Detection and analysis of genetic alterations in normal skin and skin tumours

The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin. Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis. After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC\u92s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique. Keywords:single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-rasNR 2014080

Bemötande av patienter med psykisk ohälsa : - patienters upplevelser

Bakgrund:Psykisk ohälsa är ett ökande folkhälsoproblem. Genom att stärka patienters självkänsla läggs en grund för möte och behandling. Syfte: var att ur patientperspektiv belysa hälso- och sjukvårdens bemötande av patienter med psykisk ohälsa. Metod: Artiklar söktes i PubMed och PsycInfo. En textnära kvalitativ innehållsanalys gjordes. Åtta subkategorier identifierades: information, osynlighet, ej öppen dialog, utanförskap, respektlöshet, bristande socialt nätverk, låg självkänsla och brist på autonomi. Dessa sorterades under tre kategorier: lågt människovärde, kommunikation och brist på empowerment. Resultat: Patienter upplevde brister i kommunikation och bemötande samt okunskap hos vårdspersonal. Ett bra bemötande möjliggjorde en öppen och ärlig kommunikation där tillit och förtroende skapades. Diskussion: En tydligare holistisk människosyn kan skapa förutsättningar för bättre kommunikation mellan patient och vårdspersonal vilket i sin tur kan leda till ömsesidig förståelse

Internet Marketing and Internet Marketing Development in Latvia.

Bakalaura darbā tiek pētīta interneta mārketinga būtība, tā metožu pielietojums un efektivitāte praksē, kā arī, interneta mārketinga attīstība un nākotnes perspektīvas Latvijā. Bakalaura darba mērķis ir, pamatojoties uz teorijā sniegtajām atziņām par interneta mārketingu un nozari raksturojošo statistisko datu analīzi, izvērtēt interneta mārketinga attīstības tendences un nozares potenciālu Latvijā, kā arī, izstrādāt priekšlikumus interneta mārketinga nozares attīstības veicināšanai Latvijā. Darba izstrādē tiks veikta referatīvā interneta mārketinga literatūras analīze, statistisko un ekonomisko datu analīze, SIA „Infinitum 8” sniegto pakalpojumu izpēte, nozares vadošo speciālistu interviju analīze un projekta „kredituabc.lv” analīze. Darba apjoms ir 153 lpp., bakalaura darbā ir 8 tabulas, 54 attēli un 6 pielikumi. Atslēgvārdi: interneta mārketings, SEO (Optimizēšana meklētājsistēmām), Google AdWords, partnermārketings(Affiliate), sociālo tīklu mārketings, e-pasta mārketings.This bachelor thesis is a research about the nature of Internet marketing, practical use and efficiency of internet marketing methods, as well as development and future perspectives of Internet marketing in Latvia. The aim of the thesis is, by analyzing both the theoretical opinions about the Internet marketing and the industry-specific statistics, to evaluate the development tendency and industry potential of Internet marketing in Latvia, as well as to give suggestions for the development of the Internet marketing industry in Latvia. During the development of the thesis the following has been done: the analysis of internet marketing-related literature, statistics and economics-related data, the research and analysis of the services provided by Infinitum 8 Ltd., interviews of industry leading professionals, as well as the analysis of the project „kredituabc.lv”. The thesis consists of 153 pages, 8 tables, 54 images and 6 appendixes. Keywords: Internet marketing, SEO (Search engine optimization), Google AdWords, affiliate marketing, social network marketing, email marketing

Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of &gt;60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of &gt;150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.QC 20150203</p

Tgfbr3l—an uncharacterised pituitary specific membrane protein detected in the gonadotroph cells in non-neoplastic and tumour tissue

Here, we report the investigation of transforming growth factor beta-receptor 3 like (TGFBR3L), an uncharacterised pituitary specific membrane protein, in non-neoplastic anterior pituitary gland and pituitary neuroendocrine tumours. A polyclonal antibody produced within the Human Protein Atlas project (HPA074356) was used for TGFBR3L staining and combined with SF1 and FSH for a 3-plex fluorescent protocol, providing more details about the cell lineage specificity of TGFBR3L expression. A cohort of 230 pituitary neuroendocrine tumours were analysed. In a subgroup of previously characterised gonadotroph tumours, correlation with expression of FSH/LH, E-cadherin, oestrogen (ER) and somatostatin receptors (SSTR) was explored. TGFBR3L showed membranous immunolabeling and was found to be gonadotroph cell lineage-specific, verified by co-expression with SF1 and FSH/LH staining in both tumour and non-neoplastic anterior pituitary tissues. TGFBR3L immunoreactivity was observed in gonadotroph tumours only and demonstrated intra-tumour heterogeneity with a perivascular location. TGFBR3L immunostaining correlated positively to both FSH (R = 0.290) and LH (R = 0.390) immunostaining, and SSTR3 (R = 0.315). TGFBR3L correlated inversely to membranous E-cadherin staining (R = −0.351) and oestrogen receptor β mRNA (R = −0.274). In conclusion, TGFBR3L is a novel pituitary gland specific protein, located in the membrane of gonadotroph cells in non-neoplastic anterior pituitary gland and in a subset of gonadotroph pituitary tumours

