Fibroblast growth factor-1 (FGF-1) as a potential therapeutic target for obesity: mechanisms of FGF-1 action in human preadipocytes

No abstract

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

The human factors of automatic speech recognition in control room systems

This thesis addresses the viability of automatic speech recognition for control room systems; with careful system design, automatic speech recognition (ASR) devices can be useful means for human computer interaction in specific types of task. These tasks can be defined as complex verbal activities, such as command and control, and can be paired with spatial tasks, such as monitoring, without detriment. It is suggested that ASR use be confined to routine plant operation, as opposed the critical incidents, due to possible problems of stress on the operators' speech.  It is proposed that using ASR will require operators to adapt a commonly used skill to cater for a novel use of speech. Before using the ASR device, new operators will require some form of training. It is shown that a demonstration by an experienced user of the device can lead to superior performance than instructions. Thus, a relatively cheap and very efficient form of operator training can be supplied by demonstration by experienced ASR operators. From a series of studies into speech based interaction with computers, it is concluded that the interaction be designed to capitalise upon the tendency of operators to use short, succinct, task specific styles of speech. From studies comparing different types of feedback, it is concluded that operators be given screen based feedback, rather than auditory feedback, for control room operation. Feedback will take two forms: the use of the ASR device will require recognition feedback, which will be best supplied using text; the performance of a process control task will require task feedback integrated into the mimic display. This latter feedback can be either textual or symbolic, but it is suggested that symbolic feedback will be more beneficial. Related to both interaction style and feedback is the issue of handling recognition errors. These should be corrected by simple command repetition practices, rather than use error handling dialogues. This method of error correction is held to be non intrusive to primary command and control operations. This thesis also addresses some of the problems of user error in ASR use, and provides a number of recommendations for its reduction.

An incremental multilinear system for human face learning and recognition

This dissertation establishes a novel system for human face learning and recognition based on incremental multilinear Principal Component Analysis (PCA). Most of the existing face recognition systems need training data during the learning process. The system as proposed in this dissertation utilizes an unsupervised or weakly supervised learning approach, in which the learning phase requires a minimal amount of training data. It also overcomes the inability of traditional systems to adapt to the testing phase as the decision process for the newly acquired images continues to rely on that same old training data set. Consequently when a new training set is to be used, the traditional approach will require that the entire eigensystem will have to be generated again. However, as a means to speed up this computational process, the proposed method uses the eigensystem generated from the old training set together with the new images to generate more effectively the new eigensystem in a so-called incremental learning process. In the empirical evaluation phase, there are two key factors that are essential in evaluating the performance of the proposed method: (1) recognition accuracy and (2) computational complexity. In order to establish the most suitable algorithm for this research, a comparative analysis of the best performing methods has been carried out first. The results of the comparative analysis advocated for the initial utilization of the multilinear PCA in our research. As for the consideration of the issue of computational complexity for the subspace update procedure, a novel incremental algorithm, which combines the traditional sequential Karhunen-Loeve (SKL) algorithm with the newly developed incremental modified fast PCA algorithm, was established. In order to utilize the multilinear PCA in the incremental process, a new unfolding method was developed to affix the newly added data at the end of the previous data. The results of the incremental process based on these two methods were obtained to bear out these new theoretical improvements. Some object tracking results using video images are also provided as another challenging task to prove the soundness of this incremental multilinear learning method.

