Biodegradable amphiphilic PEG-PCL-PEI triblock copolymers designed for the self-assembly of multifunctional gene carriers

The great promise that gene therapy holds is the opportunity of directly introducing genetic material into cells for a causal therapy of yet incurable diseases. One promising way to achieve that goal is the usage of non-viral delivery vehicles, constructed from amphiphilic block copolymers. This thesis presents the establishment of a multifunctional PEG-PCL-PEI block copolymer platform, designed for multifunctional gene delivery. Across the scientific disciplines of chemistry, chemical physics, pharmacy and biomedicine the underlying work covers all aspects of non-viral gene delivery: In a first step, block copolymers were synthesised and characterised. In a systematic approach a library of compounds with varying hydrophilic/hydrophobic ratio was established. Subsequently, polymers were assembled into gene carriers followed by a non-invasive structural characterisation. Most importantly it was noticed, that polymers hydrophilic in nature formed smaller micelle-like carriers, whereas hydrophobic polymers aggregated to larger particle-like assemblies. In that vein, carrier features such as colloidal stability and toxicity were found to depend on chemical composition. Second, the nucleic acid loading process was optimised. Herein it was the overall goal to manufacture compactly condensed carrier complexes by understanding the basic principles of the electrostatic loading procedure. It was hypothesised that a more homogeneous fusion of charges is supposed to lead to superior carrier complexes. In that line, a microfluidic mixing technique, bringing cationic polymer and nucleic acid together at a constant ratio during the entire mixing process, was found to be the most promising technique. Ultimately, gene delivery carriers with superior colloidal stability, RNA protection and transfection efficiency were manufactured and process parameters were optimised with the help of a central composite design. Third, as a prerequisite for effective in vivo usage, carriers were transferred into stable ready-to-use formulations by lyophilisation with the help of glucose as a lyoprotectant. Unloaded nano-suspensions could be restored after rehydration by addition of small amounts of glucose. Upon loading of those rehydrated carriers, no significant difference in complex size or transfection efficiency was observed as compared to freshly-prepared ones. Moreover, the stabilisation of pre-formed carrier/siRNA complexes is feasible at elevated N/P and higher glucose concentrations. Fourth, most promising carriers where tested for their in vitro and in vivo transfection efficiency. Vectors constructed from rather hydrophobic block copolymers showed superior transfection efficiency, whereas poor performance was found in case of predominantly hydrophilic ones in a good correlation in vitro and in vivo. FACS studies revealed that this might possibly be due to reduced cell uptake of carriers with thicker PEG shell preventing cell interaction. In that way a yet active vector with diminished toxicity as compared to PEI homopolymers was evaluated. Fluorescent microscopy images of murine lung tissue revealed emission predominantly in the alveolar region, rendering this carrier system as promising for local treatment of airway diseases. Finally, the feasibility of multifunctional carrier co-loading and FRET-monitored nucleic acid unpacking was approved. Double-labelled nano-carriers emitted light at the acceptor’s emission wavelength upon donor excitation, proving successful FRET-effect and hence, complex integrity. The ability of dual loading is especially useful for “theranostic” purposes or co-delivery of nucleic acids and drugs. FRET-switching functionality may be advantageous for monitoring complex stability and nucleic acid unpacking. In view of prospective experiments, to circumvent the observed charge-toxicity-relationship, carriers are supposed to be taken up by the target tissue in a selective way. This can be achieved, up to a certain degree, via targeting ligands. Rather hydrophilic carriers with thicker PEG shells, increased colloidal stability and reduced toxicity represent the ideal candidates for this modification. In the long term, required work is evident: the development of more effective non-viral vectors and conquering the yet severe toxicity effects of current delivery systems. By a deeper understanding in the mechanistic aspects of the gene delivery process plus a rational vector design we are on the right track to achieve that goal

The Image of Adventure in Literature, Media, and Society: 2019 SASSI Conference Proceedings

Conference proceedings of the 2019 meeting of the Society for the Academic Study of Social Imagery (SASSI). Selected, refereed essays on the conference theme of The Image of ADVENTURE in Literature, Media, and Society

Maintaining Integrity II: Further Thoughts on Ethics and Original Literature

I have previously argued (Endres, 1987) that allowing original literature in forensics oral interpretation is a bad thing. While I remain true to that sentiment, my focus of blame is shifting from the act itself to the state of the activity, i.e. it seems that lack of policy is the primary culprit which allows the use of original literature to impugn forensics integrity. The primary focus of this essay is on the ethical concerns surrounding the use of original literature, and how the introduction of policy may help preclude unethical behavior. This analysis will first recap arguments from my previous essay, then address ethics from both a pragmatic and philosophical stance, and conclude with justification for policy development

