Recombinant cationic biopolymers for nucleic acid delivery

Delivery of nucleic acids in an efficient and safe manner remains an unmet need in the biological sciences. Whether this means using gene therapy to introduce functional genes to cells, or using RNA interference mechanisms to silence misregulated proteins, the main hindrance in these approaches to disease mitigation, is the lack of a suitable delivery platform. The first chapter is a literature review of the currently available technology for nucleic acid delivery including viral vectors as well as cationic lipids and polymers. Herein the advantages and disadvantages of both viral and non-viral delivery are discussed. In Chapter II, a biopolymer featuring a lysine-histidine (KH) condensing region fused to a targeting motif is discussed. The purpose of this study was to examine the effect of architecture on transfection efficiency of plasmid DNA through a series of in vitro biological and biochemical assays. Chapter III describes a recombinant biopolymer which features an arginine-histidine (RH) DNA condensing region, a targeting motif for HER2, and endosomal release fusogenic peptide, and a nuclear localization signal. Each domain was tested for functionality through in vitro assays. The multifunctional biopolymer demonstrated selective and efficient delivery of plasmid DNA and gene expression in SK-OV-3 cells. In Chapter IV the RH based biopolymers were characterized and evaluated in terms of their ability to deliver nucleic acids to either the cytoplasm or cell nucleus. Past delivery vectors have often emphasized extracellular barriers to entry and success meant internalization of the particle. Intracellular trafficking, however is also a key compenent in successful delivery. The objective of this study was to design a biopolymer that can be programmed via its amino acid sequence to deliver siRNA specifically to cytoplasm. By modifying the amino acid sequence, the same biopolymer can also be programmed to deliver pDNA to the cell nucleus. Intracellular trafficking was observed through fluorescent microscopy and assays were conducted to demonstrate either siRNA knockdown or delivery of a suicide gene/prodrug combination. In the research presented, we describe a non-viral biopolymer system that is able to overcome the extracellular and intracellular barriers to nucleic acid delivery

The control of blood flow in skeletal muscle arterioles

"December 2010.""A Thesis presented to the Faculty of the Graduate School at the University of Missouri--Columbia In Partial Fulfillment of the Requirements for the Degree Master of Science."Thesis supervisor: Dr. Steven Segal.Skeletal muscle arterioles dilate in response to application of acetylcholine (ACh), eliciting a conducted vasodilation (CVD) that travels along unbranched segments without decrement. CVD is known to entail cell-to-cell transmission of hyperpolarization along the endothelium via gap junction channels, a purely passive mechanism. In the present thesis I study CVD in bifurcating arteriolar networks, where the pathway for hyperpolarizing current expands compared to unbranched arterioles, to test for an active component to CVD. In a separate subset of arterioles, the effect of augmenting vasomotor tone on CVD was tested using elevated O2 or phenylephrine (PE) in the superfusion solution vs. control. Male C57BL/6 mice (n=13; 10-13 weeks old) were anesthetized with pentobarbital sodium (50mg/kg, intraperitoneal injection) and maintained at 37 [degrees]C. The cremaster was carefully exteriorized and spread onto a transparent Sylgard pedestal. The tissue was maintained at 34 [degrees]C with continuous superfusion of physiologic saline solution. Microiontophoresis of ACh evoked non-decremental CVD in both unbranched and bifurcating arterioles, supporting the role of an active component in CVD. Further, augmenting vasomotor tone with PE attenuated non-decremental CVD, whereas increasing vasomotor tone with elevated O2 did not alter CVD. In summary, arteriolar networks in the mouse cremaster muscle exhibit robust dilation to ACh, which conducts along arterioles and across branch points without decrement, suggesting the contribution of both active and passive mechanisms. In preparations exhibiting poor spontaneous vasomotor tone, elevated O2 can be used to improve resting tone without impacting CVD.Includes bibliographical references (pages 51-58)

In vitro identification and in silico utilization of interspecies sequence similarities using GeneChip(® )technology

BACKGROUND: Genomic approaches in large animal models (canine, ovine etc) are challenging due to insufficient genomic information for these species and the lack of availability of corresponding microarray platforms. To address this problem, we speculated that conserved interspecies genetic sequences can be experimentally detected by cross-species hybridization. The Affymetrix platform probe redundancy offers flexibility in selecting individual probes with high sequence similarities between related species for gene expression analysis. RESULTS: Gene expression profiles of 40 canine samples were generated using the human HG-U133A GeneChip (U133A). Due to interspecies genetic differences, only 14 ± 2% of canine transcripts were detected by U133A probe sets whereas profiling of 40 human samples detected 49 ± 6% of human transcripts. However, when these probe sets were deconstructed into individual probes and examined performance of each probe, we found that 47% of human probes were able to find their targets in canine tissues and generate a detectable hybridization signal. Therefore, we restricted gene expression analysis to these probes and observed the 60% increase in the number of identified canine transcripts. These results were validated by comparison of transcripts identified by our restricted analysis of cross-species hybridization with transcripts identified by hybridization of total lung canine mRNA to new Affymetrix Canine GeneChip(®). CONCLUSION: The experimental identification and restriction of gene expression analysis to probes with detectable hybridization signal drastically increases transcript detection of canine-human hybridization suggesting the possibility of broad utilization of cross-hybridizations of related species using GeneChip technology

Difficulties in estimating the human burden of canine rabies

Current passive surveillance data for canine rabies, particularly for the regions where the burden is highest, are inadequate for appropriate decision making on control efforts. Poor enforcement of existing legislation and poor implementation of international guidance reduce the effectiveness of surveillance systems, but another set of problems relates to the fact that canine rabies is an untreatable condition which affects very poor sectors of society. This results in an unknown, but potentially large proportion of rabies victims dying outside the health system, deaths that are unlikely to be recorded by surveillance systems based on health center records. This article critically evaluates the potential sources of information on the number of human deaths attributable to canine rabies, and how we might improve the estimates required to move towards the goal of global canine rabies elimination

Canine Haiku: Yellow Ball

Poetry by Julie Andreyev and Tom

Declining Visceral Leishmaniasis in Malta

Aims: To study visceral leishmaniasis (VL) trends in Malta. Methods: Analysis of epidemiological and clinical trends, a veterinary questionnaire and questioning a canine laboratory testing facility. Results: A decline in VL in the past 25 years (1980-2005) was noted for both paediatric (p<0.001) and adult (p=0.002) populations. No seasonal variation in infection rates was found. Serological testing was reliable and a useful adjunct to bone marrow diagnosis. Treatment with sodium stibogluconate was highly effective and there were no permanent sequelae associated with disease or treatment. Vets are encountering less canine VL with no differences in localities Discussion: The decline in VL was paralleled by a decline in the stray canine reservoir population and by better management of canine infection in both sanctuaries and in domestic settings.peer-reviewe