Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity.

Hitsaustuotannon kehittäminen pk-konepajassa

Kustannusten pienentäminen hitsauksessa on loputon tie. Pääosa hitsauksen kustannuksista muodostuu hitsaukseen käytetystä ajasta. Tämä diplomityö on tarkoitettu avustamaan yrityksiä mekanisoinnin ja robotisoinnin käyttöönotossa sekä mekanisointilaitteiden ja hitsausrobottien investoinneissa ja niiden kannattavuuden arvioimisessa. Tämä diplomityö on jatkoa alueella aikaisemmin tehdyille selvityksille, joiden perusteella havaittiin Savonlinnan seudun yritysverkoston hitsaavilla pk-konepajoilla olevan kehitystarpeita tuotannossaan ja toiminnassaan. Tässä diplomityössä tarkastellaan ensin teoriatasolla hitsaustuotannon tehostamista eri keinoin, joista tarkemmin tarkastellaan hitsauksen mekanisointia ja robotisointia. Lisäksi selvitetään kolmella yritys-case-esimerkillä hitsausajan vähentämisen vaikutusta kustannuksiin. Samalla esitetään sopivia laitteistoinvestointeja, joilla hitsausajan vähentäminen on mahdollista, sekä tarkastellaan näiden investointien kannattavuutta sekä syntyviä säästöjä.