Expression of Fos protein in the rat central nervous system in response to noxious stimulation: effects of chronic inflammation of the superior cervical ganglion

The aim of this study was to investigate the possible interactions between the nociceptive system, the sympathetic system and the inflammatory process. Thus, the superior cervical ganglion of rats was submitted to chronic inflammation and Fos expression was used as a marker for neuronal activity throughout central neurons following painful peripheral stimulation. The painful stimulus consisted of subcutaneously injected formalin applied to the supra-ocular region. Fos-positive neurons were identified by conventional immunohistochemical techniques, and analyzed from the obex through the cervical levels of the spinal cord. In the caudal sub-nucleus of the spinal trigeminal nuclear complex, the number of Fos-positive neurons was much higher in rats with inflammation of the superior cervical ganglion than in control rats, either sham-operated or with saline applied to the ganglion. There was a highly significant difference in the density of Fos-positive neurons between the inflamed and control groups. No significant difference was found between control groups. These results suggest that the inflammation of the superior cervical ganglion generated an increased responsiveness to painful stimuli, which may have been due to a diminished sympathetic influence upon the sensory peripheral innervation.

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

Poly-DL-lactide-co-glycolide microspheres as a controlled release antigen delivery system

Successful vaccine application means maximum protection with minimal number of administrations. A rational development of vaccines involves studies of the nature of the antigen as well as of the adjuvant to be used to improve the immune responses. This has provided the impetus for studies to design the degradable devices and for different approaches to antigen delivery by different routes of administration. The development of controlled release systems based on polymeric devices that permit a sustained or pulsed release of encapsulated antigens has attracted much interest. Polymeric delivery systems consist of polymers that release their content continuously in a controlled manner over a period of time. The development of a biocompatible delivery system for parenteral administration offers several advantages in terms of immunoadjuvanticity over other compounds. It was found that, in contrast to other carriers, microspheres are more stable, thus permitting administration by the oral or parenteral route. In the present study, we describe the main characteristics and potentialities of this new immunoadjuvant for oral and parenteral administration.

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitope-protein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

Frequent occurrence of nicotinic acetylcholine receptors in GABAergic neurons of the chick visual system

Double-labeling immunohistochemical methods were used to investigate the occurrence of the alpha8 and alpha5 nicotinic receptor subunits in presumptive GABAergic neurons of the chick nervous system. Nicotinic receptor immunoreactivity was often found in cells exhibiting GABA-like immunoreactivity, especially in the visual system. The alpha8 subunit appeared to be present in presumptive GABAergic cells of the ventral lateral geniculate nucleus, nucleus of the basal optic root of the accessory optic system, and the optic tectum, among several other structures. The alpha5 subunit was also found in GABA-positive neurons, as observed in the lentiform nucleus of the mesencephalon and other pretectal nuclei. The numbers of alpha8- and alpha5-positive neurons that were also GABA-positive represented high percentages of the total number of neurons containing nicotinic receptor labeling in several brain areas, which indicates that most of the alpha8 and alpha5 nicotinic receptor subunits are present in GABAergic cells. Taken together with data from other studies, our results indicate an important role of the nicotinic acetylcholine receptors in the functional organization of GABAergic circuits in the visual system.

Complement system in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) consists 20% of keratinocytederived non-melanoma skin cancers (NMSC), the incidence of which is increasing globally. cSCC is the most common metastatic skin cancer and it causes approximately 20% of skin cancer-related deaths. At present, there are no molecular markers for predicting which cSCC lesions are aggressive or metastasize rapidly. UV radiation is the most important risk factor for cSCC. During the development of cSCC, normal epidermal keratinocytes are transformed and form actinic keratosis (AK), which progresses to cSCC in situ (cSCCIS, Bowen’s disease) and finally to invasive and metastatic cSCC. Inflammatory factors and cells are a part of cancer microenvironment and cSCC can develop in the chronically irritated skin or in the context of chronic inflammation. The complement system is a central part of innate immunity and it regulates normal immunological and inflammatory processes. In this study, the role of complement system components and inhibitors were studied in the progression of cSCC in culture and in vivo. Elevated expression of complement factor H (CFH), complement factor I (CFI), complement component C3 and complement factor B (CFB) was noted in cSCC cells in culture. The analysis with immunohistochemistry (IHC) revealed that the expression of CFH, CFI, C3 and CFB was specifically noted in tumor cells in vivo. The staining intensity of CFH, CFI, C3 and CFB was also stronger in invasive cSCC than in AK or cSCCIS samples. The knockdown of CFH, CFI and CFB with specific siRNAs decreased cSCC cell viability and migration, whereas the knockdown of C3 reduced only cSCC cell migration. Moreover, the knockdown of CFI, C3 and CFB inhibited growth of cSCC xenograft tumors established in SCID mice in vivo. In these tumors, CFI, C3 and CFB knockdown decreased the number of proliferating cells. Moreover, the knockdown of CFI increased local inflammation and complement activation. This study provides evidence for the roles of CFH, CFI, C3 and CFB in the tumor progression indicating these as molecular biomarkers and putative therapeutic targets of cSCC.

