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TRPA1, also known as the wasabi receptor, is a non-selective cation ion channel that plays an essential role in the detection of noxious substances and irritants. Hylobius abietis, the pine weevil, is a pest that causes substantial damage to the forestry industry. The structure of the pine weevils TRPA1 (HaTRPA1) is so far unknown. If the structure were to be determined, an agonist could be developed, which could be used as a repellent against the pine weevil. Membrane proteins are naturally expressed at lower levels and can be highly unstable in certain conditions. Heterologous expression and different protocols must often be attempted and evaluated to produce a high-yield, stable membrane protein sample. The aim of this study is to optimize and evaluate different protocols to express and purify HaTRPA1, including using a truncated version of HaTRPA1 and a cleavable green fluorescent protein tag. HaTRPA1 was expressed through large-scale fermentation in Pichia pastoris. The cells were broken, and the membrane was isolated and washed. The protein was solubilized using Fos-choline 14, purified using Immobilized metal ion affinity chromatography (IMAC), cleavage with the tobacco etch virus protease, reverse IMAC, and separated using size exclusion chromatography. Steps throughout the expression and purification were evaluated using SDS-PAGE. The protocols were attempted for two constructs of HaTRPA1, the full-length construct and a truncated version labelled C2. The results showed that degradation was the major issue with the expression and purification of HaTRPA1. Both the full-length construct and the truncated C2 showed substantial degradation. A decrease in degradation was observed when the induction time during expression was reduced. Implementing a urea wash in the membrane preparation was beneficial in reducing the amount of degradation. The conclusion of this paper was that there is little difference in stability between the two constructs of HaTRPA1. To preve, Have you ever put too much wasabi on a piece of sushi? The painful sensation you feel that causes instant regret is generated by the so-called Transient Receptor Potential Channel, subfamily A, member 1 (TRPA1). The protein TRPA1 plays an essential role in detecting irritants and noxious substances, like our dear friend wasabi. While wasabi may be harmless, there are many substances that can be highly damaging, and our bodies need a way to tell us to stay away from them. Therefore, once TRPA1 is activated by a noxious substance, a signal is sent to our nervous system to generate a painful sensation. While human TRPA1 has been the subject of many studies, it is also worth studying TRPA1 in other organisms. Hylobius abietis, or the pine weevil, is a major pest in the commercial forest industry. It eats the bark of seedlings causing them to die. The pine weevil is responsible for millions of euros in damages each year. Currently, the fight against the pine weevil has involved plenty of harmful insecticides, and with a greater focus on environmentally friendly forestry, there is an interest in other alternatives. If the 3D structure of H. abietis TRPA1 was resolved, it would be possible to develop an activator of TRPA1 that would trigger a similar response in the pine weevil as our overindulgence in wasabi caused. This study attempted different conditions and protocols to produce a substantial amount of TRPA1 from the pine weevil for structural studies. Two different constructs were studied: the full-length construct and a truncated version. Both constructs of the protein were tagged with a green fluorescent protein to enable tracking of the protein throughout the purification and expression. The protein was expressed in the yeast species Pichia pastoris. Afterwards, the protein was solubilized and purified under different conditions to find the optimal protocol.

This degree project aims to develop standard methods for determination of the size distribution in different samples using laser diffraction, as well as to create methodologies for development of new methods for unknown samples. The size distribution is crucial to understand for pharmaceutical products since it affects properties such as the stability and bioavailability of the product. A way to measure the size distribution is to use laser diffraction. Laser diffraction is a technique in which laser beams are directed through a sample flow. When the laser beam hits a particle in the sample flow, the light scatters at different angles and intensities depending on the size and shape of the particle. The scattered light is then detected and translated into a size distribution, in this degree project by using the Mie theory. Different parameters suspected to affect the size distribution when using laser diffraction were analysed and evaluated for both emulsion- and powder samples. Due to the small droplet- and particle sizes, the sample preparation technique was believed to have a great impact on the size distribution. Other than this, the parameters pump speed, sonication intensity, obscuration and delay time were evaluated. The pump speed as well as the sonication time and intensity were found to affect the size distribution the most, while the delay time had a slight effect, and the obscuration barely affected the size distribution. It was further found that the powder samples were less sensitive than the