There are approximately 600,000 cases of delayed or aberrant fracture healing in people each year, with a small subset of these fractures experiencing disunion. Dietary phosphate deficiency has been shown to impair oxidative phosphorylation and decrease BMP-2 mediated chondrogenic differentiation during fracture healing. Prior studies using pre-committed chondro-progenitor ATDC5 cell line grown in phosphate deficient media showed that energy consumption was linked to protein production and collagen hydroxylation but inversely related to matrix mineralization. The goal of this study was to further define the relationship between energy consumption and BMP-2 mediated stem cell chondrogenic differentiation and further examine how dietary phosphate, and promotion of collagen hydroxylation via ascorbate availability effected these processes. C3H10T1/2 murine cells, a multi-potential cell line, were expanded in pre-differentiation growth medium (DMEM with 10% FBS and 1% Pen/Strep). Once cells reached 60% confluence (day 0), they were grown in differentiating media (α-MEM with 5% FBS and 1X insulin-transferrin-selenium) containing either 100% (1mM) or 25% (0.25mM) inorganic phosphate (Pi), ± 200ng/mL BMP-2(BMP), and ±0.2 mM L-ascorbic acid (AA). In total, there were 8 groups with varying combinations of these three substances. Intracellular lipid, total DNA, protein, and hydroxyproline (HP) content were examined. Chondrocyte gene expression (Col2a1, Acan, ColXa1) and adipocyte gene expression (Pparg, Plin1, Ucp1) were measured to check for cell lineage commitment and specific differentiation of the C3H10T1/2. All measurements were acquired at day 8. The +BMP differentiation media groups contained significantly less DNA content and more protein content than the –BMP differentiation media groups (both p<0.0001). There was also a significant interaction between phosphate and ascorbic acid treatment (p=0.0296), with 25% Pi +AA groups producing significantly more protein than 100% Pi +AA groups. Hydroxyproline production was not different in 100% Pi or 25% Pi conditions (p=0.2951). AA presence in culture media led to greater HP production than culture media lacking AA (p=0.0035) There was a trend of an interaction between phosphate content and AA availability (p=0.0744). 100% Pi ±AA groups produced significantly different amounts of HP while 25% Pi ±AA groups did not produce significantly different amount of HP. Col2a1, Acan, and ColXa1 expression were all increased in +BMP groups. Ascorbic acid treatment groups expressed significantly more Col2a1and Acan than –AA groups. 100% Pi media led to greater Acan expression over 25% Pi groups (p=0.0009), whereas 25% Pi media trended to lead to greater ColXa1 expression over 100% Pi groups (p=0.0734). Pparg and Plin1 expression were increased in the 25% Pi condition. There were no significant differences in expression of Ucp1. C3H10T1/2 cells were significantly affected by phosphate concentration, BMP-2 treatment, and ascorbic acid supplementation. Phosphate deficiency hindered maturation of early chondrocytes into proliferating chondrocytes while also promoting MSC differentiation into the adipocyte cell lineage. Hypertrophic chondrocyte expression was decreased in phosphate deficient media, which may coincide with increased protein production observed in low phosphate conditions. BMP-2 promoted chondrogenesis which resulted in increased protein production. Whereas, lack of ascorbic acid in cell culture media led to decreased hydroxyproline production.

Tobacco SABP2, a 29. kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-. S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance.

ACP interacts with diverse proteins in an unknown way. Possibly there is a similar mode of interaction between ACP and all ACP-binding proteins, the amphiphilic helix. The hydrophobicities of helices from 4 different ACPs were compared. Hydrophobic moment plots were prepared for ACP helices and those of many EF hand calcium-binding proteins. Both groups of proteins occupied the same region of the plot.

Tobacco SABP2 has been shown to display high affinity for salicylic acid (SA) and methylsalicylate (MeSA) and plays an important role in SAR signal development. Using biochemical approach, SABP2 has been shown to demonstrate strong esterase activity in converting MeSA to SA. Recent study shows that tetra fluoroacetophenone, a synthetic analog of SA, competitively inhibits SABP2 esterase activity as well as suppresses SAR signal development in tobacco mosaic virus (TMV)-infected tobacco plants. Not much has been studied on the effect of pesticides on plant defenses. Because both AChE and SABP2 are esterase-like proteins belonging to α/β hydroxylase superfamily, we hypothesize that pesticides may inhibit the MeSA esterase activity of SABP2 and block SAR development. Biochemical and molecular biology techniques were used to test this hypothesis. SAR in tobacco-TMV plant-pathogen system is measured by significant decrease in TMV-induced lesion sizes in secondarily inoculated distal leaves.

