Models for the optical dispersion relations of amorphous semiconductors

The development of empirical expressions for the spectral dependence of the real and imaginary components of the dielectric function have proven useful over the years. These expressions can be applied in the analysis of optical data, including the interpretation of transmittance, reflectance, and ellipsometric spectra obtained from thin film semiconductors. Also, they can be used in performance simulations of optoelectronic devices, such as solar cells. In this thesis, a critical comparison of a number of empirical models for the real and imaginary components of the dielectric function is performed. A number of crystalline and disordered semiconductors of interest are considered in this analysis. Also, a new means of numerically evaluating the real component of the dielectric function from the imaginary component is developed based on a Kramers-Kronig relation. Applications of this approach are discussed

Minimal siRNA set cover heuristic for gene family knockdown

RNA interference (RNAi) is a highly evolutionally conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homologous mRNA sequences, siRNA (small interfering RNA are a class of 20-25 nucleotide-long double-stranded RNA molecules) is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. We focus on the problem of gene family knockdown by using the minimal number of siRNAs. The problem is to determine the minimal number of siRNAs required to knockdown a family of genes targeted by these siRNAs. This is a minimal set covering problem, and hence it is NP-hard. In this thesis, we explore a number of heuristic optimization methods for the minimal siRNA covering problem. Such methods include evolutionary heuristics, as well as novel greedy methods, applied for the first time to the minimal siRNA cover problem. Preliminary experiments with genetic algorithms show significant reduction in the siRNA cover size, when compared with branch & bound and probabilistic greedy. We are currently implementing novel greedy methods which are variants of well-known feature subset selection algorithms. In such methods, we define criterion functions over a collection of siRNA subsets to help us decide which subset is best to be included in a candidate solution. We use three gene families: the FREP genes from Biomphalaria glabrata and the olfactory genes from Caenorhabditis elegans. We also conducted experiments on one artificial data set

Contributions of Angiomotin-Like-1 on Astrocytic Morphology: Potential Roles in Regulating Connexin-43-Based Astrocytic Gap Junctions, Remodeling the Actin Cytoskeleton and Influencing Cellular Polarity

Indiana University-Purdue University Indianapolis (IUPUI)Glioblastoma is a lethal cancer that arises from support cells in the nervous system and kills around 20,000 people in the United States each year. While much is known about the highly malignant primary glioblastoma, the natural history of lower grade glioma (LGG) is less understood. While the majority of LGGs are initiated by a mutation in isocitrate dehydrogenase, the events leading to their malignant progression into a grade IV tumor are not known. Analysis of primary tumor sample data has revealed that low transcript levels of Angiomotin-like-1 (AmotL1) strongly associate with poor outcomes of patients with these cancers. Follow-up RNA-sequencing of human embryonic astrocytes with AmotL1 silencing revealed the downregulation of many transcripts that encode proteins mediating gap junctions (GJ) between astrocytes, especially connexin-43 (Cx43). Cx43 protein oligomerizes to form functional channels comprising the astrocytic GJ. AmotL1 knockdown through RNA interference decreases Cx43 transcript and protein levels while increasing its distribution to GJs. This suggests increased GJ formation and intercellular communication, as similar localization patterns are observed in differentiated astrocytes. Astrocytes with AmotL1 knockdown also display a pronounced pancake-like morphology, suggesting that the actin cytoskeleton is affected. Imaging reveals that cells with reduced AmotL1 have characteristic losses in both stress fibers and focal actin under the cell body but notable increases in cortical F-actin. Consistent with previous studies, AmotL1 may promote increases in the number and thickness of F-actin fibers. Because actin binding to related angiomotins is inhibited by phosphorylation from the LATs kinases, I define the effects of expressing wildtype AmotL1 versus mutants that mimic or prevent phosphorylation by LATs1/2. Interestingly, expression of AmotL1 S262D in combination with NEDD4-1, a ubiquitin ligase, results in a profound loss of actin stress fibers. Dependence on NEDD4-1 suggests that this phenotype is due to the induced degradation of proteins that promote F-actin, e.g. RhoA. These results directly support a model in which phosphorylated AmotL1 specifically inhibits F-actin formation as opposed to unphosphorylated AmotL1 which is known to promote stress fiber formation. Thus, in addition to regulating polarity and YAP/TAZ transcriptional co-activators, AmotL1 plays major functions in dictating cellular F-actin dynamics.2021-01-0