emulsions and hence could withstand more energy in terms of higher pump speeds and sonication intensities. This also contributed to different optimal dispersing techniques during sample preparation; the powders were dispersed by sonication while the emulsions were dispersed by stirring on a magnetic stirrer plate. Other than this, the temperature was found to greatly impact the size distribution of the emulsions. In short, it was concluded that the pump speed and s, Metodutveckling för Mätning av Dropp- och Partikelstorleksfördelning med hjälp av Laserdiffraktion Partiklar finns i princip överallt runtomkring oss, och har en stor betydelse inom många industrier. Partikelstorleken i målarfärg avgör dess kvalitet, partiklar i miljön påverkar vår hälsa, och partikelstorleken i läkemedel påverkar dess effektivitet och stabilitet, för att nämna några exempel. Fokus för detta examensarbete har varit att bestämma partikelstorleken i olika läkemedel, såsom emulsioner och torra pulver. Två olika typer av emulsioner analyserades; hudkräm och hudlotion. I dessa emulsioner har en oljefas blandats i en vattenfas, likt beredningen av majonnäs. Oljefasen bildar då små oljedroppar i vattenfasen, eftersom olja inte löser sig i vatten. Droppstorleken av oljedropparna är viktig att känna till då den påverkar formuleringens egenskaper, såsom hur den känns på huden och hur snabbt den absorberas. Utöver emulsionerna analyserades två pulver; laktos och mometasonfuroat. Laktos kan användas i inhalationsprodukter medan mometasonfuroat kan användas som antiinflammatorisk komponent i exempelvis hudkrämer. Det är viktigt att veta partikelstorleken för inhalationsprodukter eftersom de måste vara tillräckligt små för att ta sig ner i lungorna i stället för att exempelvis andas ut, medan partikelstorleken för det antiinflammatoriska pulvret påverkar huruvida det kan tränga igenom huden. Vid mätningar av partikelstorlek är det väsentligt att förstå att partikelstorleken i en formulering inte har ett specifikt värde, utan snarare är en distribution av olika partikelstorlekar. Exempelvis kan det påstås att partikelstorleken är 5 µm, men i verkligheten kan partiklarnas storlek variera från 3 till 7 µm. För att bestämma distributionen är laserdiffraktion en passande teknik. Principen bakom laserdiffraktion är att en laserstråle riktas mot ett prov beståendes av tusentals partiklar, varpå laserstrålen kommer att ändra riktning när den träffar en partikel. Små part

Even though the first coronavirus pandemic was registered in 1965, SARS-CoV-2 was better acknowledged when it caused the worldwide pandemic in 2019 in which millions of individuals lost their lives. Due to its facile way of spreading from one individual to another, the number of infected cases has gone up to more than 750 million to this day. DENV is a very dangerous virus due to its asymptomatic profile in most of the infected cases. If left untreated it can lead to dengue hemorrhagic fever and for individuals who get infected with the virus for the second time, the consequences can be lethal. Since there is currently no treatment for dengue infection, and due to the fast spreading of SARS-CoV-2 that still leads to deaths every day, it is important to understand how proteases are involved in viral replication and thus to find a way to stop this process in order to avoid spreading these viruses further. In this report, 2 proteins (6LZE_A and NS2B-NS3) from these viruses were produced in Escherichia coli and their biological activity has been tested on DL-BAPNA substrate. Both of these proteins are structures of the main proteases that cleave the polyproteins, this step being very important in the replication process. Furthermore, computational modeling was used to understand the differences between their structures and how their catalytic mechanisms work. The results showed that working with these proteins has been proven difficult at times due to several failures and repetitions either because very few colonies grew on the Agar plates, cells died during the cultivation process, low concentrations, or failed purification. Hence, more time and research would be necessary to fully understand the reasons behind these issues. However, this report managed to highlight the different production times between the proteins and how SARS-CoV-2 seems to have better environmental stability than DENV due to its structure and docking characteristics., Viruses are microscopic organisms that need a host, which usually is a human or an animal, in order to reproduce. The coronavirus is known for causing the worldwide pandemic in 2019 in which millions of individuals lost their lives due to its facile way of spreading from one individual to another. Dengue virus is also dangerous because in most cases does not show any symptoms and it can even lead to death. Since there is currently no treatment for dengue infection, and due to the fast spreading of coronavirus it is important to understand how these viruses reproduce and to find a way to stop this process. It is known that proteases are enzymes that are involved in breaking down proteins and helping viruses to replicate. In this paper, 2 such enzymes were produced in a host bacteria called Escherichia coli and then a substrate was used to assess if they are active or not. Their structure was also visualized using specific software such as Chimera and Yasara. The experiments were conducted at Lund University, Kemicentrum, Biotechnology