Target tissue influences the peripheral trajectory of mouse primary sensory olfactory axons

Primary olfactory neurons situated in the nasal septum project axons within fascicles along a highly stereotypical trajectory en route to the olfactory bulb. The ventral fascicles make a distinct dorsovental turn at the rear of the septum so as to reach the olfactory bulb. In the present study we have used a brain and nasal septum coculture system to examine the role of target tissue on the peripheral trajectory of olfactory sensory axons. In cultures of isolated embryonic nasal septa, olfactory axons form numerous parallel fascicles that project caudally in the submucosa, as they do in vivo. The ventral axon fascicles in the septum, however, often fail to turn, and do not project dorsally towards the roof of the nasal cavity. The presence of olfactory bulb, cortical, or tectal tissue apposed to the caudal end of the septum rescued this phenotype, causing the ventral fascicles to follow a normal in vivo-like trajectory. Ectopic placements of the explants revealed that brain tissue is not tropic for olfactory axons but appears to maintain the peripheral trajectory of growing axons in the nasal septum. Although primary olfactory axons are able to penetrate into olfactory bulb in vitro, they only superficially enter cortical tissue, whereas they do not grow into tectal explants. The ability of axons to differentially grow into different brain regions was shown to be unrelated to the migratory behavior of olfactory ensheathing cells, indicating that olfactory axons are directly responsive to guidance cues in the brain. (C) 2004 Wiley Periodicals, Inc.

Olfactory marker protein modulates primary olfactory axon overshooting in the olfactory bulb

Olfactory marker protein (OMP) is expressed by mature primary olfactory sensory neurons during development and in adult mice. In mice that lack OMP, olfactory sensory neurons have perturbed electrophysiological activity, and the mice exhibit altered responses and behavior to odor stimulation. To date, defects in axon guidance in mice that lack OMP have not been investigated. During development of the olfactory system in mouse, primary olfactory axons often overshoot their target glomerular layer and project into the deeper external plexiform layer. These aberrant axonal projections are normally detected within the external plexiform layer up to postnatal day 12. We have examined the projections of primary olfactory axons in OMP-tau:LacZ mice and OMP-GFP mice, two independent lines in which the OMP coding region has been replaced by reporter molecules. We found that axons overshoot their target layer and grow into the external plexiform layer in these OMP null mice as they do in wild-type animals. However, in the absence of OMP, overshooting axons are more persistent and remain prominent until 5 weeks postnatally, after which their numbers decrease. Overshooting axons are still present in these mice even at 8 months of age. In heterozygous mice, axons also overshoot into the external plexiform layer; however, there are fewer axons, and they project for shorter distances, compared with those in a homozygous environment. Our results suggest that perturbed electrophysiological responses, caused by loss of OMP in primary olfactory neurons, reduce the ability of primary olfactory axons to recognize their glomerular target. © 2005 Wiley-Liss, Inc.

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.

Implantation of a scaffold following bulbectomy induces laminar organization of regenerating olfactory axons

Primary olfactory axons expressing different odorant receptors are interspersed within the olfactory nerve. However, upon reaching the outer nerve fiber layer of the olfactory bulb they defasciculate, sort out, and refasciculate prior to targeting glomeruli in fixed topographic positions. While odorant receptors are crucial for the final targeting of axons to glomeruli, it is unclear what directs the formation of the nerve fiber and glomerular layers of the olfactory bulb. While the olfactory bulb itself may provide instructive cues for the development of these layers, it is also possible that the incoming axons may simply require the presence of a physical scaffold to establish the outer laminar cytoarchitecture. In order to begin to understand the underlying role of the olfactory bulb in development of the outer layers of the olfactory bulb, we physically ablated the olfactory bulbs in OMP-IRES-LacZ and P2-IRES-tau-LacZ neonatal mice and replaced them with artificial biological scaffolds molded into the shape of an olfactory bulb. Regenerating axons projected around the edge of the cranial cavity at the periphery of the artificial scaffold and were able to form an olfactory nerve fiber layer and, to some extent, a glomerular layer. Our results reveal that olfactory axons are able to form rudimentary cytoarchitectonic layers if they are provided with an appropriately shaped biological scaffold. Thus, the olfactory bulb does not appear to provide any tropic substance that either attracts regenerating olfactory axons into the cranial cavity or induces these axons to form a plexus around its outer surface. (c) 2006 Elsevier B.V. All rights reserved.

