Exploring deep phylogenies using protein structure : a dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry, Institute of Natural and Mathematical Sciences, Massey University, Auckland, New Zealand

Recent times have seen an exponential growth in protein sequence and structure data. The most popular way of characterising newly determined protein sequences is to compare them to well characterised sequences and predict the function of novel sequences based on homology. This practice has been highly successful for a majority of proteins. However, these sequence based methods struggle with certain deeply diverging proteins and hence cannot always recover evolutionary histories. Another feature of proteins, namely their structures, has been shown to retain evolutionary signals over longer time scales compared to the respective sequences that encode them. The structure therefore presents an opportunity to uncover the evolutionary signal that otherwise escapes conventional sequence-based methods. Structural phylogenetics refers to the comparison of protein structures to extract evolutionary relationships. The area of structural phylogenetics has been around for a number of years and multiple approaches exist to delineate evolutionary relationships from protein structures. However, once the relationships have been recovered from protein structural data, no methods exist, at present, to verify the robustness of these relationships. Because of the nature of the structural data, conventional sequence-based methods, e.g. bootstrapping, cannot be applied. This work introduces the first ever use of a molecular dynamics (MD)-based bootstrap method, which can add a measure of significance to the relationships inferred from the structure-based analysis. This work begins in Chapter 2 by thoroughly investigating the use of a protein structural comparison metric Qscore, which has previously been used to generate structural phylogenies, and highlights its strengths and weaknesses. The mechanistic exploration of the structural comparison metric reveals a size difference limit of no more than 5-10% in the sizes of protein structures being compared for accurate phylogenetic inference to be made. Chapter 2 also explores the MD-based bootstrap method to offer an interpretation of the significance values recovered. Two protein structural datasets, one relatively more conserved at the sequence level than the other and with different levels of structural conservation are used as controls to simplify the interpretation of the statistics recovered from the MD-based bootstrap method. Chapter 3 then sees the application of the Qscore metric to the aminoacyl-tRNA synthetases. The aminoacyl-tRNA synthetases are believed to have been present at the dawn of life, making them one of the most ancient protein families. Due to the important functional role they play, these proteins are conserved at both sequence and structural levels and well-characterised using both sequence and structure-based comparative methods. This family therefore offered inferences which could be informed with structural analysis using an automated method. Successful recovery of known relationships raised confidence in the ability of structural phylogenetic analysis based on Qscore to detect evolutionary signals. In Chapter 4, a structural phylogeny was created for a protein structural dataset presenting either the histone fold or its ancestral precursor. This structural dataset comprised of proteins that were significantly diverged at a sequence level, however shared a common structural motif. The structural phylogeny recovered the split between bacterial and non-bacterial proteins. Furthermore, TATA protein associated factors were found to have multiple points of origin. Moreover, some mismatch was found between the classifications of these proteins between SCOP and PFam, which also did not agree with the results from this work. Using the structural phylogeny a model outlining the evolution of these proteins was proposed. The structural phylogeny of the Ferritin-like superfamily has previously been generated using the Qscore metric and supported qualitatively. Chapter 5 recovers the structural phylogeny of the Ferritin-like superfamily and finds quantitative support for the inferred relationships from the first ever implementation of the MD-based bootstrap method. The use of the MD-based bootstrap method simultaneously allows for the resolution of polytomies in structural databases. Some limitations of the MD-based bootstrap method, highlighted in Chapter 2, are revisited in Chapter 5. This work indicates that evolutionary signals can be successfully extracted from protein structures for deeply diverging proteins and that the MD-based bootstrap method can be used to gauge the robustness of relationships inferred

Analysis of Land and Sea Temperatures Trend During 1985-2021 Period to Understand Local or Global Warming Effect in Bengkulu City

Global warming is a phenomenon where the earth's temperature rises drastically. The temperature increase causes negative impacts on the environment globally. Bengkulu City, Indonesia, is situated with a growing population and land-use change that may cause temperature rise. This research aimed to analyze the temperature change in the land and sea area of Bengkulu City. To understand the local or global factors influencing temperature changes in Bengkulu City, we also studied the correlation between land and sea temperatures. The temperature data were obtained from BMKG and NOAA PSL. Firstly, we analyzed the temperature trendlines for the last 36 years. Then we evaluated the coefficient determination (R2) value to determine the correlation between sea and land temperatures. The results show that during the last 36 years, the sea temperature is increased by 0.40 °C, while the land temperature is increased by 1.07 °C. Moreover, we found a relatively weak correlation between sea and land temperature, with a 10.7% correlation. We argued that the increased temperature in Bengkulu City land is associated with land change use and rising population in the last few decades, which means the local factor affected the land temperature changes. On the other hand, global phenomena (IOD and ENSO) influenced sea temperature changes, which means the global factor affected the sea temperature changes. The rising land temperature is relatively high; hence it is necessary to understand better what parameters are causing temperature changes that may affect the physical environment in Bengkulu, Indonesia

