Mammalian cell culture is typically operated at the physiological temperature of 37°C. Low temperature cell culture at 30-33°C, in particular for CHO cells, increased the specific productivity of many recombinant proteins amongst many other benefits. However, the cell density is lower, thus reducing the total protein yield. Of the 17 mammalian cold-stress genes reported to be up- or down-regulated at low temperature, CIRP shows potential as a gene target for improving recombinant protein production, as its expression levels were reported to affect both growth and specific productivity. In this study, it was shown that over-expression of the cold-stress gene CIRP did not cause growth arrest in CHO cells, in contrast to previous reports. However, over-expression of CIRP successfully improved the specific productivity and total yield of a recombinant interferon-γ CHO cell-line at 37°C by 25%. / Singapore-MIT Alliance (SMA)

No description available.

Salmonella is a food-borne pathogen that leads to substantial illness worldwide. The clinical syndromes associated with Salmonella infection are enteric (typhoid) fever and gastroenteritis, in healthy humans. Typhoid fever is caused by host-adapted S. Typhi and S. Paratyphi. Gastroenteritis is caused by serovars usually referred to as non typhoidal Salmonellae (NTS). In recent years, an increasing number of outbreaks due to NTS, despite increased efforts in food safety, were reported because of persistence of Salmonella in the food chain. Thus I hypothesized that Salmonella is able to withstand stresses in the environment and treatments used during food processing for its elimination and thereby able to develop resistance against subsequent stress encounters. The effect of cold, peroxide, and acid was tested on survival of S. Typhimurium and the survival was persistent under cold stress (5°C) for up to 240 h. Pre-adaptation to cold stress (5°C, 5 h) also increased survival of S. Typhimurium during subsequent exposure to acid stress (pH 4.0, 90 min) by repressing hydroxyl radical formation. Cold stress (5°C, 48 h) to S. Typhimurium significantly (p < 0.05) increased its adhesion and invasion in intestinal iv epithelial cells. This phenotype was attributed to a pair of protein-protein interactorsacting as receptors on microbial (STM2699) and host cell surface (SPTAN1). Cold stress significantly (q < 0.05) induced STM2699 in S. Typhimurium and SPTAN1 was significantly (q < 0.05) induced in pithelial cells upon infection with cold-stressed S. Typhimurium. Cold stress to S. Typhimurium also significantly (q < 0.05) induced genes related to virulence such as type 3 secretion system apparatus and effectors genes, prophage genes, and plasmid genes and they remain induced upon infection of epithelial cells with additional induction of spv genes on the plasmid. Infection of epithelial cells with cold-stressed S. Typhimurium significantly (p < 0.05) increased activation of caspase 9 and 3/7. Cold-stressed S. Typhimurium switched metabolism from aerobic respiration to fermentation and it persisted during infection of epithelial cells. As a result, short chain fatty acids formate and acetate, which act as diffusible signal for invasion, were detected in significantly (q < 0.05) high amounts in extracellular media of cells infected with cold-stressed S. Typhimurium supporting the phenotype of high adhesion and invasion of cold-stressed S. Typhimurium in epithelial cells.

The widespread emergence of antibiotic resistance determinants for nearly all drug classes threatens human health on a global scale. It is therefore essential to discover antibiotics with novel functions that are less likely to be influenced by pre-existing resistance mechanisms. An emerging approach to identify inhibitors of investigator-defined cellular processes involves screening compounds for antimicrobial activity under non-standard growth conditions. Indeed, by growing cells under conditions of stress, inhibitors of specific cellular targets can be enriched, thereby allowing for the identification of molecules with predictable activities in the complex environment of the cell. Here, I exploit cold stress in Escherichia coli to identify molecules targeting ribosome biogenesis and outer membrane biosynthesis. First, through a screen of 30,000 small molecules for growth inhibition exclusively at 15°C, I was able to identify the first small molecule inhibitor of bacterial ribosome biogenesis, lamotrigine. Second, by leveraging the idiosyncratic cold sensitivity of E. coli to vancomycin, I developed a novel screening technology designed to enrich for non-lethal inhibitors of Gram- negative outer membrane biosynthesis. From this platform, I identified pentamidine as an efficient outer membrane perturbant that was able to potentiate Gram-positive antibiotics against Gram-negative pathogens, similar to the polymyxins. Remarkably, however, this compound was able to overcome mcr-1 mediated polymyxin resistance. Together, this thesis highlights the utility of exploiting the bacterial cold stress response in antibiotic discovery. / Thesis / Doctor of Philosophy (PhD)

