Studying Key Amino Acids in the Function of Ras Interference 1 (Rinl) SH2 and Vps9 Domains

Receptor mediated endocytosis effectively removes the "ears" with which a cell would "hear" a signal conveyed by extracellular signaling molecules, but does not necessarily block the signaling pathway in which the endocytosed receptor participates. In the process of signal attenuation, this newly formed vesicle is fused with a phagosome and the receptor molecules are degraded. Receptor mediated endocytosis as a way to attenuate epidermal growth factor (EGF) and insulin signaling will be the focus here. Ras Interference 1 (Rin 1) is a multifunctional protein involved in intracellular membrane trafficking and receptor mediated endocytosis through its Rab5 Guanine Exchange Factor and SH2 domains. The goal of this investigation is to determine the role of key amino acids involved in the interaction of Rinl with Epidermal Growth Factor Receptor and Rab5. To elucidate this role, a number of point mutations have been created and the effects of each mutation on Rin 1 function will be investigated. Key amino acids in the SH2 and Vps9 Domain were identified and effects of mutations on rate of endocytosis were observed.

The Role of Ras Oncogene in Differentiation of 3T3-L1 Preadipocytes

Ras is a proto-oncogene that codes for a small GTPase and is responsible for linking several extracellular signals to intracellular mechanisms that involve cell growth, differentiation and cell-programmed death in normal and diseased cells. In all these processes, Ras has been extensively investigated. However, the role of Ras GTPases is still poorly understood during the differentiation of 3T3-L1 preadipocytes. In this study I investigated the role of the H-Ras defective mutant, Ras:G12V on the differentiation of 3T3-L1 preadipocytes. Preadipocytes were differentiated in vitro to adipocytes (fat cells) by adding an induction medium containing several factors including glucose and insulin. The formation of fat cells evidenced by the visualization of lipid drops as well as by quantifying the accumulation of Oil red O into lipid drops. To examine the role of Ras:G12V mutant, several selective mutations were introduced in order to determine the signaling transduction pathways (i.e., PI3(K)kinase and MAP(K)Kinase) responsible for the Ras-dependent adipogenesis. Cells expressing Ras:G12V mutant stimulated 3T3-L1 preadipocyte differentiation without he need for induction media, suggesting that Ras activation is an essential factor required for 3T3-L1 preadipocyte differentiation. Introduction of a second mutation on Ras:G12V (i.e., Ras:G12V;E37G), which blocks the activation of the MAPKinase pathway, strongly inhibited the 3T3-L1 preadipocyte differentiation. It is also important to note Ras:G12V:E37G double mutant does not inhibit the activation of the PI3kinase pathway. Other Ras double mutants (Ras:G12V;S35T, and V12G;C40Y) showed a modest inhibition of the 3T3-L1 preadipocyte differentiation. Taken together, these observations indicate that Ras plays a selective role in 3T3-L1 preadipocyte differentiation. Thus, understanding which specific pathway Ras employs during preadipocyte differentiation could clarify some of the uncertainties surrounding fat production.

An Analytical Workflow for Metagenomic Data and its Application to the Study of Chronic Obstructive Pulmonary Disease (COPD)

Metagenomics is the culture-independent study of genetic material obtained directly from environmental samples. It has become a realistic approach to understanding microbial communities thanks to advances in high-throughput DNA sequencing technologies over the past decade. Current research has shown that different sites of the human body house varied bacterial communities. There is a strong correlation between an individual’s microbial community profile at a given site and disease. Metagenomics is being applied more often as a means of comparing microbial profiles in biomedical studies. The analysis of the data collected using metagenomics can be quite challenging and there exist a plethora of tools for interpreting the results. An automatic analytical workflow for metagenomic analyses has been implemented and tested using synthetic datasets of varying quality. It is able to accurately classify bacteria by taxa and correctly estimate the richness and diversity of each set. The workflow was then applied to the study of the airways microbiome in Chronic Obstructive Pulmonary Disease (COPD). COPD is a progressive lung disease resulting in narrowing of the airways and restricted airflow. Despite being the third leading cause of death in the United States, little is known about the differences in the lung microbial community profiles of healthy individuals and COPD patients. Bronchoalveolar lavage (BAL) samples were collected from COPD patients, active or ex-smokers, and never smokers and sequenced by 454 pyrosequencing. A total of 56 individuals were recruited for the study. Substantial colonization of the lungs was found in all subjects and differentially abundant genera in each group were identified. These discoveries are promising and may further our understanding of how the structure of the lung microbiome is modified as COPD progresses. It is also anticipated that the results will eventually lead to improved treatments for COPD.