Enzymatic profiling of cellulosomal enzymes from the human gut bacterium, Ruminococcus champanellensis, reveals a fine-tuned system for cohesin-dockerin recognition

Funded by United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel Israel Science Foundation (ISF). Grant Number: 1349 Israel Science Foundation Israel Strategic Alternative Energy Foundation (I-SAEF) BBSRC. Grant Number: BB/L009951/1 Scottish Government Food, Land and People program Society for Applied Microbiology

Enzymatic profiling of cellulosomal enzymes from the human gut bacterium, Ruminococcus champanellensis, reveals a fine-tuned system for cohesin-dockerin recognition

Funded by United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel Israel Science Foundation (ISF). Grant Number: 1349 Israel Science Foundation Israel Strategic Alternative Energy Foundation (I-SAEF) BBSRC. Grant Number: BB/L009951/1 Scottish Government Food, Land and People program Society for Applied Microbiology

Contributions Of the Human Medial Prefrontal Cortex To Associative Recognition Memory: Evidence From Functional Neuroimaging

Neuroimaging studies of episodic memory, or memory of events from our personal past, have predominantly focused their attention on medial temporal lobe (MTL). There is growing acknowledgement however, from the cognitive neuroscience of memory literature, that regions outside the MTL can support episodic memory processes. The medial prefrontal cortex is one such region garnering increasing interest from researchers. Using behavioral and functional magnetic resonance imaging measures, over two studies, this thesis provides evidence of a mnemonic role of the medial PFC. In the first study, participants were scanned while judging the extent to which they agreed or disagreed with the sociopolitical views of unfamiliar individuals. Behavioral tests of associative recognition revealed that participants remembered with high confidence viewpoints previously linked with judgments of strong agreement/disagreement. Neurally, the medial PFC mediated the interaction between high-confidence associative recognition memory and beliefs associated with strong agree/disagree judgments. In an effort to generalize this finding to well-established associative information, in the second study, we investigated associative recognition memory for real-world concepts. Object-scene pairs congruent or incongruent with a preexisting schema were presented to participants in a cued-recall paradigm. Behavioral tests of conceptual and perceptual recognition revealed memory enhancements arising from strong resonance between presented pairs and preexisting schemas. Neurally, the medial PFC tracked increases in visual recall of schema-congruent pairs whereas the MTL tracked increases in visual recall of schema-incongruent pairs. Additionally, ventral areas of the medial PFC tracked conceptual components of visual recall specifically for schema-congruent pairs. These findings are consistent with a recent theoretical proposal of medial PFC contributions to memory for schema-related content. Collectively, these studies provide evidence of a role for the medial PFC in associative recognition memory persisting for associative information deployed in our daily social interactions and for those associations formed over multiple learning episodes. Additionally, this set of findings advance our understanding of the cognitive contributions of the medial PFC beyond its canonical role in processes underlying social cognition.

Object detection and recognition in complex scenes

Dissertação de Mestrado, Engenharia Informática, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Antitumor effect and mechanism of action of polysaccharides extracted from Polygonum perfoliatum L whole plant in human lung carcinoma A549 cell line

Purpose: To optimize the extraction conditions of polysaccharides from Polygonum perfoliatum L. (PSDP) and to evaluate their anti-tumor activities on A549 cell line. Methods: Extraction of PSDP was optimized using Box-Behnken design (BBD). Three factors of response surface methodology (RSM) including extraction time, ratio of water to raw material and number of extractions were employed to optimize the yield of PSDP. The cytotoxic effect of PSDP on human lung carcinoma A549 cell line was evaluated in vivo, while its effects on expressions of caspase3, caspase-9, Bcl-2 and Bax were determined by western blot assay. Result: BBD was significant and applicable to PSDP extraction. Based on the contour plots, response surface plots and variance analysis, it predicted that the optimum conditions for PSDP extraction were: 1.58 h (extraction time); 30.18 mL/g (ratio of water to raw material); and 2.02 (number of extractions). PSDP had significant inhibitory effect on the growth of A549 cells in a concentration- and timedependent manner (p < 0.05). After treatment with PSDP, caspase-3, caspase-9 and Bax were significantly up-regulated (p < 0.05), whereas Bcl-2 was down-regulated, all concentration-dependently. Conclusion: RSM analysis is an appropriate method to optimize PSDP extraction. The results also indicate that PSDP has significant anti-tumor effect against A549 cells, most likely via inducing mitochondria-mediated apoptosis.