On the adoption decision and the intensity of use of complementary innovations: The intra firm diffusion of management practices across UK establishments

This paper contributes to the literature on the intra-firm diffusion of innovations by investigating the factors that affect the firm’s decision to adopt and use sets of complementary innovations. We define complementary innovations those innovations whose joint use generates super additive gains, i.e. the gain from the joint adoption is higher than the sum of the gains derived from the adoption of each innovation in isolation. From a theoretical perspective, we present a simple decision model, whereby the firm decides ‘whether’ and ‘how much’ to invest in each of the innovations under investigation based upon the expected profit gain from each possible combination of adoption and use. The model shows how the extent of complementarity among the innovations can affect the firm’s profit gains and therefore the likelihood that the firm will adopt these innovations jointly, rather than individually. From an empirical perspective, we focus on four sets of management practices, namely operating (OMP), monitoring (MMP), targets (TMP) and incentives (IMP) management practices. We show that these sets of practices, although to a different extent, are complementary to each other. Then, we construct a synthetic indicator of the depth of their use. The resulting intra-firm index is built to reflect not only the number of practices adopted but also the depth of their individual use and the extent of their complementarity. The empirical testing of the decision model is carried out using the evidence from the adoption behaviour of a sample of 1,238 UK establishments present in the 2004 Workplace Employment Relations Survey (WERS). Our empirical results show that the intra-firm profitability based model is a good model in that it can explain more of the variability of joint adoption than models based upon the variability of adoption and use of individual practices. We also investigate whether a number of firm specific and market characteristics by affecting the size of the gains (which the joint adoption of innovations can generate) may drive the intensity of use of the four innovations. We find that establishment size, whether foreign owned, whether exposed to an international market and the degree of homogeneity of the final product are important determinants of the intensity of the joint adoption of the four innovations. Most importantly, our results point out that the factors that the economics of innovation literature has been showing to affect the intensity of use of a technological innovation do also affect the intensity of use of sets of innovative management practices. However, they can explain only a small part of the diversity of their joint adoption use by the firms in the sample.

Studies of the nature of Burkholderia cepacia in cystic fibrosis

Burkholderia cepacia is an opportunistic pathogen that colonises of the lungs of cystic fibrosis (CF) patients, with a frequently fatal outcome. Antibiotic resistance is common and highly transmissible epidemic strains have been described in the UK. 37 B. cepacia isolates from clinical and botanical sources were characterised via metabolic capabilities, antibiotic sensitivity, fatty acid methyl ester (FAME) profiles restriction digest analysis of chromosomal DNA by pulsed-gel electrophoresis (PFGE) (with the use of two separate restriction enzymes) and outer membrane protein (OMP) profiles. This revealed isolates of the UK CF epidemic strain to form a distinct group with a specific OMP profile. Cluster analysis of PFGE and FAME profiles revealed the species Burkholderia gladioli and Burkholderia vietnamiensis to be more closely related to each other and to laboratory strains of B. cepacia than to the CF epidemic strain considered a member of the latter species. The epidemic strain of B. cepacia may therefore be worthy of species definition in its own right. All the strains studied showed a high level of resistance to antibiotics, including the carbapenems. Considering this, carbapenemase production by isolates of B. cepacia was investigated. A metallo-β-lactamase from a clinical strain of B. cepacia was isolated and partially purified of using Cibacron blue F3GA-coupled agarose. The resulting preparation showed a single band of β-lactamase activity (pI 8.45) after analytical isoelectric focusing. The enzyme was particularly effective in the hydrolysis of imipenem. Meropenem, biapenem, cephaloridine, ceftazidime, benzylpenicillin, ampicillin and carbenicillin were hydrolysed at a lower rate. An unusual inhibition profile was noted. Inhibition by the metal ion chelators ethylene diamine tetra acetic acid and o-phenanthroline was reversed by addition of zinc, indicating a metallo-enzyme, whilst >90% inhibition was attainable with 0.1mM concentrations of tazobactam and clavulanic acid. A study of 8 other clinical isolates showed an enzyme of pI 8.45 to be present and inducible by imipenem in each case. This enzyme was assigned PCM-I (Pseudomonas cepacia metalloenzyme I).

The effect of haem limitation and iron restriction on outer membrane proteins and on respiratory systems of non typable Haemophilus influenzae

