No description available.

No description available.

Skeletal development is a tightly regulated homeostatic process that requires proper functioning of osteoblasts, the bone forming cells, and osteoclasts, the bone resorbing cells. Improper functioning of either of these cell types results in diseases such as osteoporosis, osteogenesis imperfecta as well as Paget's disease. Crucial for the proper maintenance of the skeleton is the bone matrix, which encompasses both organic and inorganic components. Osteopontin (Opn) is an example of a major non-collagenous protein present in bone. Its expression is crucial for bone remodeling since it functions in recruiting osteoclasts for bone resorption and facilitates their adhesion to the bone matrix. Osteopontin is expressed in variety of cells and functions in facilitating signal transduction upon engagement of integrin. Osteopontin binds to the αvß3 (vitronectin receptor) the major integrin expressed on osteoclasts thereby mediating cell adhesion and migration. As a model to study osteopontin-mediated adhesion we have employed commercially available osteopontin and the HEK 293 cells that stably overexpress the vitronectin receptor (Vnr cells). We studied the ability of the Vnr cells to adhere to different extracellular matrices including osteopontin. / Bioengineering

In order to initiate a genetic dissection of the Drosophila melanogaster larval response to light, two new single larva assays were designed: the Checker and ON/OFF assays. Each assay allows quantification of different aspects of the larval visual response by permitting the study of discrete behaviours in a single larva. Results of this study indicate that larvae respond to light by modulating their locomotion. In the Checker assay this can be seen as an increase in residence time spent in dark checks. In the ON/OFF assay this can be measured as a decrease in distance travelled during the light pulse, due at least in part to an increase in head swinging. Concomitantly, the larva exhibits a sharp change in direction from its original path when the lights are turned on. When the lights are turned off, the change in direction in the larval path, although smaller than at lights on, is still greater than in the absence of light transitions. Many of the components previously described to function in adult phototransduction and visual system specification, also have roles in the larval photoresponse as mutations in the genes that encode these components, are able to abolish light perception as measured in both the Checker and ON/OFF assays. However, these mutations disrupt only subsets of the behaviours associated with the larval perception of light, thus suggesting the existence of light detecting mechanism independent of the main visual pathway described for the adult visual system. / Thesis / Master of Science (MS)

Three issues were investigated; first, the influence of virus on pigment production in diseased plants; second, the increase of virus as indicated by a resulting change in carotene concentration; third, the function of pigments other than chlorophyll in photosynthesis in young potato leaves. Results on the first issue demonstrated an increase in carotene and xanthophyll concentration, and a decrease in chlorophyll concentration in diseased relative to normal plants. Evidence on the second issue suggested a close relationship between virus increase and carotene concentration. Data on the third issue indicated a soil and possibly significant photosynthesis due to leaf carotenoids / Thesis / Master of Science (MS)