Over the past 5 years, the use of titanium implants as temporary anchorage devices (TADs) has become an important tool in clinical orthodontic practices. The use of TADs have provided orthodontists a way of moving teeth against fixed objects rather than against the surrounding teeth, which tend to counteract desired motion. At present, viable attachment of TADs involves direct insertion through gingival tissue and piercing of the bone. Surface modifications such as sandblasted and acid-etched treatment or bone morphogenetic protein surface treatment, however, can be applied to the TADs to promote enhanced osseointegration, thereby allowing the TADs to serve as stable anchors while avoiding bone puncture. In this study, a comparison was made between sandblasted/acid-etched TADs and sandblasted/acid-etched/recombinant human bone morphogenetic protein-2 (rhBMP-2) treated TADs to determine whether rhBMP-2 promotes enhanced osseointegration. A total of 10 rats (4 controls and 6 treated with rhBMP-2) were used in the study, with 1 TAD placed on the skull of each rat. At the end of 6 weeks, the animals were euthanized by carbon dioxide asphyxiation, and bone blocks, each containing a TAD, were prepared for histological examination and biomechanical characterization. The results of this study showed that TADs treated with rhBMP-2 had greater bone formation at the bone-implant interface and an increase in total implant stability.

Although cytoplasmic lipid droplets (LDs) are the major reserves for energy-dense neutral lipids in plants, the cellular mechanisms for packaging neutral lipids into LDs remain poorly understood. To gain insights into the cellular processes of neutral lipid accumulation and compartmentalization, a necessary step forward would be to characterize functional roles of lipogenic proteins that participate in the compartmentalization of neutral lipids in plant cells. In this study, the lipogenic proteins, Arabidopsis thaliana SEIPIN homologues and mouse (Mus Musculus) fat storage-inducing transmembrane protein 2 (FIT2), were characterized for their functional roles in the biogenesis of cytoplasmic LDs in various plant tissues. Both Arabidopsis SEIPINs and mouse FIT2 supported the accumulation of neutral lipids and cytoplasmic LDs in plants. The three Arabidopsis SEIPIN isoforms play distinct roles in compartmentalizing neutral lipids by enhancing the numbers and sizes of LDs in various plant tissues and developmental stages. Further, the potential applications of Arabidopsis SEIPINs and mouse FIT2 in engineering neutral lipids and terpenes in plant vegetative tissues were evaluated by co-expressing these and other lipogenic proteins in Nicotiana benthamiana leaves. Arabidopsis SEIPINs and mouse FIT2 represent effective tools that may complement ongoing strategies to enhance the accumulation of desired neutral lipids and terpenes in plant vegetative tissues. Collectively, our findings in this study expand our knowledge of the broader cellular mechanisms of LD biogenesis that are partially conserved in eukaryotes and distinct in plants and suggest novel targets that can be introduced into plants to collaborate with other factors in lipid metabolism and elevate oil content in plant tissues.

Master of Science / Department of Clinical Sciences / David E. Anderson / Drug delivery systems for time release of recombinant human bone morphogenetic protein-2 (rhBMP-2) and antibiotics in orthopedic surgeries continue to be developed. Recently, a biodegradable novel polymeric matrix has been developed for this purpose. We hypothesized that impregnation of the matrix with rhBMP-2 would enhance bone healing. The objectives of the study were to characterize elution of rhBMP-2 and two antimicrobials (tigecycline, tobramycin) from the matrix, and bone response to the matrix in the presence or absence of rhBMP-2 and antimicrobials. In vitro elution of tigecycline, tobramycin, and rhBMP-2 from the matrix was investigated. Drug concentration in media were measured on days 1-6, 8, 10, 13, 15, 17, 21, 25, 28, and 30 using high pressure liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS; antimicrobials) and ELISA (rhBMP-2). In vivo testing was done using a unicortical defect created into each tibia of twenty adult goats. Animals were randomly assigned to one of 5 groups: 1) control (untreated defect); 2) matrix; 3) matrix+ antimicrobials (tigecycline+tobramycin); 4) matrix+rhBMP-2; and 5) matrix+antimicrobials+rhBMP-2. Plasma concentration of tigecycline and tobramycin and serum concentration of rhBMP-2 were measured by the above techniques on days 1-7, 9, 11, 13, 15, 17, 22, 26, and 30. Bone response was assessed on days 0, 14, and 30 using radiographic scoring and dual energy X-ray absorptiometry (bone mineral density [BMD]). After euthanasia on day 30, histomorphologic analyses of the bone defects were done. Categorical variables were analyzed using a generalized linear model, and continuous variables using an ANOVA with P < 0.05 considered significant. In vitro elution was characterized by a rapid release on day 1 followed by a slow release until day 30 for both antimicrobials and rhBMP-2. Plasma antimicrobial concentrations showed continued release throughout the study period. Serum rhBMP-2 concentration, radiographic scores and BMD were not significantly different between groups. Periosteal and endosteal reaction surface areas were significantly greater surrounding the defects in group 4 (matrix+rhBMP-2). There was no significant difference between the groups for the percent of bone filling the defect. The matrix served as an appropriate antimicrobial and rhBMP-2 delivery system and successfully stimulated bone production when rhBMP-2 was present.

