Articular cartilage lines the surfaces of load bearing joints and has limited capabilities for self-repair due to its alymphatic and avascular structure. Attempts at making repairs to this tissue has resulted in substandard materials and/or causing further injury to the patient making this tissue a prime candidate for tissue engineering studies incorporating adult stem cells. These studies have given rise to some answers and many more questions including a search for alternative stem cell sources and what biochemical changes the cells undergo during the differentiation of these stem cells into chondrocytes, the cells which make up articular cartilage. Recently, periodontal dental ligament stem cells (PDLs) have come to the forefront as a practical alternative to other adult stem cells as well as the involvement of the mitogen-activated protein kinases (MAPKs) in stem cell differentiation via mechanical stimulation. During dynamic unconfined compression, levels of p42/44 MAPK increased by 50% (p<0.05). Additionally, the expression of the chondrogenic differentiation factor SRY (sex determining region Y)-box 9 (SOX-9) increased by 3-fold (p<0.05) as well as the chondrocyte marker aggrecan by over 2-fold after 4h of dynamic unconfined compression. Addition of the p42/44 phosphorylation inhibitor PD98059, along with compression, yielded no change in SOX-9 or aggrecan expression levels from basal levels in uncompressed controls. Inhibition of p38 MAPK or JNK phosphorylation during unconfined compression had no effect on the elevated expression of SOX-9 and aggrecan as compared to compressed cells without the addition of an inhibitor. It is therefore the overall findings of this study that PDLs possess the ability to differentiate into chondrocytes by mechanical compression and this differentiation is mediated by the p42/44 MAPK cascade.

The investigations conducted within this dissertation centers around the use of electric cell-substrate impedance sensing (ECIS). This system is able to characterize in real-time analysis, the adhesion of cells to their substrate and neighboring cells. With this, valuable information can be gathered with in-vitro experiments regarding a tissue culture's response to physiological stimulation. This dissertation has taken advantage of ECIS' ability to analyze toxicology, barrier function, and cancer invasion on a tissue culture. With proper analysis modifications, trans-epethelial resistance (TER) can be used as a cytotoxicity assay with higher sensitivity than previously thought. In vitro assessment of cytotoxicity based on TER needs more quantitative methods to analyze the alteration of cell morphology and motility. Here, we applied ECIS to evaluate dose-dependent responses of human umbilical vein endothelial cells (HUVEC) and mouse embryonic fibroblasts (NIH 3T3) exposed to cytochalasin B and protein kinase inhibitor H7. To detect subtle changes in cell morphology, the frequency-dependent impedance data of the cell monolayer were measured and analyzed with a theoretical cell-electrode model. To detect the alternation of cell micromotion in response to cytochalasin B and H7 challenge, time-series impedance fluctuations of cell-covered electrodes were monitored and the values of power spectrum, variance, and variance of the increment were calculated to verify the difference. While a dose-dependent relationship was generally observed from the overall resistance of the cell monolayer, the analysis of frequency-dependent impedance and impedance fluctuations distinguished cytochalasin B levels as low as 0.1µM and H7 levels as low as 10 µM for HUVEC and 3T3 layers. Even though overall resistance values are relatively small for 3T3 layers, and frequency scan measurements are negligible, impedance fluctuation analysis reveals significant micromotion for cytotoxic detection. Our results show that cytochalasin B and H7 causes a decrease of junctional resistance between cells and an increase of membrane capacitance. Cigarette smoke is cytotoxic and tumorigenic. Initial studies were conducted to evaluate the cytotoxicity of cigarette smoke condensate (CSC) on HUVEC layers. The focus was then turned to investigations involving in vitro cancer invasion assays with CSC on HUVEC layers. ECIS is an excellent investigative device that can be utilized to observe cancer invasion on normal tissue cultures due to the significantly higher impedance signature of cancer cells. The investigation in this dissertation focused on cigarette smoke's influence on cellular mechanics of endothelial cells and the invasive potential of two ovarian cancer cell lines (ALST and OVCA429) against a fully active endothelium. The HUVEC cultures responded to CSC with an increase in junctional binding, where as ALST and OVCA429 relieved adhesion thereby providing an improved motility when evaluated in wound healing assays. Transmigration of the HUVEC layer by ALST cells exhibit a pre-CSC exposure time-dependence affecting the effectiveness of ALST transmigration. The HUVEC layer's decreased tight junction binding that resulted from CSC exposure, allowed for a more aggressive ALST layer formation that occurred during simulated intravasation. Increased HUVEC layer tight junction binding that occurred in the first five hours in response to CSC during extravasation contributes to impeding ALST transmigration at high concentrations of CSC. Overall, CSC has an impeding effect on ALST transmigration during extravasation while causing aggressive transmigration during intravasation.