Department. The Coronavirus outbreak started in Wuhan, China, in December 2019. From there, it spread worldwide from human to human resulting in the covid pandemic declared in March 2020 by the World Health Organization. The origin of the infection and the transmission way is unknown but it is believed to be caused by exposure to infected animals. However, the first coronavirus pandemic was actually registered in 1965, followed by another one in 2003 and 2012. This virus is considered highly infectious due to its rapid spreading and large number of infected cases. Worldwide, there are over 750 million registered cases and almost 7 million deaths. The dengue virus can cause dengue fever through mosquito bites. It is assumed that it was transmitted from primates to humans approximately 1000 years ago in Africa or Asia. Between 100 and 400 million infected cases of dengue fever are registered yearly in tropical or subtropical regions world

Plastics, derived from fossil fuels, have raised considerable environmental concerns. This has prompted the search for sustainable and safer plastic precursors alternatives. One alternative explored in the present study is vanillin, a phenolic compound derived from lignin, an abundant and renewable natural polymer. Vanillin and its derivatives have shown promise as eco-friendly substitutes for conventional industrial chemicals. The present study focused on the microbial production of chemical building blocks from vanillyl amine and vanillic acid using recombinant yeast, with a specific focus on the evaluation of amide-forming enzymes. The study also involved the screening of various co-substrates to optimize the production of vanillyl amine and vanillic acid. Our findings revealed that the choice of co-substrate significantly influenced the production of vanilloids, with specific co-substrates favoring the production of specific vanilloids., Plastics, derived from fossil fuels, pose significant environmental challenges, fueling a quest for sustainable alternatives. In this study, vanillin, a phenolic compound derived from lignin which is an abundant and renewable natural polymer, was explored as a potential eco-friendly substitute for traditional industrial chemicals. The focus was on microbial production of chemical building blocks from vanillin using recombinant yeast, with a specific emphasis on evaluating amide-forming enzymes. The study also investigated the influence of different co-substrates on optimizing the production of vanilloids, compounds derived from vanillin. Despite promising prospects, the study encountered challenges in the biosynthesis of chemical building blocks from vanillyl amine and vanillic acid. Specifically, the strains showed no detectable product in the analysis, raising questions about their capability or the methodology used. The study suggested that the inert nature of vanillyl amine and vanillic acid within yeast indicated the absence of discernible side reactions, making yeast a potential platform for future amide-forming reactions. In the investigation of co-substrates effects on vanilloid production however, we saw that the choice of co-substrate played a crucial role in determining the main product formed during the bioconversion of vanillin. Different co-substrates affected the redox balance within cells, influencing the type and quantity of vanilloids produced. Notably, monosaccharides, disaccharides, and oligosaccharides led to vanillyl alcohol production, while glycerol, ribose, and lactate led to a more balanced production of vanilloids.

T-cell administration via a scaffold is a promising tool for treatment of inoperable solid tumours. Traditional scaffold materials lack the complexity of the extracellular matrix (ECM) that cells normally grow in. An alternative is to instead use decellularised ECM (dECM) from biological tissue to create the scaffold. By making the scaffold via 3D-printing, its construction can also be a part of making it suitable for cells. This thesis aims to develop a protocol for decellularising a variety of tissues and using the dECM to make a printable biogel. The protocol was tested on three types of porcine tissue: aorta, left ventricle of heart, and skin. To be printable, it should behave as a liquid at 4 ℃ but display a storage modulus of at least 100 Pa when heated to 37 ℃. It should also have a shear thinning behaviour. All these objectives were attained. For characterisation of the dECM, assays to quantify the DNA content and glycosaminoglycane content were performed in order to determine decellularisation- and proteoglycans level respectively. However, no conclusive results were found in these assays., Administration av T-celler via en artificiell vävnadsrekonstruktion är en lovande metod för att behandla inoperabla tumörer. Traditionella vävnadsrekonstruktionsmaterial saknar den komplexitet som finns hos det extracellulära matrixet (ECM), där celler vanligtvis lever. Ett alternativ är då att använda decellulariserat ECM (dECM) som material när man skapar den artificiella vävnadsrekonstruktionen. Genom att använda en 3D-skrivare kan vävnadsrekonstruktionen ytterligare skräddarsys för cellers behov. Den här uppsatsen ämnar skapa ett protokoll för decellularisering som fungerar på flera olika typer av vävnad. dECM:et ska också användas till att skapa en biologisk gel som kan användas i en 3D-skrivare. Protokollet testades på tre olika typer av grisvävnad: aorta, vänster