An Examination of the Sabbatical Year in Leviticus 25 and Its Implications for Academic Practice

This article examines the concepts of Sabbatical Year and its connections with the concept as practiced in academia. First, the article examines the sabbatical year as portrayed in the Hebrew scriptures. Next, definitions and practices of the sabbatical year in academia is outlined. Finally, connections between the two forms of sabbatical is analyzed, with conclusions drawn about the role the Leviticus sabbatical can play in the understanding and execution of academic leave. While the purpose of the academic sabbatical year is quite different, academicians can learn from the lessons of Leviticus, and approach their leaves of absence with a more obligatory yet relational sense of its purpose, and a more flexible and transformation-focused sense of its practice

The Image of Rebirth in Literature, Media, and Society: 2017 SASSI Conference Proceedings

Conference proceedings of the 2017 meeting of the Society for the Academic Study of Social Imagery (SASSI). Selected, refereed essays on the conference theme of The Image of REBIRTH in Literature, Media, and Society

The Image of Redemption in Literature, Media, and Society: 2018 SASSI Conference Proceedings

Conference proceedings of the 2018 meeting of the Society for the Academic Study of Social Imagery (SASSI). Selected, refereed essays on the conference theme of The Image of REDEMPTION in Literature, Media, and Society

Imprecise Imputation: A Nonparametric Micro Approach Reflecting the Natural Uncertainty of Statistical Matching with Categorical Data

We develop the first statistical matching micro approach reflecting the natural uncer- tainty arising during the integration of categorical data. A complete synthetic file is obtained by imprecise imputation, replacing missing entries by sets of suitable values. We discuss three imprecise imputation strategies and raise ideas on potential refine- ments by logical constraints or likelihood-based arguments. Additionally, we show how imprecise imputation can be embedded into the theory of finite random sets, providing tight lower and upper bounds for parameters. Our simulation results corroborate that their narrowness is practically relevant and that they almost always cover the true parameters

Imprecise Imputation: A Nonparametric Micro Approach Reflecting the Natural Uncertainty of Statistical Matching with Categorical Data

We develop the first statistical matching micro approach reflecting the natural uncer- tainty arising during the integration of categorical data. A complete synthetic file is obtained by imprecise imputation, replacing missing entries by sets of suitable values. We discuss three imprecise imputation strategies and raise ideas on potential refine- ments by logical constraints or likelihood-based arguments. Additionally, we show how imprecise imputation can be embedded into the theory of finite random sets, providing tight lower and upper bounds for parameters. Our simulation results corroborate that their narrowness is practically relevant and that they almost always cover the true parameters

Codon-biased translation can be regulated by wobble-base tRNA modification systems during cellular stress responses

tRNA (tRNA) is a key molecule used for protein synthesis, with multiple points of stress-induced regulation that can include transcription, transcript processing, localization and ribonucleoside base modification. Enzyme-catalyzed modification of tRNA occurs at a number of base and sugar positions and has the potential to influence specific anticodon-codon interactions and regulate translation. Notably, altered tRNA modification has been linked to mitochondrial diseases and cancer progression. In this review, specific to Eukaryotic systems, we discuss how recent systems-level analyses using a bioanalytical platform have revealed that there is extensive reprogramming of tRNA modifications in response to cellular stress and during cell cycle progression. Combined with genome-wide codon bias analytics and gene expression studies, a model emerges in which stress-induced reprogramming of tRNA drives the translational regulation of critical response proteins whose transcripts display a distinct codon bias. Termed Modification Tunable Transcripts (MoTTs), we define them as (1) transcripts that use specific degenerate codons and codon biases to encode critical stress response proteins, and (2) transcripts whose translation is influenced by changes in wobble base tRNA modification. In this review we note that the MoTTs translational model is also applicable to the process of stop-codon recoding for selenocysteine incorporation, as stop-codon recoding involves a selective codon bias and modified tRNA to decode selenocysteine during the translation of a key subset of oxidative stress response proteins. Further, we discuss how in addition to RNA modification analytics, the comprehensive characterization of translational regulation of specific transcripts requires a variety of tools, including high coverage codon-reporters, ribosome profiling and linked genomic and proteomic approaches. Together these tools will yield important new insights into the role of translational elongation in cell stress response.National Science Foundation (U.S.) (CHE-1308839)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology Center. Infectious Disease Research Program