The effects of haem limitation and iron restriction on cells of non typable Haemophilus influenzae were investigated. Haem limitation was achieved by adding concentrations of haem to growth media which resulted in substantial decreases in final cell yields. Iron restriction was achieved by substituting protoporphyrin IX (PPIX) for haem in the growth medium and adding an iron chelator to the system. The effect of these nutrient limitations on a) outer membrane composition, and b) respiratory systems of non typable H.influenzae was investigated. Several of the strains examined produced new PPIX-specific outer membrane proteins when cultured utilising PPIX as a porphyrin source. The immune response of patients with bronchiectasis to outer membrane antigens of H.influenzae cultured under iron-restricted conditions was analysed by ELISA and immunoblotting techniques. ELISA analysis revealed that individuals with severe bronchiectasis had high titres of antibodies directed against H.influenzae OMs in both serum and sputum. Immunoblotting with homologous serum showed that where PPIX-specific OMPs were produced they were antigenic and were recognised by patients' serum. This suggested that these H.influenzae OMPs may be expressed in vivo. Additionally, the development of the immune responses to non typable H.influenzae outer membrane antigens was investigated using a rat lung model. Bacteria encased in agar beads were inoculated intratracheally into rat lungs, infection was established, and the immune response monitored for 6 weeks. The animals developed antibodies to PPIX-specific OMPs during the course of infection, providing further evidence that H.influenzae express these novel OMP antigens when growing in vivo. Studies in vitro on respiratory systems of phenotypically altered H.influenzae showed that bacteria grown utilising PPIX as a porphyrin source, or under conditions of iron-restriction produced ten fold fewer cytochromes than cells grown in nutrient excess, while haem limited H.influenzae produced no detectable cytochromes. Respiration of various substrates was depressed in haem limited and in PPIX-grown cultures as compared with cells grown in nutrient excess.

Influence of iron on bacterial infections in leukaemia

The influence of iron metabolism, both on the invading bacterial pathogen and in the host is widespread and often appears to be crucial in determining the outcome of an infection. This study involved the investigation of leukaemia, a clinical disease where abnormal availability of iron may play a part in predisposing patients to bacterial infection. The iron status throughout a Gram-negative septicaemia and in 20 random, newly diagnosed leukaemic patients was assessed. The results revealed that the majority of the patients exhibited high serum iron levels and serum transferrin saturation often at 100%, with an inability to reduce the latter to within normal values during an infection episode. The antibody response to P.aeruginosa, E.coli and K.pneumoniae outer membrane protein (OMP) antigens were investigated by immunoblotting with sequential serum samples during infection in the leukaemic host. Antibodies to all the major OMPs, were observed, although recognition of iron-regulated membrane proteins (IRMPs) was in many cases weak. Results from the enzyme-linked immunosorbent assay indicated that in all patients antibody titre in response to infection was poor. Sub-MICs of mitomycin C significantly altered the surface characteristics of P.aeruginosa. The silver-stained SDS-PAGE gels of proteinase K digested whole cell lysates of strains PAO1, 6750, M7 and PAJ indicated that core LPS was affected in the presence of mitomycin C. In contrast, the rough strain AK1012 showed no observable differences. Results obtained using quantitative gas-liquid chromatographic analysis showed the amount of LPS fatty acids to be unaffected, however, the KDO and carbohydrate content in strains PAO1, 6750 and M7 under Fe+ and Fe- growth conditions were decreased by up to 4-fold in the presence of mitomycin C, indicating perturbed expression of LPS. The cell surface became significantly more hydrophobic in the P.aeruginosa strains, except AK1012 which was comparatively unaffected. The induction of protein G (OprG) in P.aeruginosa was found to be a sensitive indicator of media iron. The data indicated that expression of OprG can be modulated by growth rate/phase, availability of iron and by the presence of ciprofloxacin in the growth medium.

Sparse image representation with encryption

In this thesis we present an overview of sparse approximations of grey level images. The sparse representations are realized by classic, Matching Pursuit (MP) based, greedy selection strategies. One such technique, termed Orthogonal Matching Pursuit (OMP), is shown to be suitable for producing sparse approximations of images, if they are processed in small blocks. When the blocks are enlarged, the proposed Self Projected Matching Pursuit (SPMP) algorithm, successfully renders equivalent results to OMP. A simple coding algorithm is then proposed to store these sparse approximations. This is shown, under certain conditions, to be competitive with JPEG2000 image compression standard. An application termed image folding, which partially secures the approximated images is then proposed. This is extended to produce a self contained folded image, containing all the information required to perform image recovery. Finally a modified OMP selection technique is applied to produce sparse approximations of Red Green Blue (RGB) images. These RGB approximations are then folded with the self contained approach.

