In this laboratory study, the role of juvenile hormone (JH) and ecdysone in regulating wing polyphenism was investigated in the non-model organism Gerris buenoi. Topical application of the JH analog methoprene elicited reduced pronotum, wing defects, and nymphal-adult intermediates but no changes to wing morph. Similarly, while microinjection of the ecdysone derivative 20-hydroxyecdysone (20E) elicited aberrant phenotypes there was again no influence on the wing morph. Using data from a transcriptomics experiment, RNAi knockdown of the differentially expressed 20E induced receptor gene, Hr4, caused high mortality rates (> 90 %) which resulted in a sample size too small to draw any inferences of Hr4’s involvement in G. buenoi wing polyphenism. My results indicate that both JH and ecdysone are involved in several developmental processes including wing development, but they do not seem to be important for determining wing polyphenism. However, several factors are important to consider in future research which means that the potential role of JH and ecdysone in G. buenoi wing polyphenism should not be dismissed at this stage.

No description available.

Primary cell walls are a composite of cellulose microfibrilsand hemicelluloses. Xyloglucan is the principal hemicelluloseof primary cell walls of dicotyledons. Xyloglucanendotransglycosylases (XETs) cleave and religate xyloglucanpolymers in plant cell walls. A XET (PttXET16A) from hybridaspen has been heterologously expressed and characterized inour lab. To study XETs enzymology on a molecular level a series ofnovel xyloglucan oligosaccharides (XGOs) have been synthesized.The chromogenic 2-nitrophenol XGO and fluorogenic XGOs havebeen used as kinetic probes for PttXET16A. The first 3-Dstructure of the XET and of the enzyme-substrate complexrevealed new insights into the requirements fortransglycosylation. Cellulose fibers are an important raw material for manyindustries. In a novel chemo-enzymatic approach, thetransglycosylating activity of XET was used for biomimeticfiber surface modification. The aminoalditol XGO derivate wasused as key intermediate to incorporate novel chemicalfunctionality into xyloglucan. TheXGO derivatives wereintegrated into xyloglucan with PttXET16A. The resultingmodified xyloglucan was used as a versatile tool fiber surfacemodification.

A solid phase extraction (SPE) method was developed for simultaneous extraction of dicarboxylic acids and diols formed during hydrolysis of poly(butylene succinate), PBS, and poly(butylene adipate), PBA. The developed SPE method and subsequent GC-MS analysis were used to extract, identify and quantify low molecular weight products migrating from linear and branched poly(butylene adipate) (PBA) and poly(butylene succinate) (PBS) during aging in aqueous media. The combination of SPE and GC-MS proved to be a sensitive tool, able to detect small differences in the degradation rate during early stages of hydrolysis before any significant differences were observed by weight loss and molecular weight measurements. The detected low molecular weight products included monomers i.e. adipic acid and 1,4-butanediol for the PBA polymers and succinic acid and 1,4-butanediol for PBS. Several dimers and trimers i.e. hydroxybutyl adipate, hydroxybutyl succinate, di(hydroxybutyl) adipate, di(hydroxybutyl) succinate and hydroxybutyl disuccinate were also detected. Best extraction efficiency for 1,4-butanediol and succinic acid was achieved with a hydroxylated polystyrene-divinylbenzene resin as solid phase. Linear range for the extracted analytes was 1-500 ng/ml for adipic acid and 2-500 ng/ml for 1,4-butanediol and succinic acid. Detection and quantification limits for all analytes were between 1-2 ng/ml (S/N=3) and 2-7 ng/ml (S/N=10) respectively. Relative standard deviations were between 3 % and 7 %. Comparison of measured weight loss and the amount of monomeric products showed that weight loss during early stages of hydrolysis was mainly caused by the release of water-soluble oligomers that on prolonged ageing were further hydrolyzed to monomeric species. Significant differences in degradation rate could be assigned to degree of branching, molecular weight, aging temperature and degradation medium. Linear and branched PBA was mixed with PVC in solution cast films to study the effects of molecular weight and branching on plasticizer efficiency. Used as polymeric plasticizer, PBA formed a semi-miscible two-phase system with PVC where the amorphous part exhibited one single glass transition temperature and the degree of polyester crystallinity was dependent on molecular weight, degree of branching and blend composition. Plasticizing efficiency was favored by higher degree of branching and a 40 weight-percent polyester composition. / QC 20101209

