Indiana University-Purdue University Indianapolis (IUPUI) / Hydrodynamic delivery is a powerful tool that allows delivery of macromolecules to the kidney culminating in gene expression. This finding is important in the fight against kidney disease. Current therapy for kidney injury, specifically acute kidney injury, is lacking. Supportive care in the form of IV fluids and medications aimed at restoring Glomerular Filtration Rate (GFR) and urine output are currently used. However, even with these treatments, prognoses of patients diagnosed with this disease remains poor. We believe that hydrodynamic delivery provides a mechanism that can be used to reverse and prevent AKI. Hydrodynamic delivery following ischemic injuries leads to reductions in serum creatinine and infiltrating mononuclear cells, as well as increased renal blood flow and survival. These changes are due to reductions in vascular congestion and inflammation typically seen following injury. To determine the underlying mechanisms of gene delivery preventing AKI, we used candidate genes identified in a proteomic screen on kidneys that recovered from AKI. We selected Isocitrate Dehydrogenase II (IDH2) and Sulfotransferase 1C2 (SULT1C2) for study and found that delivery prior to injury prevents serum creatinine increase and reduces cell death. We found that gene delivery of IDH2 prevents a glycolytic shift typically seen following ischemic injuries. The mechanism underlying the prevention of this shift are seen in increased ATP stores and spare respiratory capacity allowing the cell to remain in an oxidative state. Additionally, we show that SULT1C2 post-translationally modifies the mitochondria membrane, increasing oxidative phosphorylation providing the cell with additional energy needed in times of oxidative stress. These candidate genes allow cells to remain in an oxidative state preventing the activation of cell death pathways typically activated following injury, thereby preserving normal kidney function.

The ACL is one of the major ligaments in the knee connecting the femur to the tibia which provides stability by resisting shear in sagittal plane. ACL tears occur in 1 out of 3000, and due to inability to heal, reconstructive surgeries are performed at a rate of 200,000/year. Final graft fixation tension during surgery has been shown to wane due to stress-relaxation which has been correlated with negative clinical outcomes. Therefore, preconditioning, which currently is an isometric load (88N), is performed to remove stress-relaxation after the final tension has been applied in vivo. Three preconditioning protocols, creep, stress relaxation, and none, were tested to show significant differences and variance in graft tension after 30 minutes. The results suggest that the current preconditioning protocol may not be efficient enough to remove stress-relaxation after final fixation, and that a creep protocol causes less variability than the other preconditions performed.

Doctor of Philosophy / Department of Animal Sciences and Industry / KC Olson / We evaluated effects of preconditioning on performance and health of beef calves raised and finished in the Great Plains. In experiment 1, calves were preconditioned for 0, 15, or 45 d and vaccinated against BRD-causing pathogens 14 d before maternal separation or after feedlot arrival. During receiving and finishing, preconditioned calves had greater DMI and ADG than non-preconditioned calves; however, timing of BRD vaccination did not affect animal health. In experiment 2, calves were vaccinated against BRD pathogens 0, 1, 2, or 3 times during a 30-d preconditioning program. Vaccination for BRD, regardless of degree, improved health during preconditioning; however, DMI, ADG, and G:F during preconditioning, receiving, and finishing were unaffected by degree of vaccination. In experiment 3, calves were preconditioned for 30 d, shipped 4 h to an auction facility, commingled for 12 h, and transported 4, 8, or 12 h to a feedlot. Feedlot performance and health of beef calves were not affected by transport of up to 12 h following auction-market commingling. In experiment 4, beef calves were subjected to 1 of 3 ranch-of-origin preconditioning programs: drylot weaning + abrupt dam separation, pasture weaning + fence-line contact with dams, and pasture weaning + fence-line contact with dams + supplemental feed delivered in a bunk. Drylot-weaned calves gained more weight during preconditioning. Unsupplemented, pasture-weaned calves had the least ADG during receiving but had greater ADG during finishing and had carcass characteristics similar to other treatments. In experiment 5, we evaluated performance of early-weaned beef calves fed grain-based diets with DMI adjusted to achieve ADG of 0.45, 0.91, or 1.36 kg/d during an 84-d growing period. Calves fed at restricted rates did not exhibit improved G:F relative to full-fed counterparts. In addition, there appeared to be limitations associated with predicting DMI and ADG of light-weight, early-weaned calves fed a grain-based diet.

