A study on protective effect of BDNF-MSC in Rett syndrome model

김효정 가천대학교 대학원 2018 국내박사

고교생의 유전자 관련 오개념 분석 및 염색체 모형(模型) 활용 수업 방안

나광석 高麗大學校 敎育大學院 2011 국내석사

유전자 관련 교수․학습 과정에서 유전자의 행동은 직접 눈으로 관찰할 수 있는 방법이 극히 제한되어 있기 때문에 학생들이 오개념을 가질 가능성이 매우 높다. 본 연구에서는 서울지역의 일반계 고등학교 1~3학년 333명을 대상으로 유전자에 관한 이해도를 알아보는 검사를 실시하여 공통된 오개념 유형을 찾았으며, 이러한 오개념의 발생을 줄이고 과학적 개념 형성을 촉진하기 위해 염색체를 구조화한 모형을 활용한 수업 방안을 연구하였다. 본 연구의 결과를 종합하여 얻은 결론은 다음과 같다. 첫째, 학생들은 염색체를 이루고 있는 DNA가 유전자의 본체이며 세포 분열 시 관찰되는 염색체가 동일한 유전적 구성의 두 염색분체로 이루어져 있다는 사실에 대해서 비교적 잘 이해하고 있었다. 그러나 하나의 염색체에 다수의 유전자가 존재한다는 사실은 모르고 있거나, 염색체가 다수의 DNA 분자로 이루어져 있을 것이라는 오개념을 갖고 있었다. 둘째, 상동염색체에 대해 단순히 크기, 모양이 같은 1쌍의 염색체 정도로만 이해하고 있을 뿐, 상동염색체에 위치한 대립유전자 간의 차이를 정확히 알고 있는 학생의 비율은 낮았다. 이러한 오개념은 감수분열 과정에서 염색체의 수 변화와 감수분열을 통해 생성되는 생식세포들 간의 유전적 다양성을 이해하는데 큰 장애 요인으로 작용하게 된다. 셋째, 감수 분열을 통한 정자 형성 시 성염색체 XY가 1쌍의 상동염색체처럼 분리되어 각각의 정자로 들어간다는 사실을 모르는 경우가 많았으며, 정자는 남성의 생식세포이므로 모든 정자는 Y염색체를 가질 것이라는 오개념을 갖고 있었다. 유전자와 관련된 오개념의 발생을 줄이기 위한 방안으로 실시한 염색체 모형 활용 수업의 결론은 다음과 같다. 염색체 모형을 활용한 수업을 실시한 집단은 상동염색체와 대립 유전자의 관계 및 염색체를 구성하는 두 염색분체의 관계에 대해 일반 수업을 실시한 집단에 비해 오개념의 발생이 감소하였다. 또한 유전자가 표지된 염색체 모형을 활용하여 감수분열 과정을 재현해 봄으로써 양성 잡종의 유전에서 독립과 연관에 따른 생식 세포의 유전적 다양성을 쉽게 이해하는 것으로 나타났다. 따라서 염색체 모형은 세포 분열과 유전 학습 과정에서 유전자와 염색체를 가시화 하고 구체적 조작을 가능하게 함으로써 유전자와 관련된 오개념을 줄이고 과학적 개념의 형성을 촉진하는 것으로 나타났다.