Higiene Sanitasi dan Analisis Zat Pemanis Buatan pada Dodol yang di Produksi di Kecamatan Panyabungan Kabupaten Mandailing Natal Tahun 2012

Hygiene sanitation and analysis of artificial sweeteners on the produce of dodol on Kecamatan Panyabungan Kabupaten Mandailing Natal in 2012. Dodol is a kind of snack which is popular and spread among the Indonesian community. The process which is home industry made the control of sweetener USAge is unclear, based on the producer desire. More over the process is still traditionally, as of the workers do not care enough to the hygiene sanitation of the dodol making process. The purpose of this research was to obtain the description of hygiene sanitation process and the analysis of artificial sweeteners content on the produce of dodol snack in Kecamatan Panyabungan Kabupaten Mandailing Natal in 2012. The type of this research was descriptive survey by observing sanitation hygiene and laboratory analysis to obtain artificial sweetener contents on dodol. The result of the research showsed that hygiene sanitation principal on dodol making process which comply with Ministry of Health Decree of Indonesia Number 942 / Menkes / SK / VII/2003 was storage of raw. From the 7 samples of dodol which inspected, there were 3 samples which contain of artificial sweeteners with saccharin grading are 00274 mg/kg, 0.0073 mg/kg, and 0.0054 mg/kg which comply with health permit, according to Permenkes RI No.722/Menkes/Per / IX/1988. The Health Department needs to conduct a thorough counseling and supervision by the related institution (Department of Health) on the importance of food hygiene sanitation, also counseling to the producer in the USAge of food sweeteners

Analisis Potensi Peningkatan Nilai Jual Objek Pajak Akibat Pengaruh dari Pembangunanplaza Asia Sumedang Menggunakan Sig (Studi Kasus : Kec. Sumedang Utara, Kab. Sumedang )

Perkembangan pembangunan di suatu daerah dari waktu ke waktu kian meningkat. Salah satunya pembangunan pusat perbelanjaan pada daerah yang sedang berkembang. Pembangunan yang ada diharapkan memberikan dampak positif terhadap pertumbuhan perekonomian pada suatu daerah. Adanya pembangunan pusat perbelanjaan terbesar di Kabupaten Sumedang yaitu Plaza Asia Sumedang akan memberikan dampak terhadap perkembangan harga tanah dan bangunan yang berada dalam lingkup kawasan sekitarnya. Dari perkembangan harga tanah tersebut akan berdampak juga pada pendapatan asli daerah tersebut misalnya dalam segi pajak bumi. Dikarenakan alasan tersebut maka diperlukan pengelolaan informasi nilai tanah yang berkelanjutan agar dapat menjadi pertimbangan dasar pengenaan Pajak Bumi dan Bangunan yaitu NJOP. Informasi nilai tanah dapat dipresentasikan dalam bentuk peta Zona Nilai Tanah.Metode pengumpulan data dengan cara wawancara menggunakan formulir SPT. 112 –A dari Badan Pertanahan Nasional. Data yang digunakan adalah 430 sampel dan dokumen data NJOP bumi Kecamatan Sumedang Utara tahun 2014. Metode pengolahan data yang digunakan adalah regresi linier berganda, menghitung pengaruh aksesibilitas dan faktor lokasi terhadap sampel harga tanah. Serta menghitung peningkatan Pajak Bumi dan Bangunan khususnya pada sisi pajak Bumi (tanah) dengan cara membandingkan antara NIR tahun 2016 dengan NJOP dari DPPKAD tahun 2014.Hasil perhitungan Perubahan harga tanah yang terjadi di Kecamatan Sumedang Utara sesudah pembangunan Plaza Asia Sumedang berkisar antara 10 % sampai 87 % dari harga sebelumnya. Hasil analisis faktor aksesibilitas dan faktor lokasi secara statistik menunjukkan bahwa dari tiga variabel yaitu jarak terhadap jalan arteri dan kolektor, jarak terhadap kantor kabupaten dan jarak terhadap CBD memiliki korelasi negatif sedangkan variabel lebar jalan memiliki korelasi positif. Hasil dari perhitungan peningkatan NJOP berkisar 83% di desa Kebonjati, 226% di Kelurahan Kotakaler dan 323% di desa Situ. Hal ini menunjukkan bahwa Kecamatan Sumedang Utara masih memiliki potensi Pajak Bumi dan Bangunan yang tinggi khususnya pada pajak Bumi (tanah)

Correlation Among Rainfall, Humidity, and The El Niño-Southern Oscillation (ENSO) Phenomena in Bengkulu City During the Period from 1985-2020

