Lake Baikal in Russia is a large, ancient lake that has been the site of a major radiation of amphipod crustaceans. Nearly 400 named species are known in this single lake, and it is thought that many more await description. The size and depth of Lake Baikal, in particular, may have contributed to the radiation of endemic amphipods by providing a large number of microhabitats for species to invade and subsequently experience reproductive isolation. Here we investigate the possibility that large-scale genomic changes have also accompanied diversification in these crustaceans. Specifically, we report genome size estimates for 36 species of Baikal amphipods, and examine the relationship between genome size, body size, and the maximum depths at which the amphipods are found in the lake. Genome sizes ranged nearly 8-fold in this sample of amphipod species, from 2.15 to 16.63 pg, and there were significant, positive, phylogenetically corrected relationships between genome size, body size, maximum depth, and diversification rate among these species. Our results suggest that major genomic changes, including transposable element proliferation, have accompanied speciation that was driven by selection for differences in body size and habitat preference in Lake Baikal amphipods.

<p>This project is undergoing the patent application, so it is confidential and should not be disclosed. Further questions can be asked by contacting Dr. Jeroen Goos, whose contact information was shown in the supervisor section.</p>

Obesity is a disease that increases risk of developing metabolic diseases including insulin resistance (IR), metabolic syndrome (MS), and type 2 diabetes (T2D). Adipose tissue expansion during obesity leads to immune cell infiltration, causing local inflammation and disruption of lipid homeostasis. There is an association between exposure to environmental chemicals, like p,p’-DDE, a metabolite of p,p’-DDT, and diagnosis of obesity, dyslipidemia, IR, and prevalence of MS and T2D. DDE accumulates in fatty tissues and has been shown to have immunomodulatory properties, affecting macrophage and T cell populations. Potential mechanisms were studied by which DDE could modulate adipocyte and immune cell function and facilitate an increased risk of obesity and immune dysregulation, potentially through cyclooxygenase-2 (COX-2). 3T3-L1 preadipocytes and J774A.1 macrophages were studied for the effects of DDE on adipogenesis and macrophage reactivity, respectively. 3T3-L1 cells were induced to differentiate to adipocytes using a sub-optimal differentiation cocktail with increasing concentrations of DDE (0.5uM-100uM). It was determined that DDE enhanced adipogenesis in a concentration dependent manner and the expression of adipogenic and lipogenic genes, indicating that DDE enhances adipogenesis. In J774A.1 cells, the ability of DDE or 10uM NS-398, a specific COX-2 inhibitor, to inhibit the production of the prostaglandins PGE2, PGD2, PGF2a, was assessed in vitro and in a cellree system. DDE or NS-398 followed by immune challenge reduced cellular PG secretion and reduced PG production in a cell free system, indicating that DDE may interfere with lipid mediator signaling. Additionally, DDE or NS-398 exposure altered gene expression in J774A.1 cells following M1 or M2 polarization stimulus. Lastly, male C57Bl mice were exposed to 2mg/kg DDE for 5 days and the macrophage population of the adipose stromal vascular fraction was analyzed by flow cytometry. Adipose from DDE treated animals contained approximately 40% F4/80+CD11b+ macrophages. These results indicate that DDE may alter the homeostasis of adipose tissue by both enhancing adipogenesis and altering the reactivity of the resident macrophage population in a manner that may contribute to adipose dysfunction. These data suggest a possible mechanism by which DDE exposure may contribute to adiposity and adipose tissue dysfunction commonly seen in metabolic disease.

Perennial warm-season grasses constitute the backbone of many forage production systems, whether for grazing or harvested feed. North American native plants, specifically grasses, forbs and legumes offer unique ecosystem benefits along with forage quality and digestibility that are unmatched by introduced species. The disparity in breeding and research focused on improvement of introduced species as opposed to native genera has led to inflated use of introduced species as forage types in lieu of native options, due to their unimproved nature. Eastern gamagrass [Tripsacum dactyloides (L.) L.] is proven to be a widely adapted, highly productive forage species in the southeast, Great Plains and northeast United States. A major limitation to more widespread use of eastern gamagrass is high seed dormancy, which leads to increased seed cost. Here, research used recurrent phenotypic selection breeding methods to reduce seed dormancy, with the ultimate goal of developing a population of individuals that produce non-dormant eastern gamagrass seed.

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.

No description available.

