ2: T. maritima: T. petrophila: T. neapolitana, respectively. Composition in polysaccharide batch culture was similar to glucose continuous culture. Transcriptional response analysis provided clues to interspecies interactions. In glucose mixed culture, the ORFs encoding a phage tail-like bacteriocin (TM0785), lon proteases (TM1633, TM1869), Ã¯Â Â³E (TM1598), and a putative bacteriocin (TM1300) related to subtilosin A from Bacillus subtilis, were up-regulated relative to pure cultures. Differential regulation of several ORFs encoding HicAB Toxin-Antitoxin pairs (TM1310a-1313, TM1320-21) was noted, suggesting a potential role in interspecies interactions. Comparisons of growth on glucose and polysaccharides revealed changes in both core and non-core ORF transcription. All cultures exhibited upregulation of core genome xylan (TM0056-61, TM0070-77) and ÃŽÂ²-mannan utilization operons (TM1218-1223) on polysaccharide culture. An unclassified ABC transporter operon found only in T. neapolitana and T. sp RQ2 (TRQ2_0970-75) and a ÃŽÂ²-linked exopolysaccharide operon found only in T. maritima (TM0622-30) were up-regulated on glucose. A ÃŽÂ²-mannan utilization operon (TM1746-51), found only in T. maritima and T. sp RQ2, was up-regulated on polysaccharide culture. To investigate the potential of Thermotoga for biofuels production, biohydrogen generation through carbohydrate fermentation was examined for both pure and mixed Thermotoga cultures. T. maritima showed that, unlike the hyperthermophilic archaeon, Pyrococcus furiosus, which uses similar fermentative metabolism, sulfur had minimal effect on transcription. Furthermore, T. maritima preferred cellobiose over maltose, perhaps related to superior bioenergetics mediated by a cellobiose phosphorylase (TM1848) encoded in its genome. Volumetric H2 production rates (~1.2x10-3 mol H2 liter-1 hour-1) were similar for pure and mixed cultures, perhaps related to the function of metabolic pathways comprising the core genome. This work demonstrates the usefulness of multi-species arrays for examining closely related Thermotoga. The results indicted that, while differences in transcription were noted among pure and mixed cultures, culture growth and H2 production levels are not affected by species, substrate, or competition.
Leading Edge Dynamics during Spreading and Migration
(2008-10-20) Krajcovic, Matej; Balaji Rao, Committee Member; David F. Ollis, Committee Member; Jason M. Haugh, Committee Chair
Cell migration is an essential part of wound healing and cancer metastasis. Cells interpret adhesive and soluble stimuli from the environment into directed movement. During cell migration, cells protrude in the direction of motion, forming a leading edge, and retract at the trailing edge. The signaling events at the leading edge are crucial for efficient migration. Cells bind to extracellular matrix (ECM) via integrin receptors. Phosphoinositide 3 kinase (PI3K) is activated in fibroblasts during spreading on fibronectin and poly-lysine. Blocking of integrin receptors by Î²1-integrin antibody inhibits adhesion on fibronectin but not on poly-lysine. PI3K activity is not affected by integrin blocking on poly-lysine. Active Rac, a Rho GTPase, stimulates formation of lamellipodia at the leading edge. We used fluorescent probes for Rac and PI3K in conjunction with total internal fluorescence microscopy (TIRF) to examine spatial distribution of Rac and PI3K. In randomly migrating fibroblasts, Rac and PI3K are co-localized most times in the cell protrusions. The mechanisms governing the formation and disassembly of integrin-ECM adhesion complexes at the protruding leading edge are central to the understanding of cell migration. We have developed a simplified mathematical model that incorporates adhesion and protrusion dynamics mediated by Rac/Pak (p21-activated kinase) signaling at the leading edge. Nascent adhesions either disassemble (turn over) or mature into stable focal adhesions. Stable adhesions can be involved in two distinct pathways of protrusion inhibition. We performed a bifurcation analysis with respect to density of ECM and existence of two possible steady states has been shown. A stochastic version of the model employing the Gillepsie algorithm was developed to model the effect of diffusion in a linear geometry. At certain conditions, multiple states of activation are present within the leading edge.
Relating phosphoinositide 3-kinase (PI3K) signaling and cell motility dynamics during PDGF-stimulated chemotaxis
(2010-04-02) Melvin, Adam Thomas; Jason Haugh, Committee Chair; Robert Kelly, Committee Member; Balaji Rao, Committee Member; Sam Jones, Committee Member
Cell migration is essential for wound healing, the immune response, embryogenesis, and cancer metastasis. Chemotaxis, or cell migration directed by an external gradient of chemoattractant, is encountered in various physiological and natural settings and is a means by which cellular processes are coordinated in space and time. This cyclical process of protrusion, adhesion, and retraction is characterized by an asymmetric polarization of 3' phosphoinositides at the leading edge of a migrating cell and is common across multiple cell types. Activated by the phosphoinositide 3-kinase (PI3K) pathway, these 3' PIs act as membrane-bound second messengers which recruit additional signaling proteins, such as the Rho family GTPases, to initiate actin polymerization and membrane protrusion. These protrusions are capable of both cellular translocation as well as gradient sensing. The characteristics of chemotaxis depend on cell type. Amoeboid cells such as neutrophils and the slime mold, Dictyostelium discoideum, respond to shallow gradients of chemoattractants (~1% difference in concentration across cell dimensions) and migrate rapidly (~20 Âµm/min). Mesenchymal cells such as fibroblasts, on the other hand, require much steeper chemoattractant gradients to evoke similar polarization of PI3K signaling, but the sensitivity of chemotactic movement has yet to be quantitatively characterized. Using a 3' PI-specific, fluorescent biosensor and total internal reflection fluorescence (TIRF) microscopy, the spatial and temporal dynamics of PI3K signaling were monitored during chemotaxis of fibroblasts responding to gradients of the chemoattractant platelet-derived growth factor (PDGF). We show that the asymmetry of PI3K signaling positively correlates with cell directionality during chemotaxis and, consistent with previous work, that highly persistent movement aligned with the external gradient requires a steep gradient (> 10%) in PDGF receptor binding across the cell. Additionally, we present the development of novel microfluidic devices that generate linear gradients of chemoattractant and their integration with TIRF microscopy. In one particular device, fibroblasts exhibited similar characteristics of PI3K signaling asymmetry as compared with other methods. Finally, we evaluate the long-term viability of fibroblasts and methods to minimize toxicity in these devices.
Systematic Analysis of Crosstalk in the PDGF Receptor Signal Transduction Network
(2009-08-19) Wang, Chun-Chao; Carla Mattos, Committee Member; Jason M. Haugh, Committee Chair; Robert M. Kelly, Committee Member; Balaji Rao, Committee Member
Intracellular signal transduction is traditionally characterized in terms of pathways, comprised of serial activation processes. Although it is appreciated that canonical signaling pathways are simply dominant routes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway crosstalk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated crosstalk mechanisms in the platelet-derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3-kinase (PI3K) and Ras/extracellular signal-regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from crosstalk, PI3K enhances Erk activation in multiple ways. Whereas simultaneously blocking Ras and PI3K abolishes PDGF-stimulated Erk phosphorylation, each pathway makes an independent contribution to Erk activation, and PI3K affects Ras activation as well. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K-dependent and -independent modes of Ras/Erk activation.