hjärtkammare och hud. För att kunna användas i en 3D-skrivare bör gelen vara flytande vid 4 ℃, men hårdna när den värms upp till 37 ℃. Viskositeten på gelen bör även minska när den utsätts för en kraft. Alla dessa mål uppfylls av gelen. Analys av DNA- och glygosaminoglykaninnehållet i dECM:et gjordes, dock utan brukbara resultat., Cancer är en av de vanligaste dödsorsakerna i Sverige idag och forskning om nya behandlingar utvecklas ständigt. En av dessa är immunterapi, där kroppens egna immunceller används för att attackera cancercellerna. I det här projektet har olika typer av grisvävnad använts för att skapa en gel som kan hjälpa immuncellerna nå tumörer. Socialstyrelsen spår att mer än var tredje person kommer få en cancerdiagnos någon gång i livet. Tack vare forskningen har vi idag flera olika typer av behandling mot cancer, och cancerformer som tidigare ansågs obotliga har idag betydligt godare utsikter. Cancerforskningen utvecklas ständigt, och en relativt ny behandlingsmetod är så kallad immunterapi. Den går ut på att man odlar och tillför celler från kroppens egna immunförsvar som kan attackera cancercellerna. Det är framförallt en framgångsrik metod för att behandla olika typer av blodcancer. Ett problem uppstår dock när det gäller solida tumörer. Den koncentrerade samlingen cancerceller skapar en miljö som verkar hämmande på immuncellernas förmåga att attackera tumören. Men det kan finnas ett sätt att kringgå problemet: Artificiellt vävnadsrekonstruktionsmaterial. Det går ut på att, istället för att injicera immunceller direkt till tumörens närområde, låta dem växa i en typ av gel. Den gelen kan sedan opereras in vid tumören. Immuncellerna har då en gynnsam miljö varifrån de kan migrera till tumören. Men vilket material kan passa för att få cellerna att trivas? Vävnaden i kroppen har två beståndsdelar. Den första är celler. Men det finns även någonting som kallas extracellulärt matrix (ECM). Det är den del av vävnaden som cellerna befinner sig i. Trots att ECM inte lever i någon biologisk mening kan den på kemisk nivå kommunicera med de celler som den omger. Den kommunikationen kan uppmuntra till bland annat celldelning och -migration. Vid traditionell artificiell vävnadsrekonstruktion används material som är betydligt mindre komplext än ECM och saknar dessa kommunikationsvägar. I d

Neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s Disease (AD), and Multiple Sclerosis (MS) emphasise the need of effective diagnostics tools to provide early diagnosis and disease prognosis as well as treatment. The Neurofilament Light Chain (NfL) antigen is a promising biomarker in immunoassays for neurodegenerative disease diagnosis due to its elevated concentration in Cerebrospinal Fluid (CSF) during axonal damage. In this study, novel antibody-based reagents for an automated chemiluminescence immunoassay (CLIA) were developed towards the NfL antigen in CSF using bioconjugation techniques. The diagnostic potential of the immunological test was evaluated by comparison with an established CSF NfL assay of another CLIA instrument, Lumipulse. Capturing antibodies were coated to paramagnetic particles (PMP) and detecting antibodies were conjugated to Alkaline Phosphate molecule (ALP). When combined in five different combinations, the results showed varying recovery rates from 0 – 161% compared to the predicate device. Two reagent combinations showed promising results, with mean recovery rates of 91% and 113% respectively, indicating potential future optimisation of the test. It was established that the reagents showed diagnostic potential with the right assay configuration and pairing., Imagine a world where brain diseases like Alzheimer's, ALS, and MS, collectively affecting millions worldwide, could be met with an early diagnosis and thereby timely treatment. Over 50 million individuals live with dementias, granting many more affected by the diseases. As advancements in treating these diseases emerge, the spotlight intensifies on the value of early diagnosis. Early detection could not only extend lives but contributes to a richer, longer journey shared among family and friends. In our immune system, antibodies act as an alarm system which tracks down invading agents like viruses and thereby makes sure that we humans stay healthy. Each antibody has sites on its surface which recognize specific sites on the invading agent. Scientific progress has made sure that we can use antibodies and their specific alarm mechanism outside of the body, both in diagnostics and in treatments. The use of antibodies to detect compounds in a sample is called an immunoassay. In this thesis, antibodies are used in an immunoassay to detect a compound called Neurofilament Light (NfL). Why NfL? It is because when suffering from brain-affecting diseases like Alzheimer’s and others, the brain cells get damaged and NfL compounds emerge from the neurons to the blood and cerebrospinal fluid (CSF). By measuring the concentration of NfL in a person’s blood or CSF, one can determine if this person is sick in a brain-affecting disease. The study showed some positive results proving that the antibodies used could indeed detect the NfL compound in different patient samples and that further optimization of the tests are possible. Maybe this small piece of science can be a contribution to the utopic world were brain disease is detected even before its first sign and were we conquer it with early and correct treatment.