Thesis advisor: Tim van Opijnen / Thesis advisor: Michelle Meyer / Although bacteria are often studied as planktonic or free-living organisms, they frequently grow in complex surface-attached communities known as biofilms. Biofilms are communities of microorganisms attached to surfaces and embedded in a self-produced extracellular matrix. Biofilms are dynamic structures analogous to human settlements shaped by space and environment. These microbial communities fulfill critical roles in multiple infections in the human body. Streptococcuspneumoniae is a human pathogen that can cause biofilm-associated infections in various tissues and organs. This thesis offers a unique outlook for the study of S. pneumoniae biofilms by combining in vitro, genome-wide, and in vivo experiments to elucidate the complex population dynamics of S. pneumoniae biofilms. Existing methods to cultivate S. pneumoniae biofilms fail to fully capture the complexity of these communities, and most studies are limited to short periods of time. We developed a robust in vitro assay to grow S. pneumoniae biofilms. This assay can be maintained forever rather than days. We then use this robust assay to study their behavior in vivo and monitor disease outcomes. After establishing clear differences in biofilm and dispersal samples, we monitor population dynamics using genome-wide techniques (Tn-seq, RNA-seq and WGS) to provide some insights into this complex mode of growth. This work includes the first global identification of genetic requirements during biofilm establishment in two different S. pneumoniae strains using Tn-Seq. Coupled with our transcriptomic analysis, we found that genes involved in multiple pathways, such as capsule biosynthesis, nucleotide metabolism, and stress response, contributed to biofilm growth. Lastly, we studied the development of antibiotic resistance to three different types of antibiotics under S. pneumoniae biofilm conditions. We revealed common adaptive pathways to achieve biofilm growth and antibiotic resistance (antibiotic target genes), as well as novel routes of adaptation to develop resistance. Our findings add to the growing body of knowledge in the field of bacterial genetics and antimicrobial resistance, paving the way for future research and therapeutic advancement. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Managing turf for high aesthetic value is costly. Such management usually involves mowing, disease prevention, insect control, and weed control. Mowing is the most expensive practice on golf courses and annual bluegrass (Poa annua L) is the most challenging weed problem in professional turf. The plant growth regulators trinexapac-ethyl and paclobutrazol are commonly used in VA for these two costly and challenging jobs. Prohexadione calcium (PC) is an experimental chemical that inhibits the same enzyme (3ß-hydroxyalase) as trinexapac-ethyl and may selectively suppress annual bluegrass. Experiments were conducted at the Virginia Tech Turfgrass Research Center and Glade Road Research Facility to determine the PC rate required to reduce clipping biomass of four turfgrass species as effectively as trinexapac-ethyl. Prohexadione calcium reduced clipping biomass of bermudagrass (Cynodon dactylon (L.) Pers.), Kentucky bluegrass (Poa pratenis L.), perennial ryegrass (Lolium perenne L.), and zoysiagrass (Zoysia japonica Steud.) equivalent to trinexapac-ethyl at 0.70, 0.22, 0.60, and 0.27 kg a.i./ha -1, respectively. Further experiments conducted at three locations across Virginia determined that PC was comparable to paclobutrazol for annual bluegrass suppression. Since turfgrass response to PC was different between annual bluegrass, Kentucky bluegrass, and perennial ryegrass, 14C labeled PC was used to assess absorption, translocation, and metabolism of PC between annual and Kentucky bluegrass, creeping bentgrass (Agrostis stolonifera L.), and perennial ryegrass. Annual and Kentucky bluegrass absorbed more PC than creeping bentgrass or perennial ryegrass and partially explained the selectivity between these species. Translocation and metabolism of PC did not differ between species. Our final objective launched experiments characterizing possible resistance to acetolactate synthase (ALS) inhibiting herbicides in annual bluegrass. Several selective herbicides for annual bluegrass control inhibit ALS. Since many weeds have developed resistance to ALS-inhibiting herbicides, the ALS gene in annual bluegrass was sequenced and derived amino acid sequences were at least 87% similar to other previously sequenced grass species. This sequencing data will be used in future experiments to predict the likelihood of ALS resistance in annual bluegrass. / Ph. D.

In metabolic engineering of prokaryotes, combinatorial approaches have developed recently that induce random genetic perturbations to achieve a desired cell phenotype. A screening strategy follows the randomized genetic manipulations to select strain(s) with the more optimal phenotype of interest. This screening strategy is often divided into two categories: (i) a growth competition assay and (ii) selection by high-throughput screening. The growth competition assay involves culturing strains together. The strain with the highest growth rate will ultimately dominate the culture. This strategy is ideal for selecting strain with cellular fitness (e.g., solvent tolerance), but it does not work for selecting a strain that can over-produce a product (e.g., an amino acid). For the case of selecting highly productive phenotypes, high-throughput screening is used. This method analyzes strains individually and is costly and time-consuming. In this research, a synthetic genetic circuit was developed to select highly productive phenotypes using a growth competition assay rather than high-throughput screening. This novel system is called Feed-back Inhibition of Transcription for Growth Selection (FITSelect), and it uses a natural feedback inhibition mechanism in the L-arginine production pathway to select strains (transformed with a random genomic library) that can over-produce L-arginine in E. coli DH10B. With FITSelect, the cell can thrive in the growth competition assay when L-arginine is over-produced (i.e., growth is tied to L-arginine production). Cell death or reduced growth results if L-arginine is not over-produced by the cell. This system was created by including an L-arginine concentration responsive argF promoter to control a ccdB cell death gene in the FITSelect system. The effects of ccdB were modulated by the antidote ccdA gene under control of an L-tryptophan responsive trp promoter. Several insights and construction strategies were required to build a system that ties the growth rate of the cell to L-arginine concentrations. / Master of Science

