Molecular Biology and Biochemistry

Understanding eukaryotic replication is important, because during our lifetimes we copy approximately a lightyear’s worth of DNA, and how the different components of the molecular machinery (the replisome) work together to achieve this successfully is an area of highly active research.  In our lab, we take on the exciting challenge of understanding the dynamics of DNA replication of this process by studying the activity of eukaryotic replisome at the single-molecule level on both bare DNA and chromatin.

In this PhD project, you will learn a diverse set of techniques (synthesizing DNA constructs, purifying proteins, state-of-the-art single-molecule microscopy and measurements, in-depth quantitative analysis) and work together with others in an interdisciplinary team comprised of biologists, (bio)physicists, biochemists, and data scientists.  You will be taught how to perform high-quality experiments and then you will be invited to develop new ones of your own, making use of your training and insights! This research, carried out together with collaborators at the University of Oxford, the Francis Crick Institute, the Hubrecht Institute, and elsewhere, should lead to new discoveries and insights that inform our quantitative understanding of DNA replication and advance this exciting field while contributing to the next generation of in vitro single-molecule methods.   

The Cambridge Lactation Laboratory (https://www.cambridgelactationlab.com/) is seeking enthusiastic and motivated prospective PhD candidates to support the Cambridgeshire Multiomics of Milk (CAMB MOM) study. Join a dynamic and growing research group in the Department of Biochemistry and Pharmacology at the University of Cambridge, under the leadership of Dr Alecia-Jane Twigger. The team is passionate about women’s and infant health hosting both experimental (wet lab) and bioinformatic (dry lab) research. Here, you will have the opportunity to receive training in both disciplines.  Despite the compelling evidence supporting the benefits of breastfeeding, there are significant gaps in our knowledge about how the mammary gland matures to perform its function of milk synthesis and secretion. Within the CAMB MOM study, we conduct multiomics analyses (lipidomics, metabolomics, proteomics, and transcriptomics) on samples from a cohort of breastfeeding participants in Cambridgeshire. The insights gained from gene-gene interaction networks will be tested using in vitro mammary organoid models and integrated into computational models. You will be able to choose which aspect of the study you are most interested in and together we will develop a tailored, dynamic and exciting research programme. The overarching aim of the project is to investigate the molecular pathways of human milk production, to resolve breastfeeding challenges and promote optimal long-term health for mothers and infants.

The G1/S transition is a critical checkpoint in the cell cycle, controlling the decision of cells to either proceed into DNA replication or enter quiescence. Disruption of this checkpoint is a hallmark of cancer, often driven by hyperactivation of CDK4/6, which is known for its role in phosphorylating the retinoblastoma protein (Rb). However, recent evidence suggests that CDK4/6 targets other substrates beyond Rb that play important but less explored roles in regulating the G1/S checkpoint. In this project, we aim to identify and characterize novel CDK4/6 substrates and their phosphorylation patterns, exploring how these mechanisms contribute to cell cycle control and tumorigenesis. Through a combination of cutting-edge biochemical techniques and quantitative live-cell imaging, we will investigate how these new CDK4/6 substrates modulate the decision-making process during cell division in both normal and cancerous cells. The PhD candidate will have the opportunity to develop a multidisciplinary skill set, combining advanced molecular biology, cell biology, and state-of-the-art microscopy. The project will include extensive biochemical assays to define phosphorylation events, CRISPR/Cas9-mediated gene editing to study the functional impact of these substrates, and live-cell imaging to assess the dynamics of G1/S transition in real-time. Our ultimate goal is to uncover how dysregulation of these novel substrates drives aberrant cell proliferation in cancers, potentially opening up new therapeutic strategies targeting the CDK4/6 axis. The candidate will benefit from a collaborative environment, receiving mentorship across disciplines and contributing to a highly impactful area of cancer research.

The aberrant modification status of proteins is universally recognized as a crucial component of disease. In order to develop therapeutic agents to combat disease, we need to understand the role that posttranslational modifications (PTMs) play within pathological systems. Focusing on infectious diseases using mutant cell lines, mouse models and patient data, we will study the link between PTM status and subcellular location which has been so far poorly captured in the majority of experimental workflows. The knowledge of the PTM affecting relocalization of the protein and, in turn, its function, will be pivotal to the correct drug design. This project combines development of state of the art quantitative proteomics methodologies, computational workflows and whole cell modelling which will be used to decipher the mechanism of immunity to infection and propose new ways of treatment. 

