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The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell . Although many intracellular proteins are synthesized in the cytoplasm and membrane @-@ bound or secreted proteins in the endoplasmic reticulum , the specifics of how proteins are targeted to specific organelles or cellular structures is often unclear . A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion protein or chimera consisting of the natural protein of interest linked to a " reporter " such as green fluorescent protein ( GFP ) . The fused protein 's position within the cell can be cleanly and efficiently visualized using microscopy , as shown in the figure opposite .
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Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER , the Golgi , lysosomes or vacuoles , mitochondria , chloroplasts , plasma membrane , etc . With the use of fluorescently tagged versions of these markers or of antibodies to known markers , it becomes much simpler to identify the localization of a protein of interest . For example , indirect immunofluorescence will allow for fluorescence colocalization and demonstration of location . Fluorescent dyes are used to label cellular compartments for a similar purpose .
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Other possibilities exist , as well . For example , immunohistochemistry usually utilizes an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or <unk> signals that can be compared between samples , allowing for localization information . Another applicable technique is <unk> in sucrose ( or other material ) gradients using <unk> centrifugation . While this technique does not prove colocalization of a compartment of known density and the protein of interest , it does increase the likelihood , and is more amenable to large @-@ scale studies .
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Finally , the gold @-@ standard method of cellular localization is <unk> microscopy . This technique also uses an antibody to the protein of interest , along with classical electron microscopy techniques . The sample is prepared for normal electron microscopic examination , and then treated with an antibody to the protein of interest that is conjugated to an extremely electro @-@ dense material , usually gold . This allows for the localization of both ultrastructural details as well as the protein of interest .
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Through another genetic engineering application known as site @-@ directed mutagenesis , researchers can alter the protein sequence and hence its structure , cellular localization , and susceptibility to regulation . This technique even allows the incorporation of unnatural amino acids into proteins , using modified tRNAs , and may allow the rational design of new proteins with novel properties .
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= = = Proteomics = = =
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The total complement of proteins present at a time in a cell or cell type is known as its proteome , and the study of such large @-@ scale data sets defines the field of proteomics , named by analogy to the related field of genomics . Key experimental techniques in proteomics include 2D electrophoresis , which allows the separation of a large number of proteins , mass spectrometry , which allows rapid high @-@ throughput identification of proteins and sequencing of peptides ( most often after in @-@ gel digestion ) , protein microarrays , which allow the detection of the relative levels of a large number of proteins present in a cell , and two @-@ hybrid screening , which allows the systematic exploration of protein – protein interactions . The total complement of biologically possible such interactions is known as the interactome . A systematic attempt to determine the structures of proteins representing every possible fold is known as structural genomics .
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= = = Bioinformatics = = =
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A vast array of computational methods have been developed to analyze the structure , function , and evolution of proteins .
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The development of such tools has been driven by the large amount of genomic and proteomic data available for a variety of organisms , including the human genome . It is simply impossible to study all proteins experimentally , hence only a few are subjected to laboratory experiments while computational tools are used to extrapolate to similar proteins . Such homologous proteins can be efficiently identified in distantly related organisms by sequence alignment . Genome and gene sequences can be searched by a variety of tools for certain properties . Sequence profiling tools can find restriction enzyme sites , open reading frames in nucleotide sequences , and predict secondary structures . Phylogenetic trees can be constructed and evolutionary hypotheses developed using special software like ClustalW regarding the ancestry of modern organisms and the genes they express . The field of bioinformatics is now indispensable for the analysis of genes and proteins .
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= = = = Structure prediction and simulation = = = =
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Complementary to the field of structural genomics , protein structure prediction seeks to develop efficient ways to provide plausible models for proteins whose structures have not yet been determined experimentally . The most successful type of structure prediction , known as homology modeling , relies on the existence of a " template " structure with sequence similarity to the protein being modeled ; structural genomics ' goal is to provide sufficient representation in solved structures to model most of those that remain . Although producing accurate models remains a challenge when only distantly related template structures are available , it has been suggested that sequence alignment is the bottleneck in this process , as quite accurate models can be produced if a " perfect " sequence alignment is known . Many structure prediction methods have served to inform the emerging field of protein engineering , in which novel protein folds have already been designed . A more complex computational problem is the prediction of intermolecular interactions , such as in molecular docking and protein – protein interaction prediction .