Published Article / A study involving two incubation experiments and a germination experiment in sandy soil was conducted to determine the influence of Multiplied Effective Micro-organisms (M-EM) that were exposed to different levels of irradiation and temperature fluctuation as well as prolonged storage, on the germination and seedling vigour of maize, sorghum and sunflower. Irrespective of poor handling and/or prolonged storage of M-EM, seed treatment with M-EM improved germination under optimal conditions for all crops compared to the control. Increased planting depths and cold stress were used to create conditions where seed treatment with M-EM may improve germination and seedling growth. Seed treatment with M-EM significantly improved germination and seedling vigour of the stress-tolerant maize cultivar following cold stress. Similar improved seedling vigour results were observed for the stress-tolerant sunflower cultivar at increased planting depth. It was concluded that the beneficial influence of M-EM as a seed treatment was remarkable, even after exposure of M-EM to unfavourable environmental conditions and prolonged storage before application. Further research under field conditions and in different cultivation systems are required before the large scale application of M-EM as seed treatment can be recommended.

Master of Science / Department of Agronomy / Tesfaye Tesso / Cold temperature stress is an important abiotic constraint to grain sorghum production in temperate regions. In the United States, low temperature in late spring and early fall has limited sorghum production to a narrow growing period. Deployment of cold tolerance traits may widen this window and hence contribute to increased production. The objectives of this study were (1) to determine the effect of early and mid-season cold temperature stress on growth, phenology and yield components of sorghum, and identify key traits that are most sensitive to cold stress at seedling and flowering stages, and (2) to identify new sources of cold tolerance for use in breeding programs. Series of controlled environment (greenhouse/growth chamber) and field experiments were carried out. Three sorghum genotypes of variable response, Shan Qui Red (tolerant), SRN39 (susceptible) and Pioneer 84G62 (unknown) were subjected to cold (15/13ºC day/night) and normal (25/23ºC day/night) temperature at seedling (Experiment I) and flowering (Experiment II) stages. The genotypes were planted in a greenhouse using a 5L polytainer pots. Each pot consisted of a single plant and each plot was represented by three pots. A split-plot design with three replications was used in both experiments with temperature regimes as main plots and genotypes as sub-plots. Three days after emergence, experiment I plants were moved to the growth chamber and subjected to the designated temperature treatments. For experiment II, the treatments were assigned at heading stage immediately before anthesis had begun. The treatments lasted 10 d in both experiments. Data were collected on seedling characteristics and leaf chlorophyll content in experiment I, days to flowering, maturity, and yield components in both experiments, and anthesis duration in experiment II. For the field experiment, 150 sorghum germplasm collections of potential cold tolerance along with tolerant and susceptible checks were evaluated for emergence and seedling traits under early planting (April 13) at soil temperature of 20.1/13.4 ºC max/min. The normal temperature treatment was applied by planting at regular season (May 26) at soil temperature of 30.0/20.4ºC max/min. Twenty-four genotypes selected based on field emergence and seedling vigor were further screened under controlled environment. Early-season stress significantly reduced leaf chlorophyll content, all seedling traits (height, vigor and dry weight), and also delayed flowering and maturity. But it had no effect on final leaf number, plant height and yield components. Genotypic response to early stress was significant for all traits with the susceptible checks having the lowest score for all seedling traits. Mid-season cold stress prolonged anthesis duration, delayed maturity and highly reduced all yield components. Several genotypes among the 150 had higher seedling vigor and emergence than the tolerant check, Shan Qui Red. In conclusion, reduced seedling vigor as a result of early stress had no effect on final yield provided that stand establishment was not compromised while mid season stress is damaging to yield. The wide genetic variation for the traits indicates the potential for improvement of cold tolerance in sorghum.

