This paper presents the generation and evaluation of a novel potential drug delivery platform for biologics, based on recombinant spider silk. Targeting CD40 for activation of antigen presenting cells, in order to overcome tumor induced T cell tolerance, have shown promising results in cell and animal models. However, further trials have gained limited results due to severe side reactions. To overcome this, we have investigated a strategy for a localized CD40 activation. A CD40 agonist based on a single chain variable fragment (scFv<inf>CD40</inf>) was enzymatically coupled to silk structures, that were then used to stimulate cells in vitro. A reporter cell line responsive to CD40 agonists was used to evaluate the bioactivity of the developed scFv<inf>CD40</inf>-silk, and to optimize the method. Once the bioactivity was confirmed, human primary B cells derived from healthy donors were stimulated with the scFv<inf>CD40</inf>-silk construct. The resulting B cell response was characterized both by upregulated surface expression of the activation marker CD86 (3 fold), suggesting an improved antigen-presenting capacity, and by B cell proliferation (4 fold) generating an expanded B cell population. The detected upregulation of the costimulatory molecule CD86 on the B cells implies a potential of the functionalized silk to steer the tumor-specific T cell response from tolerance to immune activation, including the onset of appropriate effector functions. Finally, we investigated the usability of the novel silk format microspheres for CD40-mediated cell activation in vitro. Here, we were able to demonstrate that scFv<inf>CD40</inf>-coupled silk microspheres gave a pronounced activation of the CD40-expressing reporter cell line, supporting the suitability of silk microspheres for the delivery of biologics with immune modulatory purposes.

Protein nanofibrils (PNFs), especially those from the whey protein β-lactoglobulin, hold the promise for applications in food technology, medicine, and sustainable materials. In this work, we explore the mechanisms underlying the sol-gel transition of whey protein isolate triggered by pre-fragmented whey fibrils (seeds) at low pH and high temperature. We show that, under these conditions, the formed hydrogels are constructed from PNFs. The presented results suggest that the seeds amplify the fibril growth process by providing active ends that capture peptide monomers produced via acid hydrolysis. This changes the fibrils' length distribution (up to 10-fold increase of their average contour length), and the samples reach the percolation threshold at a much lower mass concentration of fibrils. We also note that seeding has a strong impact on morphology and catalyzes a conversion of short, curved fibrils into long straight ones, which also contribute to the lower percolation limit. Rheological measurements indicate that attractive inter-fibrillar forces stabilize the PNF network. This is further evidenced by the gels’ resistance to disassembly across a wide pH range, implying that other forces than electrostatics are important for stabilizing the fibrillar network. Finally, we discuss the nature of the sol-gel transition based on continuum percolation theory, which corroborates the observed relationship between PNF length distribution and the sol-gel transition.

Access to efficient and affordable materials for water purification is of fundamental importance for the sustainable development of our society. Materials based on protein nanofibrils (PNFs) from agricultural waste- or side streams have recently been shown to have excellent adsorption properties for organic as well as inorganic pollutants. We here investigate the role of the nanostructure in aerogels made from whey protein isolate for the removal of a model pollutant (ibuprofen) from water. Water stable aerogels were produced using a recently developed approach for intrinsic crosslinking of protein materials without requiring additives. By comparing materials made from PNFs and from non-fibrillar whey protein we find that the fibrils have dual roles in enhancing the ibuprofen binding capacity. The PNFs do have a higher direct binding affinity but they also remodel the cell wall structures of the aerogels, resulting in a mesoporous network with enhanced ability of pollutant adsorption.

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into β-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Proteins are one of the fundamental building blocks of life as we know it. They are central to various biological processes and pivotal parts of essential procedures in the healthcare, food and sustainability industries. Over the past few years, significant research efforts have been made to employ bio-based strategies as alternative pathways to shift away from human dependency on petroleum-based polymers, placing protein as central building blocks for sustainable material development. In this framework, this thesis explores a specific class of material building blocks derived from the food we eat, referred to as protein nanofibrils (PNFs). Particular attention is paid to PNFs from soy protein isolate (SPI) and whey protein isolate (WPI) formed at low pH and high temperature, mirroring processes commonly found in food processing and cooking. The presented work focuses explicitly on how these fibrils assemble from acid hydrolysis of the initial food proteins into smaller aggregation-prone species, how aggregation occurs and how we can potentially process fibrils as valuable materials for society. 