A correlation analysis is needed to see the relations among the three variables, rainfall with El Niño-Southern Oscillation (ENSO) and Humidity with ENSO. The instability of rainfall and the decrease in the humidity in Bengkulu City can be affected by La Nina and El Nino phenomena. This research aimed to analyze the correlations among rainfall, humidity, and ENSO with Pearson correlation analysis. The rainfall and humidity data were collected from BMKG, and the ENSO data were obtained from NOAA PSL for the 1985-2020 period. The results of the correlation analysis of rainfall and ENSO for the last 35 years generally show a moderately strong correlation. However, a strong correlation in the La Nina phase specifically occurred in 1988-1989, 1998-1999, 1999-2000, 2007-2008 and 2010-2011 with values of 58.6, 40.1, 61.4, 96, 4, and 89.2%. Meanwhile, the correlation >80% (very strong) during the El Nino phase occurred in 1987, 1991-1992, 1997, 2015. The correlation analysis results of humidity and ENSO show little correlation in the La Nina phase of >70% (1988-1989, 1998-1999, 2010-2011) and the El Nino phase of <70% (1997, 2015, 2016). The humidity correlations with ENSO do not influence the humidity change in Bengkulu City. The decrease in the humidity in Bengkulu City can be affected by other factors, such as land use change to housing due to population growth in Bengkulu City

Endogenous U2.U5.U6 snRNA complexes in S. pombe are intron lariat spliceosomes

Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2.U5.U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing

Cost estimation alongside a multi-regional, multi-country randomized trial of antenatal ultrasound in five low-and-middle-income countries

Background: Improving maternal health has been a primary goal of international health agencies for many years, with the aim of reducing maternal and child deaths and improving access to antenatal care (ANC) services, particularly in low-and-middle-income countries (LMICs). Health interventions with these aims have received more attention from a clinical effectiveness perspective than for cost impact and economic efficiency.Methods: We collected data on resource use and costs as part of a large, multi-country study assessing the use of routine antenatal screening ultrasound (US) with the aim of considering the implications for economic efficiency. We assessed typical antenatal outpatient and hospital-based (facility) care for pregnant women, in general, with selective complication-related data collection in women participating in a large maternal health registry and clinical trial in five LMICs. We estimated average costs from a facility/health system perspective for outpatient and inpatient services. We converted all country-level currency cost estimates to 2015 United States dollars (USD). We compared average costs across countries for ANC visits, deliveries, higher-risk pregnancies, and complications, and conducted sensitivity analyses.Results: Our study included sites in five countries representing different regions. Overall, the relative cost of individual ANC and delivery-related healthcare use was consistent among countries, generally corresponding to country-specific income levels. ANC outpatient visit cost estimates per patient among countries ranged from 15 to 30 USD, based on average counts for visits with and without US. Estimates for antenatal screening US visits were more costly than non-US visits. Costs associated with higher-risk pregnancies were influenced by rates of hospital delivery by cesarean section (mean per person delivery cost estimate range: 25-65 USD).Conclusions: Despite substantial differences among countries in infrastructures and health system capacity, there were similarities in resource allocation, delivery location, and country-level challenges. Overall, there was no clear suggestion that adding antenatal screening US would result in either major cost savings or major cost increases. However, antenatal screening US would have higher training and maintenance costs. Given the lack of clinical effectiveness evidence and greater resource constraints of LMICs, it is unlikely that introducing antenatal screening US would be economically efficient in these settings--on the demand side (i.e., patients) or supply side (i.e., healthcare providers).Trial registration: Trial number: NCT01990625 (First posted: November 21, 2013 on https://clinicaltrials.gov )

FLT1 and transcriptome-wide polyadenylation site (PAS) analysis in preeclampsia

Maternal symptoms of preeclampsia (PE) are primarily driven by excess anti-angiogenic factors originating from the placenta. Chief among these are soluble Flt1 proteins (sFlt1s) produced from alternatively polyadenylated mRNA isoforms. Here we used polyadenylation site sequencing (PAS-Seq) of RNA from normal and PE human placentae to interrogate transcriptome-wide gene expression and alternative polyadenylation signatures associated with early-onset PE (EO-PE; symptom onset 34 weeks) cohorts. While we observed no general shift in alternative polyadenylation associated with PE, the EO-PE and LO-PE cohorts do exhibit gene expression profiles distinct from both each other and from normal placentae. The only two genes upregulated across all transcriptome-wide PE analyses to date (microarray, RNA-Seq and PAS-Seq) are NRIP1 (RIP140), a transcriptional co-regulator linked to metabolic syndromes associated with obesity, and Flt1. Consistent with sFlt1 overproduction being a significant driver of clinical symptoms, placental Flt1 mRNA levels strongly correlate with maternal blood pressure. For Flt1, just three mRNA isoforms account for > 94% of all transcripts, with increased transcription of the entire locus driving Flt1 upregulation in both EO-PE and LO-PE. These three isoforms thus represent potential targets for therapeutic RNA interference (RNAi) in both early and late presentations