An attempt has been made to demonstrate an association between the carcinogenic activity of a chemical compound and its capacity to induce DNA damage and chromosome aberrations which may result in mutations and/or neoplastic transformation. Twenty-five 4-nitroquinoline 1-oxide (4NQO) derivatives and five related compounds of 4-nitropyridine 1-oxide (4NPO) of varying carcinogenicity were examined. [Formulae omitted] The induction of DNA damage, chromosome aberrations and clone forming capacity were used as end points. Monolayer cultures of embryonal Syrian-hamster cells and an established line of baby hamster kidney cells (BHK-21) were employed in this study. DNA damage, as measured by the unscheduled incorporation of tritiated thymidine (³H-TdR), was assayed by the autoradiographic procedure. To distinguish DNA repair synthesis from DNA replication synthesis at S-phase, cultured embryonal hamster cells were arrested at G₁ by growing them in an arginine deficient medium (ADM) prior to the application of the various carcinogens. The unscheduled uptake of radioisotope was estimated by counting the number of grains per diploid nucleus of carcinogen treated cells. The highly oncogenic derivatives of 4NQO and 4NPO elicited an elevated level of unscheduled ³H-TdR incorporation in treated cells, while the weakly oncogenic compounds induced only a smaller amount of DNA repair synthesis. The non-oncogenic N-oxides failed to provoke any detectable ³H-TdR uptake. Chromosome aberrations were studied in ADM-arrested cells which were exposed to the various compounds and then triggered into division by transferring them into the regular growth medium. A direct proportionality was observed between the degree of carcinogenicity of a compound and the frequency of induced chromosome aberrations. The clone forming ability of treated cells was employed as a means to compare the cytotoxicity of the 4NQO and 4NPO derivatives. Potent carcinogens were highly cytotoxic; weakly carcinogenic compounds showed only a slight lethal effect and non-oncogenic derivatives did not affect cell survival. This study demonstrated the capacity of carcinogens to induce alterations at the chromosome and DNA level. The possible role of DNA repair and chromosome aberrations in neoplastic transformation was discussed. The use of DNA repair synthes as an economic and relevant tool for identifying mutagens and/or carcinogens has been suggested. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Cell duplication in Escherichia coli involves complex events, coordinated with chromosome replication. Because of the importance of chromosomes in perpetuating the normal cell cycle the initiation of their replication must be coordinated with cellular division. Following initiation, the cell must replicate and segregate its chromosomes, create a site necessary for septation and divide. These events could be coordinated by either; (1) biochemical reactions involving diffusible enzymes, or (2) multienzyme complexes which are localized at the site of DNA replication and cell division. In the latter case, the cyclic events of replication, segregation and cell division may be coordinated by physical-chemica1 or biochemical means. In any case, physical association implies pleiotropic effects. To test this hypothesis, cell division of the initiator mutant of E. coli , isolated by Kohiyama (1968) was studied. The temperature-sensitive initiator mutant E. coli CR 34T83 (ts DnaA) grew normally at 30 C, and at the restrictive temperature (42 C). The DNA replication as measured by radioactive precursor uptake, stopped after approximately 40 minutes and was equivalent to completion of rounds of replication started. Measurement of ribo- and deoxyribonucleotide triphosphate pools by thin-layer chromatography at 30 C and 42 C indicated residual DNA synthesis was not due to a limitation in the DNA precursors. Using a combination of density and differential radioactive labelling for the starts and ends of chromosomes, a preferred place for reinitiation of new replication cycles was shown. It was shown that DNA replication at 42 C terminated at a fixed region of the chromosome, and was identical to