앱타머 기반의 환경 속 리튬과 네오디뮴 회수 시스템 개발

강하나 충북대학교 2019 국내석사

비자연적 조건에서 계대배양된 수두-대상포진 바이러스의 변이

연상훈 충북대학교 2019 국내석사

Varicella-Zoster Virus (VZV) is included in herpesviruses and contains double-stranded DNA genome with approximately 12.5 Kbp. VZV has a narrow host range restricted to cells of human. Primary infection of VZV causes varicella and often leads to zoster after reactivation from latency. Both varicella and zoster can be prevented by live attenuated vaccines, but the molecular mechanism of attenuation is not exactly understood. In this study, I attempted to understand mechanism of attenuating mutation in VZV by in vitro passaging under unnatural conditions such as low temperature or xenogeneic cell. Clinical strain YC02 was passaged in vitro up to 100 times. The infectivity of YC02 cultured under natural condition increased, but YC02 cultured under unnatural conditions exhibited decreased rate of infectivity. Next generation sequencing (NGS) of the YC02 genome cultured under various conditions identified specific mutations occurred in unnatural conditions. The specific mutations in low temperature culture were 87656, 91239, 110360 position and the specific mutations in xenogeneic cell culture were 1337, 63539, 94135, 97748 position. The number of genetically polymorphic sites increased when cultured under natural condition, but did not increase when cultured under unnatural conditions. Further studies on mutations and genetic polymorphism of clinical strain cultured under unnatural conditions will help to elucidate the molecular mechanism of VZV attenuation.

동해 심층수로부터 분리한 Pelagicola 속 신규 세균의 동정

최윤수 충북대학교 2017 국내석사

A Gram-negative, strictly aerobic, non-motile, club-shaped bacterial strain, designated DSW4-44, was isolated from deep sea water from East Sea Donghae of Korea. Strain DSW4-44 was found to grow optimally at 25℃, at pH 7.5 ∼ 8.0 and in the presence of 3.0 ∼ 4.0% (w/v) NaCl. Neighbour-joining phylogenetic trees based on 16S rRNA gene sequences revealed that strain DSW4-44 clustered with the type strain of Pelagicola litoralis showing 96.25% and Pelagicola litorisediminis showing 95.49% sequence similarity. 16S rRNA gene sequences of the type strains of other species exhibited lower similarity values. Strain DSW4-44 was determined to contain C18:1 ω7c (82.97%) as the predominant fatty acid. The major polar lipids of strain DSW4-44 were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid, two unidentified lipids and minor amounts of three unidentified phospholipids. The DNA G+C content of strain DSW4-44 was determined to be 52.74 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain DSW4-44 is separate from Pelagicola litoralis and Pelagicola litorisediminis. On the basis of the data presented, strain DSW4-44 is considered to represent a novel species of the genus Pelagicola. The type strain is DSW4-44 (=KCCM 43261).

회전 원판 장치와 점감포기를 이용한 식품 페수 처리 공정의 세균 군집 분석

정순재 충북대학교 2008 국내석사

The bacterial communities of the food wastewater treatment system combined with rotating biological contactor (RBC) and tapered aeration reactors (TAR) were investigated by terminal restriction fragment length polymorphism (T-RFLP) and phylogenetic analysis of 16S rRNA genes. Wastewater of food industry contains high concentration of nitrogen and phosphorus as well as organic carbon. A pilot-scale wastewater treatment plant was operated with the wastewater discharged from a food factory for 150 days. Removal efficiency of chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) was more than 92% during the stable operation period. Sludge volume index (SVI) maintained an average of 160 mg/L for the operation period. Bacterial community at the initial operation stage was clearly distinguished from that of the stable operation stage. The most predominant phylum in the sample of stable stage was Bacteroidetes (44.5%). Major population of operation period was Haliscomenobacter, Sphaerotilus and candidate division TM7, however sludge bulking caused by filamentous bacteria was not observed. This result is a contrast to other wastewater treatment systems because Proteobacteria have been reported as a major group commonly in most of other wastewater treatment systems. The population that has a close relationship with Haliscomenobacter increased during the stable operation stage, emerging as the most predominant group. These results suggest the importance of filamentous bacteria, which have been regarded as a cause of sludge bulking. Further studies on this bacterial group may provide information particularly useful for the development of a novel cost-effective wastewater treatment system.

