Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface.  The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection.  In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future. / <p>QC 20170904</p>

The present thesis discusses how gene regulation by transforming growth factor β (TGFβ) family cytokines is affected by post-translational modifications of different transcription factors. The thesis also focuses on gene regulation by transcription factors involved in TGFβ signaling. The importance of the poly ADP-ribose polymerase (PARP) family in controlling gene expression in response to TGFβ and bone morphogenetic protein (BMP) is analyzed first. PARP2, along with PARP1, ADP-ribosylates Smad2 and Smad3, the signaling mediators of TGFβ. On the other hand, poly ADP-ribose glycohydrolase (PARG) removes the ADP-ribose from Smad2/3 and antagonizes PARP1 and PARP2. ADP-ribosylation of Smads in turn affects their DNA binding capacity. We then illustrate how PARP1 and PARG can regulate gene expression in response to BMP that signals via Smad1, 5. Over-expression of PARP1 suppressed the transcriptional activity of Smad1/5. Knockdown of PARP1 or over-expression of PARG enhanced the transcriptional activity of BMP-Smads on target genes. Hence our data suggest that ADP-ribosylation of Smad proteins controls both TGFβ and BMP signaling.  I then focus on elucidating novel genes that are regulated by ZEB1 and Snail1, two key transcriptional factors in TGFβ signaling, known for their ability to induce EMT and cancer metastasis. Chromatin immunoprecipitation-sequencing (ChIP-seq) and targeted whole genome transcriptomics in triple negative breast cancer cells were used, to find binding regions and the functional impact of ZEB1 and Snail1 throughout the genome. ZEB1 binds to the regulatory sequences of a wide range of genes, not only related to cell invasion, pointing to new functions of ZEB1. On the other hand, Snail1 regulated only a few genes, especially related to signal transduction and cellular movement. Further functional analysis revealed that ZEB1 could regulate the anchorage-independent growth of the triple negative breast cancer cells, whereas Snail1 could regulate the expression of BMP6 in these cells. We have therefore elucidated novel functional roles of the two transcription factors, Snail1 and ZEB1 in triple negative breast cancer cells.

Alzheimer’s disease (AD), the most common form of dementia is associated with fibril formation of amyloid-ß peptides (Aß). Aß, proteolytically derived from Aß precursor protein (AßPP), is the major component of amyloid plaques in AD brains. Familial British and Danish dementias (FBD and FDD) share pathological and clinical characteristics with AD, and the underlying mechanisms are associated with amyloid formation of mutant peptides released from the Bri2 protein. Bri2 interacts with AßPP and its BRICHOS domain has been shown to delay Aß40 and Aß42 fibril formation and toxicity in vitro and in vivo. This makes Bri2 BRICHOS a promising anti-amyloid chaperone and a potential treatment strategy for AD. Furthermore, Bri2 BRICHOS possesses a general chaperone activity as it suppresses non-fibrillar aggregation of destabilized citrate synthase (CS). Recent findings show that Bri2 BRICHOS produced in E.coli can form different molecular weight assemblies, ranging from monomers to dimers and poly-disperse oligomers. The oligomers inhibit CS aggregation, whereas the monomers and dimers are more efficient against Aß42 fibrillation and neurotoxicity, respectively. The work in this thesis shows that similar Bri2 BRICHOS quaternary structures are formed in eukaryotic cells as in E.coli. Larger BRICHOS oligomers were found in cell media, derived from proteolytically processed endogenous Bri2 in SH-SY5Y cells, as well as in human embryonic kidney (HEK293) cells transfected with a Bri2 BRICHOS construct. Recombinant human Bri2 BRICHOS mutants with one or none of the two strictly conserved cysteine residues were studied. All mutant monomers become proteolytically degraded during purification, but form stable oligomers. Single Cys to Ser mutants form stable disulfide-dependent dimers that differ in ability to prevent Aß42 fibrillation, the most stable mutant (C164S) being even more efficient than the wildtype Bri2 BRICHOS dimer. This result suggests that intra or intermolecular disulfide(s) and oligomerization affect Bri2 BRICHOS stability and activity towards Aß42 fibril formation.