the 150 μg/ml chloramphenicol sensitive step involved in the process of initiation of chromosome replication in E. coli. A cessation of cellular division was noted by measurement of cell growth by Coulter Counter, at a shift from 30 C to 42 C, resulting in filamentous growth. Upon a return to 30 C, the cells resume division after approximately 15 - 20 min. The pleiotropic behaviour, that is, the cessation of cell division and initiation of DNA replication was a result of a point mutation in the gene DnaA, coding for a membrane bound protein involved in initiation. This mutation was mapped by transduction and was located at the isoleucine-valine region of the E. coli map. When this gene was transduced to different strains of E. coli K(12) the same pleiotropy was observed. This pleiotropy could be uncoupled, however, at 30 C by inhibitors of DNA synthesis or initiation. During recovery at 30 C from growth under 42 C, expression of cell division was proportional to cell equivalents generated at the restrictive temperature. RNA and protein synthesis, for 10 minutes during the recovery period, was obligatory for initiation of new rounds of replication, but not for the expression of cell division. A cell division "potential" protein was present under the restrictive growth condition. This "potential" was made at a derepressed rate and underwent a rapid degradation if kept at 42 C. At any given time, when returning from 42 C to 30 C, this "potential" allowed expression cell division based on DNA/mass or normal cell equivalents generated at 42 C. The half-life for decay of the division "potential" was estimated to be 1.4 minutes. The results were interpreted, in terms of an enzyme complex, which is common to the initiation of DNA replication and cellular division. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

<p>Porcine reproductive and respiratory syndrome virus (PRRSV) is estimated to cost the US swine industry $664 million in annual production losses. Therefore, the objective of this project was to evaluate the effect of MCFA on PRRSV replication using in vitro and in-vivo studies. The overarching hypothesis was that MCFA would inhibit or reduce viral replication of PRRSV infection in vitro and reduce viral load in-vivo. In the first experiment (Chapter 2), MARC-145 cells were used to determine the effects of individual MCFA (C6, C8, C10, and C12) exposure at concentrations ranging from 1-1000 µg/mL prior to and following inoculation of North American Type II (P-129) or European Type I (Lelystad) PRRSV. Viral replication was determined using FITC labeled IgG anti-PRRSV monoclonal antibody and TCID₅₀was calculated for each concentration. Data were analyzed using the Proc Mixed procedure of SAS. Incubation of MARC-145 cells with caproic acid (C6) at concentrations of 1-1000 µg/mL prior to and after inoculation with Type II North American (P129) or Type I European (Lelystad) PRRSV did not alter viral replication (<i>P</i> > 0.10). However, incubation of MARC-145 cells with caprylic (C8), capric (C10), and lauric (C12) acid prior to and after inoculation with Type I and Type II PRRSV did reduce viral replication at concentrations ranging from 100-1000 µg/mL. In general, the effective dose required to reduce (<i>P </i>< 0.05) viral replication (Log₁₀TCID₅₀/mL)decreased as MCFA chain length increased. In experiment 2 (Chapter 3), the use of MCFA combinations (C8:C10; C8:C12; C10:C12; and C8:C10:C12) to reduce viral replication of PRRSV in MARC-145 cells was investigated. The MCFA combinations were analyzed at six different concentrations ranging from 50-500 µg/mL with North American Type II (P-129) and European Type I (Lelystad) PRRSV. Viral replication was determined as described in experiment 1 (Chapter 2) using FITC labeled IgG anti-PRRSV monoclonal antibody and Log₁₀TCID₅₀/mL was calculated for each concentration. Data were analyzed using the Proc Mixed procedure of SAS. Incubation of MARC-145 cells with MCFA combinations prior to and after inoculation with Type II North American (P129) and Type I European (Lelystad) PRRSV resulted in reduced viral replication at MCFA concentrations of 200-500 µg/mL and was concentration