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The processes of protein folding and binding can be simulated using such technique as molecular mechanics , in particular , molecular dynamics and Monte Carlo , which increasingly take advantage of parallel and distributed computing ( Folding @ home project ; molecular modeling on GPU ) . The folding of small α @-@ helical protein domains such as the villin headpiece and the HIV accessory protein have been successfully simulated in silico , and hybrid methods that combine standard molecular dynamics with quantum mechanics calculations have allowed exploration of the electronic states of rhodopsins .
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= = = = Protein disorder and <unk> prediction = = = =
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Many proteins ( in <unk> ~ 33 % ) contain large unstructured but biologically functional segments and can be classified as intrinsically disordered proteins . Predicting and analysing protein disorder is , therefore , an important part of protein structure characterisation .
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= = Nutrition = =
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Most microorganisms and plants can biosynthesize all 20 standard amino acids , while animals ( including humans ) must obtain some of the amino acids from the diet . The amino acids that an organism cannot synthesize on its own are referred to as essential amino acids . Key enzymes that synthesize certain amino acids are not present in animals — such as <unk> , which catalyses the first step in the synthesis of lysine , methionine , and threonine from aspartate . If amino acids are present in the environment , microorganisms can conserve energy by taking up the amino acids from their surroundings and downregulating their biosynthetic pathways .
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In animals , amino acids are obtained through the consumption of foods containing protein . Ingested proteins are then broken down into amino acids through digestion , which typically involves denaturation of the protein through exposure to acid and hydrolysis by enzymes called proteases . Some ingested amino acids are used for protein biosynthesis , while others are converted to glucose through gluconeogenesis , or fed into the citric acid cycle . This use of protein as a fuel is particularly important under starvation conditions as it allows the body 's own proteins to be used to support life , particularly those found in muscle . Amino acids are also an important dietary source of nitrogen .
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= = History and etymology = =
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Proteins were recognized as a distinct class of biological molecules in the eighteenth century by Antoine Fourcroy and others , distinguished by the molecules ' ability to coagulate or <unk> under treatments with heat or acid . Noted examples at the time included albumin from egg whites , blood serum albumin , fibrin , and wheat gluten .
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Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius in 1838 . Mulder carried out elemental analysis of common proteins and found that nearly all proteins had the same empirical formula , <unk> . He came to the erroneous conclusion that they might be composed of a single type of ( very large ) molecule . The term " protein " to describe these molecules was proposed by Mulder 's associate Berzelius ; protein is derived from the Greek word <unk> ( <unk> ) , meaning " primary " , " in the lead " , or " standing in front " , + -in . Mulder went on to identify the products of protein degradation such as the amino acid leucine for which he found a ( nearly correct ) molecular weight of 131 Da .
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Early nutritional scientists such as the German Carl von Voit believed that protein was the most important nutrient for maintaining the structure of the body , because it was generally believed that " flesh makes flesh . " Karl Heinrich <unk> extended known protein forms with the identification of glutamic acid . At the Connecticut Agricultural Experiment Station a detailed review of the vegetable proteins was compiled by Thomas Burr Osborne . Working with Lafayette Mendel and applying Liebig 's law of the minimum in feeding laboratory rats , the nutritionally essential amino acids were established . The work was continued and communicated by William Cumming Rose . The understanding of proteins as polypeptides came through the work of Franz Hofmeister and Hermann Emil Fischer . The central role of proteins as enzymes in living organisms was not fully appreciated until 1926 , when James B. Sumner showed that the enzyme urease was in fact a protein .
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The difficulty in purifying proteins in large quantities made them very difficult for early protein biochemists to study . Hence , early studies focused on proteins that could be purified in large quantities , e.g. , those of blood , egg white , various toxins , and digestive / metabolic enzymes obtained from slaughterhouses . In the 1950s , the Armour Hot Dog Co. purified 1 kg of pure bovine pancreatic ribonuclease A and made it freely available to scientists ; this gesture helped ribonuclease A become a major target for biochemical study for the following decades .
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Linus Pauling is credited with the successful prediction of regular protein secondary structures based on hydrogen bonding , an idea first put forth by William Astbury in 1933 . Later work by Walter <unk> on denaturation , based partly on previous studies by Kaj Linderstrøm @-@ Lang , contributed an understanding of protein folding and structure mediated by hydrophobic interactions .