Ovarian cancer is a disease with a high rate of mortality where the need for novel treatments will increase in the near future as older and populous generations reach the age where the cancer is usually diagnosed. Once treated, ovarian cancer tends to recur and display a newfound resistance against the platinum-based chemotherapeutic drugs that are used to treat the disease. Therefore, devising new methods of treatment is of utmost importance. Treatment with oncolytic viruses like LOAd703 offers an alternative treatment option that is more specific, causes immunogenic cell death in tumor cells, can stimulate the patient’s own immune system into fighting the cancer, and also has the potential to induce long term immune memory. In this project, the oncolytic and immunogenic capacity of LOAd703 in three different ovarian cancer cell lines was tested in conjunction with the standard-of-care chemotherapeutical drugs paclitaxel, cisplatin and carboplatin. The chemotherapy did not inhibit the replication, transgene expression or oncolysis of the LOAd703 virus. LOAd703 was able to effectively induce oncolysis in all three cell lines. The oncolytic capacity was generally increased when combined with chemotherapeutics. In cells resistant to chemotherapeutics, combination therapy with LOAd703 increased the killing capacity. While combination therapy proved effective, it did leave behind a small population of tumor cells that appeared to be resistant to both chemotherapy and viral oncolysis but longer culturing times may be tested to evaluate if complete killing will occur or if it is a primary resistance to these treatments in the cell lines. Further, if there is resistance to oncolysis or chemotherapy-mediated killing, employing tumor-immune cell co-cultures or in vivo studies might be necessary in order to assess whether the immunostimulatory effects of LOAd703 will lead to a complete eradication of the remaining tumor cells. The treatments also caused an increase in the expression of certain cell surface markers, like PD-L1 and CD262, which might open the door for future trials combining chemotherapy and LOAd703 with anti-PD-L1 inhibitors or soluble TRAIL.

A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases Candida antarctica lipase B and Rhizomucor miehei lipase showed large chemoselectivity ratios, defined as (kcat/KM)OH / (kcat/KM)SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (paper I). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times higher for Candida antarctica lipase B and ten times higher for Rhizomucor miehei lipase. The KM towards the thiol was more than two orders of magnitude higher than the KM towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, Candida antarctica lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (paper II). Poly(e-caprolactone) with a free thiol at one of the ends was synthesized in an enzymatic ring-opening polymerization initiated with mercaptoethanol or terminated with either 3-mercaptopropionic acid or g-thiobutyrolactone.