Direct studies of speciation are possible in the superspecies complex of Drosophila paulistorum, which consists of six different semispecies undergoing incipient speciation. Strict maternal inheritance of mitochondria is the most common pattern of mitochondrial inheritance in animals. Here I show that paternal transmission of mitochondrial DNA occurs in the heteroplasmic Orinocan semispecies and is not limited to hybrid offspring. Inheritance of one mitotype is mainly maternal while the other is mainly paternal; a highly unusual pattern of mitochondrial inheritance. I used absolute quantification real-time PCR on DNA extracted from eggs and imagoes from the Amazonian and Orinocan semispecies, as well as hybrids between these two semispecies. In crosses performed between F1 hybrids with a combination of mitotypes not found in any of the parents, no F2 hybrids were acquired. One possible explanation for this is that differences in mitotypes and inheritance patterns of mitochondrial DNA may cause incompatibilities between the genomes of D. paulistorum. This may be one cause of hybrid inviability and genetic isolation between semispecies, a necessary part of the speciation process. This further complicates the story of the ongoing speciation process in the D. paulistorum superspecies complex, which offers much to learn about speciation, mitochondrial inheritance and interactions between multiple genomes in the same organism.

DuMPLING is a newly developed high-throughput method to study singlecellphenotypes in a pooled and barcoded library using a microfluidicchip. The chip enables parallel biophysical measurements of singlecells, after which in-situ genotyping connects the cells to a certainstrain of the library. The method has been previously applied with abarcoded library, where genotyping was performed on barcodes presenton high copy number plasmids. In this project, I apply and developthe Rolling Circle Amplification method to amplify the signal frombarcodes present on the E. coli chromosome. A small librarycontaining three different chromosomal barcodes is investigated. Veryhigh efficiency of signal generation is achieved for the firstbarcode, good efficiency is achieved for the second, and no signal isachieved for the third. Genotyping is also successfully performed ona strain with two different barcodes present on the chromosome. Thegenotyping method described herein can be applied to screen foradditional barcodes that may be incorporated in a larger library thatin turn can be used to ask important biological questions, forexample using the high throughput DuMPLING method.

Alzheimer’s Disease (AD) is the leading cause of dementia in the world and the World Health Organization has recognized AD as a global public health priority. One of the pathological hallmarks of AD is amyloid plaques formed from amyloid β (Aβ) fibrils. Aβ is formed when amyloid precursor protein is cleaved by secretase enzymes. Cleavage by different secretases causes Aβ to occur in different forms, mainly as 40 and 42 residue long proteins, called Aβ1-40 and Aβ1-42, where Aβ1-42 is more likely to form amyloid fibrils and is therefore considered more harmful. Fluorescent probes are currently used to stain Aβ fibrils for their detection and characterization.  We performed a literature study analysing which fluorescent probes are used for imaging of amyloid fibrils and present both the most commonly used probes but also newer probes that have been recently synthesized. Fluorescence spectra of a selection of probes were analysed in order to suggest some new combinations of probes for double-staining with the aim to be able to distinguish between Aβ1-40 and Aβ1-42. Microscopy images of the probe combinations were obtained in order to analyse the double staining results and the fluorescence intensities of the probes were plotted in different ways. All selected combinations were able to distinguish between Aβ1-40 and Aβ1-42, because of differently stained fibrils, and also displayed differences in fluorescence intensity at peak emission wavelength. The obtained results show that double-staining of amyloid fibrils with fluorescent probes can give additional information compared to staining fibrils with only one probe.