Co 및 Mo 분산촉매를 이용한 열가소성수지의 저온열분해

박준규 충북대학교 2017 국내박사

The energy market is pursuing acceptable government policy in its own right for the purpose of energy security, diversification of energy sources and reduction of greenhouse gas emission from long-term perspective in addition to the economic aspects. Particularly in Korea, which is a resource-poor country, continuous investment and invest in recycling technology is required as part of this policy. This study investigated the conversion and the yields of oil products from polyethylene, polypropylene and polystyrene by using dispersed Co and Mo catalyst on reaction time and concentration change for knowledging of characteristics at low temperature (425, 450, 475 ℃) pyrolysis and reaction time(20∼80 min, 15 min interval) in a batch reactor. It will be showed the conditions for optimum pyrolysis at reaction temperature 450 ℃ and reaction time 65 min in the polyethylene, 50 min in the polypropylene, 35 min polystyrene. The main components of converted liquid oil from polyethylene and polypropylene were alkane, alkene and polystyrene were styrene and benzene derivatives by GC/MS. The oil products formed during pyrolysis were classified into gas, gasoline, kerosene, diesel and heavy oil according to the domestic specification of petroleum products. The conversion rate was showed as Mo catalyst > Co catalyst > Thermal in all reaction time at reaction temperature 450 ℃ in the case of polyethylene and polypropylene. but in the case of polystyrene showed as Co catalyst > Mo catalyst > Thermal. The conversion and product yields of thermoplastic resins were measured by varying the catalyst concentration of Co and Mo. The conversion of all resins was slightly increased but the effect was not significant. The yield of products was not influenced by the concentration change in products other than specific products(ex, gasoline, kerosene). The conversion rate and yields of the pyrolysis products in polyethylene and polypropylene were highest when Co and Mo catalyst ratio was 50:50. And the conversion rate and yields of the pyrolysis products in polystyrene was highest when Co catalyst ratio was 100 %

압축 탑상 증기를 에너지원으로 이용한 증류 공정의 설계 및 경제성 분석

박원석 충북대학교 대학원 2013 국내석사

The energy saving and economic analysis when using overhead vapor of distillation process as energy source was investigated. To study the above work, the distillation process for benzene-toluene mixture was adopted. To design the process, Aspen HYSYS was used. For comparative analysis, both general distillation process and improved process were designed by Aspen HYSYS. The price information of equipments such as condenser, overhead vapor super heater, reboiler and heat exchanger was obtained from manufacturer. As a result, the improved process, compared to the general process, is slightly more expensive. However, saving in operation cost can in the first 1 or 2 years offset the price difference, and after that, the improved process can yield greater profit than general process.