Produktionen av rekombinanta biofarmaceutiska läkemedel, exempelvis monoklonala antikroppar, är en oumbärlig men ansträngande process. Under 2019 var sju av de tio mest sålda läkemedlen globalt baserade på rekombinant producerade monoklonala antikroppar. En betydande flaskhals i utvecklingen av stabila cellinjer är screening och selektion av högproducerande singelcellkloner. Tekniker som utspädningskloning (limiting dilution) och fluorescensaktiverad cellsortering (FACS) når idag medelmåttiga resultat som bäst, men saknar förmåga att både isolera produktiva singelcellkloner och hålla cellviabilitet på en acceptabel nivå. Samtidigt kan CellCelectorn™, ett helt automatiserat instrument utformat för att detektera, selektera och isolera singelcellkloner, screena celler för att kartlägga deras produktivitet. CellCelectorn™ är utrustad både med brightfield- och fluorescerande kameror och kan ranka samtliga cellkloner i en provplatta efter produktivitet för att sedan överföra de mest lovande klonerna till en destinationsplatta för vidare analyser och singelklonexpansion. Med avstamp i detta instrument var syftet med projektet att utveckla ett automatiserat arbetsflöde för screening och selektion av högproducerande singelcellkloner med hög genomströmning vid generering av stabila cellinjer. Inledande tester med redan utvecklade och produktiva cellinjer genomfördes för att undersöka CellCelectorns™ prestanda och för att upprätta och optimera sekundära parametrar relevanta för instrumentets kapacitet. Då dessa inledande tester var framgångsrika utfördes en polyklonal selektion i syfte att utveckla tre stabila cellinjer producerandes olika antikroppar. Dessa cellinjer skulle sedan användas för att undersöka CellCelectorns™ förmåga att isolera och överföra de mest lovande singelklonerna till en destinationsplatta. Dessa test kunde tyvärr inte genomföras, då kontaminering i de polyklonala poolerna hade uppstått, men de övergripande resultaten av detta projekt indikerar att CellCelectorn™ är kapabel till att screena för och ranka singelkloner efter deras produktivitet samt att selektera högproducerande klonkandidater under en enda arbetsvecka. Framtida tester är dock nödvändiga för att säkerställa CellCelectorns™ förmåga att isolera singelcellkloner med hög genomsnittlig cellviabilitet för att kunna genomföra singelklonexpansion. / The production of recombinant biopharmaceuticals, such as monoclonal antibodies (mAbs), from stable mammalian cell lines is an indispensable yet strenuous process. In 2019, seven of the top ten most sold drugs globally were based on monoclonal antibodies produced recombinantly. A prominent bottleneck in stable cell line development is the screening and selection of high target protein producing single clone candidates. Today, techniques such as limiting dilution and Fluorescence Activated Cell Sorting (FACS) receive moderate success at best at isolating single clones while keeping cell viability high. Simultaneously, the CellCelector™ is a fully automated instrument designed for the detection, selection, and isolation of single cell clones. Containing both a brightfield and fluorescence-detecting camera, the CellCelector™ can screen cells to measure their productivity and rank them accordingly, meaning high target protein producing clones can be selected and transferred to a destination plate for single clone expansion. Thus, this project aimed at developing an automated high-throughput workflow using the CellCelector™ to streamline the screening and selection steps of stable mammalian cell line generation. Several tests with stable proof-of-concept cells were performed to evaluate the performance of the CellCelector™ and to establish favorable secondary parameter settings. As initial proof-of-concept tests showed promise, a polyclonal selection to obtain three stable Human Embryonic Kidney (HEK) cultures expressing different antibodies was carried out in order to test the CellCelector™ proficiency in clone selection and picking. Unfortunately, no clone picking or single clone expansion could be executed due to bacterial contamination in the cell cultures. Nevertheless, the overall results of this project indicate high potential of the CellCelector™ to detect and identify promising stable clone candidates for single clone expansion over the course of a single work week. Future tests are however required to solidify CellCelector™ ability to isolate monoclonal clones while preserving cell viability for single clone expansion.

Klinisk kemiska analyser har hög klinisk relevans. I serum/plasma kan olika parametrar kvantifieras. Dessa parametrar kan vara proteiner, enzymer, joner, metaller, lipider och kolhydrater. Med hjälp av referensintervall kan veterinärer ställa diagnos, följa behandling och sjukdomsförlopp. Parametrar detekteras med olika analysprinciper/metoder; kolorimetri, immunturbidimetri, enzymatisk metod och potentiometri. Djursjukhuset, AniCura, i Hässleholm mottagas både hundar och katter. Cirka 120 kemianalyser analyseras varje dag. AniCura har köpt in ett nytt våtkemiiinstrument, Indiko Plus, som ska ersätta Cobas C111. Med Indiko Plus tillkommer fler provpositioner, 8 nya analyser, ökad kapaciteten och underlättad användning.  Syftet med denna studie var att jämföra instrumenten och ta fram eget referensintervall som jämfördes med referensintervall framtaget av Thermo Fisher. Verifiering av det nya instrumentet genomfördes med precisionsstudie och linjäritetsstudie. Provtagning på hundar och katter utfördes av personal på AniCura. Jämförelsen gjordes med patientprover och referensintervall togs fram med hjälp av prover från friska hundar och katter. Jämförelsen visade att 9 av 13 analyser hade statistisk signifikant skillnad. Orsaken till det beror troligen på skillnaden av reagens, instrumentens ålder och tid mellan mätningar. Ett nytt referensintervall utarbetades och skiljde sig inte mycket från Thermo Fishers intervall. Vidare validering på grund av liten population rekommenderades. Precisionen för Indiko Plus blev godkänd. Linjäriteten blev icke linjär och berodde troligen på en dålig pipett och bör göras om. / Clinical chemical analyzes has high clinical relevance. In serum/plasma, different parameters can be quantified. Parameters can be proteins, enzymes, ions, metals, lipids, and carbohydrates. With reference intervals, veterinarians can set diagnosis, follow treatment and development of the disease. Parameters are detected with different analysis principles/methods; colorimetry, immunoturbidimetry, enzymatic method and potentiometry. The animal hospital, AniCura, in Hässleholm accept dogs and cats. About 120 chemical analyzes are analyzed every day. AniCura purchased a new instrument, Indiko Plus, which will replace Cobas C111. Indiko Plus provide, more sample positions, 8 new analyzes, increased capacity, and facilitated use. The purpose of this study was to compare the instruments and produce a new reference interval which was compared to the reference interval provided by Thermo Fisher. To verify Indiko Plus, a precision and linearity study were conducted. Blood sampling of dogs and cats was performed by staff at AniCura. The comparison was made with patient samples and the reference intervals were obtained using samples from healthy animals. The comparison showed 9 of 13 analyzes had a statistically significant difference. The reason for this is probably due to the difference in reagents, the age of the instruments and the time between measurements. A new reference interval was developed and did not differ much from the Thermo Fisher interval. Further validation due to low population was recommended. The precision for Indiko Plus was approved. The linearity study shows not linear trend but was likely due to a bad pipette and should be redone.