Local Binary Patterns in Focal-Plane Processing. Analysis and Applications

Feature extraction is the part of pattern recognition, where the sensor data is transformed into a more suitable form for the machine to interpret. The purpose of this step is also to reduce the amount of information passed to the next stages of the system, and to preserve the essential information in the view of discriminating the data into different classes. For instance, in the case of image analysis the actual image intensities are vulnerable to various environmental effects, such as lighting changes and the feature extraction can be used as means for detecting features, which are invariant to certain types of illumination changes. Finally, classification tries to make decisions based on the previously transformed data. The main focus of this thesis is on developing new methods for the embedded feature extraction based on local non-parametric image descriptors. Also, feature analysis is carried out for the selected image features. Low-level Local Binary Pattern (LBP) based features are in a main role in the analysis. In the embedded domain, the pattern recognition system must usually meet strict performance constraints, such as high speed, compact size and low power consumption. The characteristics of the final system can be seen as a trade-off between these metrics, which is largely affected by the decisions made during the implementation phase. The implementation alternatives of the LBP based feature extraction are explored in the embedded domain in the context of focal-plane vision processors. In particular, the thesis demonstrates the LBP extraction with MIPA4k massively parallel focal-plane processor IC. Also higher level processing is incorporated to this framework, by means of a framework for implementing a single chip face recognition system. Furthermore, a new method for determining optical flow based on LBPs, designed in particular to the embedded domain is presented. Inspired by some of the principles observed through the feature analysis of the Local Binary Patterns, an extension to the well known non-parametric rank transform is proposed, and its performance is evaluated in face recognition experiments with a standard dataset. Finally, an a priori model where the LBPs are seen as combinations of n-tuples is also presented

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

HIGH PRESSURE VAPOR-LIQUID EQUILIBRIA OF PALM FATTY ACIDS DISTILLATES-CARBON DIOXIDE SYSTEM

Vapor-Liquid equilibria of palm fatty acids distillates/carbon dioxide system has been investigated experimentally at temperatures of 333, 353, and 373 K and pressures of 20, 23, 26, and 29 MPa using the static method. Experimental data for the quasi-binary system palm fatty acids distillates/carbon dioxide has been correlated with Redlich-Kwong-Aspen equation of state. Modeling shows good agreement with experimental data. Selectivity obtained indicates that supercritical carbon dioxide is a reasonable solvent for separating saturated (palmitic acid) and unsaturated (oleic+linoleic acids) fatty acids from palm fatty acids distillates in a continuous multistage countercurrent column.

SENSORY ANALYSIS OF A MODEL SYSTEM USING 5'-IMP AND CYSTEINE AT DIFFERENT pH

Sensory analysis was used to get an overall flavour description of a reaction mixtures containing 5'-IMP and Cysteine. Ribose/cysteine systems were used as reference systems. Results from triangle and aroma profiling show a clear correlation between the terms used and the volatile analysis described in literature for these model systems. For instance reactions at pH 3.0 and 4.5 for 5'-IMP/cysteine systems, which were described as "meaty" and "boiled meat" by panellists, presented, in the literature, the higher number of "meaty" compounds in volatile analysis (1, 7, 8, 20) .

A Bayesian Approach for Forecasting Heat Load in a District Heating System

The growing population in cities increases the energy demand and affects the environment by increasing carbon emissions. Information and communications technology solutions which enable energy optimization are needed to address this growing energy demand in cities and to reduce carbon emissions. District heating systems optimize the energy production by reusing waste energy with combined heat and power plants. Forecasting the heat load demand in residential buildings assists in optimizing energy production and consumption in a district heating system. However, the presence of a large number of factors such as weather forecast, district heating operational parameters and user behavioural parameters, make heat load forecasting a challenging task. This thesis proposes a probabilistic machine learning model using a Naive Bayes classifier, to forecast the hourly heat load demand for three residential buildings in the city of Skellefteå, Sweden over a period of winter and spring seasons. The district heating data collected from the sensors equipped at the residential buildings in Skellefteå, is utilized to build the Bayesian network to forecast the heat load demand for horizons of 1, 2, 3, 6 and 24 hours. The proposed model is validated by using four cases to study the influence of various parameters on the heat load forecast by carrying out trace driven analysis in Weka and GeNIe. Results show that current heat load consumption and outdoor temperature forecast are the two parameters with most influence on the heat load forecast. The proposed model achieves average accuracies of 81.23 % and 76.74 % for a forecast horizon of 1 hour in the three buildings for winter and spring seasons respectively. The model also achieves an average accuracy of 77.97 % for three buildings across both seasons for the forecast horizon of 1 hour by utilizing only 10 % of the training data. The results indicate that even a simple model like Naive Bayes classifier can forecast the heat load demand by utilizing less training data.