Intracellular IL-4, IL-10, IFN-γ Levels of Leukemic Cells and Bone Marrow T Cells in Acute Leukemia

박훈희 고려대학교 대학원 2006 국내박사

급성백혈병환자를 대상으로 골수T 세포와 백혈병세포에서 cytokine(IL-4, IL-10, IFN-γ)함량을 flow cytometry을 이용해 측정하였다. 본 연구의 목적은 급성백혈병내 골수T세포에서 발현하는 cytokine의 역할과 화학요법을 받은후 완전관해가 된 상태에 발현차이를 비교분석하였다. 연구대상은 급성백혈병으로 진단받은 환자 131명 (급성골수모세포성백혈병 95명, 급성림프모세포성백혈병 25명, 급성혼합형백혈병 11명)이었다. 백혈병세포에서 IL-4의 함량은 급성단구성백혈병(1734.9±1055.8)에서 높았으며, dysplastic AML(959.8± 544.6)에서는 낮았다. 그리고 IFN-γ 함량은 급성전골수성백혈병 (494.5±158.4)에서는 높았으며, 급성림프모세포성 백혈병(251.7±119.3)에서는 낮게 나타났다. 그러나 IL-10 함량은 각 진단별 그룹에서 유의하게 차이가 나타나지 않았다. 골수T세포 에서 IL-10 함량은 dysplastic AML (971.7±1048.7)에서 높았으며, 급성전골수성백혈병(396.9±351.8)에서는 낮게 나타났다. 급성백혈병중 dysplastic AML은 백혈병세포에서 cytokine 함량이 가장 낮게 나타났으며, 골수T 세포 상의 cytokine 함량은 가장 높게 나타났다. 또한 2년간의 생존율, 완전관해율 및 재발율간의 cytokine 함량의 상관관계는 특징적 유의성이 없는 것으로 나타났다(P>0.05). 완전관해후 골수T 세포의 cytokine 함량은 어느 진단군에서나 모두 정상과 유사한 농도로 감소되었다.(P>0.05). 결론적으로 백혈병세포와 골수T 세포내 IL-4, IL-10, IFN-γ 함량은 급성백혈병중 각 진단별로 특징적으로 다르게 나타났으며, 완전관해후 bmT cell 내의 cytokine 함량은 정상농도로 감소하였다. 이러한 연구결과는 앞으로 cytokine 을 발현하는 T세포를 이용한 급성백혈병의 세포치료법 개발에 기여할것으로 기대된다. To determine the role of cytokines in the leukemic cells and the bone marrow T lymphocytes in acute leukemia, quantitative intracellular cytokine(IL-4, IL-10, and IFN-γ) levels(number of bound PE molecule) of leukemic cells and bone marrow T cells(bmT cells) were analyzed in acute leukemia patients with flow cytometry. One hundred thirty one(95 AML, 25 ALL, 11 ABL) acute leukemia patients and 11 bone marrow transplantation donors(normal control) were studied. The leukemic cell IL-4 level was highest in the monocytic AML group(DX2, 1734.9±1055.8) and it was lowest in the dysplastic AML group (DX4, 959.8± 544.6), and the IFN-γ level was highest in the acute promyelocytic leukemia(APL) group (DX3, 494.5±158.4) and it was lowest in the ALL group (DX6, 251.7±119.3). However, the IL-10 level was not significantly different among the diagnosis groups (P=0.182). The bmT cell IL-10 level was highest in the dysplastic AML group (DX4, 971.7±1048.7) and it was lowest in the APL group (DX3, 396.9±351.8). The leukemic cell cytokine levels were lowest(817.37±550.92) and bmT cell cytokine levels were highest(661.26±667.32) in the dysplastic AML group among acute leukemia groups. There were no significant correlations of these cytokine levels with the 2-year survival rate, the complete remission(CR) rate and the relapse rate (P>0.05). The cytokine levels of the bmT cells at the time of CR were similarly decreased as normal control and were not different among diagnosis groups(P>0.05). In conclusion, the leukemic cell and bmT cell cytoplasmic expression profiles of IL-4, IL-10 and IFN-γ are characteristic for each diagnostic group of acute leukemia patients and those of the bmT cells are remarkably decreased at the time of CR.