Accumulation of misfolded proteins and aberrant protein aggregates are hallmarks of a wide range of pathologies such as neurodegenerative diseases and cancer. Under normal conditions, these potentially toxic protein species are kept at low levels due to a variety of quality control mechanisms that detect and selectively promote their degradation. Our lab investigates these protein quality control processes with a particular focus on ER-associated degradation (ERAD), that looks after membrane and secreted proteins. The ERAD pathway is evolutionarily conserved and in mammals, targets thousands of proteins influencing a wide range of cellular processes, from lipid homeostasis and stress responses to cell signaling and communication.

We investigate the mechanisms of ERAD using multidisciplinary approaches both in human and yeast cells. Using CRISPR-based genome-wide genetic screens and light microscopy experiments we identify and characterize molecular components involved in the degradation of disease-relevant toxic proteins. In parallel, we use biochemical tools to dissect mechanistically the various steps of the ERAD pathways. In this collaborative project with the Lea lab we will use structural approaches such as cryo-electron microscopy to gain insight into the molecular mechanisms of ERAD.

These studies, by providing mechanistic understanding of the ERAD process, may shed light on human diseases impacting ER function and may ultimately contribute to better therapeutics. 

Recent work in the Nitschke group has produced cages potentially capable of encapsulating proteins or nucleic acids. This project will develop the encapsulation of these biomolecules, and study their properties and potential therapeutic applications.

Creatine is an important energy storage and transfer molecule in muscle and brain but is synthesized primarily in the kidneys and liver. Hence, creatine uptake in skeletal muscle, brain, and heart is dependent on the creatine transporter (CrT or SLC6A8). Loss-of-function mutations in CrT are the second most common cause of X-linked intellectual disability and low tissue creatine levels result in skeletal muscle atrophy and are closely associated with heart failure. Cells down-regulate expression of CrT when creatine levels are high, but the mechanisms underlying this autoregulation and the importance to normal physiology and disease are unknown.

Recently, we found that creatine feedback inhibits translation of the CrT mRNA to control transporter production, and we identified elements in the CrT mRNA that are important for this control. This PhD project will involve molecular genetic analyses to more fully characterize the translational control mechanism(s) by which creatine feedback inhibits its own cellular uptake. In addition, CRISPR-Cas technology will be used to eliminate the translational control mechanisms in mice, and then physiological studies of the mice will be used to characterize the role of CrT autoregulation.

A signalling pathway linking nutrient availability to changes in gene expression that hinges on the phosphorylation of translation initiation 2 (eIF2) has long been known to exist. Recognized initially as the yeast General Control Response, recent convergent lines of research have implicated its metazoan counterpart, the Integrated Stress Response, in diverse physiological processes ranging from immunity to memory formation.

This PhD programme will exploit our emerging detailed understanding of translation initiation and termination to shed light on unanticipated mechanistic aspects of the ISR. An understanding of these details may inform efforts to target the ISR to therapeutic ends.

The goal of our research is to understand how cells use various protein quality control (PQC) strategies to eliminate misfolded proteins, and how defects in these processes lead to aging-associated neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Specifically, we study the molecular mechanisms underlying protein translocation-associated quality control at the endoplasmic reticulum (ER), the export of misfolded proteins via unconventional protein secretion, and cell-to-cell transmission of misfolded alpha-Synuclein and Tau aggregates. We envision that a thorough characterization of these protein quality control systems may one day improve both diagnosis and treatment of aging-associated neurodegenerative diseases.

The project will use X-ray crystallography, cryoEM, microbial genetics and molecular biology to explore how small RNAs and small proteins act as regulators with speed and precision in diverse bacteria.

The impact of proteins and their modification on disease states is now being recognized as crucial, but there are knowledge gaps that have to be filled to develop therapeutic agents to combat disease. Focusing on infectious diseases using mutant cell lines, mouse models and patient data we will study the link between PTM status and subcellular location which has been so far poorly captured in the majority of experimental workflows. The knowledge of the PTM affecting relocalization of the protein and, in turn, its function, may be pivotal to the correct drug design. This project combines development of state of the art quantitative proteomics methodologies, computational workflows and whole cell modelling which will be used to decipher the mechanism of immunity to infection and propose new ways of treatment.