Polycationic Polymers in Nucleic Acid Delivery: Membrane Interaction and Cellular Responses.

In order to optimize the design of gene delivery vector systems using polycationic polymers, their internalization pathway needs to be extensively studied. In chapter 2, Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers (G7-NH2) with amine termination were prepared to investigate polymer/cell membrane interaction. C6 cells were employed in this experiment because of their unique characteristic of inherently lacking GM1 but still possessing downstream requirements for successful GM1 mediated endocytosis. This resulted in the reported binary experiment where C6 cells without and with GM1 did not show differential G7-NH2 uptake supporting the hypothesis that GM1 mediated endocytosis is not the primary pathway for G7-NH2 internalization. In addition to understanding uptake mechanisms, efforts to improve the polymer-based vector systems have assumed that cells play a passive role and don’t actively respond to these polymer vector systems. In chapter 3, I will show how cells respond to different polymer systems by activating different levels of cytosolic nucleases and how this nuclease activity can be measured with the help of Molecular Beacon (MB). We also learn that membrane porosity is not the reason for nuclease activation. This nuclease activation decreases gene expression, ultimately decreasing polymer efficiency. In doing this, I show that it is possible to screen multiple polymer-based vector systems, thereby increasing the probability of identifying and designing an efficient polymer vector system. In chapter 4, I will concentrate on DNA degradation characteristics of these cytosolic nucleases. High-throughput sequencing was used to identify and quantify degradation patterns of these cytosolic nucleases on a plasmid. We will also see that S1 nuclease (a known nuclease) has similar degradation pattern as cytosolic nucleases. Designing polymer vector systems that protect these labile sites on DNA can improve gene expression. In chapter 6, invention of The Endocutter, a gastrointestinal device that cuts clots, cuts costs and saves lives is described. This device was designed to be compatible with both single and dual channel endoscope for easy adaptation. Endocutter is easy to use and has been shown to be safe and efficient in removing blood clots both in-vitro and in-vivo systems (http://www.youtube.com/watch?v=gxnB_kuzOsQ )PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/102455/1/rrattan_1.pd

Quantification of cytosolic plasmid DNA degradation using high‐throughput sequencing: implications for gene delivery

Background Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Methods Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and sequencing approaches. Results The poly(A) region between the β‐lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex‐treated cytosol. The second most likely site, at the 5' end of the β‐lactamase gene, was identified by gel electrophoresis and sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β‐lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β‐lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication ( OriC ). Conclusions High‐throughput plasmid sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression. Copyright © 2014 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106947/1/jgm2761.pd

Multi-objective optimization of TW-ECSM process parameters for machining of advanced non-conducting material

52-61Travelling wire electrochemical spark machining (TW-ECSM) is newly evolved and developed hybrid machining process for the machining of advanced non-conducting materials which possess significant values of the properties like high strength, high wear and fatigue resistance, high refractoriness and high strength to weight ratio, etc. The control parameters like voltage, wire feed rate, electrolyte concentration and inter-electrode gap were selected as Input Parameters and Material Removal Rate (MRR), and Surface Roughness (SR) were the corresponding output responses. In present work, for multiobjective optimization and purpose of better control of machining parameters, three approaches, grey-relational analysisprincipal component analysis (GRA-PCA), fuzzy logic and desirability function approach are used to determine the optimal combination of TW-ECSM process variables. Results of fuzzy logic and GRA-PCA approach are found comparable while desirability function approach is found to be capable of predicting the optimal responses at such levels of process variables also at which experiments are not performed. Consequences of the applied approach in the present work are also validated by conducting the confirmatory experiments and results are found in well agreement with the predicted results

Structural Health Monitoring of Existing Reinforced Cement Concrete Buildings and Bridge Using Nondestructive Evaluation with Repair Methodology

Sustainable development means the utilization of resources at a rate less than the rate at which they are renewing. In India infrastructure industry is growing rapidly due to globalization and raising awareness. In the present study, challenges faced by countries like India are to sustain the existing expectations with limited resources available. Reinforced Concrete (RC) structure may suffer several types of defects that may jeopardize their service life. This chapter deals with condition assessment and repair of RCC (G+3) building situated at Northern part of the country. There are various techniques available for repair and rehabilitation of reinforced concrete structures. From a maintenance point of view, it is essential to take up the strength assessment of an existing structure. So, to find out the reason behind the deterioration of the concrete structures some of the NDT and partially destructive technique are used. The NDT tests conducted during this study are Rebound Hammer, Ultra-sonic Pulse Velocity, Concrete resistivity Meter, Ferro-scanning and Carbonation, etc. This chapter helps to explains, how identified the different parameters of distress building like strength, density, level of corrosion and amount of reinforcement. On basis of these results, apply a repair methodology to revert back the strength parameters of the buildings

