Immunoregulatory Receptor Genetics, Expression, and Splicing Studies in Alzheimer’s Disease

Microglia are the resident immune cells of the brain, undertaking many critical tissue maintenance functions such as immune surveillance and phagocytosis. Microglial dysfunction has recently been identified as a multi-stage signature of many neurodegenerative diseases, including late-onset Alzheimer’s Disease (LOAD). Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in over thirty genes that modulate risk of developing LOAD. In the central nervous system, roughly half of these LOAD-associated genes are primarily expressed in microglia. The proteins encoded by these genes include cell surface receptors that contain either immunomodulatory tyrosine-phosphorylated activating motifs (ITAMs) or inhibitory motifs (ITIMs), including TREM2, CD33, and SIGLEC14. Here, I studied the molecular genetics underlying these three genes and their respective contributions to LOAD risk. First, I found that TREM2 undergoes extensive alternative splicing in multiple tissues, including brain. Total TREM2 expression is not different as a function of LOAD diagnosis (p = 0.1268), but TREM2 expression is increased by 34% in tissues with higher National Institute on Aging/Reagan Institute (NIARI) scores (p = 0.0033). I also found that a novel TREM2 isoform lacking exon 2, D2-TREM2, accounts for 11% of the total TREM2 mRNA in human brain, and that this splicing efficiency is not altered as a function of AD status (p = 0.4909) or brain pathology (p = 0.9502). I also found that the D2-TREM2 protein has similar subcellular localization to its parent TREM2 protein, as both are primarily retained in the Golgi apparatus. Next, I studied the exon 2-lacking CD33 isoform, D2-CD33. I developed an in vitro model to study the function of the D2-CD33 using a CRISPR-Cas9 approach in the U937 human monocyte cell line. After validating this model with sequencing, qPCR, and flow cytometry, I found that a nearby pseudogene, SIGLEC22P, was used as a repair template in approximately 10% of edited cells. This finding also provided the highest resolution to date of the clinically relevant anti-CD33 P67.6 antibody clone, gemtuzumab. Finally, I combined a recent LOAD GWAS with a protein quantitative trait loci (pQTL) study to uncover SIGLEC14 as a potentially overlooked LOAD risk factor. I found that a previously described SIGLEC14 genetic deletion occurs within a 692 bp crossover region. I also found additional copy number variation not previously described using both qPCR-based and in silico assays, with copy numbers identified ranging from zero to four. While SIGLEC14 deletion does correlate well with a proxy single nucleotide polymorphism (SNP), rs1106476, additional SIGLEC14 genomic copies do not correlate with this SNP. Further, the SIGLEC14 genomic deletions correlate stepwise with decreased SIGLEC14 expression (p = 0.0002), and also correlate significantly with decreased SIGLEC5 expression (p = 0.0389). In conclusion, microglial cell surface receptors are heavily implicated in the risk of developing LOAD, and these studies advance the field by adding to the molecular mechanisms which underlie their risk contribution. Further studies will be needed to address whether these findings can be translated clinically to either potential druggable targets or biomarkers

A Novel Forensic DNA Profiling Method Based On Molecular Beacons Without Dna Purification

Analysis of polymorphisms in nucleic acid sequences provides the basis for identification of individuals and their genetic deficiencies. Currently, the accepted method of analysis for profiling is Short Tandem Repeat (STR) profiling. This is a lengthy process, typically taking up to 3 days. The time necessary to generate an STR profile, along with the ever-increasing reliance on DNA to solve crimes, has led to a large DNA sample backlog, with violent crime turnaround taking an average of 103 days. The time and resource investment required for STR analysis is significant, and not all samples generate useful profiles. The current methods for use of STR technologies require an isolated template sample. This isolation typically requires hours of extractions and incubations, followed by still more time for analysis. The considerable length of time necessary for this process makes it inherently expensive, while also increasing the backlog. A universal protocol allowing amplification from various, frequently used samples would allow extremely rapid sampling and results. Further, these templates are faster and easier to amplify than standard STRs, which reduces the risk of resources and time on a sample which may not amplify. Common forensic samples include blood, hair, saliva, and buccal swabs. Using a single, universal protocol to prepare these samples for analysis without extensive isolation allows the simultaneous preparation of multiple samples. Accordingly, this work explores the development of a preparatory method for multiple forensic samples coupled with the optimization of polymerase chain reaction conditions to facilitate the real-time monitoring of the interaction of molecular beacons (MBs) with the template. These MBs can then be used to identify the presence or absence of specific nucleotide polymorphisms. This increase in throughput has extensive application in forensic and medical applications