Cross-reactivity occurs when an antibody binds to the epitope of a protein that is not the targeted antigen. This is problematic in the analysis of immunoassay diagnostics. Detecting a protein incorrectly might cause issues such as incorrect mapping of metabolic conditions for research or diagnosis. In this study, articles have been collected within two main fields. The first of which is focused on bioinformatic tools to predict cross-reactivity risk and the second field investigates how single substitutions affect the antibody-antigen binding. The results from the collected articles were analyzed with the aim of providing as much information surrounding the topic as possible, to gain a further understanding of how protein similarities impact cross-reactivity. FASTA alignments proved to be efficient in classifying cross-reactive proteins based on sequence similarity. Moreover, epitope analysis, using PD tool or Cross-React, can provide an even more precise subset of proteins with risk of causing cross-reactivity. Individual residues of the epitopes of the subset can then be analyzed. Specific residue’s physicochemical properties such as hydrophobicity, polarity, size and charge have proven to be relevant for the binding affinity, with charge having the largest impact. The position of an amino acid has also shown great importance. More centrally located amino acids within the epitope contribute more to paratope affinity than those on the outer positions. However, a conclusive classifier based on specific residues within epitopes is difficult to implement in cross-reactivity analysis. A workflow of the different prediction steps has been constructed into a workflow that may be implemented as an automated pipeline in the future.

Characteristics as unknown cause, complicated pathophysiology and a great amount of complexity are describing systemic lupus erythematosus (SLE) more than well. It’s an autoimmune disease that is almost exclusive for women in their reproductive years and are believed to correlate with both genetics and environmental factors. Risk factors like stress, usage of cigarettes or birth controls with estrogen and infections are believed to trigger the progression of SLE. The spectra of therapeutic drugs are narrow due to the complexity and requirement of financial resources for scientific causes. Treatment is mainly symptomatic. The alternative splicing (AS) is a highly complex mechanism that is essential and are able to generate a great diversity of proteins encoded by the same gene are referred to as isoforms. Splicing occurs after the transcription that generates pre-mRNA because the exons need to fuse together and excision of introns. In patients with cancer diagnosis, they have observed that progression of disease and AS are correlated by the means of isoforms and splicing regulators. In studies, the relevance of alternative splicing events in SLE has been shown for both splicing regulators like SRSF1 and different isoforms for example CD44 and CD45. The aim of this study was to evaluate the potential biomarker AS in SLE.  This study of literature started with looking for clinical trials within databases like PubMed and Web of Science, that matched the aim of the study. Usage of terms like ‘SLE and biomarker’ och ‘SLE and alternative splicing’ etcetera. After inclusion of six scientific articles the author started the work with this literature of study.  Results gave strong indications that usage of alternative splicing as biomarker do have strong potential. Although the need of more goal-oriented scientific studies is required. Results from all six studies can be summarized by the line of argument that AS, in different ways, are somehow involved in the pathogenesis and progression of SLE. Both spliceosome, isoforms, splicing factors, other proteins and is also a possible, in the future, therapeutic target for example monoclonal antibodies. Other therapeutic targets maybe against phosphatases and kinases. New strategies are going to bring hope for the patients that are suffering from SLE, especially when the disease is active for 2–3 years. Being able to individualize treatment are going to generate a better quality of life for many SLE patients and usage of AS as a biomarker for disease severity.

Alternative splicing is the process of producing a variety of transcripts from one and the same gene. This adds further possible variability to gene expression and can in theory mean that one protein coding gene can produce multiple proteins with potentially different functions. Therefore, to understand the function of a gene, alternative splicing must be accounted for. However, this is made more complex by the fact that the existence of different messenger RNA isoforms does not necessarily entail different protein isoforms, which in turn means that an analysis of both the transcripts and final protein is necessary. Far Upstream Element Binding Protein 1 Like 1 (FUBL-1, or C12D8.1) is an RNA binding protein in Caenorhabditis elegans which is believed to take part in gene regulation, and which seemingly interacts within an argonaut effector pathway called ERGO-1. The gene has five proposed isoforms for which there are varying amounts of RNA data but only the first isoform, FUBL-1a has proteomics data available. In other words, different messenger RNA isoforms exist but it is unclear which are translated into protein. In this study, I have looked at fubl-1 and its isoforms to gain further understanding of this protein. This entailed both analysing long read RNA sequencing data to identify messenger RNA isoforms as well as a laboratory analysis of the protein to look for protein isoforms. I found evidence for all isoforms existing as messenger RNAs, and fubl-1a was by far the most highly expressed. In my protein analysis, I found indications of different isoforms, but not conclusive evidence.

Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).