The Application of Fluorescent Nanocrystals to Study the Distribution of Neuropeptides in the Aedes Aegypti Mosquito

Semiconductor nanocrystals, also known as quantum dots (QDs), have been used in studies involving mice and human tissues, but never before in research on insects. We used QDs to study the distribution of two neuropeptides in the Aedes aegypti mosquito, the vector of both dengue and yellow fever. These neuropeptides play a significant role in the production of juvenile hormone, a hormone that controls biting behavior, metamorphosis, and reproduction throughout the life of the mosquito. The two neuropeptides allatostatin-C (AS-C) and allatotropin (AT) function as inhibitory (AS-C) and stimulatory (AT) regulators of juvenile hormone synthesis in the corpus allatum gland. In other insects, they also affect heart rate, gut movement, and nutrient uptake. Conjugating these neuropeptides to quantum dots via a streptavidinlbiotin link, we were able to expose the mosquito corpus allatum and abdomen to allatostatin-C and allatotropin and then to visualize their distribution under UV light using confocal and compound light microscopy. Histological sections of the whole mosquito, incubations of tissues with conjugates (in vitro), and microinjections of conjugates into the mosquito (in vivo) were performed. The results showed that quantum dots can be used to detect neuropeptide distribution in the mosquito. The more we understand about these neuropeptides and juvenile hormone, the more we can contribute to stopping the spread of infectious diseases, such as dengue and yellow fever.

The Role of PP2A Regulatory Subunit B56 in Dictyostelium Discoideum Motility

Protein Phosphatase 2A, PP2A, is a heterotrimeric threonine/serine phosphatase system that is involved in a variety of cellular processes. This phosphatase is composed ofthree subunits: a catalytic subunit (C subunit), a scaffolding subunit (A subunit), and a regulatory subunit (B subunit). The regulatory subunit B is divided into four subclasses, B, B' (B56), B'' and B'' '. Studies showed that PP2A/B56 complexes regulate development of Dictyostelium and other metazoan cells. In addition to development, our experimental data suggest that PP2A/B56 complex also plays an important role in Dictyostelium cell motility. Cells lacking B56 was generated previously in our laboratory (Lee et al., 2008). Further studies showed that b56- cells are compromised in random cell motility compared to the wild type (AX3) cells. In contrast, b56 cells with re-introduced B56 displayed wild-type like motilities. Furthermore, one of the colleagues in our laboratory found that one of the Dictyostelium Ras species, RasG, associates with PP2A/B56 complex and RasG activation is compromised in b56- cells. Considering that Ras proteins are central in cellular motility regulation, PP2A/B56 complex may modulate cell motility through regulating Ras. We propose to determine if an introduction of constitutive active RasG proteins improves compromised b56- cell motility.

The Effect of Natural Products and Small Gtpases in Adipogenesis

Insulin signaling is one of the main initiators of adipogenesis, the conversion from pre-adipocyte to adipocyte or lipid droplet. Rab proteins are the master regulator of intracellular trafficking and endosome fusion in endocytosis, making them potential regulators of insulin signaling in adipogenesis. Pre-adipocytes 3T3-Ll cells expressing several Rab5 constructs were used to examine the effect of dehydroleucodine (DhL ), a sesquiterpene lactone isolated from aerial parts of Artemisia douglasiana Besser. The results obtained identify Rab5 deactivation as a key step for adipogenesis by forming signaling endosomes. The addition of DhL significantly inhibited the lipid droplet accumulation in a dose-dependent manner and dramatically attenuated the synthesis of adipogenic transcriptional factors, C/EBPa and PPARy. Activation of AMPKa, Erk and Akt during adipocytic differentiation was not inhibited by treatment with DhL. This data suggest that DhL has an important role in Rab5 dependent adipogenesis by regulating several transcriptional factors including PP ARy expression, which is known to play an essential role during fat formation.