Novel Sequence-Based Method for Identifying Transcription Factor Binding Sites in Prokaryotic Genomes

Computational techniques for microbial genomic sequence analysis are becoming increasingly important. With next–generation sequencing technology and the human microbiome project underway, current sequencing capacity is significantly greater than the speed at which organisms of interest can be experimentally probed. We have developed a method that will primarily use available sequence data in order to determine prokaryotic transcription factor binding specificities. The prototypical prokaryotic transcription factor: TF) contains a helix–turn–helix: HTH) fold and bind DNA as homodimers, leading to their palindromic motif specificities. The connection between the TF and its promoter is based on the autoregulation phenomenon noticed in E. coli. Approximately 55% of the TFs analyzed were estimated to be autoregulated. Our preliminary analysis using RegulonDB indicates that this value increases to 79% if one considers the neighboring operons. Given the TF family of interest, it is necessary to find the relevant TF proteins and their associated genomes. Due to the scale–free network topology of prokaryotic systems, many of the transcriptional regulators regulate only one or a few operons. Within a single genome, there would not be enough sequence–based signal to determine the binding site using standard computational methods. Therefore, multiple bacterial genomes are used to overcome this lack of signal within a single genome. We use a distance–based criteria to define the operon boundaries and their respective promoters. Several TF–DNA crystal structures are then used to determine the residues that interact with the DNA. These key residues are the basis for the TF comparison metric; the assumption being that similar residues should impart similar DNA binding specificities. After defining the sets of TF clusters using this metric, their respective promoters are used as input to a motif finding procedure. This method has currently been tested on the LacI and TetR TF families with successful results. On external validation sets, the specificity of prediction is ∼80%. These results are important in developing methods to define the DNA binding preferences of the TF protein residues, known as the “recognition code”. This “recognition code” would allow computational design and prediction of novel DNA–binding specificities, enabling protein-engineering and synthetic biology applications

Non-viral nanoparticles for RNA interference : Principles of design and practical guidelines

Ribonucleic acid interference (RNAi) is an innovative treatment strategy for a myriad of indications. Non-viral synthetic nanoparticles (NPs) have drawn extensive attention as vectors for RNAi due to their potential advantages, including improved safety, high delivery efficiency and economic feasibility. However, the complex natural process of RNAi and the susceptible nature of oligonucleotides render the NPs subject to particular design principles and requirements for practical fabrication. Here, we summarize the requirements and obstacles for fabricating non-viral nano-vectors for efficient RNAi. To address the delivery challenges, we discuss practical guidelines for materials selection and NP synthesis in order to maximize RNA encapsulation efficiency and protection against degradation, and to facilitate the cytosolic release of oligonucleotides. The current status of clinical translation of RNAi-based therapies and further perspectives for reducing the potential side effects are also reviewed. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

A screening for novel immune-checkpoints identifies CAMK1D as a kinase responsible for immune resistance in multiple myeloma