Jesus\u27 Resurrection: A Historical Investigation

This thesis will investigate Jesus\u27 resurrection from a historical perspective. This investigation will begin by evaluating historical criteria that help historians determine the authenticity of past events. The historical approach to this investigation will be the minimal facts approach as pioneered by Gary Habermas. Using this approach, twelve historical facts relevant to the resurrection will be explored. We will evaluate why these twelve facts are agreed upon by a large variety of critical scholars. Next, an argument will be made demonstrating that the earliest reporting of eyewitnesses who experienced the resurrection can be traced to within three years of Jesus\u27 death on the cross. Lastly, a chapter will be devoted to Bart Ehrman and his various objections regarding the historian\u27s ability to determine whether or not a miracle has occurred and other limitations of historical inquiry related to the resurrection of Jesus

Philosophy of History, Historical Jesus Studies, and Miracles: Three Roadblocks to Resurrection Research

Jesus’ resurrection is considered by many to be a historical event, but objections are often raised regarding to such inquiry into the past. Philosophy of history is thus an important field in which various roadblocks to resurrection research have been raised. These philosophical questions related to the study of the Jesus’ resurrection have become more prominent recently and seek to undermine the very act of historical inquiry into Jesus’ resurrection specifically and the past more generally. Accordingly, the issues addressed here have implications beyond resurrection research. This work seeks to identify and assess three common roadblocks to such research. The first is the question related to the subjectivity historian and whether or not they can have objective knowledge of the past or whether our knowledge of the past is ultimately a mere construction of the historian. We note that both are possible and that what differentiates objective knowledge of the past or a construction of the past is whether or not virtues or vices have been cultivated by the historian. Second, since we can have knowledge of the past, two ways in which it is possible for one to have this knowledge of the past are then presented. We present the Minimal Facts Approach as one possible avenue and note the application of various historical criteria as a second. These are not the only two methods, but two that we believe to provide secure historical knowledge. Lastly, we argue that historians could, in principle, conclude that a miracle has occurred. After offering some philosophical analysis of the issue of miracles and the historian’s craft, we identify and assess to objections to our conclusion. We ultimately conclude that these are more like bumps in the road rather than actual roadblocks that prevent investigation into the past. They should be considered in historical inquiry, but they certainly do not prevent one from investigating Jesus’ resurrection in particular or the past in general

History and Eschatology by N. T. Wright: A Review

Wright, N. T. History and Eschatology: Jesus and the Promise of Natural Theology. Waco, TX: Baylor University Press, 2019. 365 pp. $34.9

Crucifixion in the Ancient World: A Historical Analysis

Cook, John Granger. Crucifixion in the Mediterranean World. 2nd ed. Vol. 327. Wissenschaftliche Untersuchungen Zum Neuen Testament. Tübingen: Mohr Siebeck, 2019. Pp 549 pp. 79,00 €

Analysis of Genetic Variants Associated with Levels of Immune Modulating Proteins for Impact on Alzheimer’s Disease Risk Reveal a Potential Role for SIGLEC14