Diving Behaviour of Red-Eared Sliders (Trachemys Scripta Elegans) Under the Risk of Predation

Predation risk influences a variety of behavioral decisions of many organisms and results in animals having to trade-offs safety with other behaviors. The effects of predation, however, have been largely ignored in the study of vertebrates that forage underwater (divers). I tested the predictions of an on optimal diving model that incorporates the risk of predation, using red eared slider turtles (Trachemys scripta elegans). Specifically, I tested the hypothesis that divers will increase their surface time when instantaneous risk decreases with time at the surface. By using a model aerial predator and exposing turtles to both risk and no risk treatments, I tested how turtles perceive risk at the surface and whether they increase or decrease their surface time depending on how they assess risk. The model's predictions for situations in which risk at the surface is decreasing with time spent there-likely to be the case for aerial predation-were supported by the results. I found that surface time and time spent submerged per dive were significantly greater when turtles were at risk and that turtles also spent more time resting at the bottom when exposed to this treatment. Interestingly, turtles under risk engaged in vigilance behaviors while on the bottom just prior to surfacing. This behavior could have implications for model predictions and future experiments are needed to test whether subsurface vigilance may alter diving decisions made under risk.

Cardiac Neural Crest Cells Affect the Development of the Myocardium and Conduction System in the Mouse

Neural crest cells originate from the dorsal most region of the embryonic neural tube. These cells migrate into several embryonic locations and differentiate into a variety of cell types. Cardiac neural crest (CNC) cells are a set of neural crest progenitors that aid in the proper formation of the cardiac septum, which separates the pulmonary from the systemic circulation. We have used Splotch mice to investigate whether the murine CNC cells play a role during the development oft he myocardium and the conduction system. Splotch mice carry a mutation in the P AX3 transcription factor, and display a problem in CNC cell migration. A scanning-electron-microscopy analysis of Splotch mutant-embryonic-hearts reveals abnormalities in the interventricular septum. In addition, the right and left ventricular cavities appear dilated relative to a wild type heart. Hoechst nuclei staining of Splotch heart cryosections demonstrates a decreased number of cardiomyocytes and a corresponding thinner ventricular wall. The absence of Connexin 40 in the ventricles of Splotch mutants, suggests conduction system defects. These results support the evidence that CNC cell signaling plays a role in modulating the growth and development of murine cardiomyocytes and their differentiation into conductile cells.

The Expression of Rap6 in the Adult Mouse Brain and Early Mouse Embryonic Development

The previously identified RAP6 (Rab5 activating protein 6) was associated with plasma membrane mediated endocytosis and contains a Rab5 guanine nucleotide exchange factor (GEF) domain. RAP6 has been shown to act a Ras activating protein (GAP) domain. The identification of RAP6 and its crucial role in both receptors mediated endocytosis and fluid phase endocytosis presents the opportunity to investigate its role in murine embryonic development and in the adult brain. To confirm and characterize the presence of RAP6 during embryonic development and in the adult brain, the current study examined the expression of both the RGD and the Vps9 domains of RAP6 through in situ hybridization. We present an extensive evaluation of the expression for both RAP6 domains through in situ hybridization of 12.5 and 14.5 weeks old C67 mouse embryos and adult C67 mouse brain. The current study confirms the presence of both RAP6 domains and presents an extensive evaluation its expression in embryonic development and the adult brain. These data together support the role of RAP6 in receptor mediated endocytosis and fluid phase endocytosis relevant active during murine embryonic development and adult brain processes.

