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Weeks 6, 7 and 8

Final Weeks I decided to upload just one post covering the final 3 weeks, as they were very similar in that I spent them repeating the macrophage phagocytosis assay and analysing the images I obtained from microscopy. Laboratory Work Macrophage Phagocytosis - Calcofluor White and Concavalin A + FITC Staining This protocol was very similar as I described for week 5, except it involved additional cell permeabilization and second staining steps. Following infection with Candida yeast, samples were stained with calcofluor white, meaning that all external Candida were coloured blue. Then, I fixed the samples and permeabilized the cells before applying the second stain, which was concavalin A conjugated to FITC, which coloured both internal and external Candida green. As concavalin A binds to residues present in the macrophage as well as the Candida , these also became visible with the conA + FITC staining. External Candida Stained with Calcofluor White External and Int
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Recent posts

Week 5

First Week in the Johnston Laboratory My time in the Johnston laboratory will focus on one particular experiment, the macrophage phagocytosis assay. This week I first shadowed Robbie, the post-doc in Dr. Johnston's lab, and then had a go at doing the assay myself. Laboratory Work Macrophage Phagocytosis Assay This experiment aims to determine if and how wild-type and apm4 Δ mutant strains of Candida differ in their interaction with macrophages, a major cellular component of the innate immune system. Macrophages most prominently phagocytose, or "eat", and destroy pathogens in an attempt to protect the body from invading organisms. Due to the fact that: (i) we suspect that the apm4 Δ mutant may have a different cell wall composition in comparison to wild-type, some of the components of which I have been examining over the past 4 weeks (e.g. chitin); and (ii) cell wall components are integral for macrophage recognition of Candida ; this experiment gives a pathophy
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Week 4

My Last Week Solely in the Ayscough Laboratory This week was my last week in the Ayscough laboratory, as I will be doing experiments in the Johnston laboratory starting from Monday. However, I will still be growing up my Candida with Harriet and would like to squeeze in another cell wall microscopy session if possible; so it is not a permanent farewell just yet. Laboratory Work Experiment 1: Caspofungin Growth Curve Take Two This weeks experiment was almost identical to that of last week, except I used a different concentration of caspofungin, namely 0.2  µg/mL instead of 5 µg/mL. Remarkably, despite the staggered timings and attempting the experiment solo, everything ran smoothly. Unfortunately, my results were not the same as last weeks. Although I got very similar looking trends, these were the reverse of last weeks findings, in that the wild-type strain grew "better" than the apm4 Δ  mutant strain this time around. However, once again none of the differences were
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Week 3

Laboratory Work Experiment 1: Caspofungin Growth Curve The caspofungin plate spotting assay we set up last week did not show any differences between wild-type and apm4 Δ  mutant strains in their ability to grow in the presence of various concentrations of caspofungin. Wild-type and apm4Δ Strains Appear to Grow Similarly  in the Presence of Caspofungin. Wild-type (1st and 3rd rows) and apm4Δ mutant (2nd and 4th rows) strains were grown at 37˚C on a YPD plate supplemented with 0.16 µg/mL caspofungin. Because of this, we decided to do a literature search to find other caspofungin sensitivity assays in order to double check our results. With help from Harriet, I adapted an experiment which measured the OD 600  of cells growing in YPD media supplemented with various concentrations of caspofungin. As it would not be accurate to measure the OD of hyphal cells as an indication of total population size, we could only do this experiment with yeast. In addition, due to the fact that
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Week 2

BMS Away Day Firth Court Entrance At the beginning of the week I attended the Biomedical Science (BMS) research away day, which was held in Firth Hall. The day consisted of numerous talks and poster presentations given by various members of the four research areas which exist within the BMS department at the University of Sheffield. Namely, cell biology and cancer, developmental biology and disease, neuroscience, and stem cell and regenerative medicine. The day was really interesting as it allowed me to explore the work that is going on within the department; in addition to giving me an indication as to how to approach poster and flash talk presentations when I (hopefully) become a PhD student.   Laboratory Work Experiment 1: Microscopy This week I had my first go at microscopy; using standard transmitted light before moving on to stain some samples and use fluorescence. However, I wasn't able to gain any results from these latter experiments as our cells were co
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Week 1

Arrival View of Firth Court from the Laboratory Everyone that I have come into contact with so far has been incredibly friendly and welcoming, which has helped me settle into the laboratory in a relatively short space of time. My supervisor for this project is a fantastic second year PhD student named Harriet, who has involved me with experimental planning as well as helping me with practical laboratory work. On the first day, I attended a seminar given by  Dr. Evan Reid   on the role of spastin in membrane trafficking; and how this may contribute to hereditary spastic paraplegias (HSPs). This is an area of research that I have barely touched upon during my undergraduate study; which meant the talk itself was very informative as well as interesting.  Laboratory Work Experiment 1: Cell Wall Stress Plates This involved preparing YPD (the media used to support Candida growth) plates which contained various chemical compounds that exerted stress onto particular cell wall comp
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Introduction to the Project: The Role of Endocytosis in Regulating Cell Wall Composition in Candida Albicans

Background Candida Albicans (C. albicans) C. albicans is the most common human fungal pathogen, which causes various types of Candidiasis depending on the anatomical site of disease manifestation. It is perhaps most readily associated with thrush, which occurs in the throat and vagina. Both in vitro  and in vivo, Candida are capable of growing in a variety of morphogenic forms, namely yeast, pseudohyphae and true hyphae. These forms can be selectively induced by particular experimental conditions; for example, growing C. albicans at 28 o C and 37 o C induces yeast and true hyphal forms, respectively. Pseudohyphal, Yeast, and True Hyphal Forms of Candida albicans . Source: Sudbery, 2011   Endocytosis, AP-2 and Polarised Growth Yeast budding and hyphal growth are highly polarised events, that are co-ordinated in-part by the regulation of cell surface composition via endocytosis (membrane internalisation) and exocytosis (secretion). Both processes are highly complex
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