There are many scientific applications in which there is a need to solve very large linear systems. The preconditioned Krylove subspace methods are considered the preferred methods in this field. The preconditioners employed in the preconditioned iterative solvers usually determine the overall convergence rate. However, choosing a good preconditioner for a specific sparse linear system arising from a particular application is the combination of art and science, and presents a formidable challenge for many design engineers and application scientists who do not have much knowledge of preconditioned iterative methods. We tackled the problem of choosing suitable preconditioners for particular applications from a nontraditional point of view. We used the techniques and ideas in knowledge discovery and data mining to extract useful information and special features from unstructured sparse matrices and analyze the relationship between these features and the solving status of the spearse linear systems generated from these sparse matrices. We have designed an Intelligent Preconditioner Recommendation System, which can provide advice on choosing a high performance preconditioner as well as suitable parameters for a given sparse linear system. This work opened a new research direction for a very important topic in large scale high performance scientific computing. The performance of the various data mining algorithms applied in the recommendation system is directly related to the set of matrix features used in the system. We have extracted more than 60 features to represent a sparse matrix. We have proposed to use data mining techniques to predict some expensive matrix features like the condition number. We have also proposed to use the combination of the clustering and classification methods to predict the solving status of a sparse linear system. For the preconditioners with multiple parameters, we may predict the possible combinations of the values of the parameters with which a given sparse linear system may be successfully solved. Furthermore, we have proposed an algorithm to find out which preconditioners work best for a certain sparse linear system with what parameters.

Master of Science / Food Science / Sajid Alavi / Validation studies on process equipment are an important step for effective pathogenic control during dry expanded pet food manufacturing. The preconditioner is used to hydrate, mix and pre-cook raw materials before extrusion of pet food. The High-Intensity-Preconditioner (HIP) was designed with two independently driven shafts, thus offering control of both shaft speed and rotational direction with potential for improving residence time and thus pathogen inactivation. Residence time distribution (RTD) of raw dog food mix was impacted by the HIP process parameters (average residence time varying between 104-178 s for dry experiment and 65-177 s with steam addition) depending on shaft speed and direction. In general, increase in shaft speed resulted in shorter residence time with the larger shaft having a greater impact than the smaller shaft. Rotational direction of shafts also had an effect on average residence time (a maximum difference of 37 s was noticed between treatments with different shaft directions and the same speed). The uniformity of residence time distribution (difference of 97-132 s between 15 and 85 percentiles of the cumulative RTD) also varied considerably with process conditions, with uniformity increasing with shaft speed.  Enterococcus faecium (ATCC® 8459™) was chosen as a surrogate for Salmonella for microbial inactivation studies on the HIP. Both HIP shaft speed (200 and 300 rpm) and process temperature (67-70°C and 89-91°C) impacted E.faecium survival. Lower shaft speed (corresponding to longer residence time) or higher temperature led to greater E.faecium inactivation. A 5 log CFU/g of E.faecium was reduced using selective agar (m-Enterococcus or mE agar) after treatment with high temperature, but approximately 3.5 log CFU/g of E.faecium reduced on non-selective agar (Brain Heart Infusion or BHI agar). Uneven heat distribution, inadequate residence time and system instability might have negatively affected the inactivation. Microbial inactivation, with E.faecium as surrogate, was also studied for the complete dry expanded pet food process using a pilot-scale single-screw extruder with a regular double shaft preconditioner. Meal was inoculated with E.faecium at 6 log CFU/g and processed. Preconditioner downspout temperature ranged from 89-94°C and extrusion die temperature was between 120-140°C. Complete inactivation was observed after extrusion.

Typically, dormant seedlings are transplanted when revegetating nonirrigated disturbed lands in order to prevent transplant shock triggered by water stress. Since dormant seedlings have to be used, this limits the duration of the transplant season. It may be possible to increase this limited season by inducing acclimation responses that would increase drought tolerance. Preconditioning actively growing seedlings to water stress prior to transplanting could induce acclimation responses such as solute accumulation and/or stomatal modulation. Under greenhouse conditions, A. cana and A. intermedium seedlings were subjected to three water stress preconditioning treatments: a well watered control, one dry-down cycle, and three dry-down cycles. After conditioning, seedlings were either allowed to dry-down in their containers until leaf senescence, or were transplanted to disturbed land sites. Plant water potential components, relative water content, and leaf mortality were measured. Immediately following treatments, water relations parameters of preconditioned seedlings were not markedly different from controls in either species. At the end of the final dry-down, water stress preconditioning had not induced active or passive solute accumulation, prolonged leaf survival when exposed to lethal drought conditions, or resulted in differences in transplant survival rates under the experimental conditions of this study.

