A caracterização da síntese de fosfatidilserina (PS) em Leishmania (Leishmania) amazonensis mostrou a presença de uma única via de síntese de PS, pela ação da enzima fosfatidilserina sintetase II (PSS II). A seqüência que codifica essa enzima, presente em cópia única no genoma, apresentou uma identidade de 38% e uma similaridade de 55% com seu homólogo humano. Tentativas de se nocautear esse gene não foram bem sucedidas, indicando que o gene é essencial à sobrevivência do parasita. Ensaios de incorporação de fosfolipídios marcados mostraram que o parasita captura do meio fosfatidiletanolamina, substrato da PSS II, mas a incorporação de PS se dá em uma taxa muito baixa. Também foi feita a caracterização do transporte de serina pelo parasita, que mostrou a existência de um único transportador com pH ótimo de 7,5, dependente da aitvidade metabólica da célula, com um Km de 0,826 +/- 0,183 mM e Vmax de 355,37 +/- 19,41 pmol/min * 2 * 107 promastigotas, que se mantém ativo a até 45 oC. / The characterization of phosphatidylserine (PS) synthesis in Leishmania (Leishmania) amazonensis revealed a single pathway that presented phosphatidylserine synthase II (PSS II) activity. The single copy gene that encoded this enzyme showed 38% of identity and 55% of homology to the human homolog. Attempts for the gene knockout were not successful, indicating that the gene is essential for the parasite survival. Incorporporation assays of labeled phospholipids showed that the parasite take phosphatidylethanolamine, substract for the PSS II, from the medium, but the rate of incorporation of PS occurred at a very low rate. The biochemical characterization of the serine incorporation by the parasite showed the existence of a single transport system, with an optimum pH of 7.5, which was dependent of metabolic activity of the cell, that showed a Km of 0.826 +/- 0.183 mM and Vmax of 355.37 +/- 19.41 pmol/min *2 * 107 promastigotes, that remained active up to 45oC.

Thesis (M.S.)--The Chinese University of Hong Kong. / Bibliography: leaves 68-71. / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PRINCIPLE OF NORMAL COORDINATE ANALYSIS --- p.5 / Chapter CHAPTER 3 --- THE UREY-BRADLEY FORCE FIELDS OF HEXAHALIDE IONS OF NIOBIUM AND TANTALUM --- p.20 / Chapter CHAPTER 4 --- THE UREY-BRADLEY FORCE FIELDS OF PERHALYL FLUORIDE --- p.34 / Chapter CHAPTER 5 --- THE UREY-BRADLEY FORCE FIELDS OF ISOTOPIC CHLORYL FLUORIDES --- p.47 / Chapter CHAPTER 6 --- CONCLUSION --- p.66 / BIBLIOGRAPHY --- p.68 / APPENDIX --- p.72

The examination and processing of sexual assault evidence within the forensic science community has presented many challenges. Sexual assault evidence is often submitted as a heterogeneous mixture that requires separation of cell types for further analysis. The utilization of differential extractions provides a separation technique based on structural differences between epithelial cells (E-cells) and spermatozoa. Differential extraction does not separate cell types completely, as there may be carry over in both fractions. A protocol using several proteases was designed to separate cell types, making use of structural differences between spermatozoa and epithelial cells. The purpose of this study included optimizing the protease extraction process to produce the greatest DNA yield with focus on the following variables: concentration of enzymes, concentration of semen, (plus/ minus) addition of the ZyGEM Buffer BLUE, addition of proteases together or separately at the start of the thermal cycler program, reduction of final reaction volume, and digestion time of both enzymes. When initiated, the total process to prepare DNA from sperm was 90 minutes; this time was reduced to 45 minutes. The protocol is capable of use over a wide range of semen concentrations; a final serial dilution including 9 concentrations ranging from 1:50 to 1:3200 was prepared with DNA extracted from each concentration. For this protocol to be further utilized the epithelial cell digest optimization is also needed. An additional concern when processing sexual assault evidence is the ability to locate spermatozoa quickly and efficiently after their separation from the evidentiary substrate. Of the numerous cytological and immunohistochemical staining protocols it is important to find a quick and efficient way to fluoresce sperm heads. The current fluorescent techniques require many wash steps with long incubation times. Using digitonin, tris(2-carboxyethyl)phosphine (TCEP), and the thiol-reactive probe N-(7-Dimenthylamino-4-Methylcoumarin-3-yl)) Maleimide (DACM), a tentative protocol for fluorescently labeling sperm heads has been produced. Future work with this protocol will include optimization of the reagent concentrations, time of incubation, and sufficient control of sperm pelleting through the entirety of the procedure. / 2018-11-03T00:00:00Z

