Ph. D. Thesis Abstracts

Tolga Bilgen, Ph.D. Biology 1998

Differential gene expression in two cold hardy insects in response to low temperatures

 

Abstract:

The two cold hardy gall insects, Epiblema scudderiana andEurosta solidaginis, employ freeze avoidance and freeze tolerance, respectively, in coping with low seasonal temperatures. Biochemical and metabolic changes in these animals in response to low temperatures have been well documented, but the possibility that cold and subzero temperatures could upregulate genes in these animals has not been investigated. In this study, cDNA library screening and differential display PCR were used to address this question. Animals were sampled over a successive cold (2 weeks at 4°C) and subzero temperature (one week at -20°C) time course. Differential screening/PCR against RNA from control (15°C) animals and Northern analyses revealed a number of upregulated transcripts. All clones were sequenced, and all but two were unidentifiable through GenBank database comparisons, due to the novel and incomplete nature of their sequences. The two identifiable sequences had strong homologies to Drosophila genes mlp6OA andrpAl, encoding respectively, a factor crucial for myogenesis and an acidic ribosomal protein. Implicated in embryonic and adult development, the upregulation of these genes suggests a unique developmental regime in E. scudderiana and F. solidaginis,both of which overwinter as final instar larvae. These animals may be preparing in advance for impending morphogenic changes, allowing for a head-start with the onset of spring.

Shaobo Wu, Ph.D. Biology 1999

Differential gene expression under environmental stress in the freeze tolerant wood frog, Rana sylvatica

 

Abstract:

Freeze survival of wood frog, Rana sylvatica, involves adaptations including control over extracellular ice formation, production of glucose cryoprotectant, and resistance to freezing-caused intracellular dehydration and ischemia. Gene expression associated with stress survival was investigated in this freeze tolerant species. Freeze-inducible genes were found by differential screening of frog brain and liver cDNA libraries; these included mitochondrial genes [encoding ATPase subunit 6 & 8, 16S rRNA and NADH-ubiquinone oxidoreductase subunit 4 (ND4)], the phosphoglycerate kinase 1 (PGK1) gene, and genes whose products are involved in translational processes [acidic ribosomal phosphoprotein (P0), elongation factor 1 gamma subunit (EF-1 gamma)]. Another ribosome-associated gene, encoding ribosomal protein L7 (RPL7), was identified in skin via differential display of polymerase chain reaction (DD-PCR). This gene was up-regulated in skin of cold-acclimated frogs and brain of freeze-exposed frogs. Freezing stimulated the upregulation of the above genes in selected frog organs. Tissue-specific gene expression also occurred in frog brain and liver in response to anoxia or dehydration. Anoxia stimulated P0, PGK1, RPL7, 16S rRNA and ATPase 6 & 8 gene expression and modulated ND4and EF-l gamma expression, whereas dehydration enhanced the expression of genes such as PGK1.Upregulation of genes whose products are directly involved in energy generation (PGK1, ATPase subunit 6 & 8, ND4) and whose products are related to protein biosynthesis suggested that maintenance of minimal ATP levels and functional translation machinery may be critical for freezing survival. Freezing-induced ischemia may be a primary signal that triggers the upregulation of most of the isolated genes. However, low temperature seemed to play a role in the expression of ribosome associated protein genes, whereas freezing-related cell water stress may also regulate other selected genes (e.g. PGK1).Immunoblotting confirmed that elevated PGK1 transcripts resulted in increased enzyme protein and showed the potential physiological significance of up-regulated genes in response to stress. Immunoblotting also showed the elevation of Ca2+/ca1modulin-dependent protein kinase and phosphatase under freezing, anoxia and/or dehydration stresses which suggests that a Ca2~signaling pathway plays a role in stress-mediated gene expression. These cellular responses may play an important role in survival of environmental stress.