사람 역분화줄기세포를 이용한 분화특이 세포주 개발에 관한 연구

이재원 가천대학교 대학원 2023 국내석사

GATA6(GATA binding protein 6)는 18번 염색체(18q11.2)에 위치하며 Zinc finger transcription factors의 small family member에 속하며 척추동물의 발달과정에서 세포 분화 및 기관 형성을 조절하고 endo-mesoderm으로 분화되는 세포에 주로 발현된다. 본 연구에서는 clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9(이하 CRISPR/Cas9이라 함)을 이용해 GATA6-EGFP knock-in 세포주(이하 GATA6 리포터 세포주라 함)를 제작하였다. 제작된 GATA6 리포터 세포주는 definitive endoderm(이하 DE라 함)으로 분화시켜 GATA6의 내재적 프로모터에 의한 형광단백질의 발현을 확인하였다. CRISPR/Cas9으로 균질한 단일 세포로 제작된 GATA6 리포터 세포주는 분화 모니터링을 이용한 분화조건 최적화, 줄기세포의 발생 및 분화기전 연구, 분화된 세포의 분리와 같은 다양한 줄기세포 연구에 활용될 수 있다

혈액응고9인자 분비 유도만능 줄기세포 유래 간세포의 B형 혈우병 치료효과 연구

최준원 가천대학교 대학원 2022 국내박사

B형 혈우병은 X 염색체상에 존재하는 Factor IX (이하 FIX이라 함) 유전자의 구조 이상 돌연변이에 의해 발생하는 열성 유전병으로 항응고인자9 의 결핍으로 혈액이 응고되지 않는 치명적인 유전 질환이다. 혈장 유래 제제나 유전자 재조합 제제의 항응고 단백질을 이용한 기존의 치료는 혈전성 미세혈관 병증이 보고 되었으며, 약 20 - 30%의 혈우병 환자에서 항응고 단백질 제제에 대한 면역반응에 따른 부작용이 보고되었다. 또한, 항응고 단백질 제제의 짧은 반감기와 비싼 치료비용은 빈번한 출혈이 발생하는 혈우병 질환의 특성상 효과적인 치료법이 되기 어렵다. 정상 세포의 기능을 보조 및 대체하는 세포치료제는 유전 질환의 근본적 치료를 위한 대안으로 다양한 연구가 진행되고 있다. 유도만능 줄기세포 (induced pluripotent stem cell, iPSC)는 분화 만능성을 지니고 환자의 혈액이나 피부세포와 같은 체세포를 이용하여 이식시 환자에서 나타나는 면역거부반응을 해결할 수 있다. 또한, 체세포를 역분화시켜 제작하기 때문에 윤리적 걸림돌로부터 자유롭다는 장점이 있다. 유도만능 줄기세포를 이용한 세포치료제는 돌연변이가 발생한 유전자의 부위의 서열을 편집 수정하거나 AAVS1 site와 같은 safe harbor site에 정상 유전서열을 삽입하여 FIX 단백질 분비하도록 한다. 유도만능 줄기세포의 직접 이식은 teratoma 발생 가능성이 있어, 간세포로의 분화 및 단일 세포 배양 후 이식을 통해 종양원성을 제거하고 효과적인 생착을 통해 혈액 응고 단백질을 원활하게 분비할 수 있다. 한편, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins9 (Cas9) (이하 CRISPR-Cas9 이라 함)으로 대표되는 유전자 편집기술은 유전자 서열의 효과적인 편집을 가능하게 해주었다. 기존의 viral vector를 이용한 유전자 편집방법은 낮은 유전자 전달 효율과 무작위적인 유전자 삽입 (Random integration)의 위험성이 존재했다. Zinc finger nucleases (이하 ZFNs이라 함), transcription activator-like effector nucleases (이하 TALENs이라 함)는 단백질 생산이 복잡하고 상대적으로 긴 시간이 소요되는 반면 편집효율이 CRISPR-Cas9에 비해 낮은 단점이 있었다. 본 연구에서는 3’-NGG-5’의 구조를 가지는 protospacer adjacent motif (이하 PAM라 함) 서열을 인식하는 RNA-guided endonucleases (이하 RGENs이라 함)을 이용한 유전자 편집 방식인 CRISPR-Cas9을 이용하여 환자 모방 세포주를 제작하였고 이를 이용하여 FIX knock-in 유도만능 줄기 세포주를 제작하였다. FIX 서열의 PAM 서열을 타겟으로 제작한 single guide RNA(이하 sgRNA라 함)는 CRISPR RGEN tool을 이용하여 예상되는 off-target을 줄여 높은 유전자 교정률과 안정적인 유전자 서열 편집이 가능하도록 개선하였다. 또한, FIX knock-in 유도만능 줄기세포주를 간세포로 분화시켜 FIX 단백질을 분비하는 유도만능 줄기세포 유래 FIX 단백질 분비 간세포 (iPSC derived-hepatocyte like cell, 이하 iPSC-HLCs라 함)를 제작하였다. 본 연구에서 제작한 iPSC-HLCs는 safe harbor site로 알려져 있는 간세포의 APOC3 유전자의 전사 부위를 타겟하여 환자모방 유도만능 줄기세포주에 FIX 유전자를 knock-in 하였다. iPSC-HLCs는 정상 세포에 근접한 FIX 유전자 발현율을 polymerase chain reaction (이하 PCR 이라 함)을 통해 검증하였으며, human FIX ELISA 분석을 통해 FIX 단백질의 정상 분비를 확인하였다. 또한, 면역 억제 생쥐모델인 NOD/SCID 생쥐와 B형 혈우병 생쥐모델을 통해 in vivo 에서 iPSC-HLCs의 체내 생착과 분비를 면역 화학염색 및 면역 형광염색을 통해 확인하였고 혈액 내 FIX 단백질을 human FIX ELISA를 이용하여 검증하였다. 최종적으로 B형 혈우병 생쥐 모델에서 activated partial thromboplastin time(이하 aPTT 라함)을 통해 혈액응고 효과를 확인하였다. 본 연구를 통해 CRISPR-Cas9을 통한 유도만능 줄기세포의 FIX 유전자 교정과 간세포 분화 및 동물모델에 이식을 통한 혈액응고 효과는 향후 B형 혈우병의 예방적 혹은 치료적 세포치료제로 활용될 수 있다고 생각한다.