Acute myeloid leukaemia (AML) is a highly aggressive type of blood cancer. In particular, elderly and medically unfit patients face a poor prognosis. Treatment with venetoclax and azacitidine improved the outcome for these patients, yet drug resistance remains a significant issue. Combining the poly (ADP-ribose) polymerase (PARP)1/2 inhibitor talazoparib with venetoclax and azacitidine was suggested as a potential treatment option. However, PARP2 inhibition is associated with haematological toxicities. This study explored the therapeutic potential of the selective PARP1 inhibitor saruparib compared to talazoparib in overcoming venetoclax resistance when administered combined with venetoclax and/or azacitidine. Cytotoxic effects in venetoclax resistant AML cell lines were studied in drug sensitivity assays and time to progression assays mimicking the clinical administration of the drugs. Induction of DNA damage and apoptotic signalling were measured to assess molecular mechanisms upon treatment. The triple combination of saruparib, venetoclax and azacitidine effectively induced DNA damage and apoptotic signalling leading to cell death. Talazoparib combined with venetoclax and/or azacitidine induced slightly higher levels of DNA damage and apoptotic signalling, resulting in lower cell viability. In time to progression assays, the triple combination with saruparib, venetoclax and azacitidine effectively inhibited cell expansion but only for the duration of azacitidine administration, suggesting azacitidine as an essential cytotoxic factor. Conversely, the talazoparib triple combination effectively eradicated AML cells with intrinsic venetoclax resistance, possibly due to the additional effect of PARP2 and/or PARP16 inhibition. This indicates that PARP1 inhibition in combination with venetoclax and azacitidine might not be sufficient to overcome venetoclax resistance., One of the major challenges in treating cancer is drug resistance. Either patients do not respond initially to treatments, or they develop resistance after a few treatment cycles. In this project, the new drug saruparib was tested as a way to overcome drug resistance in acute myeloid leukaemia. While saruparib alone was not effective, combining saruparib with the currently used drugs venetoclax and azacitidine significantly decreased the number of cancer cells. Acute myeloid leukaemia is a highly aggressive type of blood cancer that mainly affects the elderly population. Without treatment, the disease is fatal within a few months. The standard of care is intensive chemotherapy, but this is not tolerated by elderly and medically unfit patients. These patients are instead treated with a combination of azacitidine for 7 days and venetoclax for 28 days. However, most patients end up developing resistance to venetoclax. Therefore, new treatment options are needed for these patients. One possible treatment is the new drug saruparib which selectively targets PARP1, a protein that is involved in DNA damage repair. It has previously been shown that venetoclax causes DNA damage in venetoclax resistant cells. Therefore, targeting DNA repair pathways should lead to accumulation of DNA breaks which should eventually become intolerable for the cancer cells and lead to cell death. This project tested if saruparib increased DNA damage and if this led to cell death. Saruparib was tested alone and combined with venetoclax and/or azacitidine to see if these enhanced the effect of saruparib. It was found that saruparib alone did not increase DNA damage or cause cell death. However, combining saruparib with venetoclax and/or azacitidine enhanced the effect of saruparib. The triple combination with saruparib, venetoclax and azacitidine led to the highest levels of DNA damage and cell death. Azacitidine enhanced the effect of saruparib more than venetoclax since both drugs led to increase

Forensic sexual assault samples typically contain a mixture of a small quantity of sperm cells and a high quantity of epithelial cells. This relationship complicates the generation of a DNA profile, which prompts the need for differential lysis and extraction of the DNA. Differential extraction is a lengthy method that includes multiple tube transfers and requires a purification step. Amicon Ultra centrifugal filters are employed to purify samples; however, these filters are costly and susceptible to potential breaks in the supply chain. The objective of this project was to examine alternative methods utilizing “off-the-shelf” reagents for sperm cell lysis or sample purification, with the goal of streamlining the current differential extraction protocol. Three methods for sperm cell lysis were studied: alkaline lysis, lysis with the nonionic detergent NP-40, and lysis with a natural decondensation assay. Isopropanol precipitation was examined as an alternative purification method. Additionally, reducing the number of washing steps in the differential extraction protocol was investigated. The results showed that optimized alkaline lysis (25 µL NaOH (0.2 M), 25 µL Tris-HCl (0.2 M) and TE buffer) was comparable to the differential extraction protocol in terms of DNA recovery. Alkaline lysis was also comparable to the differential extraction when performed on mock sexual assault samples. Isopropanol precipitation likewise showed equal DNA yields, complete DNA profiles and peak heights compared to Amicon filtration. Lastly, it was shown that reducing the number of washing steps of the reference protocol produced comparable DNA profiles. In conclusion, the findings suggest that the differential extraction protocol can be modified by using alkaline lysis for sperm cell lysis or isopropanol for purification, as well as by performing only one washing step., Sperm cells are usually the most interesting biological material to analyze in forensic sexual assault samples. However, today’s method for extracting DNA from sperm is both time-consuming and requires purification with expensive filters. It is therefore relevant to investigate alternative protocols that can help streamline the process. The term “sexual offense” it the Swedish legal umbrella term for crimes of a sexual nature, with major crimes including sexual assault. Typically, the biological material of highest interest in sexual assault samples is