Cys2-His2 (C2H2) zinc finger domains were originally identified as DNA binding domains, and uncharacterized domains are typically assumed to bind DNA. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. Over 100 C2H2 zinc finger-protein interactions have been described. This study uses common bioinformatics tools to identify sequence features that predict a DNA- or protein-binding function. Several issues, including uncertainties about the full functional capabilities of the zinc fingers, complicated these efforts. Therefore, an unbiased approach which directly examined the potential for zinc fingers to facilitate DNA or protein interactions was used to determine the full functional capabilities of the C2H2 domains in two model proteins, human OLF-1/EBF associated zinc finger (OAZ) protein and Zif268. OAZ contains 30 zinc fingers in six clusters, some of which have been previously indicated in DNA or protein interactions. Zif268 is a well-known DNA binding protein with three C2H2 domains. DNA binding was assessed using a target site selection (CAST) assay, and protein binding was assessed using a yeast two-hybrid assay. Results indicate that clusters known to bind DNA could facilitate specific protein interactions, but clusters known to bind protein did not facilitate specific DNA interactions, indicating that DNA binding is a more restricted function of zinc fingers than has previously been recognized. These results also suggest that the role of C2H2 zinc finger domains in protein interactions has probably been underestimated. The implication of these findings for the prediction of zinc finger function is discussed.

Each member of the protein kinase C (PKC) family activates cell signaling pathways with different and sometimes opposing cell functions, such as cell division, migration, or death. Because of the importance of these processes in human diseases and disorders like cancer, stroke, and Alzheimer’s disease, there is a need for drugs which modify the action of PKC. However, drug design is difficult due to the complicated nature of PKC regulation. To better understand the differential regulation of PKC activity, these studies probe the structure, dynamics, and reactivity of one of the domains responsible for PKC regulation, C1B. C1B binds signaling molecules and translocates PKC to membranes in order to release the kinase domain from inhibition. Mutagenesis and ligand-binding assays monitored with fluorescence and nuclear magnetic resonance (NMR) techniques show that a single variable residue in C1B dramatically affects the sensitivity to signal activators. Investigation of the domain structure and dynamics using NMR revealed the identity of this residue alters the dynamics of the activator binding loops, without changing the structure. NMR studies of the C1B variants in membrane-mimicking micelles showed this residue also changes the interaction of the regulatory domain with lipids. These results demonstrate PKC isoforms have evolved specific functions by tuning dynamics and membrane affinity. Alternatively, PKC can be activated by reactive oxygen species by a mechanism that does not require binding of signaling molecules or membrane localization. To investigate the role of C1B in this type of signaling, the regulatory domain reactivity is monitored via NMR and gel electrophoresis. These studies reveal a particular cysteine residue in C1B that is most reactive, an alternative conformation of C1B in which this residue is more exposed, and modification of C1B leads to unfolding and zinc loss. Because the regulatory domains are responsible for auto-inhibition of the kinase domain, C1B unfolding provides a plausible explanation for activation of PKC by reactive oxygen species. The relation of the intrinsic C1B properties to the activation of PKC can be used to develop drugs with a single mechanism and to better understand how closely related signaling proteins develop specific functions.

The impact of lactate producing direct-fed microbial (DFM) on growth performance and rumen fermentation in beef cattle was explored in four studies. Experiment 1 studied the interaction between DFM and degradable intake protein (DIP) supply in receiving cattle. No differences (P≥0.06) in intake, morbidity, or immune response were observed; however, during the first 28 d gain and efficiency responses to DFM were dependent on DIP (DIP×DFM P≤0.05). Experiment 2 showed that in vitro gas production and select endpoint metabolites differed (P≤0.04) with DFM application. Experiment 3 compared lactate producing DFM to a lactate producing/utilizing DFM in finishing cattle. No differences (P≥0.14) in intake, gain, efficiency, or carcass characteristics were observed between control and lactate DFM; however, gain and growth efficiency differed (P≤0.05) between the lactate producing and lactate producing/utilizing DFM during the later portions of feeding. Experiment 4 studied the impact of DFM on ruminal fermentation, lactate utilization, and total tract digestibility. Ruminal pH and molar proportions of acetate were increased (P≤0.05) with DFM; however, lactate utilization and total tract digestibility did not differ (P≥0.33). The studies suggest that DFM improve growth performance during receiving and responses are at least partly mediated through differences in ruminal fermentation.