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The first protein to be sequenced was insulin , by Frederick Sanger , in 1949 . Sanger correctly determined the amino acid sequence of insulin , thus conclusively demonstrating that proteins consisted of linear polymers of amino acids rather than branched chains , colloids , or cyclols . He won the Nobel Prize for this achievement in 1958 .
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The first protein structures to be solved were hemoglobin and myoglobin , by Max Perutz and Sir John Cowdery Kendrew , respectively , in 1958 . As of 2016 , the Protein Data Bank has over 115 @,@ 000 atomic @-@ resolution structures of proteins . In more recent times , cryo @-@ electron microscopy of large macromolecular assemblies and computational protein structure prediction of small protein domains are two methods approaching atomic resolution .
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= = Textbooks = =
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= = = Databases and projects = = =
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The Protein Naming Utility
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Human Protein Atlas
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NCBI Entrez Protein database
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NCBI Protein Structure database
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Human Protein Reference Database
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Human <unk>
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Folding @ Home ( Stanford University )
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Comparative <unk> Database curates protein – chemical interactions , as well as gene / protein – disease relationships and chemical @-@ disease relationships .
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Bioinformatic Harvester A Meta search engine ( 29 databases ) for gene and protein information .
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Protein Databank in Europe ( see also <unk> , short articles and tutorials on interesting PDB structures )
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Research Collaboratory for Structural Bioinformatics ( see also Molecule of the Month , presenting short accounts on selected proteins from the PDB )
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Proteopedia – Life in 3D : rotatable , zoomable 3D model with wiki annotations for every known protein molecular structure .
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<unk> the Universal Protein Resource
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<unk> – Exploring the universe of human proteins : human @-@ centric protein knowledge resource
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Multi @-@ <unk> Profiling Expression Database : <unk> human and model organism protein / gene knowledge and expression data
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= = = Tutorials and educational websites = = =
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" An Introduction to Proteins " from <unk> ( Huntington 's Disease Outreach Project for Education at Stanford )
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Proteins : Biogenesis to Degradation – The Virtual Library of Biochemistry and Cell Biology
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Alphabet of Protein Structures
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= LiSA ( Japanese musician , born 1987 ) =
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Risa Oribe ( <unk> <unk> , Oribe Risa , born June 24 , 1987 ) , better known by her stage name LiSA ( an acronym of Love is Same All ) , is a Japanese pop singer @-@ songwriter from Seki , Gifu , signed to Aniplex under Sony Music Artists . After aspiring to become a musician early in life , she started her musical career as the vocalist of the indie band Chucky . Following Chucky 's disbandment in 2005 , LiSA moved to Tokyo in order to pursue a solo career , making her major debut in 2010 singing songs for the anime television series Angel Beats ! as one of two vocalists for the fictional band Girls Dead Monster . In April 2011 , she made her solo debut with the release of her mini @-@ album Letters to U. She performed at Animelo Summer Live in August 2010 , Anime Expo in 2012 , and is a regular guest at Anime Festival Asia .
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LiSA 's songs have been featured as theme music for various anime such as Fate / Zero and Sword Art Online . Her singles have regularly been in the top ten of the Oricon weekly charts , with " Crossing Field " being certified platinum by the Recording Industry Association of Japan and " Oath Sign " being certified gold . She performed at the Nippon Budokan in 2014 and 2015 . In 2015 , she made her acting debut as Madge Nelson in the Japanese dub of the animated film Minions .
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= = Career = =
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= = = Early years and major debut = = =
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Oribe 's musical experience began at the age of three when , upon her mother 's suggestions , she took private piano lessons . Although she was a shy student in kindergarten , she enjoyed her music lessons . In primary school , she was inspired to sing after seeing the band Speed on television . She later took dance and vocal lessons , which continued through her junior high school years . While in elementary , she participated in an audition held at the Nippon Budokan , and it was during this time she decided to become an artist . In junior high school , she formed a band which covered songs by Avril Lavigne , Love <unk> , and Ego @-@ <unk> ' .
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Oribe began her singing career in 2005 during her first year in high school when she formed the indie rock band Chucky , which mainly covered songs by other artists . During her second year , the band received advice from their peers that they should start making their own songs ; eventually the band 's reputation grew enough that they performed in Osaka and Nagoya in addition to Gifu . Although her grades in high school were good , she decided not to go to university , despite the recommendations of her teachers , in order to focus on her work with Chucky . Nevertheless , after graduation , due to varying schedules among the band 's members , it became difficult to continue performing . Following the band 's disbandment in July 2008 , she moved to Tokyo in order to continue her singing career .