dependent. Reduction of viral replication with MCFA was further evaluated by independently incubating MARC-145 cells or PRRSV. Results indicated that viral replication was reduced when MARC-145 cells were incubated with MCFA and not when PRRSV was incubated with MCFA. In experiment 3 (Chapter 4), 112 mixed sex pigs (PIC 1050 females x PIC 359 sire), weaned at 21 d of age, weighing 7.5 ± 0.68 kg, were used in a 33d PRRSV challenge study. Pigs were blocked by body weight and sex and randomly assigned to one of four treatments in a 2x2 factorial design with pigs receiving 0 or 0.30% MCFA in the diet and placebo or PRRSV inoculation. Following a 5 d adjustment to diets and rooms, pigs were inoculated with either a placebo (sterile PBS) or Type II North American (P129) PRRSV (1 x 10⁵,TCID₅₀/mL) given in 1 mL each intranasal and IM injection. Each room contained 4 pens with 7 pigs per pen and an equal ratio of barrows to gilts within treatment. Diets were formulated to meet or exceed all nutritional requirements (NRC, 2012) and were fed in 4 nursery phases. Feed budgets by phase were 1.13 kg/pig in phase 1, 2.72 kg/pig in phase 2, 6.35 kg/pig in phase 3, and phase 4 fed until the end of the experiment. MCFA (C8:C12) were mixed in a 1:1 ratio (wt:wt), and then mixed with finely ground corn to prepare a premix added to diets at 0.60% to provide 0.30% total MCFA. Control diets used soybean oil mixed with finely ground corn at the same 0.60% inclusion to keep ME levels constant across treatments. Body weights, feed intakes, blood samples, and temperatures were determined or collected on d 0, 3, 7, 10, 14, 21, and 28 post inoculation. Sections of tonsil, lung, and intestines were collected at d 10 post-inoculation from 1 pig per pen and at d 28 from all remaining pigs. Data were analyzed using the PROC Mixed procedure of SAS with pen as the experimental unit for growth and performance measurements and pig as the experimental unit for viral load analysis. Serum viral load confirmed PRRSV was only detectable in challenged pigs. Body weights were not different (<i>P</i> > 0.05) between treatments prior to d 14 post inoculation. Body weights from d 14 to 28 post inoculation were reduced (<i>P</i> < 0.05) in PRRSV infected pigs compared to non-infected pigs. Overall ADG and ADFI were reduced (<i>P </i>< 0.05) for PRRSV infected pigs compared to non-infected pigs by an average of 18 and 28%, respectively. Body temperatures were not different between treatments. Viral load measured in the lung was not different (<i>P </i>> 0.05) between PRRSV infected treatments. Tonsil viral load was not different (<i>P</i> > 0.10) between PRRSV treatments. However, there was a trend (<i>P</i> ≤ 0.10) for an effect of day post inoculation with control-fed, PRRSV-infected pigs having higher viral loads at d 10 post inoculation compared to d 28 post inoculation. Overall, no effects of MCFA on PRRSV viral load or performance were observed during the in-vivo trial. MCFA was effective at reducing viral replication of PRRSV in MARC-145 cells in vitro. However, the results could not be confirmed in the in-vivo experiment. Porcine alveolar macrophages should be used to confirm the in vitro inhibition of PRRSV replication observed in MARC-145 cells. In order to fully understand the application of MCFA to inhibit PRRSV infection in pigs, more studies should be conducted to evaluate the form of MCFA as well as viral inoculation with field strains of PRRSV. </p>

Numerous factors that affect herpes simplex virus 1 (HSV1)-mediated pathogenesis have been identified. Such factors directly impact the replication of HSV1 as well as modulate host immune responses following HSV1 infection. In this work, I characterize how HSV1 replication is impacted by expression of the protein encoded by C21orf91, or “Cold Sore Susceptibility Gene” (CSSG1), that has been linked to HSV1 reactivation in humans. I investigated expression of CSSG1 mRNA expression in various tissues and found that CSSG1 mRNA was present in several tissues of importance in HSV1 disease, including brain, trigeminal ganglia (TG), cornea and spleen. Western blot analysis demonstrated that CSSG1 protein is expressed in