Implementing Green IT approach for transferring Big Data over Parallel Data Link

The whole research of the current Master Thesis project is related to Big Data transfer over Parallel Data Link and my main objective is to assist the Saint-Petersburg National Research University ITMO research team to accomplish this project and apply Green IT methods for the data transfer system. The goal of the team is to transfer Big Data by using parallel data links with SDN Openflow approach. My task as a team member was to compare existing data transfer applications in case to verify which results the highest data transfer speed in which occasions and explain the reasons. In the context of this thesis work a comparison between 5 different utilities was done, which including Fast Data Transfer (FDT), BBCP, BBFTP, GridFTP, and FTS3. A number of scripts where developed which consist of creating random binary data to be incompressible to have fair comparison between utilities, execute the Utilities with specified parameters, create log files, results, system parameters, and plot graphs to compare the results. Transferring such an enormous variety of data can take a long time, and hence, the necessity appears to reduce the energy consumption to make them greener. In the context of Green IT approach, our team used Cloud Computing infrastructure called OpenStack. It’s more efficient to allocated specific amount of hardware resources to test different scenarios rather than using the whole resources from our testbed. Testing our implementation with OpenStack infrastructure results that the virtual channel does not consist of any traffic and we can achieve the highest possible throughput. After receiving the final results we are in place to identify which utilities produce faster data transfer in different scenarios with specific TCP parameters and we can use them in real network data links.

Evaluation of the BAX® system for the detection of Salmonella spp. in naturally contaminated chicken meat

The aim of this study was to verify the efficiency of the BAX® system for the detection of Salmonella spp. in raw chicken meat. The conventional culture method (IN 62, MAP) was used as a reference method. A total of 8,813 chicken carcass samples were analyzed. In the first part of the study, 1,200 samples were analyzed using the BAX® System and the conventional culture method. In the second part, 7,613 samples were analyzed by the BAX® system, and the conventional method was used only for samples that tested positive for Salmonella spp. by the BAX® system. The sensitivity, specificity, relative accuracy, positive predictive value, and negative predictive value obtained in the first part of this study were 100%, 92.3%, 96.4%, 53.3% and 100%, respectively. The BAX® system showed no false-negative results and reduced the time to obtain presumptive positive results. It is a suitable method for use in laboratories that perform a large number of food samples analyses daily. However, the conventional method is still required to confirm the presence of Salmonella spp. in samples that test positive using the BAX® system.

Energy Management System for LVDC Island Networks

Recent developments in power electronics technology have made it possible to develop competitive and reliable low-voltage DC (LVDC) distribution networks. Further, islanded microgrids—isolated small-scale localized distribution networks— have been proposed to reliably supply power using distributed generations. However, islanded operations face many issues such as power quality, voltage regulation, network stability, and protection. In this thesis, an energy management system (EMS) that ensures efficient energy and power balancing and voltage regulation has been proposed for an LVDC island network utilizing solar panels for electricity production and lead-acid batteries for energy storage. The EMS uses the master/slave method with robust communication infrastructure to control the production, storage, and loads. The logical basis for the EMS operations has been established by proposing functionalities of the network components as well as by defining appropriate operation modes that encompass all situations. During loss-of-powersupply periods, load prioritizations and disconnections are employed to maintain the power supply to at least some loads. The proposed EMS ensures optimal energy balance in the network. A sizing method based on discrete-event simulations has also been proposed to obtain reliable capacities of the photovoltaic array and battery. In addition, an algorithm to determine the number of hours of electric power supply that can be guaranteed to the customers at any given location has been developed. The successful performances of all the proposed algorithms have been demonstrated by simulations.

Lolium multiflorum seeds in the soil: I. Soil seed bank dynamics in a no til system

The objective of the study was to characterize annual ryegrass seed population dynamics, managed for natural re-sowing, in no til systems in rotation with soybean, in different chronosequences An area was cultivated for two years with soybean, left as fallow land for the next two years and then cultivated again with soybean for the next two years. The four chronosequences represented different management periods, two with soybean (6 and 8 years old) and the other two resting (3 and 9 years old). Soil samples were taken every month during one year and divided into two depths (0-5 and 5-10 cm). Vegetation dynamics were also evaluated (number of plants, inflorescences and seedlings). Soil seed bank (SSB) dynamics showed structural patterns in time, with a "storage period" in summer, an "exhausting period" during autumn and a "transition period" in winter and spring. Pasture establishment by natural re-sowing was totally dependent on the annual recruitment of seeds from the soil. The influence of the management practices on the SSB was more important than the number of years that these practices had been implemented. Places where soybean was sown showed the largest SSBs. Most of the seeds overcame dormancy and germinated at the end of the summer and beginning of the autumn, showing a typically transitory SSB, but with a small proportion of persistent seeds