Andreas Fahlman, Ph.D. Biology 2000

On the physiology of hydrogen diving and its implication for hydrogen biochemical decompression

 

Abstract:

Biochemical decompression, a novel approach for decreasing decompression sickness (DCS) risk by increasing the tissue washout rate of the inert gas, was tested in pigs during simulated H2dives. Since there is only limited physiological data on the use of H2as a diving gas, direct calorimetry and respirometry were used to determine whether physiological responses to hyperbaric H2 and He are different in guinea pigs. The data suggested that responses in hyperbaric heliox and hydrox cannot be explained solely by the thermal properties of the two gas mixtures. To increase the washout rate of H2, a H2-metabolizing microbe (Methanobrevibacter smithii) was tested that converts H2 to H2O and CH4. Using pigs (Sus scrofa) comparisons were made between untreated controls, saline-injected controls, and animals injected with M. smithiiinto the large intestine. To simulate a H2 dive, pigs were placed in a dry hyperbaric chamber and compressed to different pressures (22.3-25.7 atm) for times of 30-1440 min. Subsequently, pigs were decompressed to 11 atm at varying rates (0.45-1.80 atm · min-1), and observed for severe symptoms of DCS for 1 h. Chamber gases (O2, N2, He, H2, CH4) were monitored using gas chromatography throughout the dive. Release of CH4 in untreated pigs indicated that H2 was being metabolized by native intestinal microbes andresults indicated that native H2-metabolizing microbes may provide some protection against DCS following hyperbaric H2exposure. M. smithii injection further enhanced CH4 output and lowered DCS incidence. A probabilistic model estimated the effect of H2-metabolism on the probability of DCS, P(DCS), after hyperbaric H2 exposure. The data set included varying compression and decompression sequences for controls and animals with intestinal injections of H2metabolizing microbes. The model showed that increasing total activity of M. smithii injected into the animals reduced P(DCS). Reducing the tissue concentration of the inert gas significantly reduced the risk of DCS in pigs, further supporting the hypothesis that DCS is primarily caused by elevated tissue inert gas tension. The data provide promising support for the development of biochemical decompression as an aid to human diving.

Tamara E. English, Ph.D. Biology 2000

Differential gene expression in response to freezing and anoxia in the intertidal marine gastropod, Littorina littorea.

 

Abstract:

The intertidal zone is a highly variable environment where temperature, salinity and oxygen availability all fluctuate on a daily and seasonal basis. As a result, animals that inhabit this zone possess a high degree of metabolic plasticity. Littorina littorea, an intertidal marine gastropod, is tolerant of both freezing and anoxia. This study searched for changes in gene expression that may underlie the animal’s ability to endure these stresses. Screening of cDNA libraries was employed to identify freezing- and anoxia-induced genes in foot muscle of L.littorea; two libraries were synthesized from mRNA isolated from foot muscle of animals exposed to either 1, 12 and 24 hours of freezing or 1, 12 and 24 hours of anoxia. Differential screening of the frozen or anoxic libraries with mRNA isolated from stressed versus control (5°C)snails, followed by northern blot analysis, resulted in six transcripts that were confirmed as stress-upregulated. DNA sequence analysis identified 3 clones as myosin heavy chain, beta actin and cytochrome oxidase subunit 2. These clones were isolated from the freezing library but showed greatest transcript accumulation during recovery after stress. The translated amino acid sequence of a fourth clone, LLMET, elicited a putative identification of metallothionein, a heavy metal binding protein with a possible antioxidant role. The remaining two clones, LLGRP and LLAFW, were novel, showing little or no homology to DNA or amino acid sequences in various databases. Analysis of their translated amino acid sequences indicated that both proteins possessed a secretory signal at the N terminus, suggesting that they had either a membrane location or are excreted from the cell. Structural predictions based on previously analyzed proteins, suggested that LLGRP is a membrane channel protein of the porin class whereas LLAFW fits the criteria of an anti-parallel bundle (apb) protein of the alpha-class. None of the six genes/proteins found by this study has a cellular function that integrates well with any of the previously-identified biochemical adaptations that support anoxia or freezing tolerance but each suggests that there is still much more to learn about the types of molecular adjustments that are needed to support natural stress tolerance.