Periodontium consists of gingiva, alveolar mucosa, alveolar bone, cementum and periodontal ligament (PDL). PERIOSTIN, which is expressed in fibroblastic cells in PDL and in osteoblastic cells on the alveolar bone surface, is a key extracellular matrix protein that maintains homeostasis of periodontal tissue. During the process for FAM20C protein purification, we discovered that PERIOSTIN is co-purified with FAM20C. Immunolocalization of both FAM20C and PERIOSTIN were observed in the PDL extracellular matrix of murine periodontal tissues. PERIOSTIN bound with FAM20C in vitro. We further identified the binding domain of FAM20C in PERIOSTIN, which mapped within Fasciclin (FAS) I domain 1-4 (RD 1-4) of PERIOSTIN. PERIOSTIN has four conserved S-X-E motifs in both human and mouse FAS domain, which allowed us to use the murine model determine whether PERIOSTIN is phosphorylated by FAM20C and whether this phosphorylation might contribute to the integrity of periodontium tissues. / 2019-04-12T00:00:00Z

The increased sensitivities of recently developed polymerase chain reaction (PCR) and separation techniques have afforded forensic deoxyribonucleic acid (DNA) analysts an opportunity to detect low template deoxyribonucleic acid (LT-DNA) samples. However, with LT-DNA samples stochastic effects become more prevalent, compromising the reliability and robustness of these techniques. In addition, these innovations have presented analysts with an increased incidence of higher-order mixtures. These types of mixtures, confounded by LT-DNA effects, continue to test the interpretation step of the DNA analysis pipeline. The combination of allele drop-out, allele drop-in and allele sharing create such complex samples that it necessitates the transition from traditional, threshold-based, interpretational methods to a probabilistic approach. Therefore, this study has two objectives: 1) to optimize the computational tool NOCIt, designed to provide a probability distribution on the number of contributors (NOC) to a sample and 2) to investigate the source of the variability present in quantitative real-time polymerase chain reaction (qPCR) in hopes of minimizing variations observed when determining the quantity of an unknown DNA sample. The NOCIt graphical user interface (GUI) was validated during its developmental phase. With no current forensic guidelines for the validation of computational tools, the protocol was designed based on a guide developed by the Center for Devices and Radiological Health for the Food and Health Administration (FDA). The protocol required using a variety of test types and detailed documentation of the methods used, the inputs, outputs and results. A total of 325 tests were completed across 11 different software distributions. As a critical software system, NOCIt’s settings also had to be optimized because of its ability to substantially influence the interpretation, the statistical conclusions and the accuracy of the results. Two different settings (Condition 3 and Condition 4) were tested on AmpFlstr® Identifiler® Plus and PowerPlex® 16 HS PCR amplified samples. The differences between the two conditions were the parameter Number of Samples in Batch and the Multiplicative Factor. All other settings were kept the same. The reproducibility and accuracy were examined to determine which condition was more reliable. Both settings had similar results, with both performing better in different categories. As with interpretation, quantification is an integral part of the human identification pipeline. Previous studies have shown that the Quantifiler® recommended protocol of generating a standard curve for every quantification (referred to in this study as the Recommended Method) introduces additional sources of variability compared to protocols that utilize one, external, validated curve (referred to in this study as the Experimental Method). To identify the major source of variability inherent in the quantitative polymerase chain reaction (qPCR) process, these two methods of determining the quantity of a DNA sample were investigated using the Quantifiler® Duo and Quantifiler® Trio DNA Quantification assays. Four tenfold serial dilutions were quantified in five independent runs using the Quantifiler® Duo DNA Quantification kit and five independent runs using the Quantifiler® Trio DNA Quantification kit. Quantification with Quantifiler® Duo reported less variability using the Experimental Method than the Recommended Method. Conversely, quantification with Quantifiler® Trio exhibited approximately equal variability between both methods. To assess whether the errors associated with generating a calibration played a substantive role in introducing additional variability, the test samples were also quantified using digital polymerase chain reaction (dPCR). The data for the more dilute samples were indistinguishable from the noise associated with the instrument. The more concentrated samples showed less variability than the samples quantified with Quantifiler® Duo and approximately the same as those amplified with Quantifiler® Trio. This suggests that both qPCR and dPCR processes can be used to quantify DNA amounts, however, fundamental differences in the ways each determines the values suggests that noise is an inherent and measurable part of dPCR. Thus, for purposes of DNA quantification signal thresholds will need to be determined prior to implementation.