농토 유래의 group I.1.a 계통의 암모니아 산화 고세균의 특성 분석

Islam, Md. Arafat 충북대학교 2018 국내석사

An ammonia-oxidizing archaeon (AOA), having chemolithoautotrophic, mesophilic and neutrophilic characteristics, designated as strain MY1T, was isolated from an agricultural soil of Republic of Korea. Microscopic observation of isolated strain resulted in short-rod shape with a diameter of 0.3 to 0.5 µm and 0.6 to 1.0 µm in length. In addition, the strain MY1T had no flagella and pilus at the surface of the cell. Strain MY1T produced glycerol dialkyl glycerol tetraether (GDGT) membrane lipids such as crenarchaeol and GDGT-0 to GDGT-4 as major lipid. The isolated organism metabolized ammonium aerobically with nitrite production and thus had the ability to fix CO₂. Addition of keto acid compounds, such as pyruvate, α-ketoglutarate and oxaloacetate was necessary to sustain the growth of strain MY1T. Optimal growth conditions were 25 °C temperature with pH of 7.0 and salinity 0.2 to 0.4 ‰ respectively. The levels of ammonia and nitrite concentrations that the strain MY1T tolerated were up to 10 mM and up to 5 mM, individually. The genome of strain MY1T was 1.6 Mb in size and had a DNA G+C content of 32.7 mol%. Phylogenetic analysis of 16S rRNA genes showed that strain MY1T was affiliated with the phylum Thaumarchaeota, sharing 96.9 % 16S rRNA gene sequence identity with the Nitrosopumilus maritimus SCM1T, a marine AOA, and 84 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T, a soil AOA. Based on the studies of phenotypic, phylogenetic and genomic structures of strain MY1T, I propose the name Nitrosoarchaeum koreensis sp. nov. to be assigned as strain MY1T. 암모니아 산화 고세균은 화학무기독립영양체, 중온성, 호중성의 특징을 가졌으며, 대한민국의 농토에서 분리된 균주 MY1T가 발표되었다. 분리된 균주를 현미경으로 관찰한 결과, 지름 0.3∼0.5 µm, 길이 0.6∼1.0 µm의 짧은 막대기 모양으로 관찰되었다. 균주 MY1T은 세포 표면에 편모와 섬모가 없다. 균주 MY1T은 운동성 뿐 아니라 편모와 주화성과 관련된 유전자를 가지고 있지만 관찰되지 않는다. 균주 MY1T은 crenarchaeol, GDGT-0 ∼ GDGT-4와 같은 glycerol dialkyl glycerol tetraether (GDGT) 막지질이 주된 지질로 보여진다. 분리된 이 고세균은 호기성으로 아질산염 생산과 함께 암모니아를 대사하며, 이산화탄소를 고정한다. 또한 pyruvate, α-ketoglutarate, oxaloacetate과 같은 케토산 화합물 (keto acid compound)을 균주 MY1T의 생장을 위해 필요로 한다. 균주 MY1T의 최적 생장 조건은 25 °C, pH 7.0, 염도 0.2-0.4 ‰이다. 또한 균주 MY1T은 암모니아 농도 10 mM, 아질산염 농도 5 mM까지 내성이 있다. 균주 MY1T의 유전체는 1.6 Mb, DNA G+C 함량은 32.7 % 이다. 16S rRNA 계통수 분석 결과, 균주 MY1T은 Thaumarchaeota 문에 속하며, 16S rRNA를 기반으로 Nitrosopumilus maritimus SCM1T (해양 암모니아 산화 고세균) 와 96.9 %의 유사도를 보이며, Nitrososphaera viennensis EN76T (토양 암모니아 산화 고세균) 와 84 % 의 유사도를 보인다. 표현형, 계통발생학적, 유전체 연구를 기반으로 균주 MY1T을 Nitrosoarchaeium koreensis sp. nov로 명명하였다.