sperm cells, as DNA can be extracted and potentially linked to a suspect. However, due to the nature of most sexual assault samples, where sperm cells are present in lower quantities in comparison to other cell types (e.g. epithelial cells), successful extraction and analysis of sperm cell DNA is difficult. Because of this, a method called differential extraction is typically performed, where the sperm cells and other cells in the mixture are treated separately. Differential extraction is a lengthy protocol that requires multiple transfers of material between tubes, which can lead to loss of DNA and contamination. On top of this, it uses the strong reagent dithiothreitol (DTT) to successfully break open the sperm cells and release the DNA. DTT interferes with downstream analysis and DNA profiling, for which reason the samples must be purified. At the National Forensic Centre (NFC), purification of samples is done with centrifugal filters called Amicon filters. As there is only one supplier of these filters, the process is susceptible to disruptions in the supply chain, which occurred during the Covid-19 pandemic. Furthermore, Amicon filters are expensive. Therefore, it was relevant to examine alternative methods for sperm cell disruption (lysis) and sample purification that could simplify the current method, preferably while using easily accessible reagents. This project examined three alternative methods for sperm ce

Although microalgae have been considered for the biological treatment of wastewater since the 1950’s, their potential to efficiently remove pollutants from various streams remains largely untapped and is yet to be utilized at large scale. The production of algal biomass and extraction of high-value compounds has, however, become increasingly popular with large scale farms and biorefineries around the world producing many promising alternatives to petroleum-based products and processes. While microalgae systems rival traditional production in terms of environmental sustainability and health benefits, the economic viability remains a challenge. This study aimed to investigate the potential of a biorefinery concept using wastewater as growth medium for the production of natural pigments by cyanobacterium, Arthrospira platensis. Using effluent streams as a growth media drastically cuts costs of production and with this circular approach, we are able to address challenges facing the wastewater treatment industry while providing a sustainable alternative to petroleum-based products. During phase one, the growth of A. platensis was monitored on different amounts of wastewater supplemented with standard growth media. Biomass productivity and growth rate ranged from 233,12 – 2000 mg.L-1.d-1 and 0,19 – 0,37 d-1, with 50% FWW supplemented with 50% Zarrouk media resulting in the highest values and thus proving to be the preferred growth media. However, cultures grown on 75% FWW supplemented with 25% Zarrouk media resulted in the highest PC content and thus was chosen as the growth media for further investigation. Phase two investigated the effect of different photoperiods (14h:10h and 16h:8h) on the production of PC, while assessing the scale-up potential of the system and monitoring nutrient removal. Photoperiod had no significant effect on PC content, however, a decrease in total PC production was seen between phases, suggesting further optimization for scale-up to be viable., Harnessing Microalgae: Transforming Wastewater into Valuable Pigments Innovative solutions are constantly sought to address the challenges of wastewater treatment and the increasing demand for sustainable products. One promising approach involves utilizing microalgae in a biorefinery system that not only cleans wastewater but also extracts valuable pigments from the biomass. This concept presents an intriguing solution that combines environmental remediation with the production of high-value products. Microalgae are microscopic organisms that harness the power of photosynthesis to convert sunlight and carbon dioxide into organic matter. They are incredibly efficient at this process, exhibiting high growth rates and accumulating various valuable compounds, including pigments. Their ability to thrive in diverse environments, including wastewater, makes them an ideal candidate for treatment and resource recovery. Traditional wastewater treatment processes consume large amounts of energy and are environmentally taxing. However, by integrating microalgae cultivation with wastewater treatment systems, we create a symbiotic relationship where the nutrients and organic matter present in the wastewater are used as a nutrient source for growth, effectively purifying the water in the process. One particularly exciting aspect is the extraction of valuable pigments from the microalgae biomass. These pigments not only provide color to the microalgae but also possess significant commercial value in various industries such as food, cosmetics, and pharmaceuticals. This study aims to investigate the potential of this biorefinery concept on local wastewater sources from the treatment facility RecoLab located in Helsingborg, Sweden. By establishing growth of a popular microalgae, Arthrospira platensis, on a food wastewater stream which is high in nutrients, we can optimize the biomass growth and assess the pigment content produced throughout cultivation. Additionally, we investigate th