Dustin S. Hittel, Ph.D. Biology, 2001

The physiological role of differentially expressed genes and their protein products in the hibernating thirteen-lined ground squirrel Spermophilus tridecemlineatus

 

Abstract:

The role of differential gene expression in supporting the survival of the hibernation phenotype was investigated using a variety of “gene discovery” techniques. A cDNA library constructed from kidney of the thirteen-lined squirrel,Spermophilus tridecemlineatus, was differentially screened for genes that were up-regulated during hibernation. A clone encoding cytochrome c oxidase subunit 1 (Cox1) was found and confirmed as up-regulated by Northern and Western blotting. This revealed the differential expression of Cox1 mRNA in multiple organs during hibernation. It is hypothesized that hibernating mammals increase the expression of the mitochondrial genome in general and Cox1specifically during torpor, to prevent or minimize the damage caused by the cold and ischemia experienced during a hibernation bout. The up-regulation of heart and adipose type fatty acid binding proteins (FABPs) was detected during hibernation in brown adipose tissue (BAT) using a commercial rat cDNA array. Full length cDNAs encoding heart and adipose-type FABPs were subsequently retrieved from a BAT cDNA library. H-FABP mRNA transcripts increased in BAT, skeletal muscle and heart of hibernating animals whereas A-FABP transcripts, which are normally expressed exclusively in adipose tissue, increased in both BAT and heart during torpor. The translation status of differentially expressed mRNAs during hibernation was also investigated in kidney and brown adipose tissue. Polysome profile analysis revealed a significant de-aggregation of polyribosomes during hibernation and a shift of housekeeping gene mRNAs and the up-regulated organic cation transporter 2 (OCT2) mRNA to the translationally silent monosome and mRNP fractions of kidney cytoplasmic extracts. In vitrotranslation rate and immunoreactive OCT2 protein were also significantly decreased in hibernating kidney. By contrast, the increased translation status of H-FABP mRNA, increase in immunoreactive H-FABP protein and unchanging in vitrotranslation rate reflect the important role of active brown adipose tissue during hibernation. The decrease in protein synthesis and de-aggregation of polysomes in kidney but not in brown adipose tissue, is linked to the phosphorylation of eIF2“ . Additionally, redistribution of Cox4 but not H-FABP mRNA to the monosome fraction in hibernating BAT may indicate a mechanism for the preferential translation of a subset of genes physiologically relevant to the survival of hibernation.

 

Kevin F. Larade, Ph.D. Biology 2002

Response to anoxia exposure by the marine snail, Littorina littorea: transcription and translation patterns of differentially expressed genes and proteins

 

Abstract:

Situated in the intertidal zone, the marine periwinkle Littorina littoreahas modified a number of biochemical mechanisms in order to endure extended periods of oxygen deprivation that accompany aerial exposure at low tide. To cope with decreased ATP production during anaerobiosis, organisms often suppress energy-consuming processes such as those involved in macromolecular synthesis. Analysis of hepatopancreas samples from snails exposed to 24-96 h anoxia showed a gradual disaggregation of polysomes into monosomes, signifying a decrease in protein synthesis, with a subsequent re-aggregation of polysomes observed after 3 h of aerobic recovery. Upon examination of protein synthesis, the rate of [3H] leucine incorporation into newly translated protein of hepatopancreas isolated from 48 h anoxic snails was determined to be 49% relative to normoxic controls. Western blots examining the phosphorylation state of eIF-2alpha, a factor involved with initiation of protein translation, also supported the proposal that metabolic suppression during anoxia in L. littorea involves a decrease in protein translation. The rate of overall mRNA synthesis in anoxia was also examined; [32P] UTP incorporation into RNA transcripts of nuclei obtained from 48 h anoxic snails was determined to be 31.7% relative to normoxic controls. Although these data show that transcription and translation are suppressed overall during anoxic exposure, specific RNA transcripts were up-regulated during anoxia as identified via differential screening of a hepatopancreas cDNA library. Several anoxia-induced clones were identified as homologues of known genes (e.g. ribosomal protein L26, ferritin heavy chain) and others were deemed novel (e.g.kvn). Northern blots showed gene-specific patterns of transcript elevation over a time course of anoxia. Nuclear run-off assays confirmed transcriptional up-regulation during anoxia, whereas organ culture experiments implicated selected second messengers and protein kinases in signal transduction pathways regulating gene expression. Examination of the protein products of anoxia-induced genes was accomplished with western blotting when antibodies to specific proteins were available. These results suggest that metabolic suppression in L. littoreainvolves a general decrease in macromolecular synthesis, whereas specific transcripts that may help cope with oxygen lack are up-regulated during anoxia exposure.