A critical step in forming fibrils at low pH and high temperature is the heat-induced acid hydrolysis of protein chains into shorter peptides. The work first uses SPI as a model protein source to understand PNF formation. By determining the activation energy for SPI hydrolysis and comparing it with the lower activation energies for the aggregation processes of one of the peptide building blocks, BB2, we establish that hydrolysis is the with the highest energy of activation. Computer simulations are then employed to replicate the hydrolysis of proteins to model peptide production and degradation from rates of protein hydrolysis. With this, a connection between monomer production and fibrillation kinetics can be established. With this, numerical simulations of BB2 at 90 °C and pH two are then attempted, demonstrating that aggregation of this peptide alone does not replicate the fibrillation behaviour of SPI.

The thesis continues focusing on the fibrillation of food proteins and their potential safety for humans, now exploring how food fibrils affect the aggregation behaviour of Aβ42 aggregation, a hallmark of Alzheimer’s disease. The results show that seeds from food amyloids do not accelerate Aβ42 aggregation, with lysozyme- and oat-derived fibrils even delaying the aggregation of Aβ42. Further kinetic analysis reveals that aggregation inhibition is likely due to interactions between Aβ42 aggregates and food-derived fibrils, likely affecting the pathway for secondary nucleation in Aβ42 assembly. 

In contrast, seeding accelerates fibrillation in WPI, leading to the formation of longer fibrils that can reach the percolation threshold necessary for gelation and material formation. However, the results also indicate that percolation theories might need to be refined to better frame and predict the behaviour of PNFs and how they interact to form macromolecular structures.

Finally, the usage of food-derived WPI fibrils in material applications is also investigated. Fibril-based aerogels exhibit distinctive microstructural differences from aerogels without fibrils and enhanced pollutant adsorption capabilities, represented using the model molecule ibuprofen. Additionally, preliminary results show that fibrillar hydrogels can be used as electrolyte matrices for energy storage, displaying a broad operational potential window and excellent rate performance. 

Combined, the results provide important insights regarding the formation of fibrils at low pH and high temperature, their potential non-hazardous nature for human consumption, and their application in areas such as water purification and energy storage.

Proteiner är några av de fundamentala byggstenarna för livet som vi känner det, centrala inte bara för variation av biologiska processer men även som avgörande delar av viktiga procedurer inom hälso-, livsmedels- och hållbarhetsindustrier. De senaste åren har stora forskningsinsatser genomförts för att använda bio-baserade strategier som alternativa vägar för att minska det mänskliga beroendet i petroleumbaserade polymerer, och istället placera proteiner som centrala byggstenar för hållbar materialutveckling. Denna avhandling  utforskar, enligt detta ramverk, en specifik klass av materielbyggstenar som härstammar från maten vi äter, även kända som protein-nanofibriller (PNF). Särskild uppmärksamhet riktas mot PNF:er från sojaproteinisolat (SPI) och vassleproteinisolat (WPI) bildade vid lågt pH-värde och hög temperatur, vilket speglar vanligt förekommande processer vid livsmedelsbearbetning och matlagning. Det presenterade arbetet fokuserar specifikt på hur dessa fibriler sammansätts genom hydrolys under sura förhållanden ur de initiala livsmedelsproteinerna till små aggregeringssbenägna peptider, hur aggreggation sker samt hur vi potentiellt skulle kunna bereda fibriller som användbara material ute i samhället.