비자연적 조건에서의 배양에 따른 수두-대상포진 바이러스의 변이 분석

박지선 충북대학교 2018 국내석사

Varicella-Zoster Virus (VZV) is a member of herpesviruses and contains double-stranded DNA genome with approximately 125,000 bp. VZV has a very narrow host range restricted almost exclusively to cells of human or simian origin. Primary infection of VZV causes varicella in about 90% of children under 12. VZV usually establishes a latent infection in sensory ganglia and the reactivation of VZV can occur with decreased immune function, which leads to zoster. Both varicella and zoster can be prevented by live attenuated vaccines, but the molecular mechanisms of attenuation are not clearly understood. In this study, I attempted to understand mechanism of attenuating mutations in VZV by in vitro passaging under unnatural conditions such as low temperature or guinea pig cells. Clinical strain YC01 isolated from Korean patients was passaged in vitro cell culture up to 61 times under various natural and unnatural conditions. The infectivity of the cultured virus was monitored by plaque assay and genome copy number was determined by real-time PCR. Replication of the clinical strains grown at 37℃ increased at higher passages, whereas replication decreased at high passages under unnatural conditions such as at 34℃ or in Guinea pig lung cells.. Next generation sequencing of the full genome of YC01 cultured under various conditions identified mutations specific for passaging at 37℃ or 34℃. The number of genetically polymophic sites increased when YC01 was passaged at 37℃, but decreased when cultured at 34℃. Further studies of mutations and genetic polymorphism in clinical strain cultured at low temperature or guinea pig cells will help to elucidate the molecular mechanisms of VZV attenuation.