Despite tremendous progresses in cancer immunotherapy, a plethora of tumor patients is still refractory to current immunotherapeutic strategies. Unresponsiveness to therapy is ascribed to the ability of cancer cells to elude the immunosurveillance. Indeed, by taking advantage of different immune-checkpoint molecules tumor cells can either dampen immune cell functionality or promote tumor cell resistance towards immune attack. The current study aimed at identifying novel tumor-associated immune-checkpoint molecules by developing a RNAi high-throughput (HTP) screening and successively corroborate candidate genes whose blockade increases anti-tumor immune response. Hence, I generated stable luciferase expressing multiple myeloma cells, transfected them with a siRNA library targeting 2887 genes (enriched for kinases and surface-associated molecules) and co-cultured them with HLA-A2-matched patient-derived marrow-infiltrating lymphocytes (MILs). T cell-mediated killing of tumor cells was assessed by measuring the remaining luciferase activity of knocked down tumor cells. The HTP screening revealed 128 genes whose knockdown increased T cell-mediated tumor cell death more efficiently than the positive control CCR9. To validate the results, candidate genes were re-tested in a secondary screening that allowed to distinguish between genes altering tumor susceptibility towards MIL-mediated killing and those impairing MIL activity. Among the candidate immune-checkpoints the serine/threonine protein kinase CAMK1D was selected for extensive validation. Knockdown of CAMK1D resulted in increased tumor susceptibility towards MIL-mediated killing. In particular, CAMK1D was shown to support intrinsic tumor resistance towards T cell attack by interfering with the apoptotic signaling cascade. By directly interacting with effector caspases, CAMK1D inhibits caspases activation and activity via phosphorylation. In line, CAMK1D depletion sensitizes tumor cells to FasLinduced apoptosis by MILs. These results obtained in the hematological malignancy were further confirmed in uveal melanoma emphasizing the relevant role of CAMK1D in different tumor entities. Taken together, this study describes the establishment of a HTP-discovery platform to unravel the arsenal of immune-checkpoint molecules used by cancer cells to escape the immune system. The molecular pathway of CAMK1D is described highlighting the importance of discovering immune-checkpoints that mediate resistance towards T cell attack

Pulling the right viral levers: Engineering, screening and application of next-generation combinatorial AAV vectors

Adeno-associated viruses (AAVs) present powerful vectors for human gene therapy and biomedical research. They enable the delivery of transgenes to a broad range of target tissues. This allows persistent expression of reporter genes, therapeutic gene replacement and the delivery of control elements for manipulating endogenous gene expression. Transduction efficiency and specificity for on-target over off-target cells are critical factors driving vector safety and applicability. In this study, I engineered AAV capsids, promoters and knockdown tools with the goal of generating efficient and specific vector components for the next generation of cell-type-specific gene therapy vectors. In the first part of this doctoral work, I utilized Cas13d (CasRx) and short-hairpin (sh)RNA effectors to assess AAV-induced RNA degradation (knockdown). While CasRx showed promising knockdown of a Renilla luciferase reporter target, it failed to silence endogenous CD44, a potential driver of metabolic (non-alcoholic) steatohepatitis. shRNA effectors, however, allowed robust target knockdown of cellular RNAs (CD44 and ACE2) as well as SARS-CoV-2 viral RNA. Direct targeting of SARS-CoV-2 genomic RNA triggered the evolution of escape mutations within the viral target sites. This mutational escape was efficiently suppressed by multiplexing of three shRNAs in a single AAV vector, thereby allowing a sustained suppression of SARS-CoV-2 infection in Vero E6 cells. In the second part, I focused on improving screening conditions for promoters and AAV capsids. By assessing eYFP reporter expression in vivo for four promoter constructs individually, I could validate the findings of a previous promoter screen. This screen had utilized high-throughput barcode sequencing for parallel readout of a library of AAV-promoter constructs. I then applied this barcoding technique to dissect the activity of the GFAP promoter and truncated versions thereof. The GFAP promoter has previously mostly been used to induce astrocyte-specific transgene expression in the central nervous system. Strikingly, though, my results demonstrate a highly efficient GFAP promoter-driven transgene expression in human and murine hepatocytes. To optimize the directed evolution of AAV capsids, I modified conventional capsid library screening by altering selection parameters. This was achieved by (i) introducing a Cas9-based negative selection for the removal of unwanted variants from the capsid library, and (ii) by exploring and applying RNA-based functional selection. I could generate an RNA-based screening platform by driving the expression of cap from the ubiquitous CMV promoter instead of the endogenous p40. Both screening approaches proved applicable in cell culture settings. As RNA-driven selection offers a functional readout from both on- and off-target cells, I applied this approach for in vivo screening of an AAV6 peptide display library in mouse non-parenchymal liver cells. CMV promoter-driven cap expression enabled RNA-based readout of variant enrichment for on- and off-target cell-types. This demonstrated improved selectivity for RNA- over conventional DNA-based screening and facilitated the identification of functional, cell-type-specific capsid candidates. In conclusion, my results show the development of efficient combinatorial shRNA-based knockdown vectors for inhibiting CD44 expression or SARS-CoV-2 infection in vitro. Furthermore, I could implement improvements in capsid and promoter screening. This allowed the detection of highly functional variants with potential future applications in the development of novel gene therapy vectors