Ett kritiskt steg i formandet av fibriller vid lågt pH-värde och hög temperatur är den värmeinducerade sura hydrolysen av proteinkedjor till kortare peptider. SPI används som en modellproteinkälla för att förstå PNF-formering. Genom att fastställa aktiveringsenergin för SPI-hydrolys och jämföra den med de lägre aktiveringsenergierna för aggregationsprocesserna för en av peptidbyggstenarna, BB2, kan det fastslås att hydrolysen är processen med den högsta aktiveringsenergin. Datorsimulationer genomförs sedan för att reproducera hydrolysen av proteiner till modellpeptidproduktion och nedbrytning från olika hastigheter av proteinhydrolys. Därmed kan en koppling mellan monomerproduktion och fibrillationskinetik etableras. Med detta görs ett försök med numersiska simulationer av BB2 vid 90 °C och pH-värde 2, som demonstrerar att aggregation av endast en peptid inte reproducerar SPI:s fibrillationsbetéende.

Avhandlingen fortsätter att fokusera på fibrillering av livsmedelsproteiner och deras potentiella säkerhet för människor, och utforskar nu hur livsmedelsfibriller påverkar aggregeringsbeteéendet i Aβ42-aggregering, ett kännetecken för Alzheimers sjukdom. Resultaten visar att frön från livsmedelsamyloider inte accelererar Aβ42-aggregering, och att fibriller från lysozym och havreprotein till och med försenar aggregeringen av Aβ42. Ytterligare kinetisk analys avslöjar att aggregationshämning sannolikt beror på interaktioner mellan Aβ42-aggregat och fibriller som härrör från livsmedel, vilket troligen påverkar vägen för sekundär kärnbildning i Aβ42-sammansättningen.

Däremot accelererar sådd fibrillering i WPI, vilket också leder till bildandet av längre fibriller som kan nå den perkolationströskel som krävs för gelbildning och materialbildning. Resultaten indikerar dock att  perkolationsteorier kan behöva förfinas för att bättre rama in och förutsäga beteendet hos PNF och hur de interagerar för att bilda makromolekylära strukturer.

Slutligen utforskas även användandet av livsmedelshärledda WPI-fibriller i materialapplikationer. Fibrilbaserade aerogeler uppvisar distinkta mikrostrukturella skillnader jämfört med aerogeler utan fibriller och förbättrad adsorptionsförmåga för föroreningar, representerad här genom att använda modellmolekylen ibuprofen. Dessutom visar preliminära resultat också att fibrillära hydrogeler kan användas som elektrolytmatriser för energilagring, och påvisar ett brett operativt potentialfönster och utmärkt hastighetsprestanda.

Sammantaget ger resultaten en viktig insyn gällande bildning av fibriller vid lågt pH-värde och hög temperatur, deras säkerhet för mänskliga konsumtion, och deras tillämpning inom områden såsom vattenrening och energilagring. 

Proteínas são moléculas orgânicas essenciais à vida e elementos centrais em inúmeros processos biológicos e industriais. Nos últimos anos, têm sido realizados esforços significativos no âmbito da investigação e desenvolvimento de estratégias para reduzir a dependência humana de polímeros à base de petróleo. Assim sendo, proteínas surgem como blocos de construção centrais para o desenvolvimento de materiais sustentáveis. Neste sentido, a presente tese de doutoramento tem como foco principal explorar a formação e utilização de proteínas nanofibrilares (PNFs), também designados de amilóides artificiais, como unidades de construção de nanomateriais. Especificamente, a tese explora a formação de PNFs a partir da hidrólise de proteínas alimentares. Neste caso, a formação de PNFs é precedida pela hidrólise das proteínas na sua forma nativa que, em termos simples, "corta" as proteínas em segmentos mais pequenos, designados de péptidos. Do vasto número de péptidos formados durante a hidrólise de proteínas, uma parte terá propensão para posteriormente se agregarem e formarem PNFs. Estas podem ter várias aplicações práticas, tais como o desenvolvimento de biomateriais para a sociedade e com utilidade pública. 