Biotin is an essential co-factor in various metabolic processes and is crucial for the growth and maintenance of living organisms. The market need for biotin is multifaceted and driven by its importance in human health, beauty and cosmetics applications, pharmaceuticals, animal nutrition, and other industries. Current methods of industrial biotin production are unsustainable and generate environmentally hazardous waste, fueling the demand for new, greener strategies. The production of biotin through microbial fermentation has thus become an attractive alternative. Biotin biosynthesis is a complex and energy-intensive process that requires several enzymatic reactions, and biotin titers in microbial production have not reached economically viable levels. Biotin Synthase (BioB), the enzyme that catalyzes the last step of this process in E. coli, the conversion of dethiobiotin to biotin, has been identified as the bottleneck for this pathway. Overexpression of the enzyme generates oxidative stress and inhibits growth, though the exact mechanism has not yet been elucidated. This thesis employed a multiplexed engineering approach via high-throughput genetic engineering, selection, and screening methods to explore whether creating E. coli strains more resistant to oxidative stress would lead to higher biotin production capabilities. Using the expression of a DNA methylase to introduce controlled genetic mutations in Biosyntia's proprietary biotin-producing E. coli strains and selecting strains with improved resistance to oxidative stress yielded a selection of strains with increased resistance, which were screened for their biotin production. The improved resistance to oxidative stress and BioB induction in the strains did not lead to higher biotin production levels., The global need for biotin in the food, feed, and cosmetics industries is increasing steadily. Because current production methods use petrochemicals as precursors for synthesis, they are unsustainable and generate environmental waste. This has fueled the demand for alternative, more sustainable strategies. Biotin production via microbial fermentation could be a greener alternative but has not reached economically viable levels and cannot compete with current production methods. E. coli can naturally produce biotin but does so in minute amounts. This thesis aims to improve biotin production levels in biotin-producing E. coli strains to contribute towards a greener, more sustainable process. A bottleneck in the biosynthetic pathway, which has limited production levels, has been identified in previous research: the enzyme BioB, which catalyzes the last step of biotin synthesis. This enzyme creates oxidative stress in the cells and thereby inhibits cell growth. It was hypothesized that creating strains more resistant to oxidative stress and higher levels of intracellular BioB could lead to higher biotin production capabilities. By generating a controlled level of genetic diversity or introducing new genes into the strains and then selecting those with higher oxidative stress resistance, this thesis aims to find a strain that can produce higher levels of biotin. By nature, the randomness of the mutagenesis process generates a high number of strains that need to be tested for their biotin production. In this thesis, the number of strains that need to be tested is systematically reduced by selecting strains with a higher oxidative stress resistance by exposing them to various stressor concentrations. Only the fittest, most resistant strains survive this and are subsequently tested for their biotin-production capabilities. The work revealed that strains with higher resistance to oxidative stress and BioB production did not produce higher levels of biotin, indicating other l

Protein stability is a property of high importance and is of interest in a variety of fields. It determines if a protein has its native fold and can be of influence in certain diseases such as Parkinson's and Alzheimer's disease. It can also be of interest in an industrial setting to optimise the stability of enzymes in certain physicochemical environments. Recent developments in machine learning have yielded novel methods able to predict protein characteristics with surprising accuracy solely from sequence information. However, few such models have been proposed for predicting protein stability. The aim of this project was to create a model able to predict protein stability from sequence information, by utilising a multiple sequence alignment based protein language model. Different models were developed to predict two quantities relating to protein stability, Gibbs free energy of unfolding and the heat denaturation temperature. Due to limited training data the performance of the models predicting Gibbs free energy was poor. One of the models predicting heat denaturation temperature, proved more promising, with higher performance than a previously published model trained on similar data. However its ability to predict conventionally obtained heat denaturation temperatures was poor., Can computers find better proteins? Finding more efficient enzymes is critical for chemical industry to become sustainable. In order to efficiently find better enzymes new tools are needed that reliably predict protein properties. The chemical industry is on a search for new green technology that can replace catalysts that are often toxic and made of scarce minerals. A promising alternative is catalytic proteins, so called enzymes, that can be produced renewably and pose no toxic threat. However, in order to be a viable alternative, the enzymes must become more effective and be able to withstand harsher conditions. To design enhanced or entirely new enzymes accurate and fast predictions regarding the protein’s properties are needed. One such highly sought after property is protein stability at high temperatures since it usually speeds up chemical reactions. A promising approach for developing predictive models are machine learning techniques, which have proved powerful in numerous fields. At the department of Biophysical Chemistry at Lund University it was tested if such a model, that predicts the thermal stability of proteins, could be created. The study concluded that the tested models could detect some features of proteins, however for the models to be more useful, they must be trained on more data of high quality. The limiting factor during development was therefore the amount of relevant and available data. This is a common problem in the machine learning field, where the models are created by training on data, the computer is taught how to make predictions by learning from a dataset. The rule of thumb is that the more data it is trained on, the better performance. But what would be the benefit of using computers over good old laboratory work? While resulting in reliable values, laboratory work is usually costly and time-consuming and requires well trained personnel. If a relatively accurate predictive model can be constructed an estimate of a protein’s stabili

