Characterization of Non-aureus Staphylococci Isolated from Intramammary Infections and Extramammary Sites on Dairy Farms

This thesis is an investigation of the diagnosis, udder health effects and sources of nonaureus staphylococcal (NAS) intramammary infections (IMI) on dairy farms. The first objective of this series of studies was to evaluate the diagnostic accuracy of different target genes (rpoB or cpn60) and reference databases (GenBank or Chaperonin) to identify NAS species. The use of both rpoB and cpn60 genes using either databases provided similar accuracy in the identification of NAS species, although the highest proportion of NAS species was correctly identified with the rpoB gene using the GenBank database. The second objective was to determine the effects of NAS species on udder health and milk yield compared with healthy quarters and quarters infected with a major pathogen. The majority of NAS species had higher somatic cell counts (SCC) than healthy quarters and lower SCC than S. aureus-positive quarters. These SCC values were within the range of what is considered a ‘healthy’ SCC level. There was no effect of NAS IMI on overall milk yield. The third objective was to characterize the distribution of NAS isolated from the environment, body sites and IMI on dairy facilities and to determine potential sources of NAS IMI by comparing isolates from milk samples and extramammary sites. Staphylococcus chromogenes was identified exclusively from milk samples and represented the largest proportion of isolates. Staphylococcus equorum was identified exclusively from body and environmental site samples. Staphylococcus xylosus was the only NAS species that was identified from all sample types and was present in both milk and extramammary sites from the same cow. The fourth set of objectives were to determine (1) the proportion of specific S. chromogenes genotypes on dairy farms; (2) the effect of these genotypes on SCC; and (3) the persistency of each genotype within quarters. Of all 4 S. chromogenes genotypes, Genotype 1 was the most predominant in the study and had the largest proportion of persistent IMI identified. There was no effect of SCC among all genotypes and SCC levels were considered to be healthy

EXAMINING THE FUNCTIONAL ROLES OF AQUAPORINS AND UREA TRANSPORTERS IN THE MOVEMENT OF UREA ACROSS THE RUMINAL EPITHELIUM

Ussing chamber studies of ruminal epithelial tissue have provided insight into the mechanisms that regulate serosal-to-mucosal urea transport. Of these mechanisms, urea transport (UT-B) proteins have been shown to facilitate urea movement across the ruminal epithelium; however, other mechanisms may be involved as well because inhibiting UT-B does not completely eliminate urea transport. Of the aquaporins (AQP), which are a family of membrane-spanning proteins that are predominantly involved in the movement of water, AQP-3, -7, and -10 are also permeable to urea, but it is not clear if they contribute to urea transport across the ruminal epithelium. My objectives were to determine the relative functional roles of UT- and AQP-mediated serosal to mucosal urea flux (Jsm-urea) in response to changes in dietary carbohydrate fermentability, as well as ruminal ammonia and blood urea concentrations. The objectives of the Chapter 2 studies were: 1) to evaluate if there are differences in the magnitude of serosal-to-mucosal urea transfer in ruminal epithelium obtained from the caudal-dorsal or ventral sacs; 2) to determine the optimum mucosal buffer pH for maximal urea transport across the bovine ruminal epithelium; 3) to determine the time that is required for steady-state isotope equilibration with bovine ruminal epithelium; and 4) to determine if NiCl2 and HgCl2 are suitable inhibitors of aquaporin-mediated urea transport in bovine ruminal epithelium. Steady-state Jsm-urea and Jsm-mannitol fluxes were observed by 45 min following isotopic additions to the serosal buffer. Epithelia collected from the caudal-dorsal sac had higher Jsm-urea (P = 0.03) and lower Jsm-mannitol (P < 0.01) than that collected from the ventral sac. Reducing mucosal buffer pH from 7.0 to 5.2 increased Jsm-urea quadratically, where Jsm-urea increased from pH 7.0 to 6.4 and thereafter decreased (P = 0.01). Both HgCl2 and NiCl2 inhibited Jsm-urea (P < 0.01); however, the addition of HgCl2 increased Tissue Conductance (Gt) when compared to NiCl2. The objectives of Chapter 3were to determine: 1) the functional roles of AQP and UT-B in the serosal-to-mucosal urea flux (Jsm-urea) across rumen epithelium; and 2) whether functional adaptation occurred in response to increased diet fermentability. Serosal addition of phloretin. The addition of phloretin or NiCl2 reduced the Jsm-urea from 116.5 to 54.0 and 89.5 nmol·cm-2·h-1, respectively across all dietary treatments. When both inhibitors were added simultaneously, Jsm-urea was further reduced to 36.8 nmol·cm-2·h-1. Phloretin-sensitive and NiCl2-sensitive Jsm-urea III were not affected by diet. The Jsm-urea tended to increase linearly as the duration of adaptation to MGD increased, with the lowest Jsm-urea being observed in animals fed CON (107.7 nmol·cm-2·h- 1) and the highest for those fed the MGD for 21 d (144.2 nmol·cm-2·h-1). Phloretin-insensitive Jsm-urea tended to increase linearly as the duration of adaptation to moderate grain diet increased, whereas there was a tendency for NiCl2-insensitive Jsm-urea to be affected by diet. Gene transcript abundance for AQP-3 and UT-B in ruminal epithelium increased linearly as the duration of MGD adaptation increased. For AQP-7 and AQP-10, gene transcript abundance in animals that were fed the MGD was greater when compared to CON animals. The objective of Chapter 4 was to determine the effect of an acute dose of NH3 on total and aquaporin (AQP)-mediated urea flux across the ruminal epithelium in Angus cross bulls and Plains bison bulls. Ruminal NH3 was not affected by species (P = 0.60) or diet (P = 0.27) while PUN tended (P = 0.055) to be greater for BIS (12.5 mg/dL) than BOV (10.8 mg/dL), but was not affected by diet (P = 0.22). The Jsm-urea tended to decrease with addition of NiCl2 (P = 0.065), while mucosal NH3 had no effect on Jsm-urea (P = 0.41). Jsm-urea and Jsm-mannitol were not affected by species (P = 0.41) or dietary treatment (P = 0.29). I evaluated the effects of mucosal NH3 and serosal urea concentrations on total and phloretin sensitive Jsm-urea in Chapter 5. High Ammonia (HA) tended to inhibit total Jsm-urea with HU, but there was no effect of either NH3 concentration on total Jsm-urea with Low Urea (LU; interaction, P = 0.055). Addition of phloretin in the presence of serosal urea or mucosal NH3 had no effect on Jsm-urea. The Jsm-mannitol was not affected by serosal urea (P = 0.86) or mucosal NH3 (P = 0.22) concentration. The Jsm-urea and Jsm-mannitol tended to be positively correlated for HA (R2 = 0.301, P = 0.08), but not Low Ammonia (LA; R2 = 0.027, P = 0.70) in combination with LU. The same pattern was observed with High Urea (HU) treatments where Jsm-urea and Jsm-mannitol tended to be positively correlated for HA (R2 = 0.329, P = 0.08), but not LA (R2 = 0.111, P = 0.32). This research provides evidence that both AQP and UT-B play an important role in the Jsm-urea. In the future, additional research will be required to determine the mechanisms involved in NH3 inhibition of Jsm-urea, as this key step is critical in the role of urea-N recycling in ruminants