Heme-activated proteins (HAPs) are transcription factors that have multiple roles in plant growth and development, such as embryogenesis, flowering time control, and drought tolerance. In the present study I found that HAP3b was also involved in controlling response to cold stress. Transcript profiling and gene expression analyses indicated that HAP3b repressed the CBF3 regulon under normal growth conditions. As a result, plants with HAP3b-overexpressed showed decreased survival rates while plants homozygous for the null allele hap3b showed an improved freezing tolerance compared to wild-type plants. To understand the mechanism of HAP3b in Arabidopsis, i.e. whether it also acts through forming a heterotrimer, the yeast two-hybrid system and the protein coimmunoprecipitation method were used to identify the proteins that could interact with HAP3b. From yeast two-hybrid analyses, it was found that HAP3b could interact with one (At3g14020) of ten HAP2s and all ten HAP5s tested. Further analyses showed that the newly identified HAP2 protein could only interact with two HAP5 proteins, those encoded by At5g63470 and At1g56170. To address whether HAPs also play important roles in major crop plants, HAP3 genes in barley (Hordeum vulgare L.) were identified and characterized. From database sequence analyses, cloning, and sequencing, it was found that barley plants have at least six full-length members in the HAP3 family. Phylogenetic analyses showed that each barley HAP3 was different, forming its own cluster with the HAP3s from other plant species. Each barley HAP3 also showed its own expression pattern in different tissues, at different developmental stages and under various environmental stresses. In particular, TC176294 showed the highest sequence similarity to HAP3b in Arabidopsis and its high expression was associated with flowering. In addition, TC176294 was upregulated by various abiotic stresses and by abscisic acid (ABA). Thus, TC176294 might be a barley ortholog of HAP3b. TC191694 showed the highest sequence similarity to HAP3c and might be a barley ortholog of HAP3c. TC191694 overexpression plants were early flowering compared to HAP3b-overexpression and wild-type plants while overexpression of TC176294 plants were not.

Several studies were designed to investigate effect of cold winter transportation on broiler meat quality and physiological parameters. The first study assessed the effect of microclimate environment during winter transportation (-27 to +11ºC) from farm to the processing plant (3-4 h) on core body temperature (CBT) and breast meat quality parameters of 522 broiler chickens using an actively ventilated transport vehicle. Temperatures below freezing during transportation resulted in significantly (P < 0.05) higher ultimate pH (pHu) and darker color breast meat, which led to 8% incidence of dark, firm and dry (DFD) breast meat (pH > 6.1; L*< 46).<p> The effects of extreme cold conditions (-18 to -4ºC) along with age (5 and 6 wk), gender and lairage (0 and 2 h) on physiology parameters, breast muscle metabolites and meat quality of 360 birds using a simulated transport system were investigated. Five and 6 wk birds exposed to temperatures below -8 and -14ºC, respectively, showed significantly (P < 0.05) lower CBT and blood glucose and higher live shrink with breast meat that was darker in color, higher in pHu, water holding and water binding capacity compared to breast meat of birds exposed to warmer temperatures. A high incidence of DFD breast meat (> 50%) was observed for 5 and 6 wk birds exposed to temperatures below -8 and -14ºC respectively. Lairage following acute cold exposure caused an increase of up to 20% in DFD breast meat following exposure of the birds to temperatures below -8ºC.<p> Characteristics of cold-induced DFD breast meat were investigated in broiler chickens. DFD breast meat was significantly darker, redder, and less yellow in color and had higher pH, water binding capacity, processing cook yield and lower thaw and cook loss compared to normal meat. However, no significant difference in initial energy reserves was observed between DFD and normal breast meat.<p> Effect of extreme cold-stress (-15 to -9ºC) was further assessed on thigh meat quality and incidence of DFD from 160 male broilers. A greater impact of cold stress was observed on muscle metabolites and pH of thigh compared to breast meat, with 85% DFD incidence in thigh compared to 42% in breast meat of cold-stressed birds.<p> Biochemical basis of cold-induced DFD breast and thigh meat was investigated by measuring muscle metabolites at different times post-mortem. Lack of energy reserves at slaughter clearly explained the incidence of DFD thigh meat, but other factors might contribute to DFD breast meat aside from glycogen reserve at slaughter. However, the factors leading to DFD breast meat still remain unresolved and association of post-mortem enzyme activity and development of DFD breast meat merits further investigation.