Design and Evaluation of Tumor‐Specific Dendrimer Epigenetic Therapeutics

Histone deacetylase inhibitors (HDACi) are promising therapeutics for cancer. HDACi alter the epigenetic state of tumors and provide a unique approach to treat cancer. Although studies with HDACi have shown promise in some cancers, variable efficacy and off‐target effects have limited their use. To overcome some of the challenges of traditional HDACi, we sought to use a tumor‐specific dendrimer scaffold to deliver HDACi directly to cancer cells. Here we report the design and evaluation of tumor‐specific dendrimer–HDACi conjugates. The HDACi was conjugated to the dendrimer using an ester linkage through its hydroxamic acid group, inactivating the HDACi until it is released from the dendrimer. Using a cancer cell model, we demonstrate the functionality of the tumor‐specific dendrimer–HDACi conjugates. Furthermore, we demonstrate that unlike traditional HDACi, dendrimer–HDACi conjugates do not affect tumor‐associated macrophages, a recently recognized mechanism through which drug resistance emerges. We anticipate that this new class of cell‐specific epigenetic therapeutics will have tremendous potential in the treatment of cancer.Targeting tumors via epigenetics: Histone deacetylase inhibitors (HDACi) alter the epigenetic state of tumors and are promising therapeutics for cancer. Although studies with HDACi have shown promise in some cancers, variable efficacy and off‐target effects have limited their use. Here we report the design and evaluation of a tumor‐specific dendrimer–HDACi.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111996/1/open201402141.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/111996/2/open201402141-sup-0001-misc_information.pd

Multi-objective optimization of TW-ECSM process parameters for machining of advanced non-conducting material

Travelling wire electrochemical spark machining (TW-ECSM) is newly evolved and developed hybrid machining process for the machining of advanced non-conducting materials which possess significant values of the properties like high strength, high wear and fatigue resistance, high refractoriness and high strength to weight ratio, etc. The control parameters like voltage, wire feed rate, electrolyte concentration and inter-electrode gap were selected as input parameters and material removal rate (MRR) and surface roughness (SR) were the corresponding output responses. In present work, for multi-objective optimization and purpose of better control of machining parameters, three approaches, grey-relational analysis-principal component analysis (GRA-PCA), fuzzy logic and desirability function approach are used to determine the optimal combination of TW-ECSM process variables. Results of fuzzy logic and GRA-PCA approach are found comparable while desirability function approach is found to be capable of predicting the optimal responses at such levels of process variables also at which experiments are not performed. Consequences of the applied approach in the present work are also validated by conducting the confirmatory experiments and results are found in well agreement with the predicted results

Polyplex-Induced Cytosolic Nuclease Activation Leads to Differential Transgene Expression

Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane’s electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI, and little activity for L-PEI. jetPEI and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp

Effects of Conservation Tillage and Nutrient Management Practices on Soil Fertility and Productivity of Rice (Oryza sativa L.)–Rice System in North Eastern Region of India

Over centuries and even today, traditional farming practices are well performed without any ecological degradation. However, management practice such as conservative tillage combined with nutrient and residue could increase the crop production as well as soil fertility. A three-year replicated study was conducted to assess the effects of agronomic modification of traditional farming practices on productivity and sustainability of rice (wet season)–rice (dry season) system (RRS). The replacement of farmers practice (T2) with conservation effective tillage (no-till (NT)) and integrated nutrient management (INM) practice along with 30% residue retention (T5) enhanced the straw, root and biomass yield of both wet season rice (WR), dry season rice (DR) and system as a whole over T2. Treatment T5 recorded significantly lower soil bulk density (ρb) and higher pH than the T2 after three years of the experiment. Further, treatment T5 increased total soil organic carbon (2.8%), total soil organic carbon stock (2.8%), carbon sequestration rate (336.5 kg ha−1 year−1), cumulative carbon stock (142.9%) and carbon retention efficiency (141.0%) over T2 of 0–20 cm depth after three year. The soil microbial biomass carbon concentration was significantly the highest under T5. Similarly, the dehydrogenase activity was the maximum under T5. Adoption of conservation tillage and nutrient management practice involving NT and INM along with residue retention can enhance the system productivity, and C and N sequestration in paddy soils is thereby contributing to the sustainability of the RRS