This thesis provides improvements to computational fluid dynamics accuracy and ef- ficiency through two main methods: a new boundary condition verification procedure and preconditioning techniques. First, a new verification approach that addresses boundary conditions was developed. In order to apply the verification approach to a large range of arbitrary boundary condi- tions, it was necessary to develop unifying mathematical formulation. A framework was developed that allows for the application of Dirichlet, Neumann, and extrapolation bound- ary condition, or in some cases the equations of motion directly. Verification of boundary condition techniques was performed using exact solutions from canonical fluid dynamic test cases. Second, to reduce computation time and improve accuracy, preconditioning algorithms were applied via artificial dissipation schemes. A new convective upwind and split pressure (CUSP) scheme was devised and was shown to be more effective than traditional precon- ditioning schemes in certain scenarios. The new scheme was compared with traditional schemes for unsteady flows for which both convective and acoustic effects dominated. Both boundary conditions and preconditioning algorithms were implemented in the context of a "strand grid" solver. While not the focus of this thesis, strand grids provide automatic viscous quality meshing and are suitable for moving mesh overset problems.

Current therapeutic options for ischemic stroke are limited to tissue plasminogen activator and mechanical clot removal therapies. Diazoxide (DZ) is a mitochondrial ATP-sensitive potassium channel opener and is protective in models of brain ischemia, but the signaling pathways involved are unknown. The mammalian target of rapamycin (mTOR) is a master regulator of protein synthesis and is involved in protection against cerebral ischemia. The neuronal nitric oxide synthase (nNOS) pathway has also been shown to provide protection from ischemic insults. Additionally, mitochondrial respiratory status has not been investigated. I examined the role of the mTOR pathway, the nNOS pathway, and mitochondrial respiration in delayed DZ-induced preconditioning of neurons. I cultured rat primary cortical neurons and simulated ischemic stroke using oxygen-glucose deprivation (OGD) for 3 h followed by re-oxygenation. Viability, mitochondrial membrane potential, reactive oxygen species (ROS) measurements, and western blots were performed. The mTOR pathway was inhibited by rapamycin, Torin-1, and S6K targeted silencing RNA. The NOS pathway was inhibited by L-NAME. NO-donors SNP and DEANONOate (DEANO) were applied to rescue the effects of L-NAME. Mitochondrial oxygen consumption rate (OCR) was measured in intact neurons by serial injections of oligomycin, FCCP, and antimycin/rotenone. OGD decreased viability by 50 percent, depolarized mitochondria, and reduced mitochondrial respiration whereas DZ improved viability to 75 percent and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Diazoxide also increased phosphorylation of protein kinase B, mTOR, and S6K. Rapamycin, Torin-1, and S6K targeted siRNA abolished the protective effects of DZ. Co-application of L-NAME with DZ prevented preconditioning whereas adding SNP or DEANO along with L-NAME and DZ restored protection. Diazoxide increased phosphorylated nNOS. Interestingly, co-application of LNAME with DZ blocked the phosphorylation of nNOS as well as S6K. The ratio of phosphorylated/total Akt and mTOR were not significantly altered with L-NAME co-application. Diazoxide altered OCR 24 and 48 h after the ischemic period. Diazoxide had no acute effect on OCR but increased ECAR significantly. Activation of the mTOR and nNOS pathways is critical for DZ preconditioning in neurons. Furthermore, OCR is modified by the DZ-induced preconditioning of neurons. / acase@tulane.edu

<p>Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives & Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury.</p><p>PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.</p>

Elliptic partial differential equations that are used to model physical phenomena give rise to large sparse linear systems. Such systems can be symmetric positive definite and can be solved by the preconditioned conjugate gradients method. In this thesis, we develop support graph preconditioners for symmetric positive definite matrices that arise from the finite element discretization of elliptic partial differential equations. An object oriented code is developed for the construction, integration and application of these preconditioners. Experimental results show that the advantages of support graph preconditioners are retained in the proposed extension to the finite element matrices.