human cells. Subcellular fractionation analysis reveals that CSSG1 is predominantly found in the cell nucleus, where it colocalizes with chromatin and with Tip60, a chromatin-binding histone modifying protein that has been shown to be essential for the replication of herpesviruses. I also discovered that CSSG1 is present in the cytosol of cells where it forms large cytosolic aggregates in presence of TRAF6, a downstream adapter that plays an important role in innate immune receptor signaling. To determine if CSSG1 directly impacts viral replication, I generated CSSG1 knockdown human cell lines. I found that HSV1 replication was reduced in CSSG1 knockdown cells compared to control cells, whereas replication of the unrelated virus, vesicular stomatitis virus (VSV), was not affected by knockdown of CSSG1. I demonstrate that CSSG1 was necessary for efficient expression of HSV1 viral proteins during infection. Western blot analysis and measurement of expression of HSV1 proteins expressed at various stages of viral replication illustrates that CSSG1 was required for HSV1 replication at very early stage of infection. I also noted that CSSG1 expression impacted the DNA damage response in HSV1 infected cells. Levels of H2AX phosphorylation, a marker of the DNA damage response, were increased in HSV1-infected CSSG1 knockdown cells compared to control cells. DNA damage responses are thought to promote HSV1 reactivation from latency and HSV1 gene expression, indicating a potential mechanism for role of CSSG1 in HSV1 replication through modulating the DNA damage response. Overall, my work demonstrates that CSSG1 affects HSV1 replication and provides insight on how CSSG1 polymorphisms in humans could affect HSV1 reactivation and replication to promote cold sores. These discoveries may also lead to a better understanding of pathogenesis of other herpesviruses in humans.

Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomaviruses (HPVs) are non-enveloped, circular double-stranded DNA viruses that infect basal keratinocytes of stratified squamous epithelia. High-risk HPV (HR-HPV) infection causes nearly all cervical cancers and an increasing number of head and neck cancers. While prophylactic vaccinations have reduced the incidence of HPV infection and attributable cancers, currently there is no cure for pre-existing HPV infection. As such, HPV remains a global health threat and a better understanding of HPV biology remains of significant medical importance for identification of novel therapeutic targets. The multi-subunit structural maintenance of chromosomes 5/6 complex (SMC5/6) is comprised of SMC5, SMC6 and NSE1-4. SMC5/6 is essential for homologous recombination DNA repair and reportedly functions as an antiviral factor during hepatitis B and herpes simplex-1 viral infections. Intriguingly, SMC5/6 has been found to associate with HR-HPV E2 proteins, which are multifunctional transcription factors essential to regulation of viral replication and transcription. The function of SMC5/6 associations with E2, as well as its role during HR-HPV infection remain unclear and we explored this question in the context of HR-HPV- 31. SMC6 interacted with HPV-31 E2 and co-immunoprecipitation of SMC6/E2 complexes required the E2 transactivation domain, inferring SMC6 association is limited to the full-length E2 isoform. Depletion of SMC6 and NSE3 increased HPV replication and transcription in keratinocytes stably maintaining episomal HPV-31, suggesting that the SMC5/6 complex represses these processes. Neither SMC6 nor NSE3 co-IP the viral E1 DNA helicase alone or E1/E2 complexes but the association of SMC6 with E2 was reduced in the presence of E1, indicating that SMC6 competes with E1 for E2 binding. This infers that SMC6 repression of the viral replicative program may involve inhibiting initiation of viral replication by disrupting E2 interactions with E1. Chromatin immunoprecipitation determined that SMC6 is present on episomal HPV-31 genomes, alluding to a possible role for SMC5/6 in modifying the chromatin state of viral DNA. Taken together, these findings describe a novel function for SMC5/6 as a repressor of the HPV-31 replicative program.