Alzheimer’s disease results from an accumulation of aggregated amyloid beta peptide into oligomeric forms. Soluble oligomers are neurotoxic species, which are believed to be the pathophysiological cause of Alzheimer’s neurodegeneration. Amyloid β species (Aβ) are formed via normal physiological cleavage of amyloid precursor protein by β and γ secretases. Cleaved isoforms aggregate further to form oligomeric configurations of Αβ peptide. To target toxic soluble Aβ oligomers, monoclonal antibodies have been synthesized. Experimental analysis demonstrates the ability of these antibodies to recognize synthetic and endogenous oligomers. In transgenic mice designed to overexpress oligomeric isoforms of Aβ, the antibodies were able to reduce the cerebral amyloid load with proceeding improvements in cognitive abilities. However, large-scale clinical trials corroborated results indicating diminished amyloid load, but failed to produce observable improvements in clinical outcome in patients with Alzheimer’s disease. Simply put, the removal of amyloidogenic species was insufficient in alleviating the associated neurodegeneration and elicited no improvement in cognitive ability, suggesting that Aβ might not be the responsible pathogen in Alzheimer’s. The successes of antibodies in in vitro and transgenic mice studies suggest the potential of antibodies in the treatment of Alzheimer’s, but the inability of these drugs to produce marked improvements in clinical trials questions the role of amyloid in the pathophysiology of the disease.

A caracterização da síntese de fosfatidilserina (PS) em Leishmania (Leishmania) amazonensis mostrou a presença de uma única via de síntese de PS, pela ação da enzima fosfatidilserina sintetase II (PSS II). A seqüência que codifica essa enzima, presente em cópia única no genoma, apresentou uma identidade de 38% e uma similaridade de 55% com seu homólogo humano. Tentativas de se nocautear esse gene não foram bem sucedidas, indicando que o gene é essencial à sobrevivência do parasita. Ensaios de incorporação de fosfolipídios marcados mostraram que o parasita captura do meio fosfatidiletanolamina, substrato da PSS II, mas a incorporação de PS se dá em uma taxa muito baixa. Também foi feita a caracterização do transporte de serina pelo parasita, que mostrou a existência de um único transportador com pH ótimo de 7,5, dependente da aitvidade metabólica da célula, com um Km de 0,826 +/- 0,183 mM e Vmax de 355,37 +/- 19,41 pmol/min * 2 * 107 promastigotas, que se mantém ativo a até 45 oC. / The characterization of phosphatidylserine (PS) synthesis in Leishmania (Leishmania) amazonensis revealed a single pathway that presented phosphatidylserine synthase II (PSS II) activity. The single copy gene that encoded this enzyme showed 38% of identity and 55% of homology to the human homolog. Attempts for the gene knockout were not successful, indicating that the gene is essential for the parasite survival. Incorporporation assays of labeled phospholipids showed that the parasite take phosphatidylethanolamine, substract for the PSS II, from the medium, but the rate of incorporation of PS occurred at a very low rate. The biochemical characterization of the serine incorporation by the parasite showed the existence of a single transport system, with an optimum pH of 7.5, which was dependent of metabolic activity of the cell, that showed a Km of 0.826 +/- 0.183 mM and Vmax of 355.37 +/- 19.41 pmol/min *2 * 107 promastigotes, that remained active up to 45oC.

Late SV40 Factor (LSF) is a ubiquitously expressed mammalian transcription factor and an oncogene in Hepatocellular Carcinoma (HCC), a deadly cancer that currently has poor treatment options. LSF is expressed at low levels in normal hepatocytes but is overexpressed in HCC making it a key target for treating HCC. Small molecule inhibitors called Factor Quinolinone Inhibitors (FQIs) that specifically target LSF have been developed as possible chemotherapeutics to treat HCC. FQI1, a lead inhibitor, reduces HCC tumor sizes in mouse models without any evident toxicity in other organs. The focus of many recent studies regarding LSF has been to characterize it as an oncogene in HCC, however little is known about how it functions in normal hepatocytes. Understanding the role of LSF in hepatocytes could provide more insight into how it drives HCC as well as give more insight into how to target it in HCC. Here, FQI1 was used as a tool to uncover phenotypes in immortalized fetal hepatocytes (FH-B) that result from inhibiting the low levels of LSF expressed in this cell line. Rapid morphological phenotypes resulting from FQI1 treatment suggested a disruption in a non-transcriptional v process, which was unexpected since LSF is a transcription factor. Further investigation revealed that LSF and a-tubulin interact in immortalized human fetal hepatocytes and that this interaction at least partially disrupted upon short treatment with FQI1 in FH-B cells. These findings suggest mechanisms for non-transcriptional roles that LSF may have in addition to its roles as a transcription factor. / 2020-02-20T00:00:00Z