Several studies were designed to investigate effect of cold winter transportation on broiler meat quality and physiological parameters. The first study assessed the effect of microclimate environment during winter transportation (-27 to +11ºC) from farm to the processing plant (3-4 h) on core body temperature (CBT) and breast meat quality parameters of 522 broiler chickens using an actively ventilated transport vehicle. Temperatures below freezing during transportation resulted in significantly (P < 0.05) higher ultimate pH (pHu) and darker color breast meat, which led to 8% incidence of dark, firm and dry (DFD) breast meat (pH > 6.1; L*< 46).<p> The effects of extreme cold conditions (-18 to -4ºC) along with age (5 and 6 wk), gender and lairage (0 and 2 h) on physiology parameters, breast muscle metabolites and meat quality of 360 birds using a simulated transport system were investigated. Five and 6 wk birds exposed to temperatures below -8 and -14ºC, respectively, showed significantly (P < 0.05) lower CBT and blood glucose and higher live shrink with breast meat that was darker in color, higher in pHu, water holding and water binding capacity compared to breast meat of birds exposed to warmer temperatures. A high incidence of DFD breast meat (> 50%) was observed for 5 and 6 wk birds exposed to temperatures below -8 and -14ºC respectively. Lairage following acute cold exposure caused an increase of up to 20% in DFD breast meat following exposure of the birds to temperatures below -8ºC.<p> Characteristics of cold-induced DFD breast meat were investigated in broiler chickens. DFD breast meat was significantly darker, redder, and less yellow in color and had higher pH, water binding capacity, processing cook yield and lower thaw and cook loss compared to normal meat. However, no significant difference in initial energy reserves was observed between DFD and normal breast meat.<p> Effect of extreme cold-stress (-15 to -9ºC) was further assessed on thigh meat quality and incidence of DFD from 160 male broilers. A greater impact of cold stress was observed on muscle metabolites and pH of thigh compared to breast meat, with 85% DFD incidence in thigh compared to 42% in breast meat of cold-stressed birds.<p> Biochemical basis of cold-induced DFD breast and thigh meat was investigated by measuring muscle metabolites at different times post-mortem. Lack of energy reserves at slaughter clearly explained the incidence of DFD thigh meat, but other factors might contribute to DFD breast meat aside from glycogen reserve at slaughter. However, the factors leading to DFD breast meat still remain unresolved and association of post-mortem enzyme activity and development of DFD breast meat merits further investigation.

The effects of moderate, long-term intake of ethanol on the hypothalamic response to cold stress were examined. The long-term experimental animals were given .25 ml of 28% ethanol or .25 ml of water orally once a day, five days a week for fourteen months. A stainless steel electrode was then surgically implanted into the paraventricular nucleus, after which the animal was subjected to cold stress (-150 C, 10 min.). Recordings were taken in the forms of frequency and activity. The data clearly indicate that: (1) alcohol fed rats exhibited a suppressed response to cold stress compared to sham-fed rats; (2) this suppression of activity occurred at the level of the hypothalamus, and (3) mortality was significantly lower in alcohol-fed males than it was in sham fed males. This study clearly points out the need for further work in the area of the beneficial effects of moderate doses of alcohol.