Chronic obstructive pulmonary disease (COPD) is the third largest cause of death worldwide, with its most prominent risk factor being inhalation of smoke and pollutants. In this project, we studied two processes that are involved in COPD: autophagy and cellular senescence. Using primary human bronchial epithelial cells (HBECs) from healthy and COPD donors, we aimed to show that cigarette smoke extract (CSE) gives rise to an impaired autophagy flux as well as increased senescence. With multiple CSE incubation times and CSE concentrations, our data shows an impaired autophagy flux at the early time points 5h and 24h. However, the senescence data is inconclusive. Further optimization of the study design could enable the development of an appropriate in vitro assay. In the end, could continued research of this kind contribute to increased knowledge and understanding of the disease, as well as enable development of medical targets in the mission of disease treatment., Rökextrakt och dess negativa påverkan på återvinningsprocesser i lungceller från KOL-patienter Kroniskt obstruktiv lungsjukdom (KOL) är en global folksjukdom och den tredje största dödsorsaken i världen. Den kan kopplas till en mängd andra sjukdomar och tillstånd som också ofta ökar risken för att utveckla KOL, t ex hög ålder, kardiovaskulära och neurosjukdomar. Men den största och mest välkända riskorsaken för KOL är rökning och exponering för föroreningar. Vi har studerat celler från KOL-patienters andningsvägar och fokuserat på två processer som är viktiga för cellens hälsa: autofagi och cellulär senescence. Vi kan utifrån våra resultat, och i likhet med tidigare publiserad forskning, konstatera att rökningen påverkar dessa processer och ger upphov till en sjuk och skadad cell. Autofagi är ett typ av återvinningssystem i cellen, där proteiner och andra cellkomponenter bryts ned och återvinns. Det är en process som alltid pågår, men som vid cellulär skada och/eller näringsbrist aktiveras ytterligare. Detta med syfte att regenerera komponenter så som aminosyror samt ta hand om de skadade celldelarna. Cellulär senescence å andra sidan, är en process som kan liknas vid cellulärt åldrande. När en cell blir tillräckligt gammal och/eller skadad finns det olika slutdestinationer. Antingen så genomgår cellen programmerad celldöd (apoptos) eller så blir den senescent. Det är ett mer eller mindre permanent tillstånd som innebär att cellen är levande men har slutat celldela sig. Processen är viktig för att hålla vävnad frisk och för att förhindra att skadade celler utvecklas till t ex cancerceller. Samtidigt har förhöjda nivåer av senescence kunnat kopplas ihop med olika sjukdomar, bland annat KOL. Vi studerade nivåerna av autofagi och cellulär senescence i celler från friska och KOL- patienters bronchier, närmare bestämt celler från det yttersta lagret av vävnaden – epitelet. Det är dessa celler som röken kommer i direktkontakt med. Genom att utsätta cellerna för rökextrakt

Development in sequencing technologies has made the analyses of genetic material much more accessible. Processing sequenced data for an accurate analysis comes with its challenges, especially with the studies in microbial in clinical in vivo samples where difficulties in the collection of these samples for sequencing could lower the quality and contamination from the human host which might affect the accuracy of downstream analysis. In this project, we use RNA-seq and different reference genomes to look at the differential gene expression of Pseudomonas aeruginosa (PA), one of the most prevalent species of bacterial pathogens in the progression of chronic pulmonary diseases such as cystic fibrosis, due to its resistance to antimicrobial treatment. In this project, we created a pangenome from 21 strains of PA and explored the use of this, its subsets (core and soft-core gene sets) and a commonly used PA genome (PAO1) as reference genomes. We compared some of the differences and similarities in the results using the four gene sets, including mapping transcripts while developing a feasible pipeline to process raw sample reads from human sputum samples for differential expression and gene ontology enrichment analysis. From the analyses, we have found that differentially expressed genes upregulated in in vivo samples were related to biofilm, which plays a role in the difficulties in the treatment of PA infections, across most of the various genome reference-based results., How a “pan-genome” can help discover which genes that bacteria use to survive inside the human body Have you ever wondered about the microscopic world that exists in our bodies? Would they attack us at our moment of weakness? For people who are affected by cystic fibrosis, a disease that affects the lungs by causing a buildup of fluids, this can become a lifetime warfare against microbes such as Pseudomonas aeruginosa (PA). This tricky species of bacteria can change its growth form, making it difficult to treat patients who are infected. Thanks to a large set of genes in its chromosome, PA can change its growth mode from free-living cells in a liquid to forming a thick layer of cells known as a biofilm, by switching on or off various genetic materials. Since different strains of PA have different sets of genes, it is difficult to study common traits between strains and the importance of each gene. Hence, we want to use a more inclusive approach to explore their gene expression. Here we create a pan-genome, a complete set of genes of different strains to use as a reference when studying how different PA strains use their particular set of genes. We used this to explore what kind of genes are more expressed in human samples from people with PA in their lungs, compared to PA grown in a lab. Improvement in sequencing technology has made the analyses of genetic material more accessible and the use of software tools has become essential in processing these data. We created a pan-genome from 21 strains of PA. From this, we identified a gene set that was shared among all these strains, i.e., the core gene set. While exploring the pan-genome, we found most genes that support their survival are found more commonly in their core gene set. To highlight the importance of which gene set that is used as a reference, we examined how the results from the analysis changed depending on which gene set we used as a reference. We did this by comparing sequenced RNA from the samples by ps