Neural differentiation of uniform-sized embryoid bodies derived from embryonic stem cells by microencapsulation in alginate bead

김종현 School of Life Sciences and Biotechnology, Korea U 2010 국내석사

Generation of neuroepithelial cells from embryonic stem cells (ESCs) is of great value for studying the mechanism of neural specification during gastrulation of embryos. The present study established a feasible microencapulation system that allows rapid and efficient derivation of an enriched population of neuron cells from uniformed embryoid body (EB) by using cell-chip-based technology. Furthermore, this study described a two-stage process for the efficient differentiation of mouse ESCs (mESCs) into neuron cells in alginate beads. The process begins by priming ESCs towards EB formation within micro-alginate beads and retinoic acid (RA) treatment for 4 days. The alginate microencapsulated mESCs using a cell-chip system generated relatively uniform sized EBs, compared to conventional EBs (242 ∂ 80 vs. 100 ∂ 5 ??m, respectively). After 4 days of RA treatment, the encapsulated EBs were efficiently differentiated into lineages of neuronal cells. Consequently, a significantly enriched population of neuron cells was generated from smaller and uniformed-sized of EBs, compared to EBs formed by conventional method (approximately 90% vs. 25%, respectively). The results obtained from this study suggested that current alginate microencapsulation system could generate uniformed-size of EBs, and the EBs were able to differentiation into neuron cells, more efficiently than conventional method of EBs generation. This microencapsulation system may facilitate the investigation in vitro models of embryonic neurogenesis and provide potential application of cell-chip-based system to cell replacement therapy for neural diseases.

Estimate of cellular damage and application of several treatments in brain and lung diseases of neonate rat induced by hypoxia/hyperoxia

성동경 Graduate School, Korea University 2010 국내박사

A very preterm infant is a baby born before 37 weeks gestationor birth weights less than 1500g. Preterm baby organs, especially brain and lung, do not enough to allow normal postnatal survival and growth. Especially premature babies can not maintain saturation percentage of dissolved oxygen because their lung is immature that can not exchange of gases between inspired air and the blood. In the brain, low oxigen level leads high risk of death or major neurological and neuro developmental abnormalities resulting from injury in the basal ganglia/thalamus and cerebral cortex. Premature infants who deliver at 26?36 wk of gestation are born in the saccular phase of lung development and are particularly susceptible to bronchopulmonary dysplasia (BPD) and pulmonary fibrosis because premature infants have been received ventilatory support for oxygen supplementation for maintain saturation percentage of dissolved oxygen. For improvement of brian and lung diseases in premature baby, there are required new therapeutic targets and methods. In this study was demonstrated brain and lung diseases animal model to improve the diseases that is caused hyper- and hypoxia in premature infant brain and lung is described. In chapter 1, loss of mitochondrial permeability detection by flow cytometry was demonstrated, and examined the neuroprotective effect of permeability transition of mitochondrial membrane chemical on hypoxic-ischemic (HI) brain injury was also described in the newborn animal. HI brain injury was performed on 7-day old Sprague Dawley (SD) rats. The rat pups were sacrificed at 24h or 48h after hypoxia-ischemia under deep anesthesia, and the whole brain tissue was obtained. Thereafter, the brain was transected in the coronal plane and assayed by TTC stain, western blot, TUNEL and FACS. In chapter 2, in vivo siRNA delivery was mentioned using PDMAEMA polymer and CTGF expression was silenced at the translational level using CTGF siRNA and whether CTGF mRNA knockdown could prevent the progression of bleomycin induced pulmonary fibrosis was also demonstrated. Four week old male SD rats were randomly exposed bleomycin or phosphate-buffered saline (PBS). siRNA-PDMAEMA complex was prepared with 100?l final volume by add deionized water and intratracheally administrated at 3h and 72h after the bleomycin treatment At P14, the harvested lungs were examined the level of CTGF, TGF-?, IL-6, and collagen content. For histological evaluation, lungs were removed and immersed in the same fixative overnight at room temperature. The fixed right lungs were embedded in paraffin. The sections were used with hematoxylin and eosin (H&E), Masson?s trichrome staining and immunostain. In chapter 3, BPD rat animal models were established and then development therapy methods in that animals were applied using stem cell transplantation and growth factor. Wild-type SD rats were randomly exposed to 95%/90% oxygen or air from birth. In the transplantation groups, a single dose UCB-derived MSCs was administered at postnatal day (P) 5. In the GCSF injection groups, GCSF treatments of 20?g/kg were administered intraperitoneally on the fourth, fifth, and sixth postnatal days. At P14, the harvested lungs were examined for the myeoloperoxidase activity, the level of tumor necrosis factor (TNF)-?, interleukin (IL-6), and transforming growth factor (TGF-?) mRNA, ?-smooth muscle actin (SMA) protein, and collagen levels. Intratracheal transplantation of human UCB-derived MSCs is more effective than intraperitoneal transplantation in attenuating the hyperoxia-induced lung injury and GCSF treatment significantly attenuated hyperoxia-induced lung injury in neonatal rats. The result is that the intratracheal rather than the intraperitoneal transplantation of human UCB-derived MSCs significantly attenuated the hyperoxia-induced lung injury such as decreased alveolarization and increased fibrosis. This protective effect appears to be mediated primarily by their anti-inflammatory effects and less possibly by their differentiation into type II pneumocytes. GCSF treatment significantly attenuated hyperoxia-induced lung injuries such as decreased alveolarization by down-modulating the oxidative stress and inflammatory responses in neonatal rats. These findings support the potential use of GCSF as a new therapeutic agent for BPD.