RCU Semantics: A First Attempt

There is not yet a formal statement of RCU (read-copy update) semantics. While this lack has thus far not been an impediment to adoption and use of RCU, it is quite possible that formal semantics would point the way towards tools that automatically validate uses of RCU or that permit RCU algorithms to be automatically generated by a parallel compiler. This paper is a first attempt to supply a formal definition of RCU. Or at least a semi-formal definition: although RCU does not yet wear a tux (though it does run in Linux), at least it might yet wear some clothes

Resizable, Scalable, Concurrent Hash Tables

We present algorithms for shrinking and expanding a hash table while allowing concurrent, wait-free, linearly scalable lookups. These resize algorithms allow the hash table to maintain constant-time performance as the number of entries grows, and reclaim memory as the number of entries decreases, without delaying or disrupting readers. We implemented our algorithms in the Linux kernel, to test their performance and scalability. Benchmarks show lookup scalability improved 125x over readerwriter locking, and 56% over the current state-of-the-art for Linux, with no performance degradation for lookups during a resize. To achieve this performance, this hash table implementation uses a new concurrent programming methodology known as relativistic programming. In particular, we make use of an existing synchronization primitive which waits for all current readers to finish, with little to no reader overhead; careful use of this primitive allows ordering of updates without read-side synchronization or memory barriers

Unbound states of 32Cl and the 31S(p,\gamma)32Cl reaction rate

The 31S(p,\gamma)32Cl reaction is expected to provide the dominant break-out path from the SiP cycle in novae and is important for understanding enrichments of sulfur observed in some nova ejecta. We studied the 32S(3He,t)32Cl charge-exchange reaction to determine properties of proton-unbound levels in 32Cl that have previously contributed significant uncertainties to the 31S(p,\gamma)32Cl reaction rate. Measured triton magnetic rigidities were used to determine excitation energies in 32Cl. Proton-branching ratios were obtained by detecting decay protons from unbound 32Cl states in coincidence with tritons. An improved 31S(p,\gamma)32Cl reaction rate was calculated including robust statistical and systematic uncertainties

A path-oriented encoding evolutionary algorithm for network coding resource minimization

Network coding is an emerging telecommunication technique, where any intermediate node is allowed to recombine incoming data if necessary. This technique helps to increase the throughput, however, very likely at the cost of huge amount of computational overhead, due to the packet recombination performed (ie coding operations). Hence, it is of practical importance to reduce coding operations while retaining the benefits that network coding brings to us. In this paper, we propose a novel evolutionary algorithm (EA) to minimize the amount of coding operations involved. Different from the state-of-the-art EAs which all use binary encodings for the problem, our EA is based on path-oriented encoding. In this new encoding scheme, each chromosome is represented by a union of paths originating from the source and terminating at one of the receivers. Employing path-oriented encoding leads to a search space where all solutions are feasible, which fundamentally facilitates more efficient search of EAs. Based on the new encoding, we develop three basic operators, that is, initialization, crossover and mutation. In addition, we design a local search operator to improve the solution quality and hence the performance of our EA. The simulation results demonstrate that our EA significantly outperforms the state-of-the-art algorithms in terms of global exploration and computational time

Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.

The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria

Attractiveness of periodic orbits in parametrically forced systemswith time-increasing friction

We consider dissipative one-dimensional systems subject to a periodic force and study numerically how a time-varying friction affects the dynamics. As a model system, particularly suited for numerical analysis, we investigate the driven cubic oscillator in the presence of friction. We find that, if the damping coefficient increases in time up to a final constant value, then the basins of attraction of the leading resonances are larger than they would have been if the coefficient had been fixed at that value since the beginning. From a quantitative point of view, the scenario depends both on the final value and the growth rate of the damping coefficient. The relevance of the results for the spin-orbit model are discussed in some detail.Comment: 30 pages, 6 figure

Factors influencing the seeking of medical attention with cancer of the colon

Thesis (M.S.)--Boston UniversityThis study was designed to determine whether education, marital status, sex, age and religion are factors influencing the time lapse between onset of symptoms and the seeking of medical attention with cancer of the colon

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.This work was supported by the intramural program of the Medical Research Council (MRC Grant U105663150 (to J. E. W.)