Aptamer-based Photodynamic Treatment for Antimicrobial Periodontal Therapy

우주아 충북대학교 2019 국내석사

Periodontitis is a disease between the gingiva and the teeth that causes bacterial attact to the tissue. The direct cause of the disease is caused by a bacterial membrane called plaque, which is constantly formed in the teeth. Therefore, the most basic principle in the treatment of periodontal disease is to prevent the propagation of bacteria. However, eating gum medicines are not helpful for treatment, and they have the problem that they acquired resistant to medicines. A strain called Capnocytophaga ochracea, which causes periodontitis, is mainly found in human oral cavity and acts as an opportunistic pathogen. Among more than 500 kinds of oral bacteria identified so far, it is a bacterium detected from the beginning of oral bacterial infectious disease. It also accelerates periodontitis and other periodontal diseases by creating Streptococcus mutans that cause tooth decay and Fusobacterium nucleatum species and plaques that cause periodontal disease and bad breath. This strain grows only in an anaerobic atmosphere with a CO 2 concentration of 5% or more, which is slow to incubate and has difficulties in independent culture. Therefore, treatment are used only antibiotics. As a means to solve these problems, we tried to use the aptamer to inhibit the strain. An aptamer has the characteristic of forming a tertiary structure of a single strand of DNA or RNA and specifically binding to a target substance. The range of target materials is very diverse, relatively easy to deform, and has high binding strength. If such inhibition by aptamer is possible, antibiotics resistance is expected to be solve. Therefore, we developed an aptamer and tried to graft PDT for wide-area dynamic treatment. Light, light photosensitizer, oxygen molecules through the treatment of three elements, free radicals, radical ions, single oxygen molecules through oxygen molecules are produced and have a sterilizing effect. It has the advantage of high sterilization power even under hypoxic and polar conditions. Methylene blue was used as a photosensitizer, and the wavelength of the absorbed light was around 670 nm. Although it is already used as a therapeutic agent, treatment with appropriate concentration treatment is necessary because of the side effects such as headache, vomiting, confusion and hypertension at high concentration. It is anticipated that if the strain can be treated through a complex with a photosensitizer combined with the developed aptamer, it will overcome the limit of other periodontal diseases in the future. 치주염은 치은과 치아사이의 홈으로 박테리아가 공격하여 조직을 손상시키는 질환이다. 이 질환의 직접적인 원인은 치아에 지속적으로 형성되는 플라크라는 세균막이 원인이다. 따라서 치주 질환의 치료에 있어서 가장 기본은 세균의 번식을 막는것이다. 하지만 먹는 잇몸 치료약은 치료에 도움이 되질 않으며, 특수 약제를 넣거나 항생제를 사용하지만 약제에 내성이 생긴다는 문제점을 가진다. 치주염을 일으키는 Capnocytophaga ochracea라는 균주는 주로 인간의 구강에서 발견이 되며 기회성 병원균으로 작용한다. 지금까지 확인된 500여종 이상의 구강 세균 중 구강세균감염성 질환의 초기부터 검출되는 균이다. 또한 충치를 발생시키는 streptococcuss mutans와 치주질환 및 구취를 발생시키는 fusobacterium nucleatum 종들과 플라크를 만들어 치주염과 다른 치주 질환을 가속화 시킨다. 이 균주는 CO2 농도가 5% 이상인 혐기상태에서만 생장하는 특성을 가지고 있기 때문에 증식이 느리고, 독립배양에 어려움을 가진다. 따라서 치료로는 항생제만을 사용하고 있는 실정이다. 이러한 문제점을 극복하기 위한 수단으로 앱타머를 이용하여 균주의 저해에 이용하고자 하였다. 앱타머는 단일가닥의 DNA나 RNA의 3차 구조를 형성시켜 표적으로 하는 물질에 특이적으로 결합하는 특징을 가진다. 표적 물질의 범위는 매우 다양하고 비교적 변형이 쉬우며, 높은 결합력을 가지는 등의 장점을 가지고 있다. 이러한 앱타머를 통한 저해가 가능하다면 항생제에 대한 내성의 어려움을 극복할 수 있을것으로 기대된다. 따라서 앱타머를 개발하여 PDT라는 광역동치료에 사용하고자 하였다. 빛과 광감광제, 산소분자인 세가지 요소를 통한 치료법으로 산소분자를 통해 free radical과 radical 이온, 단일 산소 분자가 생성되어 살균 효과를 가진다. 저산소상태나, 극성의 조건에서도 높은 살균력을 가지는 장점이 있다. 사용한 광감광제는 메틸렌블루로 흡수하는 빛의 파장은 670nm 부근이다. 이미 치료제로도 사용하고 있지만, 높은 농도에서 두통과 구토, 혼란, 고혈압과 같은 부작용이 있기 때문에 적절한 농도 처리를 통한 치료가 필요하다. 개발한 앱타머에 광감광제를 결합시킨 복합체를 통해 균주의 치료가 가능하다면 앞으로의 다른 치주 질환의 한계점을 극복할 수 있을 것으로 기대된다.

국내 환자로부터 분리된 수두-대상포진 바이러스의 특징과 계통발생학적 분석

김민호 충북대학교 2017 국내석사

Varicella-zoster virus (VZV) is a causative agent for chicken pox in primary infection and shingles after its reactivation from latency. Complete or almost complete genomic DNA sequences of various VZV strains have been reported. Recently, clinical VZV strains were isolated from Korean patients and their genome sequences were completed by high-throughput sequencing technology. The genome size among the three Korean strains was different by 17 insertion/deletion (in/del). Interestingly, all of the in/dels occurred after a run of same 6 to 10 nucleotides. In addition, 11 of the 17 in/dels were found to be located in regions where the polarity of VZV ORF changes. The single nucleotide polymorphism (SNP) of VZV strain was analyzed for the genetically characteristics of Korean clinical isolates. Phylogenetic and genotyping analyses revealed that three Korean strains, YC01, YC02 and YC03, were linked to clade 2. Comprehensive SNP analysis identified 86 sites specific for each of 5 VZV clades. Clade 2 could be further divided into 2 subgroups: subgroup 2a including strains pOka, LAX1, 092 and YC01, and subgroup 2b including strains YC02, YC03 and 84-7. Subgroup 2a and 2b differed at 6 SNP sites at positions 40130, 40370, 44995, 76131, 88867, and 100653. The separation of two subclades was confirmed by phylogenic analysis, including the inferred most recent common ancestor (MRCA). Subclade 2a strains appeared to descend earlier than subclade 2b strains from the clade 2 MRCA. Moreover, the substitution studies suggested an important role of subclade 2b-specific sequences in separation of subclade 2b strains from subclade 2a strains. Further genetic analysis with additional strains isolated from Japan, China, and other Asian countries would provide a novel insight into the significance of two distinct subclades within clade 2.