The Endoplasmic Reticulum (ER), which serves as a site for protein biogenesis in budding yeast, contains a quality control system that ensures that only proteins that have attained a native conformation are deployed to other destinations in the cell. In order to gain insight into the mechanisms that encompass the quality control system, two studies were conducted. First, I tested whether the host of chaperones and secretion machinery that is induced by the Unfolded Protein Response during ER stress can have a positive impact on protein biogenesis. My results indicate that degradation of misfolded proteins, rather than refolding, seems to be one of the major mechanisms activated by the Unfolded Protein Response that the cell uses to reduce the burden on the ER. Packaging of certain proteins into ER-derived vesicles seems to increase in order to counter balance the load in the ER during stress. Finally, the Unfolded Protein Response seems to play a role in the processing of proteins after the stress is removed; however this rescue does not appear to be dependent on the ER membrane expansion component of the Unfolded Protein Response but rather in other players like chaperones, ER-associated degradation and forward traffic. Second, a genome-wide screen was conducted to identify novel players involved in ER protein retention and export. For this purpose, extracellular secretion of the ER resident protein, Kar2p, was monitored in strains of the yeast gene deletion collection. We identified 73 strains in which deletion of a particular gene causes increased secretion of Kar2p. Secretion of Kar2p in some of these strains depended on an intact Unfolded Protein Response and moreover, deletion of some genes was synthetic lethal with deletion of HAC1, placing these genes as prime candidates to be involved in protein biogenesis. Further characterization of these strains revealed novel candidates involved in protein glycosylation, glycosylphosphatidylinositol-anchored protein maturation and quality control. These results represent a strong starting point to gain further insight in how the processes necessary for proper ER homeostasis are interrelated.

Accurate DNA replication is essential for proper cellular growth and requires an adequate and balanced supply of dNTPs. In Saccharomyces cerevisiae, de novo dNTP synthesis through nucleotide reduction by the Ribonucleotide reductase (RNR) enzyme is the sole method of production. Hence, RNR activitity is highly regulated via allosteric control, transcriptional control, differential localization of subunits, and direct inhibition of the large subunit, Rnr1, by Sml1. Loss of RNR regulation results in increased mutation due to an imbalance or an absolute change in the dNTP levels in cells. In this study, I describe how mutants in dNTP regulation, including sml1∆, play a role in telomere length homeostasis. Reduction in total dNTP concentration results in a modest decrease in telomere length, while altering the ratios between the four dNTPs has a much more pronounced effect. The altered telomere lengths correlate with the relative amount of dGTP and are dependent on telomerase. At reduced levels of relative dGTP, telomerase repeatedly stalls and dissociates from telomeres, thereby leading to shorter telomeres. Conversely, with elevated relative dGTP levels, telomerase is able to processively add nucleotides and even shows low levels of repeat addition processivity. The correlation between telomerase activity and dGTP is conserved in human telomerase, which shows increased repeat addition processivity at increased dGTP concentrations. Thus, telomere length homeostasis is also sensitive to dNTP regulation in the cell via a conserved dependence on dGTP. RNR regulation is, however, relaxed in the cell following DNA damage to allow for an increase in dNTP levels to repair the damage. In response to various forms of damage, Rad53 and Dun1 are activated and then phosphorylate numerous downstream targets, including the Rnr1 inhibitor Sml1. In this study, it was shown that the phosphorylation of Sml1 triggers its ubiquitylation by the Rad6-Ubr2-Mub1 ubiquitin ligase complex and subsequent degradation by the 26S proteasome. Furthermore, I was able to identify novel genes involved in the degradation of Sml1. Of the genes identified, many are involved in the spindle assembly checkpoint (SAC), cohesin establishment, and kinetochore integrity. The loss of SML1 in mutants of these genes resulted in synthetic growth defects that were not due to the loss of dNTP regulation, indicating a second dNTP-independent function for Sml1. Analysis of the double mutants revealed elevated chromosome loss and aberrant spindle dynamics, pointing to a role for Sml1 in the spindle/kinetochore. Through analysis of kinetochore assembly kinetics, Sml1 was found to be the functional human Mis18α ortholog involved in timely establishment of the kinetochore. Thus, Sml1 has a novel structural function at the kinetochore in addition to its role in dNTP regulation.