RNA Interference

RNA interference (RNAi), a hallmark of all biological sciences of twenty-first century, is an evolutionarily conserved and double-stranded RNA-dependent eukaryotic cell defense process. Opportunity to utilize an organisms own gene and to systematically induce and trigger RNAi for any desired sequence made RNAi an efficient approach for functional genomics, providing a solution for conventional longstanding obstacles in life sciences. RNAi research and application have significantly advanced during past two decades. This book RNA interference provides an updated knowledge and progress on RNAi in various organisms, explaining basic principles, types, and property of inducers, structural modifications, delivery systems/methodologies, and various successful bench-to-field or clinic applications and disease therapies with some aspects of limitations, alternative tools, safety, and risk assessment

Establishing the developmental function of the rhamnogalacturonan II component of pectin

The primary plant cell wall consists of a complex set of polysaccharides including pectin, cellulose and hemicelluloses that are critical for normal plant development. There are three major forms of pectin, rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HGA). Of these, the pectic polysaccharide RG-II, is the least abundant but the most complex. Despite this, RG-II is highly conserved among vascular plants, suggesting animportant function which is dependent upon structure. RG-II consists of four structurally welldefined side chains attached to a backbone of 1,4-linked galacturonic acid (GalA) residues and exists predominately as a dimer in plant cell walls. RG-II function has yet to be identified; however, mutations affecting RG-II structure have severe growth and development defects. 3-deoxy-D-manno-2-octulosonic acid (Kdo) is a rarely found sugar and is a component of the RGII side chain C. Kdo biosynthesis has been well characterised and a number of Kdo synthesis genes identified in Arabidopsis. Traditional gene knockout approaches to study the effect of disrupting Kdo biosynthesis have been limited by the apparent lethality of these mutants. Alternative approaches using partial knockout, inducible gene silencing and chemical approaches have being employed with the primary aim of specifically altering the structure of RG-II to determine the developmental function of RG-II. By combination of a GAL4/VP16 expression system and ALCR/alcA ethanol-switch to achieve temporal and spatial control of transgene expression, it has been possible to generate a genetic tool kit consisting of a series of Arabidopsis lines in which it should be possible to disrupt Kdo biosynthesis in specific tissues at strictly defined developmental stages. As a proof of concept the J0951/iKdsB line, in which expression of an AtKdsB antisense sequence is restricted to the epidermis and root cap in the presence of ethanol, is shown to be almost completely devoid of root hairs when grown under induced conditions. This result is suggestive of a role for RG-II in tip growth processes and is consistent with the phenotypes of null mutants in which a failure in pollen tube elongation results in gametophyte lethality. In silico and in vitro approaches are used to investigate the potential application of an inhibitor of AtKdsB, 2?-deoxy Kdo, as a tool for the disruption of CMP-Kdo synthesis in plants. Using homology modelling the Arabidopsis and E. coli enzymes are shown to have a near identical active site conformation. Using recombinantly expressed AtKdsB in enzyme kinetic and inhibition studies the substrate analogue 2?-deoxy-Kdo was shown to be a potent in vitro inhibitor of AtKdsB with a Ki of 1.26 ± 0.15 ??, consistent with measures of the Kd made by isothermal titration calorimetry (ITC) analysis. The 2?-deoxy-Kdo was subsequently applied in vivo and results in a severe inhibition of cell elongation of Arabidopsis root cells that can be partially rescued by either Kdo or boron. It is likely that 2?-deoxy-Kdo application disrupts CMP-Kdo biosynthesis with consequences for RG-II structure and dimer formation