Development of methods for studying drug delivery systems is of great significance for the improvement of topical formulations. Active compounds for topical drug delivery are often formulated into gels and creams, that can be applied onto skin surfaces. It is important to know the extent of the permeability of the active compounds, in order to determine the medical effect. This study examines the possibilities of using an animal-free skin equivalent for penetration and permeation experiments, i.e. a silk scaffold integrated with viable human dermaland epidermal cells. Mammalian cell culturing together with silkconstruct formation, constituted the upstream bioprocess and acquisition of the skin equivalents. Permeability of fluorescently labelled Bovine Serum Albumin and Sodium Fluorescein salt was assessed, using a Franz- cell setup incorporated with the skin equivalents. Furthermore, fluorescence analysis and SDS-PAGE was performed on the collected samples, along with cryosectioning and image analysis of the skin equivalents. The results indicate variations in tissue integrity, leading to both high and low permeability. Fluorescence intensity can be correlated with the amount of sample liquid passing through. The model is still under development, hence more research is needed to draw a conclusion regarding the cellular composition of the skin equivalents, and how it influences permeability. / NextBioForm

Each year countless lives are affected and about half a million people die from malaria, a disease caused by parasites originating from the Plasmodium family. The most virulent species of the parasite is Plasmodium falciparum (P. falciparum).   Calmodulin (CaM) is a small, 148 amino acid long, highly preserved and essential protein in all eukaryotic cells. Previous studies have determined that CaM is important for the reproduction and invasion of P. falciparum in host cells. The primary structure of human CaM (CaMhum) and CaM from P. falciparum (CaMpf) differ in merely 16 positions, making differences in their structures and ligand affinity interesting to study. Especially since possible inhibitors of CaMpf in favor of CaMhum, in extension, could give rise to new malaria treatments.   Some antagonists, functioning as inhibitors of CaM, have already been analysed in previous studies. However, there are also compounds that have not yet been studied in regards to being possible antagonists of CaM. This study regards three known antagonists; trifluoperazine (TFP), calmidazolium (CMZ) and artemisinin (ART) and also three recently created fentanyl derivatives; 3-OH-4-OMe-cyclopropylfentanyl (ligand 1), 4-OH-3OMe-4F-isobutyrylfentanyl (ligand 2) and 3-OH-4-OMe-isobutyrylfentanyl (ligand 3).   Bioinformatic methods, such as modelling and docking, were used to compare the structures of CaMhum and CaMpf as well as observe the interaction of the six ligands to CaM from both species. In addition to the differences in primary structure, distinguished with ClustalW, disparities in tertiary structure were observed. Structure analysis of CaMhum and CaMpf in PyMOL disclosed a more open conformation as well as a larger, more defined, hydrophobic cleft in CaMhum compared to CaMpf. Simulated binding of the six ligands to CaM from both species, using Autodock 4.2, indicated that TFP and ART bind with higher affinity to CaMhum which is expected. Ligand 2 and ligand 3 also bound with higher affinity and facilitated stronger binding to CaMhum, which is reasonable since their docking is based on how TFP binds to CaM. However, ligand 1 as well as CMZ both bound to CaMpf with higher affinity. Despite promising results for ligand 1 and CMZ, no decisive conclusion can be made solely based on bioinformatic studies.    To gain a better understanding on the protein-ligand interactions of the six ligands to CaMhum and CaMpf, further studies using e.g. circular dichroism and fluorescence would be advantageous. Based on the results from this study, future studies on the binding of CMZ and ligand 1 to CaM as well as ligands with similar characteristics would be especially valuable. This is because they, based on the results from this study, possibly are better inhibitors of CaMpf than CaMhum and thereby could function as possible antimalarial drugs.