This is an author-produced electronic version of an article accepted for publication in the British Journal of Psychiatry. The definitive publisher-authenticated version is available online at http://bjp.rcpsych.org. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19584号 / 医博第4091号 / 新制||医||1014(附属図書館) / 32620 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 小西 靖彦, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chapter 1 Chloroplast nucleoids (ct-nucleoids) are DNA/protein complexes involved in compacting the chloroplast genome, and may play a role in regulating DNA replication. Ct-nucleoids were isolated from young soybean plants and separated by 2-D gel electrophoresis. Gel spots were excised and analyzed by MALDI-ToF mass spectrometry, resulting in several protein identifications. The proteins identified all have functions unrelated to DNA replication. While some of these proteins may be due to contamination, it is possible that some of these proteins are dual-functional, playing direct roles in the regulation of DNA replication. Chapter 2 A 28 kDa soybean protein was isolated by sequence specific DNA affinity chromatography from total chloroplast protein isolations. Mass spectrometry analysis revealed that the 28 kDa protein contains some homology within an ssb domain of an Arabidopsis mitochondrial-targeted SSB (mtSSB) of approximately 21 kDa. N-terminal sequencing revealed that the 28 kDa soy protein is identical to a 36 amino acid region at the N-terminus of the Arabidopsis mtSSB. Protein fractions containing the 28 kDa protein shift oriA in electrophoretic mobility shift assays (EMSAs). Arabidopsis mtSSB fails to shift oriA in EMSAs run under identical conditions. Arabidopsis mtSSB causes a shift of ssDNA in EMSAs, while the ability of the 28 kDa soy protein to bind ssDNA is still unclear. Importantly, the 28 kDa soy protein was identified from total protein extracts obtained from intact chloroplasts, while in-vitro targeting experiments suggest that the Arabidopsis mtSSB localizes only to mitochondria and not to chloroplasts. BLAST searches of the available soybean genomic and EST databases do not produce any significant homologies to the 36 amino acid N-terminal sequence.

Biological organisms have evolved complex epigenetic mechanisms to tailor their gene expression programs to specific needs. These adaptations allow cells, that otherwise have identical genomes, to carry out specialized functions. In this work I develop and use data-integrative techniques to examine the mechanisms and consequences of epigenetic processes. To better understand the changes in DNA methylation landscape that accompany breast cancer molecular subtypes, I integrated DNA methylation and gene expression data from 208 breast cancer samples obtained from a Polish population-based case-control study. Using a weighted correlation network approach, I identified gene co-methylation modules and asked if the genes in these modules are preferentially methylated and silenced in a breast cancer subtype-specific manner. This approach identified two non-overlapping gene co-methylation modules. The first module is silenced in Basal breast cancers, while the second is silenced in Luminal B breast cancers. Gene-set enrichment analysis suggests that epigenetic silencing of these modules interferes with processes that maintain cellular differentiation, and that the methylation status of the Luminal B module is associated with disease prognosis. To uncover the determinants of the temporal order of metazoan genome replication, I used a reductionist model of DNA replication to test the ability of hundreds of epigenetic marks to predict replication timing. My work showed that DNA replication timing can be completely predicted from locations of DNase I hypersensitive sites. I further demonstrated the robust emergent character of DNA replication that could be understood without invoking a complex regulatory mechanism. To determine the underlying cause of cell de-differentiation in osteosarcoma, I examined the relationship between microRNA expression and the bone-cell differentiation program. Focusing on the inhibitory role of miR-23a in bone differentiation, I analyzed the effect of its over-expression in osteosarcoma cells. Extensive computational analysis led me to propose that a major mechanism by which miR-23a exerts its effect is by interfering with expression of GJA1, which encodes a gap junction channel essential for intercellular communication and external stimuli sensing in bone cells. Follow-up experiments indicate that GJA1 is sharply up-regulated during bone cell differentiation and that GJA1 inhibition significantly delays the onset of differentiation. Together, this work uses data integrative techniques to provide new insights into the decisive role of epigenetic processes in cellular differentiation. / 2019-04-01T00:00:00Z