Genome-wide association studies (GWAS) have identified immune-related genes as risk factors for Alzheimer’s disease (AD), including TREM2 and CD33, frequently passing a stringent false-discovery rate. These genes either encode or signal through immunomodulatory tyrosine-phosphorylated inhibitory motifs (ITIMs) or activation motifs (ITAMs) and govern processes critical to AD pathology, such as inflammation and amyloid phagocytosis. To investigate whether additional ITIM and ITAM-containing family members may contribute to AD risk and be overlooked due to the stringent multiple testing in GWAS, we combined protein quantitative trait loci (pQTL) data from a recent plasma proteomics study with AD associations in a recent GWAS. We found that pQTLs for genes encoding ITIM/ITAM family members were more frequently associated with AD than those for non-ITIM/ITAM genes. Further testing of one family member, SIGLEC14 which encodes an ITAM, uncovered substantial copy number variations, identified an SNP as a proxy for gene deletion, and found that gene expression correlates significantly with gene deletion. We also found that SIGLEC14 deletion increases the expression of SIGLEC5, an ITIM. We conclude that many genes in this ITIM/ITAM family likely impact AD risk, and that complex genetics including copy number variation, opposing function of encoded proteins, and coupled gene expression may mask these AD risk associations at the genome-wide level

Geographical range in liverworts: does sex really matter?

AimWhy some species exhibit larger geographical ranges than others remains a fundamental, but largely unanswered, question in ecology and biogeography. In plants, a relationship between range size and mating system was proposed over a century ago and subsequently formalized in Baker's Law. Here, we take advantage of the extensive variation in sexual systems of liverworts to test the hypothesis that dioecious species compensate for limited fertilization by producing vegetative propagules more commonly than monoecious species. As spores are assumed to contribute to random long-distance dispersal, whereas vegetative propagules contribute to colony maintenance and frequent short-distance dispersal, we further test the hypothesis that monoecious species exhibit larger geographical ranges than dioecious ones.LocationWorldwide.MethodsWe used comparative phylogenetic methods to assess the correlation between range size and life history traits related to dispersal, including mating systems, spore size and production of specialized vegetative propagules.ResultsNo significant correlation was found between dioecy and production of vegetative propagules. However, production of vegetative propagules is correlated with the size of geographical ranges across the liverwort tree of life, whereas sexuality and spores size are not. Moreover, variation in sexual systems did not have an influence on the correlation between geographical range and production of asexual propagules.Main conclusionsOur results challenge the long-held notion that spores, and not vegetative propagules, are involved in long-distance dispersal. Asexual reproduction seems to play a major role in shaping the global distribution patterns of liverworts, so that monoecious species do not tend to display, on average, broader distribution ranges than dioecious ones. Our results call for further investigation on the spatial genetic structure of bryophyte populations at different geographical scales depending on their mating systems to assess the dispersal capacities of spores and asexual propagules and determine their contribution in shaping species distribution ranges

Exogenous Short Chain Fatty Acid Effects in APP/PS1 Mice

Elucidating the impact of the gut microbiome on Alzheimer’s Disease (AD) is an area of intense interest. Short chain fatty acids (SCFAs) are major microbiota metabolites that have been implicated as a mediator of gut microbiome effects in the brain. Here, we tested the effects of SCFA-treated water vs. saline-treated water on APPswe/PSEN1dE9 mice maintained under standard laboratory conditions. Mice were treated with SCFAs from five months of age until ten months of age, when they were evaluated for microbiome profile, impaired spatial memory as evaluated with the radial arm water maze, astrocyte activation as measured by Gfap expression and amyloid burden as assessed by histochemistry and MSD ELISA. We report that SCFA treatment increased alpha-diversity and impacted the gut microbiome profile by increasing, in part, the relative abundance of several bacteria that typically produce SCFAs. However, SCFA treatment did not significantly affect behavior. Similarly, SCFAs did not affect cortical or hippocampal astrocyte activation observed in the APP/PS1 mice. Lastly, although robust levels of soluble and insoluble amyloid were present in the APP/PS1 mice, SCFA treatment had no effect on these indices. Overall, our findings are that SCFA treatment modifies the microbiome in a fashion that may increase further SCFA production. However, SCFA treatment did not alter behavior, astrocyte activation, nor amyloid neuropathology in APP/PS1 mice maintained with a conventional microbiome