 

J. Dayre McNally, Ph.D. Chemistry 2002

Response to freeze exposure by the wood frog, Rana sylvatica: investigating the freeze induced changes to transcription and translation patterns in heart and liver

 

Abstract:

In recent years changes to gene and protein expression have been shown to underlie animal adaptation to environmental stresses. Similarly, recent studies have demonstrated significant changes to transcription and translation in wood frog tissues during freeze and thawing. The primary goal of this thesis was to further investigate these changes through the application of cDNA library screening and 2-D PAGE technologies. In total 7 additional genes and one protein were identified, significantly extending the list of known freeze up-regulated genes and proteins. From this expanded list the contractile protein, MLC2; two identifiable heart transcripts (hsp27 and anac); and the novel liver transcripts (li16 and ddx8) were selected for further investigation. The results implicate the heat shock and hypertrophic gene responses as part of the cellular response to freezing, and reveal that the wood frog heart responds to freezing with changes expected to alter AP-1 mediated gene transcription. Moreover, all of the identified cardiac responses, including specific increases in gene/protein levels and changes to protein phosphorylation states could be linked to the p38 MAPK pathway, confirming that it is the major signaling pathway activated in the wood frog heart following freezing exposure. The two novel liver clones were similarly investigated and studies were carried out to obtain full open reading frames, confirm the protein coding capacity and determine the factors regulating their expression. Although the cellular functions of the novel proteins remain a mystery the results obtained suggest distinctive cellular roles, and on the whole provide further evidence that novel gene development is an important component of acquired freeze tolerance. Secondary to the determination of the specific changes in gene and protein expression patterns following freeze exposure the impact of freezing on the overall rates of transcription and translation in wood frog heart tissue were evaluated. The data from the nuclear run-on assays and western blots analysis (phosphorylation status of eIF2a ), confirmed the presumed suppression of transcription and translation in the wood frog heart during freezing. This discovery highlights the importance of the aforementioned genes and proteins, as their up-regulation takes place against a background of suppressed macromolecular synthesis.

Sean F. Eddy, Ph.D. Chemistry 2003.

Role of differential gene and protein regulation in hibernating mammals

 

Abstract:

Winter survival for many mammals involves hibernation. By strongly suppressing metabolic rate, animals conserve energy and endure long months of subzero environmental temperatures and lack of food. The little brown bat, Myotis lucifugus, and thirteen-lined ground squirrel, Spermophilus tridecemlineatus, are two such hibernating mammals. Hibernation consists of long periods (1-2 weeks) of cold torpor with body temperature near ambient interspersed with brief periods of arousal to euthermia. Precise control over gene and protein expression is needed to prevent costly overuse of essential fuel reserves while ensuring that selected specific changes are made that aid survival in the cold, torpid state. The present studies evaluated signal transduction pathways and changes in gene expression in skeletal and cardiac muscle during hibernation. The p38 MAP kinase signal transduction pathway in skeletal muscle of both species appears triggered with increased amounts of active phosphorylated p38 found during hibernation along with activation of various downstream targets of p38 including ATF-2, CREB, HSP27 and IκB. The Akt-mediated insulin-signalling pathway was, by contrast, apparently suppressed in hibernator muscle; this may help restrict the use of carbohydrate fuels.cDNA arrays were used to compare gene expression in euthermic versus hibernating states; selected genes (0.5-2%) were up-regulated during hibernation but most were unaffected. A general suppression of protein synthesis is likely during hibernation and this was supported by elevated levels of phosphorylated initiation (eIF2α) and elongation (eEF2) ribosomal proteins. Despite the probable suppression of transcription and translation, selected genes and proteins were up-regulated during hibernation including two involved in fatty acid transport and metabolism: fatty acid-binding protein and carnitine palmitoyl transferase-1. Furthermore, two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry revealed the up-regulation of a thioredoxin peroxidase type enzyme in heart of M. lucifugus; both mRNA and protein levels rose during hibernation. This, along with elevated amounts of the active forms of oxidative stress markers, HSP27 and IκB, demonstrates that oxidative stress has a role in hibernating tissues. The data enhance our knowledge of the molecular mechanisms of hibernation with novel contributions to the understanding of the roles of fatty acid metabolism, oxidative stress, and muscle atrophy.

