No description available.

Neutrophils and macrophages accumulate at sites of inflammation and cause chronic inflammation leading to various diseases. Therefore, to better understand chronic disease pathways it is important to investigate the properties of macrophage accumulation in inflamed tissues. The I-domain of the macrophage receptor integrin aDb2 plays a vital role in macrophage retention by binding to CEP (carboxyethyl pyrrole), a ligand available at inflammatory sites. This thesis mainly focuses on evaluating the binding site within integrin aDb2 that binds carboxyethyl pyrrole (CEP)-modified proteins. So, a recombinant plasmid construct containing the integrin I-domain was developed. Seven non-conserved amino acids were mutated by PCR-site-directed mutagenesis to create a mutant construct. After expressing in E. coli, the binding affinities of wild-type and mutant I-domains to CEP were analyzed using biolayer interferometry. It was found that a patch of seven positively charged amino acids contributes to the strong binding of the I domain to CEP.

No description available.

Pac1/LIS1, an essential tip tracking protein of the WD40 super family, is required to target cytoplasmic dynein to the plus ends of astral microtubules in budding yeast. Pac1/LIS1 protein is composed of two regions: a small coiled-coil domain and a highly conserved WD40 repeat domain. Because of in vivo data suggesting the motor domain of Dyn1 interacts with Pac1, I attempted to locate the region of Pac1/LIS1 essential for binding to Dyn1/HC by utilizing PCR-mediated site directed mutagenesis. PCR-generated site directed Pac1(S226P) mutant appears to bind Dyn1/HC, allowing it to localize to the microtubule plus ends; whereas, Pac1(H197R) and Pac1(D379H) mutants appear to disrupt motor localization. I further hypothesized that Dyn1/HC would bind to either the coiled-coil domain or the WD40 repeat domain. Using truncated Pac1 constructs, I have observed that neither the coiled-coil domain nor the WD40 repeat domain alone is sufficient to recruit Dyn1/DHC to the plus ends of the cytoplasmic microtubules. Additionally, if I dimerize the WD40 repeat domain with a GST fusion tag, I observed that Dyn1/HC colocalized with the truncation at the spindle pole bodies. This result indicates that Pac1 must dimerize with its coiled-coil domain prior to interacting with Dyn1/HC. Furthermore, the WD40 dimer, is unable to track microtubule plus-ends; indicating that the very N-terminus of Pac1 is important for other interactions responsible for recruiting the Pac1/Dyn1 complex to the astral microtubule plus end.

Assessment of chemical mutagenicity is essential to protecting human health from genetic disease. Current assays are limited in their ability to provide mechanistic insight into the endogenous and exogenous processes involved in mutagenesis. Duplex Sequencing (DS), a novel error-corrected sequencing technology, overcomes many of the limitations faced by conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. Furthermore, customizable panels enable assessment of the endogenous genomic features that drive mutagenesis. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. The objectives of this study were to demonstrate the potential of DS as a robust in vivo mutagenicity test and to explore its rich data to gain a better understanding of spontaneous and chemically-induced mutagenicity in somatic and germ cells. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) and germ cells of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and tissues were sampled at least 28 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in MF and distinct spectra consistent with the known mutagenic mechanisms of PRC in both tissues. Mouse PRC doses at which significant effects were observed are in range with those used for chemotherapy, suggesting that similar effects may be observed in human patients. This supports the contribution of PRC towards secondary cancers following treatment. DS results were comparable to those obtained using the gold-standard lacZ TGR assay, with DS showing greater sensitivity to detect smaller changes in MF. Analysis of mutation spectra and the genomic features that drive the mutational response revealed intrinsic differences between BM and germ cells that may underlie differences in endogenous mutagenic mechanisms and/or DNA repair pathways. The results suggest that germ cells may have intrinsic mechanisms to reduce mutation burden relative to somatic cells. While historically analysis of germ cell mutagenicity has been neglected in favour of somatic cells, our work supports the independent assessment of germ cell mutagenicity during regulatory testing. Finally, we conducted power analyses to inform the optimal DS study designs for the two tissues. We found that low intra-group variability within BM samples allows a reduction in sample size to three animals per group whilst still maintaining 80% power to detect an effect. In contrast, the relatively high intra-group variability and low background MF in germ cells suggests a minimum of eight animals per group to detect an effect. Overall, our results support the use of DS as a mutagenicity test and highlight many of the advantages it holds over conventional assays. Moreover, our study reveals the potential for mutagenic effects in PRC-treated cancer patients. Further work to test DS with more chemicals and across a wider range of tissues is recommended for future implementation as a mutagenicity test.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA suffers constant damage, leading to a variety of lesions that require repair. One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome. Necessarily, cells have evolved a number of DSB repair mechanisms. One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous template, followed by copying of the donor molecule through telomeric sequences. BIR is an important cellular process implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities. Furthermore, BIR uniquely combines processive replication involving a replication fork with DSB repair. This work employs a system in Saccharomyces cerevisiae to investigate genetic control, physical outcomes, and frameshift mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase delta (Pol delta), as well as RAD9 and RAD24, which participate in the DNA damage checkpoint response, resulted in a BIR defect characterized by decreased BIR repair and increased loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed in pol32 and rad24, as well as a rad9rad50S double mutant. HCO formation was also stimulated by addition of a replication-inhibiting drug, methyl-methane sulfonate (MMS), to cells undergoing BIR repair. Based on these data, it is proposed that interruption of BIR after it has initiated is one mechanism of HCO formation. Addition of a frameshift mutation reporter to this system allowed mutagenesis associated with BIR DNA synthesis to be measured. It is demonstrated that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2800-fold higher than normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. Pol  proofreading and mismatch repair (MMR) are confirmed to correct BIR errors. Based on these data, it is proposed that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR. Pif1p, a helicase that is non-essential for DNA replication, and elevated dNTP levels during BIR also contributed to BIR mutagenesis. Taken together, this work characterizes BIR as an essential repair process that also poses risks to a cell, including genome destabilization and hypermutagenesis.