Measurement of soil phytase activity (PA) and delineation of the impact of this important phosphomonoesterase on the P-cycling process in soil and sediments suffer from the lack of a reliable assay. A method for measuring PA in soil that promises to be accurate and reliable has been recently published. The method involves the use of a novel chromophoric analog of phytic acid, referred to as T(tethered)InsP5 (5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate). This study was conducted to measure PA in a Clymer forest soil, which contained over twice the amount of soil organic C as previously tested soils, using the TInsP5 PA assay. This investigation specifically addresses: (1) the development of a soil dilution technique for determining maximal PA, (2) identification of previously unsubstantiated soil-produced dephosphorylated intermediate probe species, (3) the impact of increasing assay buffer pH on soil PA and (4) testing stability of the probe's amide bond in a highly (bio)active forest soil. PA assays were conducted by measuring dephosphorylation of TInsP5 in citrate-acetate buffered (pH 4.2) active and autoclaved (Control) soil suspensions. Phosphorylated probe intermediates (i.e., TInsP4, TInsP3, TInsP2 and TInsP1) and T-myo-inositol were extracted from samples of soil suspension following incubation. Probe species were quantified using reversed phase high-performance liquid chromatography (RPHPLC) with UV detection. PA was calculated based on a mass balance approach. A soil dilution technique was developed to address the challenge of determining maximal PA in soils containing higher organic matter content. In the initial report on use of the TInsP5 method for measuring PA in soil, two "soil-generated" UV-adsorbing compounds (designated Y and Z) were observed, but never confirmed as probe species. The experimental evidence presented in this report supports inclusion of compound Y as a phosphorylated probe intermediate species (i.e. TInsPy), based primarily on its UV adsorption spectra (diode-array detection analysis). Compound Z could not be substantiated as a probe species based on the evidence presented in this study. PA of Claymer forest soil decreased with an increase in assay buffer pH. Further, the probe's amide bond linkage was stable in a forest soil exhibiting high PA. / Master of Science

Macrophoma rot is a general term for fruit rots of Vitis spp. caused by the fungus Neofusicoccum ribis (syn. Botryosphaeria ribis) or closely related or renamed taxa, including Botryosphaeria dothidea. While mainly observed as a fruit pathogen of muscadine grape, the disease has recently been observed on bunch grapes in Virginia. Isolates (N = 835) were collected from Petit Manseng fruit clusters from seven Virginia vineyards in 2018 and 2019. A subset of these isolates was sequenced using three primer sets (ITS, RPB2, and EF). The preliminary result showed a single taxonomic strain of N. ribis. A controlled inoculation study of Petit Manseng clusters verified that infection could occur anytime between bloom and 2 weeks post-veraison; however, both the mean cluster incidence and the severity of Macrophoma rot did not differ from each other at any growth stage during the season. A season-long cluster exposure experiment showed that any amount of sun exposure significantly increased Macrophoma rot severity compared to shaded clusters, and that full sun exposure was associated with greatest rot severity. This finding contravenes current management recommendations for Macrophoma rot, and it raises yet unanswered questions as to why exposed clusters are more susceptible to Macrophoma rot than are shaded clusters. An in vitro fungicide assay study using nine fungicides identified captan, thiophanate-methyl, and tetraconazole as potential candidates for management of Macrophoma rot which need to be investigated further. / Master of Science in Life Sciences / Macrophoma rot is a general term for fruit rots of grapes caused by the pathogenic fungi in the family Botryosphaeriaceae. The rot is mainly observed on Muscadine grapes, but recently more cases were found on a wine grape cultivar Petit Manseng in Virginia. Macrophoma rot symptoms begin as dark brown, circular lesions on the surface of the berry and look similar to sunburn and other fruit rots. As the disease progresses, the lesion envelopes the entire berry and black fruiting bodies develop. Severe cases may lead to crop loss. The same group of pathogens is also associated with rots on other crops including apple, pear, olive, and kiwis. Very little is known about the disease cycle and the control of Macrophoma rot, therefore, an investigation into this fungal pathogen was needed. Multiple studies with the wine grape variety Petit Manseng were conducted during the 2018-2019 growing seasons, including a survey, leaf removal trial, and an inoculation study. Results showed that a species called Neofusicoccum ribis was found in vineyards across northern and central Virginia based on the genetic identification of fungal isolates collected at seven vineyards in those areas. Macrophoma symptoms were observed to be more prevalent and severe in more exposed clusters based on a leaf removal experiment. An artificial inoculation experiment revealed that grape clusters are susceptible to Neofusicoccum ribis at any time during the season. Based on the screening of nine fungicides, three chemicals (captan, thiophanate-methyl, and tetraconazole) showed promising results as possible management tools for Macrophoma rot. The knowledge collected will lead to an increase in understanding of this fungal pathogen and to further studies to manage Macrophoma rot.