Flora et al. (1992, 2003) found that aversive stimulation in the form of intense noise or cold water increased the number of impulsive responses when presented concurrently to a choice task. Hurtado-Parrado et al. (2023) systematically replicated Flora et al.’s (1992) study and found that adding a visual matching-to-sample task with images from the International Affective Picture System (IAPS; Lang et al., 2008) produced a higher number of responses on the impulsive option (Sooner-Smaller - SS) near indifference, as compared to a No-images group who showed preference for the self-controlled option (Larger-Later - LL). They also found that the overall rate of correct matching across all conditions was low. Hurtado-Parrado et al. proposed that the potential aversive or distractive effect of frequent failure during the matching task, and thus frequent point loss, could have had a similar effect of the cold pressor and aversive noise on impulsive responses (SS). The present study systematically replicated Hurtado-Parrado et al.’s (2023) experiment to evaluate the effects on SS responses of different rates of correct responses during the matching-to-sample component of the experimental task across four conditions: No-images, 0%, 60%, and 100% correct matching. Fourteen college students were exposed to all conditions in a within-subject design. The prediction that the number of SS responses would increase as the percentage of correct responses in the matching task decreased was not confirmed. Participants developed one of three choice patterns during the first no matching condition (No Images), namely preference for the self-control or impulsive option, or indifference. Subsequent changes in percentages of correct matching did not affect indifference or self-control preference, but contrary to the predicted effect, decreased impulsive responses towards indifference or self-control preference. These findings are discussed in terms of possible effects of (a) motivating operations (e.g., monetary compensation in the present study differed from previous studies by Flora et al., 1992, 2003 and Hurtado-Parrado et al., 2023); (b) extra-experimental contingencies (e.g., several students participated during the last days of the semester trying to complete course credits in small amount of time); (c) rule-construction and rule-following behavior (e.g., rule-construction during the No images condition could explain insensitivity to subsequent changes in percentage of correct matching), and (d) local and molar reinforcement rate variations across conditions (e.g., an increase in rates of point gain, and thus increase in their reinforcement value, could explain the reduction in initial impulsive preference of some participants when correct matching was later manipulated).

The universal traits of cellular expression and replication in modern life point to the existence of an ancient RNA world. Leading up to the origin of life, this stage of evolution utilized RNA as the genetic material, and as a catalyst in the form of ribozymes. Although it is expected that a polymerase ribozyme was required for the efficient replication of RNA, it is also likely that the earliest form of replication took place under non-enzymatic conditions. There are several problems with the current scenarios depicting non-enzymatic RNA replication, thus we aim to examine them in more detail using computational models. We first consider the relationship between the thermodynamics of RNA base pairing and non-enzymatic nucleotide addition in an attempt to model the rate of primer extension. Our predicted rates reveal the model parameters to be too simple to produce reliably accurate results. For now, we should simply use available experimental rate data, until we have access to more data and less unknown parameters. Nevertheless, the model indicates that the primer extension rate does depend on thermodynamics of base pairing, and a more accurate model can be of great use when creating realistic complex models of RNA world scenarios. In chapter 3, we investigate non-enzymatic RNA replication under temperature cycling using computer simulations. When starting with a diverse mixture of sequences, partially matching sequences can reanneal in configurations that allow continued strand growth. This is in contrast to the case of having multiple copies of matching sequences, where reannealing occurs quickly upon cooling. We find that, starting with short oligomers, strands can grow over multiple cycles to produce long sequences over 100 nucleotides in length. The small strand extension per cycle does not produce replicates of any one specific sequence. This relates to the work done in chapter 4, where we look for the presence of a virtual circular genome within our simulations. In a virtual circle, short overlapping RNA sequences will make up a mutually catalytic set. Within the diversity of our simulation, virtual circles are rare, and require a specific level of starting mixture diversity along with no input of new sequences. Continued replication of the diverse sequence mixture and emergence of long strands may eventually lead to the creation of rolling circles and ribozymes. / Thesis / Master of Science (MSc) / The origin of biological life can be traced back by looking at the common themes between modern cellular processes. The role of RNA polymers seems to be of great importance, making us believe that an RNA world existed leading up to life’s origin. During this time, RNA would act as both a genetic material and a catalyst. To examine this theory in more detail, we use computational modeling to recreate and explore the various potential chemistries and conditions on the early Earth. Specifically, we explore the problems that exist for the replication and production of RNA polymers. Our results can be used to guide future theoretical and experimental research of the RNA world.