tRNA genes are highly transcribed and perform one of the most fundamental cellular functions. Although a universal pattern observed across all three domains of life is that highly transcribed genes tend to evolve slowly, tRNA genes have been shown previously to evolve rapidly. This rapid sequence evolution could result from relaxed selection, increased mutation rate, or a combination of both. Here, we use mutation-accumulation line sequencing data to show that tRNA genes accumulate more mutations than other gene types. Our results indicate that this elevated mutation rate is a consequence of both elevated transcription-associated mutagenesis and a lack of transcription-coupled repair in tRNA genes. We also identify the gene MSH2 as being involved in transcription-coupled repair.

Measles virus (MV) can cause severe acute diseases as well as long-lasting clinical deteriorations due to viral-induced immunosuppression and neuronal manifestation. How the virus enters the brain and manages to persist in neuronal tissue is not fully understood. Various mutations in the viral genes were found in MV strains isolated from patient brains. In this study, reverse genetics was used to introduce mutations in the fusion, matrix and polymerase genes of MV. The generated virus clones were characterized in cell culture and used to infect rat brain slice cultures. A mutation in the carboxy-terminal domain of the matrix protein (R293Q) promoted the production of progeny virions. This effect was observed in Vero cells irrespective of the expression of the signaling lymphocyte activation molecule (SLAM). Furthermore, a mutation in the fusion protein (I225M) induced syncytia formation on Vero cells in the absence of SLAM and promoted viral spread throughout the rat brain slices. In this study, a solid ex vivo model was established to elucidate the MV mutations contributing to neural manifestation.

The genetic variation among extant life forms reflects the outcomes of evolution. The fodder of evolution – germline mutations – is shaped by the interplay among evolutionary forces – notably natural selection and random genetic drift. In turn, these forces leave footprints recorded in the genetic variation of extant life forms. Characterizing these footprints to understand how evolution works is at the heart of population genetics. To this end, massive datasets of genetic variation have opened new avenues of research, around how mutation rates evolve for instance, and reinvigorated long standing questions in population genetics, notably about the genetic basis of adaptation. In turn, theoretical models of evolution inform what kind of footprints we expect evolution to leave behind in such data. Two theoretical models that investigate open questions in population genetics are described in this thesis. In Chapter 1, I consider the evolution of germline mutation rates, particularly on short evolutionary timescales, and ask if recently observed variation in mutation rates among human lineages could be explained by evolution at genetic modifiers of mutation rates. Genetic modifiers of mutation rates are expected to evolve under purifying selection: mutations at modifiers that increase mutation rates (“mutator alleles”) should be selected against, because they increase the burden of deleterious mutations in individuals who carry them. The frequencies of mutator alleles are also affected by mutation, genetic drift, and demographic processes. We model the evolution of mutator alleles under the interplay of these forces and characterize the dynamics at mutation rate modifiers as a function of the efficacy of selection acting on them. We find that modifiers under intermediate selection have the greatest contribution to variation in mutation rates between distantly related populations, but only variation at strongly selected modifiers turns over fast enough to explain variation in mutation rates among human lineages. We also predict that strongly selected modifiers could be potentially identified in the contemporary datasets of human pedigrees used to study germline mutations. In Chapter 2, I consider a central and enduring question in evolutionary biology: whether adaptation typically arises from few large effect changes or from many small effect changes. Both sides are supported by ample evidence. Yet it is unclear how to translate this evidence into general answers about the genetic basis of adaptation, in part because different methodologies have different limitations and ask different questions. Theory may offer a way out of this quagmire or at least a start. To this end, we reframe the question in terms of traits and ask: how does the genetic basis of adaptation depend on the ecological and genetic attributes of a trait? To start answering this question, I model adaptation in a simple yet highly relevant setting. I consider a trait under stabilizing selection and assume the distribution of trait values in the population is initially at mutation-selection-drift-balance. I then characterize the adaptive response that is elicited by a sudden change in the environment. I find that the adaptive response, and notably the probability that adaptation arises from the fixation of large effect alleles, depends on the size of the environmental change and the genetic architecture of the trait. These attributes are measurable and can be directly related to the disparate evidence that we have about the genetic basis of adaptation. Thus, this kind of modeling may help translate such evidence into general conclusions about how traits evolve. My thesis work was interrupted by the global COVID-19 pandemic, and in response to this pandemic, governments around the world implemented shelter-in-place protocols. However, essential workers were exempt from these protocols, potentially decreasing their efficacy. In Chapter 3, we describe our epidemiological project, aimed at understanding the impact of essential workers on epidemic control. To this end, we model three different archetypes of essential workers under a reasonably realistic SEIR model of the COVID-19 pandemic. We find that the different social interactions that essential workers maintain qualitatively changes their personal risk of infection and the spread of the overall epidemic. These results highlight the utility of not considering essential workers as a monolithic group but instead distinguishing between the impact of different types of essential workers on epidemic control.