RNA- and Antibody-Based Profiling of the Human Proteome with Focus on Chromosome 19

An important part of the Human Proteome Project is to characterize the protein complement of the genome with antibody-based profiling. Within the framework of this effort, a new version 12 of the Human Protein Atlas (www.proteinatlas.org) has been launched, including transcriptomics data for 27 tissues and 44 cell lines to complement the protein expression data from antibody-based profiling. Besides the extensive addition of transcriptomics data, the Human Protein Atlas now contains antibody-based protein profiles for 82% of the 20 329 putative protein-coding genes. The comprehensive data resulting from RNA-seq analysis and antibody-based profiling performed within the Human Protein Atlas as well as information from UniProt were used to generate evidence summary scores for each of the 20 329 genes, of which 94% now have experimental evidence at least at transcript level. The evidence scores for all individual genes are displayed with regards to both RNA- and antibody-based protein profiles, including chromosome-centric visualizations. An analysis of the human chromosome 19 shows that ∼43% of the genes are expressed at the transcript level in all 27 tissues analyzed, suggesting a “house-keeping” function, while 12% of the genes show a more tissue-specific pattern with enriched expression in one of the analyzed tissues only

Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins

The localization of proteins at a tissue- or cell-type-specific level is tightly linked to the protein function. To better understand each protein's role in cellular systems, spatial information constitutes an important complement to quantitative data. The standard methods for determining the spatial distribution of proteins in single cells of complex tissue samples make use of antibodies. For a stringent analysis of the human proteome, we used orthogonal methods and independent antibodies to validate 5981 antibodies that show the expression of 3775 human proteins across all major human tissues. This enhanced validation uncovered 56 proteins corresponding to the group of "missing proteins" and 171 proteins of unknown function. The presented strategy will facilitate further discussions around criteria for evidence of protein existence based on immunohistochemistry and serves as a useful guide to identify candidate proteins for integrative studies with quantitative proteomics methods

Reverse analysis of proteins previously characterised as glomeruli specific.

<p>4 of 20 glomeruli specific proteins selected based on literature (NPHS2, PODXL, NPHS1 and PTPRO) are found in the group of kidney enriched/enhanced proteins when compared to RNAseq data for the 27 tissues. Furthermore, 6 of the 20 genes do not display a glomeruli-specific localisation within kidney (ITGB5, GALNT10, CD2AP, SH2D4A, LAMB2, CLDN5). Numbers under each IHC image correspond to mean FPKM in kidney (left bottom), Max FPKM in next most highly expressed tissue and the tissue name (middle bottom), and tissue specificity score in kidney (right bottom).</p

Kidney enriched proteins previously not described in kidney.

<p>Immunohisto-chemically stained images of kidney enriched proteins (TS>5 FPKM) for which we found no previous description expression in kidney based searches in literatures or online databases (GeneCards, WikiGene, BioGPS). Of the 16 kidney enriched genes not described previously, IHC images confirming kidney specific localisation were available for 10 genes. In proximal tubule cells, AP000322.53 localise to the nucleus while TMEM52B, RAB11FIP3 and CRYAA show a general cytoplasmic staining, and RNF152 shows a granular cytoplasmic staining. In distal tubule, TMEM72 localises to the basolateral membrane. C9orf66 is found in all nephron segments but with different localisations. In glomeruli, it localise to the nucleus, in proximal tubular cells to nucleus and cytoplasm, while in cells in distal tubule and collecting duct only in cytoplasm. Both HMX2 and CLEC18B localises to the cytoplasm of cells in proximal and distal tubule, and also in the collecting duct, however for CLEC18B only in the intercalated cells. Numbers under each IHC image correspond to Mean FPKM in kidney (left bottom), Max FPKM in the tissue with 2^nd most highly expressed level (middle bottom), and Tissue specificity score in kidney (right bottom).</p