<p>Phospholipase Cs (PLCs) are a family of enzymes that hydrolyze membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglycerol (DAG). These second messengers activate a variety of intracellular responses, including inflammation, vascular smooth muscle contraction, and cardiac hypertrophy. While much is known about how Gaq-mediated activation of PLCb occurs, the same cannot be said for Gbg-mediated activation. Residues within the PLCb-Gbg binding interface were previously identified in interior regions of the protein, suggesting the PH domain must undergo a conformational change to allow for Gbg-mediated activation. However, the role of PH domain conformational dynamics in Gbg-mediated activation of PLCb has yet to be determined. In this work, I discuss efforts to characterize conformational dynamics of the PLCb PH domain and its role in interactions of the enzyme with liposomes and Gbg. First, I generated a disulfide crosslink between the PH domain and EF hands1/2 of PLCb3, purified under oxidizing or reducing conditions, and conducted biochemical and structural tests to determine any differences in structure and/or function of the protein as compared to wild-type. Results of these studies provided the first direct structural evidence of PLCb PH domain dynamics in solution. Then, I discuss the rationale behind the generation of a surface cysteine-less PLCb for use in solvatochromic fluorescence assays in the presence and absence of liposomes and Gbg. Initial results of these studies suggest the PLCb PH domain favors a buried conformation alone and in the presence of Gbg or liposomes, and likely exists at an equilibrium between open and closed states.</p>

A Cytokeratin 8 (K8)/Green Fluorescent Protein (GFP) fusion construct was created to better understand the behavior of K8 within cancer cells. This intermediate filament (IF) protein is a member of the cytoskeletal gene family along with actin and tubulin. IF's are normally expressed in a tissue specific and differentiation dependant manner, in which their role is more supportive than essential to the cell. Such roles include rigidity of cellular shape, protein trafficking, cellular locomotion, and cell signaling platforms. K8 mutation, over expression, and aberrant post translational modifications have been observed in various carcinoma cell lines to be the cause of several phenotypes including apoptosis inhibition, drug resistance, transformation, Mallory-Denk body (MDB) formation, localization at the plasma membrane, and secretion of the protein. In order to study these abnormal phenotypes the K8 gene was generated and inserted into the GFP over expression vector. This allowed for the study of K8 within a well defined cervical cancer cell line named Hela. This study intended to provide answers to K8's localization at the plasma membrane in carcinoma cell models while avoiding criticisms to previous immunohistochemical localization studies. A model which exhibits established phenotypes found in the literature was thus created which has the potential to address several paramount questions related to K8's role in supporting the development and progression of cancer. It could also be utilized as an assay for the discovery of K8 filament formation inhibitors, which may prove useful in combination with current chemotherapeutics. The model could also be used to provide weight to diagnostics, such as the Cancer Recognition test, which utilizes antibodies against K8 as biomarkers for malignancy via an Enzyme-Link ImmunoSorbent Assay (ELISA).

Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.

The Plasmodium vivax Duffy Binding Protein (DBP) is the ligand in the major pathway for P. vivax invasion of human reticulocytes, making it an appealing vaccine candidate. Region II of DBP (DBP-RII) is the minimal portion of the ligand that mediates recognition of the Duffy Antigen Receptor for Chemokines (DARC receptor) on the reticulocyte surface and constitutes the primary vaccine target. Analysis of natural variation in the coding sequences of DBP-RII revealed signature evidence for selective pressure driving variation in the residues of the putative receptor-binding site. We hypothesize that anti-DBP immunity in P. vivax infections is strain-specific and hindered by polymorphic residues altering sensitivity to immune antibody inhibition. To comprehend the human IgG response following P. vivax infections we investigated the specificity of IgG in Pursat Province, Western Cambodia. Using ELISAs, we quantified the antibody titer against five variant alleles of DBP-RII. We also sequenced the DBP-RII of the field isolates to determine their relationship to the variant alleles used in the ELISAs. When correlating the IgG titer between the DBP variants a strain-specific immune response was observed in patients with a high antibody titer to DBP-RII_AH as compared to the other variants. This was different from the correlation of high antibody titers between DBP-RII_P and DBP-RII_7.18 (ρ=0.88, p-value<0.0001) and DBP-RII_P and DBP-RII_O (ρ=0.87, p-value<0.0001). There appeared to be little correlation between specific polymorphic residues and IgG titer. Understanding the immune response to the polymorphisms within PvDBP will allow further identification of epitopes to enable the production of a more effective P. vivax vaccine