A pesquisa examina, particularmente, PNFs feitas a partir de isolado de proteína de soja (SPI) e isolado de proteína de soro de leite, também conhecido como whey protein (WPI). Estas PNFs são formadas em condições de pH baixo e alta temperatura. Tal estratégia é bastante comum no processamento de produtos nas indústrias alimentares e farmacêuticas. Inicialmente, esta tese explora a utilização de SPI como proteína-modelo para investigar a formação de PNFs. Ao determinar a energia de ativação relacionada com a hidrólise ácida de SPI, usando a lei de Arrhenius, comparando-a com as energias de ativação referentes aos processos de agregação de um dos blocos de construção peptídicos das fibras da soja, BB2, é estabelecido que a hidrólise é o processo que requer maior energia. Assim sendo, são utilizadas simulações computacionais para replicar a hidrólise das proteínas, modelando a produção e degradação de péptidos a partir das velocidades de hidrólise proteica determinadas para várias temperaturas. Desta forma, é possível estabelecer uma ligação entre a produção de monómeros e os microprocessos de fibrilação. Posteriormente, simulações numéricas do péptido BB2 a 90 °C e pH 2 são realizadas, demonstrando que a agregação deste péptido isoladamente não replica o comportamento de fibrilação do SPI.

A tese prossegue com uma análise detalhada dos processos de fibrilação de proteínas, com especial foco na segurança e potenciais perigos do uso de PNFs para consumo humano, focando-se em como várias fibrilas de origem alimentar afetam o comportamento de agregação de Aβ42, um péptido marcador da doença de Alzheimer. Os resultados demostram que fibrilas alimentares não aceleram a agregação da Aβ42, e fibrilas derivadas da lisozima e da aveia retardam até a agregação de Aβ42. Análises adicionais dos processos cinéticos da agregação revelam que esta inibição é, provavelmente, devida a interações entre agregados de Aβ42 e fibrilas derivadas de alimentos, afetando possivelmente a via de nucleação secundária durante à fibrilização da Aβ42. Em contraste, as fibrilas de WPI autocatalisam e aceleram a fibrilação do próprio WPI, levando à formação de fibrilas mais longas que formam géis. Estes resultados revelam que um aumento da velocidade associada ao processo de alongamento de fibras é responsável por gerar PNFs mais longas, mas que a teoria de coloides tem de ser refinada para melhor enquadrar e prever o comportamento de PNFs e a forma como elas interagem para formar estruturas macromoleculares.

Finalmente, é também investigada a utilização de fibrilas derivadas de WPI em aplicações materiais. Aerogéis baseados em fibrilas exibem diferenças microestruturais distintivas em comparação com aerogéis sem fibrilas e capacidades aprimoradas de adsorção de poluentes, representadas usando a molécula modelo ibuprofeno. Adicionalmente, resultados preliminares mostram que hidrogéis fibrilares podem ser usados como matrizes eletrolíticas para armazenamento de energia, exibindo um intervalo operacional de potential amplo e uma e excelente desempenho de taxa. 

Desta forma, estes resultados fornecem informações insights importantes acerca da a formação de fibrilas em baixo pH e alta temperatura; a sua potencial segurança para o consumo humano; e, a sua aplicação em áreas como purificação de água e armazenamento de energia.

The deposition of proteins in the form of amyloid fibrils is closely associated with several serious diseases. The events that trigger the conversion from soluble functional proteins into insoluble amyloid are not fully understood. Many proteins that are not associated with disease can form amyloid with similar structural characteristics as the disease-associated fibrils, which highlights the potential risk of cross-seeding of disease amyloid by amyloid-like structures encountered in our surrounding. Of particular interest are common food proteins that can be transformed into amyloid under conditions similar to cooking. We here investigate cross-seeding of amyloid-beta (A beta), a peptide known to form amyloid during the development of Alzheimer's disease, by 16 types of amyloid fibrils derived from food proteins or peptides. Kinetic studies using thioflavin T fluorescence as output show that none of the investigated protein fibrils accelerates the aggregation of A beta. In at least two cases (hen egg lysozyme and oat protein isolate) we observe retardation of the aggregation, which appears to originate from interactions between the food protein seeds and A beta in aggregated form. The results support the view that food-derived amyloid is not a risk factor for development of A beta pathology and Alzheimer's disease.

Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (standing for Flanking Genes) takes a list of NCBI protein accessions as input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. The web tool version of FlaGs (webFlaGs) can optionally include a BLASTP search against a reduced RefSeq database to generate an input accession list and analyse neighbourhood conservation within the same run.