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The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
Medicolegal forensic entomology is the study of insects to aid with legal investigations (Gemmellaro, 2017). Insect evidence can be used to provide information such as the post-mortem interval (PMI). Blow flies are especially useful as these insects are primary colonizers, quickly arriving at a corpse (Malainey & Anderson, 2020). The age of blow flies found at a scene is used to calculate the PMI. Blow fly age can be estimated using weather data as these insects are poikilothermic (Okpara, 2018). Morphological analysis also can be used to estimate age; however, it is more difficult with pupal samples as the pupae exterior does not change significantly as development progresses (Bala & Sharma, 2016). Gene regulation analysis can estimate the age of samples. MicroRNAs are short noncoding RNA that regulate gene expression (Cannell et al., 2008). Here, we aim to catalog miRNAs expressed during the development of three forensically relevant blow fly species preserved in several storage conditions. Results demonstrated that various miRNA sequences were differentially expressed across pupation. Expression of miR92b increased during mid pupation, aga-miR-92b expression increased during early pupation, and bantam, miR957, and dana-bantam-RA expression increased during late pupation. These results suggest that microRNA can be used to estimate the age of pupal samples as miRNA expression changes throughout pupation. Future work could develop a statistical model to accurately determine age using miRNA expression patterns.
Forensic entomology is an important field of forensic science that utilizes insect evidence in criminal investigations. Blow flies (Diptera: Calliphoridae) are among the first colonizers of remains and are therefore frequently used in determining the minimum postmortem interval (mPMI). Blow fly development, however, is influenced by a variety of factors including temperature and feeding substrate type. Unfortunately, dietary fat content remains an understudied factor on the development process, which is problematic given the relatively high rates of obesity in the United States. To study the effects of fat content on blow fly development we investigated the survivorship, adult weight and development of Lucilia sericata (Meigen; Diptera: Calliphoridae) and Phormia regina (Meigen; Diptera: Calliphoridae) on ground beef with a 10%, 20%, or 27% fat content. As fat content increased, survivorship decreased across both species with P. regina being significantly impacted. While P. regina adults were generally larger than L. sericata across all fat levels, only L. sericata demonstrated a significant (P < 0.05) difference in weight by sex. Average total development times for P. regina are comparable to averages published in other literature. Average total development times for L. sericata, however, were nearly 50 hours higher. These findings provide insight on the effect of fat content on blow fly development, a factor that should be considered when estimating a mPMI. By understanding how fat levels affect the survivorship and development of the species studied here, we can begin improving the practice of insect evidence analysis in casework.
The field of forensic science has been growing and changing with improvements in DNA analysis. One field affected is forensic entomology, which is exploring many ways in which DNA can increase the application of insects in forensic science. One application being explored is the use of insects as a source of human DNA in a criminal investigation. Using flies as a source of foreign DNA can also be utilized in ecological research to conduct surveys on the various species present in different environments. This experiment intends to determine if flies can act as a viable source of alternate DNA. This will be accomplished by an ecological survey of DNA extracted from flies. DNA extractions were performed on flies gathered from parts of the greater Phoenix area. The DNA was then amplified with primers targeting different animal species and examined to observe what animals the flies had come in contact with. Several samples had contamination due to human error and were not able to be evaluated. One DNA extraction out of fifteen yielded pig DNA, indicating flies can be used as a source of DNA. Future experiments should use different animal primers and amplify sections of DNA that can determine the different species consumed by flies. Further research into flies as a DNA source can increase the amount of information available to forensic scientists as well as improve ecologist’s observation of an environment’s biodiversity.