Purpose: Evidence has accumulated to show that elevated subclinical inflammation and impaired endothelial function has been associated with higher risk of cardiovascular disease (CVD). Despite data on these emerging risk factors, scant attention has been paid to: (1) the interactions of inflammation with endothelial function in relatively healthy African Americans; and (2) the efficacy of non-pharmacologic treatment modalities, such as exercise training, on inflammation and endothelial function. The aim of study 1 was to assess the levels of inflammatory markers, to assess the endothelial function in asymptomatic African Americans. The aim of study 2 was to determine the effects of six-months of exercise training on inflammatory markers and endothelial function in the same cohort. Methods: We recruited 79 African Americans who were sedentary, non-diabetic, non-smoking, and free of CV and renal disease. Before and after 6-month AEXT intervention, inflammatory markers (CRP, TNF-a and IL-6) were measured. Right brachial artery diameter was assessed at rest, during flow-mediated dilation (FMD), and after nitroglycerin-mediated dilation (NMD). Peak dilation was calculated as a measure of FMD and NMD, and the FMD/NMD ratio was calculated as a measure of endothelial function normalized by smooth muscle function. Fasted blood samples were obtained and were analyzed for the metabolic profile. Results: In study 1, the mean CRP for the group was 3.3 ± 0.3 mg/L which falls in the high-risk CRP category as per AHA/CDC guidelines. When divided into tertiles for CRP, low-risk (CRP 3 mg/L); VO2max was significantly higher in the low-risk category compared to average-risk category (P =0.004), and significantly higher in the low-risk category compared to high-risk category (P <0.001). Further, Cardiorespiratory fitness was significantly correlated with CRP (Figure. 1; r = -0.456, P <0.001) and BMI (r = -0.362, P = 0.002). CRP was correlated with BMI (r = 0.424, P <0.001). In a multivariable regression model that included age, gender, BMI, CVD risk factors (total cholesterol, triglycerides, HDL lipoprotein, LDL lipoprotein, plasma glucose, BP, and CRP), the following variables were significantly associated with fitness: fitness [B-coeff = -0.434 ± 0.05 (SE), P <0.001] independently predicted CRP. Fitness explained 22% of variance in CRP levels. In study 2, 6-month AEXT intervention significantly increased VO2max, (P=0.001), indicating that the prescribed exercise program may have been sufficient to elicit improvements in cardiovascular fitness. Significant reductions were observed for CRP (P =0.014). On repeated measures ANCOVA, the mean CRP values were significantly different (F (1,32) =6.703, P=0.014) between before vs. after training (Mean ± SEM; 3.1 ± 0.4 mg/L vs. 2.4 ± 0.4 mg/L), after adjusting for changed variables (BMI, mean BP and VO2max) as covariates. For endothelial measures, significant increase in endothelial function were observed for %FMDpeak (P =0.043) [Figure. 22] and FMD/NMD Ratio (P =0.047) increased post-AEXT. On repeated measures ANCOVA, the mean %FMD was statistically significantly different (F (1,16) =5.582, P=0.031) between before vs. after training (Mean ± SEM; 6.4 ± 2.6% vs. 9.4 ± 2.1%), after adjusting for changed variables (BMI, total-cholesterol and C-reactive protein) as covariates. Conclusions: The results from study 1 provide evidence of the prevalence of high levels of inflammation in the putatively healthy cohort of African Americans. When the group was categorized into tertiles for CRP and the cardio-metabolic, clinical and vascular profiles assessed, statistically significant differences, and rising trends were observed for CRP, body weight, BMI, BBF%, VO2max, SBP and DBP among the three CRP categories indicating a subclinical high cardiovascular risk profile in this cohort of putatively healthy population. Study 2, showed statistically significant improvements in inflammatory marker (CRP) and vascular measures (%FMDpeak, FMD/NMD Ratio and IMT); metabolic profile (triglycerides and FBS); clinical parameters (weight, BMI); cardiorespiratory fitness (VO2max). As we hypothesized, a marked reduction in CRP (-13.5%) post-AEXT was noticed, independent of change in(BMI, MAP and VO2max). Further, baseline CRP and BMI predicted change in CRP on regression analysis. Next, a marked increase in vascular measures, %FMDpeak (23.9%), FMD/NMD Ratio (25.4%) and IMT (-7.4%) were noticed independent of Ä(BMI, total cholesterol, MAP and VO2max). This may suggest: 1) the dominant anti-inflammatory role of exercise training. 2) that long-term exercise training improves clinical vascular measures in our cohort. / Kinesiology