During apoptosis, changes occur in T-lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A2 (sPLA2). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was first applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: initial hydrolysis rate was elevated, total reaction product was increased four-fold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by substrate availability rather than enzyme adsorption. Fluorescence experiments identified three membrane alterations that might affect substrate access to the sPLA2 active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting increased lipid spacing. Second, laurdan detected increased solvation of the lower head group region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it was proposed that the apoptotic membranes become susceptible to sPLA2 through a reduction in lipid-neighbor interactions which facilitates migration of phospholipids into the enzyme active site. This proposal was then examined in T-lymphocytes treated with glucocorticoid, a more physiologically relevant apoptotic stimulant, using similar techniques. The following observations corresponded to induction of membrane susceptibility: increased merocyanine 540 intercalation; phosphatidylserine flip-flop, detected by annexin binding; and alterations in laurdan fluorescence properties. These observations implied a relationship among sPLA2 susceptibility, lipid spacing, and phosphatidylserine exposure. To clarify this relationship, additional assays were also performed using dibutyryl-cAMP to induce apoptosis, a drug reported to induce apoptosis in S49 cells without the typical translocation of phosphatidylserine. Our results indicated that in cells treated with dibutyryl-cAMP, the merocyanine 540 response and its correlation with sPLA2 susceptibility was similar to that observed with dex-treated samples. This suggests that the underlying mechanisms which promote sPLA2 hydrolysis lead to alterations that may be facilitated by but do not require phosphatidylserine exposure. Taken together, all of the results suggest that direct regulation of the biophysical microenvironment of the membrane is the mode of control of membrane susceptibility to the hydrolytic activity of sPLA2.

Penstemon is the largest genus in North America with more than 270 reported species. However, little is known about the genome size of this genus and how this information may be useful in selecting species in developing hybrids for landscape use. Using flow cytometry, we estimated the genome size of approximately 40% of the genus (117 specimens from 104 different species.) Genome sizes for the putative diploids ranged from 2C = 0.94 – 1.89 pg (1C = 462 – 924 Mbp) and the putative polyploids ranged from 2.57 – 6.54 pg (1C = 1,257 – 3,156 Mbp). Chromosome counts were compiled and compared with the flow cytometry results for the species within this publication. Ploidy within the genus ranged from diploid to dodecaploid. These data were compared and contrasted with the current taxonomy of Penstemon and previously published ITS and cpDNA phylogenetic work. Based on genome size, reassigning P. montanus, P. cardinalis, and P. uintahensis to the subgenus Penstemon and P. personatus to the subgenus Dasanthera, would better reflect the phylogeny of the genus. Both auto- and allo-polyploidization are plausible mechanisms for increasing ploidy within the genus. The diploid species within the subgenus Saccanthera contain on average 1.09 pg (1C = 532 Mbp); however, two species within this subgenus are tetraploid and octaploid. The DNA content of subgenus Penstemon exhibits high plasticity and spans a six-fold increase. Our study found flow cytometry to be useful in species identification and verification. This represents the first published work on the genome size of Penstemon. This research will aid in future DNA sequencing experiments and breeding programs.

Infertility is a problem that could be treated by using In Vitro Fertilization (IVF). This procedure starts by increasing estrogen levels and then retrieving oocytes. However, studies have showed an increased risk for venous thromboembolism (VTE) and lung embolism that is correlated with the rising levels of estrogen. Microvesicles, vesicles formed from the cell membrane, increases in number in connection with IVF and diseases such as thrombosis. These vesicles can transport proteins, one of them is fetuin-B. Fetuin-B plays a role in fertilizing the egg by inhibiting ovastacin, a protein that leads to hardening of zona pellucida. Phosphatidylserine, a component of the cell membrane with strong binding capacity to annexin V, has also been studied while investigating VTE. The aim of this project was to analyze the amount of fetuin-B, ovastacin and phosphatidylserine in women undergoing IVF. The study included 55 women where three blood samples were collected from them at different time points. The tests were analyzed using flow cytometry and fluorescent antibodies targeting the proteins in question. The results showed a significant increase in fetuin-B and ovastacin after the treatment where double positive microvesicles (fetuin-B+ ovastacin+) had the highest significant difference, a rise of 78% after the hormone increase. (Fetuin-B+ annexin V+) increased over time, 205% after increasing estrogen levels, while no difference was seen for (ovastacin+ annexin V+). This thesis suggests that fetuin-B is strongly related to fertility and coagulation in women undergoing IVF. The protein has also a noticeable relationship to ovastacin. Most importantly, (fetuin-B+ ovastacin+) was suggested to be a better marker for women’s fertility than the other analyzed parameters.

No description available.