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After moving to Tokyo , Oribe formed the band Love is Same All with members from the indie band Parking Out and began using the stage name LiSA , which is an acronym for Love is Same All . The band performs with LiSA during the latter 's solo live performances . In 2010 , she made her major debut singing songs for the anime series Angel Beats ! as one of two vocalists for the fictional in @-@ story band Girls Dead Monster . She was the vocalist for the character Yui , and the second vocalist , Marina , sang as the character Masami Iwasawa . LiSA put out three singles and one album in 2010 under the name Girls Dead Monster on Key 's record label Key Sounds Label . The first single " Thousand Enemies " was released on May 12 ; the second single " Little Braver " came out on June 9 ; and the third single " Ichiban no Takaramono ( Yui final ver . ) " ( <unk> 〜 Yui final ver . 〜 , " My Most Precious Treasure ( Yui final ver . ) " ) was sold on December 8 . The album Keep The Beats ! was released on June 30 . LiSA made her first appearance at Animelo Summer Live during the concert 's 2010 iteration on August 28 .
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LiSA made her solo debut on April 20 , 2011 with the release of her mini @-@ album Letters to U by Aniplex under Sony Music Artists . The songs on the album were composed by dōjin and major artists , and she composed the first song " Believe in Myself " ; she wrote the album 's lyrics . On November 12 , 2011 , She made her first appearance in Singapore at Anime Festival Asia , Southeast Asia 's largest anime and pop culture convention . She released her first solo single " Oath Sign " on November 23 , 2011 , which was used as the opening theme to the 2011 anime series Fate / Zero . The single peaked at No. 5 on the Oricon weekly charts and was certified gold by the Recording Industry Association of Japan ( RIAJ ) .
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= = = 2012 – present = = =
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LiSA released her first full solo album Lover " s " mile on February 22 , 2012 ; the album peaked at No. 7 on the Oricon weekly charts . She attended the Anime Expo 2012 in Los Angeles as a guest of honor and performed her first concert in North America there on July 1 . After her success with " Oath Sign " , she was chosen to perform the first opening theme to the 2012 anime series Sword Art Online ; the single " Crossing Field " , her second single , was released on August 8 , 2012 ; the single peaked at No. 5 on Oricon and was later certified gold by the RIAJ . Her third single " Best Day , Best Way " , which peaked at No. 6 on Oricon , was released on April 3 , 2013 , and her fourth single " Träumerei " , which peaked at No. 15 on Oricon and was used as the opening theme to the 2013 anime series Day Break Illusion , was released on August 7 , 2013 . LiSA released her second solo album Landspace on October 30 , 2013 ; the album peaked at No. 2 on Oricon charts . A photobook titled Kyō mo Ii Hi da ( <unk> , " Another Great Day " ) that contains photos of her taken by Lis Ani ! magazine over three years was published on November 22 , 2013 . When asked if she would revisit the meaning of her acronym in the future in an interview with <unk> at Anime Festival Asia 2013 in Singapore , she suggested Lovely international Super Apple .
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On January 3 , 2014 , LiSA held a sold @-@ out solo concert at the Nippon Budokan . Her fifth single " Rising Hope " , which is used as the opening theme to the 2014 anime series The Irregular at Magic High School , was released on May 7 , 2014 ; the single peaked at No. 4 on Oricon . She later covered the songs " Headphone Actor " ( <unk> ) and " <unk> Yesterday " ( <unk> , " Yesterday Evening " ) from the <unk> Project franchise for the sixth episode of the 2014 anime series <unk> Actors . She released her sixth single " Bright Flight / L. Miranic " on September 17 , 2014 , which peaked at No. 8 on Oricon , and her seventh single " <unk> " ( <unk> , lit . " Sign " ) on December 10 , 2014 , which peaked at No. 3 on the Oricon ; the title song is used as the third ending theme to the 2014 anime series Sword Art Online II , and the single also includes the song " No More Time Machine " , which was used as the second ending theme to Sword Art Online II . She held her second Nippon Budokan concert on January 10 and 11 , 2015 , tickets for which were sold out . She released her third solo album Launcher on March 4 , 2015 and her eighth single " Rally Go Round " on May 27 , 2015 ; the song is used as the opening theme to the second season of the anime television series <unk> . She was cast as Madge Nelson in the Japanese dub of the animated film Minions , which premiered in Japanese theaters on July 31 , 2015 . She released her ninth single " Empty Mermaid " on September 30 , 2015 . In late 2015 , she performed " ID " , which is used as theme song of the 2015 video game Dengeki Bunko : Fighting Climax Ignition . To commemorate her fifth year as a solo artist , LiSA released her Letters To U EP as a limited edition LP on March 23 , 2016 . LiSA released a mini @-@ album titled Lucky Hi Five ! on April 20 , 2016 . She will release the single " Brave Freak Out " , which will be used as the opening theme to the 2016 anime television series <unk> Code , on August 24 , 2016 .