국내 산림바이오매스 우드펠릿 생산·수요량 예측 및 제조 원가 분석

김상선 충북대학교 2018 국내박사

Recently, interest in new renewable energy is increasing worldwide. According to the 5-year document of state administration announced by the new government, the government will nullify the construction of new nuclear power plants and increase the proportion of renewable energy generation to 20% by 2030 from 7% in 2016. The government also announced to increase the mandatory rate of its existing 10% RPS(Renewable Portfolio Standards) to 28% by 2030. With the RPS system implemented in 2012, power generation companies with more than 500MW of power generation facilities are compulsorily required to supply more than a certain amount of electricity produced from new renewable energy sources. Biomass fuel usage has increased sharply as a new renewable energy source since 2013 when it started to be used mainly among the Korean power subsidiaries that weredeveloping coal. As a result, they carried out 5.8% in 2013, 20.9% in 2014 and 18.1% of the total mandatory supply amount of RPS using wood pellets and Bio-SRF. Wood pellet industry was founded in Korea based on support project for “low-carbon Green Growth” in 2018 and in response to climate change and greenhouse gas by expanding business of new renewable energy development and distribution. Currently, world wood pellet production is 27,963,000 tons/year in 2015, 7.7% more than in 2014, and Korea accounts for 12.8% of the world's imports by importing 1,850,000 tons/year in 2014. However, in the domestic wood pellet market, only few among 23 wood pellet manufacturers operate normally by 2015. In this study, the amount of unused forest biomass is estimated based on the analysis method and the growth rate of the forest, and 20% of the byproducts generated during the processing of wood that can be used as the raw material of wood pellets. As a result, it is possible to acquire wood pellet raw materials of 4.57 million tons/year in 2016, 5.23 million tons/year in 2020 and 7.06 million tons/year in 2030. It was concluded that the production of wood pellets of 2.74 million tons/year in 2016, 3.14 million tons/year in 2020 and 4.23 million tons/year in 2030 was possible. Based on the worldwood pellet production estimation, 13 million tons of western Europe, 11 million tons of North America, 10 million tons of China and 4.4 million tons of West America by 2020 is predicted, but the domestic wood pellet industry is expected to maintain import dependency rate of 97.2%: domestic production of 53,000 tons, import of 1.72 million tons among 2016 total usage of 1.77 million tons usage in 2016. Based on the existing and licensed biomass power plant and wood pellet power plants used for “In case of mixed combustion”, the demand for wood pellets for domestic power generation was estimated to be 3.27 million tons in 2020, 5.44 million tons in 2025and 7.69 million tons in 2030. Assuming that the power generation efficiency of the power plant is 40%, the required amount even increases to be 3.91 million tons in 2020, 6.48 million tons in 2025, and 9.16 million tons in 2030. As a result of analyzing the effect of production cost and raw material supply distance of wood pellet manufacturing industry, bigger production capacity and with shorter supply distance of raw material, is more competitive. And the proportion of raw materials cost exceeds 60% of the production cost of wood pellet production of 300,000 tons. Assuming supply of raw materials at an average distance of 100km, the appropriate selling price of wood pellets excluding transportation costs was estimated at 272,528 Won/ton. As a result of comparing the price of wood pellets (305,000 Won/ton) sold domestically and industrially in major European countries with domestic prices in Korea, if facilities with production cost more than 300,000 ton/year improve the raw material cost and maintain stable supply of raw materials which accounts for more than 60%, it is possible to compete with foreign wood pellet manufacturers.