Understanding the Effect of Protein Tyrosine Phosphatase Receptor Type 0 on Mammalian Sensory Development: Behavioral Studies on PTPRO Knockout Mice

Nerve development, which includes axon outgrowth and guidance, is regulated by many protein families, including receptor protein tyrosine phosphatases (RPTP's).Protein tyrosine phosphatase receptor type 0 (PTPRO) is a type III RPTP that is important for axon growth and guidance, as observed in chicks and flies. In order to examine the effects ofPTPRO on mammalian development, standard behavioral tests were used to compare mice lacking the gene for PTPRO (ROKO mice) to wild-type (WT) mice. The ROKO mice showed a significant delay in reacting to a thermal noxious stimulus, hotplate analgesia, when compared to the WT mice suggesting deficient nociceptive function. In a rotarod test for proprioceptive function the ROKO mice exhibited a significant decrease in the amount of time spent on the rotating rod than did the WT mice. Additional proprioception tests were performed including the climb, step reflex, beam, and mesh walk tests. In the climb and step (place) test, the ROKO group had a significantly lower accuracy in performing the tests than did the WT mice. Thus, mice lacking the PTPRO gene showed behavioral deficiencies that reflect impairment in sensory function, specifically for nociception and proprioception.

Tracking the Effects of Salt Water Encroachment on South Florida Coastal Ecotones Using Mollusks

Many coastal wetland communities of south Florida have been cut off from freshwater sheet flow for decades and are migrating landward due to salt-water encroachment. A paleoecological study using mollusks was conducted to assess the rates and effects of salt-water encroachment due to freshwater diversion and sea level rise on coastal wetland basins in Biscayne National Park. Modem mollusk distributions taken from 226 surface sites were used to determine local habitat affinities which were applied to infer past environments from mollusk distributions found in soil cores. Mollusks species compositions were found to be strongly correlated to habitat and salinity, providing reliable predictions. Wetland soils were cored to bedrock at 36locations. Mollusks were abundant throughout the cores and 15 of the 20 most abundant taxa served as bioindicators of salinity and habitat. Historic accounts coupled with mollusk based inference models indicate (1) increasing salinity levels along the coast and encroaching into the interior with mangroves communities currently migrating westward, (2) replacement of a mixed graminoid-mangrove zone by a dense monoculture of dwarf mangroves, and (3) a confinement of freshwater and freshwater graminoid marsh to landward areas between urban developments and drainage canals.

The Relationship of Heteroptera Species Richness, Abundance, and Morphology to Elevation Gradients and Land-use Regimes on Mt. Kilimanjaro

Insect biodiversity is unevenly distributed on local, regional, and global scales. Elevation is a key factor in the uneven distribution of insect diversity, serving as a proxy for a host of environmental variables. My study examines the relationship of Heteroptera (true bugs) species diversity, abundance, and morphology to elevational gradients and land-use regimes on Mt. Kilimanjaro, Tanzania, East Africa. Heteroptera specimens were collected from 60 research sites covering an elevational range of 3684m (866-4550m above sea level). Thirty of the sites were classified as natural, while the remaining 30 were classified as disturbed (e.g., agricultural use or converted to grasslands). I measured aspects of the body size of adult specimens and recorded their location of origin. I used regression models to analyze the relationships of Heteroptera species richness, abundance, and body measurements to elevation and land-use regime. Richness and abundance declined with greater elevation, controlling for land use. The declines were linear or logarithmic in form, depending on the model. Richness and abundance were greater in natural than disturbed sites, controlling for elevation. According to an interaction, richness decreased more in natural than disturbed sites with rising elevation. Body length increased as a quadratic function of elevation, adjusting for land use. Body width X length decreased as a logarithmic function of elevation, while leg length/body length decreased as a quadratic function. Leg length/body length was greater in disturbed than natural sites. Interactions indicated that body length and body width X length were greater in natural than disturbed sites as elevation rose, although the general trend was downward. Future research should examine the relative importance of land area, temperature, and resource constraints for Heteroptera diversity and morphology on Mt. Kilimanjaro.