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= = Musical style and influences = =
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LiSA lists Avril Lavigne , Oasis , Green Day , Paramore , Ke $ ha , and Rihanna as among her musical influences , as well as her time in Chucky . LiSA wrote the lyrics for some of her songs in her Landspace and Launcher albums , as well as the lyrics for the singles " Bright Flight / L. Miranic " , " <unk> " , and " Rally Go Round " ; " Rally Go Round " was co @-@ written with songwriter Shin Furuya .
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LiSA 's musical style is described by Dennis Amith of J ! -ENT as a young woman with style , beautiful vocals , and the ability to take on various musical styles , may it be happy , upbeat rock music or " even something more darker . " She has used the phrase " kyō mo ii hi da " ( <unk> , " Another Great Day " ) as a theme throughout her career ; the phrase is also the name of her personal blog . In an interview with HMV Japan , she mentioned that before her major debut , she tended to sing pop songs , and because of this , she felt uneasy when making " Oath Sign " , which had a heavy and dark style , although she was able to receive several favorable responses to the single , which allowed her to make her next single " Crossing Field " with confidence . As for " Crossing Field " , she says that feels that the song grows every time she sings it , in the same way that love is nurtured through dates or love letters . In " Best Day , Best Way " , she mentioned that the song 's theme is " what you did yesterday will lead to what happens today " , and that the lyrics are delivered as if they are her own words . She also shared her experience with the single 's B @-@ sides : " I 'm a Rock Star " , which according to her represents her fulfilling her dream of becoming a singer , and " Shiroi <unk> " ( <unk> , " White Sigh " ) , which represents happy feelings .
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In an interview with Oricon , LiSA explained her experiences with " Träumerei " , where she stated that because Day Break Illusion is an original anime , she wanted the song to be presented in the color of her own work , but even in the world of anime , her music would continue to have an atmosphere of rock , as opposed to the pop style she used in " Best Day , Best Way " . She mentioned that she sang that song in a way that she felt the conflicts that were present in the story and with the strength that was part of the show 's theme . With regards to the song 's music video , she wanted to have a video which would express in color emotions , such as the use of red to represent confrontations , black to represent the feeling of being lost , and green for frightening things .
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In making the single " Bright Flight / L. Miranic " , she mentions in an interview with HMV Japan that the concept of the single is that it can be divided into two parts : pink and black . Pink is the color of " Bright Flight " , a song which is in a pop style and was written in a " cute " image in mind , while black is the color of " L. Miranic " , which is in a dark rock style and has the theme of a " bad woman " . LiSA mentioned that the name " L. Miranic " was chosen because the song is linked to criminals and that since " Miranic " is a name in some countries , adding her name to " Miranic " to make " LiSA @-@ Miranic " would mean that the criminal in the song is her .
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Dennis Amith of J ! -ENT reviewed LiSA 's album Landspace , where he describes the song " Crossing Field " as a song about " wanting to forget the weakness and securities of one past and being with someone you love who gives you the strength to become even stronger . " Meanwhile , he describes the song " Best Day , Best Way " as " a fun and happy track about believing in yourself and overcoming anything bad that have happened in the past and moving forward . " Finally , he describes " Träumerei " as " another inspirational song about one losing the rhythm in their heart and is now on the road of loneliness , but still the person wants to pierce the sky with their own light , shatter the darkness and have a fresh new start in life . " He concludes the review by saying that the album is full of upbeat and inspirational songs and showcases LiSA 's talents as a vocalist , and in contrast to many <unk> artists who are known for their cute and sweet vocals , LiSA is able to take on many musical styles .
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= = Discography = =
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The discography of LiSA includes three studio albums , one extended play , ten singles , and five video albums .
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Studio albums
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Lover " s " mile ( 2012 )
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Landspace ( 2013 )
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