Multidrug resistance protein 2 (MRP2) is the second member the C subfamily in the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) efflux transporters. MRP2 is a critical player for generation of bile acidindependent bile flow and biliary excretion of glutathione, glucuronate and sulfate conjugates of endo- and xenobiotics. Dysfunctional expression of MRP2 is associated with Dubin-Johnson Syndrome. Pathological and physiological states or xenobiotics change the MRP2 expression level. Under some conditions, expression of the human MRP2 and rat Mrp2 proteins are regulated at the translation level. There are several transcription initiation sites in MRP2/Mrp2 gene. The 5’ untranslated regions (5’UTRs) of MRP2/Mrp2 contains multiple translation start codons. The focus of this study, therefore, was investigation of the translational regulatory mechanisms mediated by the upstream open reading frames (uORF) of MRP2/Mrp2. Using in vitro translation assays and transient cotransfection assays in HepG2 cells, we showed that the rat uORF1 starting at position -109 (relative to the ATG of Mrp2) and the human uORF2 starting at position -105 (relative to the ATG of MRP2) are two major cis-acting inhibitors of translation among the rat and human multiple uORFs, respectively. Translational regulation mediated by the uORFs in the rat Mrp2 mRNA is a combined effect of the leaky scanning model and the reinitiation model, and also results from interaction of the multiple uORFs. In addition, by Ribonuclease Protection Assays (RPA), we detected multiple transcription initiation sites of MRP2/Mrp2 gene in tissues. We also found that the relative abundance of the rat Mrp2 mRNA isoforms with different 5’UTRs differed in the rat liver, kidney, jejunum, ileum, placenta, and lung. This is the first study on the translational regulatory mechanisms of the MRP2/Mrp2 gene.

Adipositas und Typ-2-Diabetes sind häufige Erkrankungen des Stoffwechsels. Zur Basistherapie der Adipositas und des Typ-2-Diabetes gehören eine gesunde Ernährungs- weise und die Erhöhung der körperlichen Aktivität unter anderem mit dem Ziel der Gewichtsreduktion. Vermehrte Bewegung führt neben der Verbesserung der körperlichen Leistungsfähigkeit zur Fettmassenreduktion, Verbesserungen der Hyperglykämie, Lipo- proteinstoffwechsels und des Adipokinprofils. Bone morphogenetic proteins (BMPs) werden im Fettgewebe produziert und spielen eine wichtige Rolle in der Adipogenese und Transdifferenzierung von Adipozyten. Während ein Zusammenhang zwischen der BMP-7-Serumkonzentration und Adipositas vor kurzem belegt wurde, ist bisher nicht bekannt, ob weitere BMPs wie BMP-2 und -4 mit Adipositas und Typ-2-Diabetes assoziiert sind. Ziel dieser Arbeit war es deshalb zu untersuchen, ob die BMP-2 und -4 Serumkonzentrationen im Zusammenhang mit Körpergewicht, Fett- verteilung und Parametern des Glukosestoffwechsels bei Patienten mit Adipositas und Typ-2-Diabetes (n=213) stehen. Im Rahmen von drei Interventionsstudien wurde der Einfluss einer hypokalorischen Ernährungsweise über sechs Monate (n=19), eines 45,3 ± 7,4 kg Gewichtsverlustes ein Jahr nach bariatrischer Chirurgie (n=32) sowie eines zwölf- wöchigen Trainingsprogramms (n=60) auf die BMP-2- und -4-Serumkonzentrationen untersucht. Zusätzlich wurde die BMP-2-und -4-mRNA-Expression in humanen omentalen und subkutanen Fettgewebsproben von 161 Patienten charakterisiert. Die BMP-2- und -4-Serumkonzentrationen und die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe korrelieren signifikant mit dem BMI und dem Körperfettgehalt. Zirkulierende BMP-4-Spiegel sind geschlechtsabhängig und bei Patienten mit T2D signifikant niedriger als bei gesunden Kontrollpatienten. Sowohl eine moderate Gewichts- reduktion durch kalorienreduzierte Ernährung als auch ein Gewichtsverlust von 45,3 ± 7,4 kg nach bariatrischer Chirurgie führen zu einer signifikanten Reduktion der zirkulierenden BMP-2- und -4-Spiegel. Das zwölfwöchige Trainingsprogramm führte lediglich zu einer signifikanten Reduktion der BMP-2-Serumkonzentration und zu signifikanten Ver- besserung der Leistungsfähigkeit, von Parametern des Glukosestoffwechsels und der Serumkonzentrationen von Adiponektin und Interleukin-6. Zusammengefasst zeigen die Daten, dass erhöhte Serumkonzentrationen von BMP-2 und 4 mit Adipositas assoziiert sind und durch Gewichtsreduktion und Erhöhung der körperlichen Aktivität verringert werden können. Die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe kann zu erhöhten Serumkonzentrationen dieser Adipokine bei viszeraler Fettverteilung beitragen.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres. / Dissertation