Chlorofluorocarbons, helium, and neon measured on water bottle samples during POLARSTERN cruise ANT-XXII/2 (ISPOL)

During Ice Station POLarstern (ISPOL; R.V. Polarstern cruise ANT XXII/2, November 2004-January 2005), hydrographic and tracer observations were obtained in the western Weddell Sea while drifting closely in front of the Larsen Ice Shelf. These observations indicate recently formed Weddell Sea Bottom Water, which contains significant contributions of glacial melt water in its upper part, and High-Salinity Shelf Water in its lower layer. The formation of this bottom water cannot be related to the known sources in the south, the Filchner-Ronne Ice Shelf. We show that this bottom water is formed in the western Weddell Sea, most likely in interaction with the Larsen C Ice Shelf. By applying an Optimum Multiparameter Analysis (OMP) using temperature, salinity, and noble gas observations (helium isotopes and neon), we obtained mean glacial melt-water fractions of about 0.1% in the bottom water. On sections across the Weddell Gyre farther north, melt-water fractions are still on the order of 0.04%. Using chlorofluorocarbons (CFCs) as age tracers, we deduced a mean transit time between the western source and the bottom water found on the slope toward the north (9±3 years). This transit time is larger and the inferred transport rate is small in comparison to previous findings. But accounting for a loss of the initially formed bottom water volume due to mixing and renewal of Weddell Sea Deep Water, a formation rate of 1.1±0.5 Sv in the western Weddell Sea is plausible. This implies a basal melt rate of 35±19 Gt/year or 0.35±0.19 m/year at the Larsen Ice Shelf. This bottom water is shallow enough that it could leave the Weddell Basin through the gaps in the South Scotia Ridge to supply Antarctic Bottom Water. These findings emphasize the role of the western Weddell Sea in deep- and bottom-water formation, particularly in view of changing environmental conditions due to climate variability, which might induce enhanced melting or even decay of ice shelves.

Single-Cell Transcriptome Analysis of Olfactory Sensory Neurons

Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.

In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.

Investigations into the mechanism of orotidine 5'-monophosphate decarboxylase: Sources of substrate destabilization and transition state stabilization

Orotidine 5′-monophosphate decarboxylase (OMPDC) achieves a rarely paralleled rate acceleration, yet the catalytic basis prompting this enhancement have yet to be fully elucidated. To accomplish decarboxylation, OMPDC must overcome the high energy barrier due to the localized anionic charge of the intermediate. Mechanistic studies employing enzyme mutagenesis and product or intermediate analogues were used to investigate possible transition state stabilization by a carbene resonance structure. Viability of the carbene structure depends upon a key hydrogen bond between O4 of the substrate and the amide backbone of a conserved serine or threonine. Substitution of the conserved residue with Pro resulted in a kcat/KM of 1 M-1s-1; deletion of the FUMP O4 resulted in a product analogue that does not undergo H6 exchange or inhibit decarboxylation. Hence, indirect evidence reveals the O4-backbone interaction plays an important role for binding and catalysis. OMPDC likely has honed multiple mechanisms to attain its remarkable catalysis. The successful crystallizations of OMPDC a decade ago sparked hypotheses that structure and sequence conserved residues induced productive strain on the substrate-enzyme complex. Here, we demonstrate a new source of stress: a hydrophobic pocket adjacent to the OMP carboxylate that exhibits kinetic parameters characteristic of substrate destabilization. Substitution of these residues with hydrophilic side-chains, by providing hydrogen-bonding partners, decreased kcat by 10 to 10^4–fold. The same substitutions display very little change in the rate of product H6 exchange, supporting that this hydrophobic pocket affects the substrate-enzyme complex before the transition state. We also provide evidence that hydrophilic residues can insert water molecules into the pocket with detrimental effects to catalysis.

Ecolog?a del concepto de volumen en una instituci?n escolar una aproximaci?n desde la tad

Este trabajo, se apoya en la Teor?a Antropol?gica de lo Did?ctico (TAD) y se plantea como un trabajo de tipo exploratorio, donde dar? cuenta de la ecolog?a que se presenta en el nivel de la Obra Matem?tica Propuesta (OMp) con relaci?n a la ense?anza del volumen en una instituci?n escolar de la ciudad de Cali. A partir del estudio de algunos fen?menos did?cticos como la atomizaci?n del proceso de ense?anza, el autismo tem?tico, dicho an?lisis pondr? en evidencia las restricciones o limitaciones que desde el nivel de la OMp son atribuidas al tratamiento que se le da en la escuela a dicho objeto matem?tico y que en gran medida delimitar? su puesta en el aula como objeto de aprendizaje. Para lo cual, se elabor? una Obra Matem?tica de Referencia (OMr) con el apoyo de algunos estudios te?ricos al respecto y que permiti? realizar el an?lisis como un contraste entre OMr y OMp. Se realiz? una revisi?n a los documentos curriculares de car?cter oficial, el plan de ?rea de matem?ticas y la gu?a escolar de grado 9? del colegio Parroquial Nuestra Se?ora de los Andes. En el desarrollo de este trabajo se evidencia la ausencia en el plan de ?rea y la gu?a escolar de elementos claves para abarcar la relaci?n del concepto de volumen (desde una visi?n est?tica o din?mica), aunque cabe resaltar que en la gu?a escolar se presenta una posible relaci?n del volumen de un cubo con las funciones c?bicas.