J.N. Amritha De Croos, Ph.D. Biology, 2003

Gene and protein regulation in liver of the freeze tolerant wood frog, Rana sylvatica.

 

Abstract:

The wood frog Rana sylvaticahas the ability to tolerate whole body freezing in nature. Biochemical mechanisms supporting freezing survival have been widely studied in this species, but much remains to be explored. The present research demonstrates the involvement of specific gene and protein level changes in the response to freezing by liver. Screening of a cDNA library prepared from liver of R. sylvatica identified a freeze-responsive clone containing a 1370 nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over time during freezing, reaching >70-fold up-regulation after 24 hours frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase inpic transcript levels during dehydration (9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers of the same family were not affected. Southern blot analysis showed that the increase in PiC was not due to an increase in mitochondrial numbers. Analysis of polysome profiles revealed disaggregation of polysomes into translationally less active monosomes upon freezing. PiC transcripts segregated into monosomes in control liver, but would aggregate into actively transcribing polysomes during freezing. Western blot analysis showed that the activity of specific transcription factors in frogs is temporally controlled. The transcription factors CREB and NFk B were significantly activated after two hours of freezing exposure whereas protein kinase B (Akt) was activated after 24 hours of freezing. Screening of a cDNA microarray identified an anti-apoptotic factor that is up-regulated during freezing in liver. Western blot analysis showed time dependent changes in anti-apoptotic activity mitigated by significant changes in AP-2a , BAX-inhibitor and p53 protein levels. These results suggest that freeze tolerance in R. sylvatica involves control of gene and protein levels through the up-regulation of selected transcripts and the activation of kinases and transcription factors. This gene and protein control, or regulation, combined with specific anti-apoptotic actions, allows hepatocyte survival of the multiple stresses imposed by freezing.

David C. McMullen, Ph.D. Chemistry 2004.

Molecular and biochemical adaptations in two cold-hardy gall insects.

 

Abstract:

Terrestrial insects have evolved molecular and biochemical adaptations that have allowed them to exploit virtually all climates on earth.  Insects living in temperate and polar climates are forced to deal with freezing temperatures head-on.  Two strategies of cold-tolerance in insects have emerged, freeze-tolerance and freeze-avoidance.  Freeze-tolerantallow extracellular ice formation, whereas freeze-avoiding species depress the supercooling point of their body fluids below temperatures that they would normally encounter as part of their life-cycle.  This research used a model species from each category of cold-tolerant insects.  The model species chosen wereEpiblema scudderiana, a freeze-avoiding gall moth, and Eurosta solidaginis, a freeze-tolerant gall fly.  Previous studies on these systems were based on measuring physiological parameters such as substrate utilization of product build-up.  Results presented here are from molecular and biochemical studies completed to further our understanding of cold-tolerance and hypometabolism. Studies also included the effects of hypoxia on the two model species because this stress is often associated with freezing.  The first step in each type of experiment was to examine how these species responded in nature and then see if we could reproduce that response under laboratory conditions to identify the trigger for such a response.  The mechanism of ion pump regulation was examined and possible mediators of control were proposed.  Three different gene expression analysis techniques were employed in an effort to identify key gene product that contribute to cold and hypoxic tolerance.  Gene expression studies and enzyme assays have indicated that the role of the mitochondria in cold and hypoxic tolerance is more dynamic that previously understood.  In addition, the MAP kinase superfamily of signal transduction has clearly been implicated in the survival success of these insects. The p38 family of MAPK is of particular interest because there were clear differences in this pathway between the freeze-tolerant and freeze-avoiding species.  Reversible phosphorylation in undoubtedly a key mechanism of control in insect hypometabolism and stress tolerance.   Finally, the role of NF-κB and the inflammatory response appears to be significant in the over winter survival of both species.