수두 백신주 간의 유전적 다양성

김석천 충북대학교 2019 국내석사

Varicella-zoster virus (VZV) is a causative agent for chickenpox in primary infection and shingles after reactivation from latency. Both varicella and zoster can be prevented by live attenuated vaccines, but the molecular mechanism of attenuation is not clearly understood. In this study, the genome sequences of three varicella vaccine strains were used to analyze the single nucleotide polymorphism (SNP) and genetic polymorphism. A total of 32 SNPs were identified frame shift and in frame insertion/deletion were further analyzed because the number of repetitions of the specific nucleotide sequence in the R region was different among the vaccine strains. The number of TA repeats in the ORI region was lower in vOka-derived vaccines each as Varilrix and Varivax than Suduvax. Genetically polymorphic sites (GPS) in the R region including R1, R2, and R3 appeared to be responsible for the genetic polymorphisms in the ORF11, 14, and 22. A relatively large number of GPS were observed in ORF31, 55, and 62, which are known to be essential for virus replication, suggesting that the attenuation of the vaccine strains may be attributed by the diversity of the these genes. In particular, GPS found in domain Ⅱ (position aa 512) of IE62 of all vaccine strains may be associated with attenuation. Comparison of the genome sequences of the vaccine strains made it possible to distinguish between Suduvax and vOka-derived vaccine strains by analyzing consensus sequence of position 109546.

국내 분리 Salmonella enterica serovar Typhimurium 및 Monophasic Variant S. I 4,[5],12:i:- 의 Extended Spectrum β-Lactamase 특성 분석

박정선 충북대학교 2018 국내석사

Non-typhoidal Salmonella infection is an important public health problem, which has caused food-borne illnesses worldwide. In recent years, antimicrobial resistance is increasing in non-typhoidal Salmonella isolates, with the emergence of resistance to third-generation cephalosporins. In this study, Salmonella enterica serovar Typhimurium and monophasic variant S. I 4,[5],12:i:- isolates from humans were characterized by their plasmids carrying extended-spectrum beta-Lactamase(ESBL) genes. The clinical samples of S. Typhimurium and S. I 4,[5],12:i:- from humans with diarrhea were collected as a part of the national surveillance system. These isolates were tested for the resistance of cephalosporins by broth microdilution methods. All the third generation cephalosporin resistant isolates were analyzed using PCR assay for the presence of ESBL genes and plasmid replicon typing. A total of thirty-two ESBL producing S. Typhimurium (n=20) and S. I 4,[5],12:i:- isolates (n=12) were identified from 2009 to 2016. All isolates were found to harbor the blaCTX-M genes: blaCTX-M-1 (n=6), blaCTX-M-14 (n=4), blaCTX-M-15 (n=3), blaCTX-M-55 (n=9), blaCTX-M-65 (n=4) and blaCTX-M-90 (n=1) with the combination of beta-lactamase genes such as blaTEM, blaCMY-II and blaDHA. The blaCTX-M were located on the IncI1 (n=9), IncA/C (n=5), IncHI2 (n=4), IncF (n=3), IncZ (n=2) and IncK (n=1) but two were non-typeable. All ESBL producing S. Typhimurium and S. I 4,[5],12:i:- isolates were characterized by their plasmids encoding the blaCTX-M genes with various genetic contexts, suggesting the possibility of further dissemination of cephalosporin-resistance genes. Therefore, it is imperative to continue the monitoring of the cephalosporins resistance and their resistant genes in S. Typhimurium and S. I 4,[5],12:i:- isolates.